Journal of Clinical Medicine

Article Phytomedicine-Based Potent Antioxidant, Fisetin Protects CNS-Insult LPS-Induced Oxidative Stress-Mediated Neurodegeneration and Memory Impairment

Ashfaq Ahmad †, Tahir Ali †, Shafiq Ur Rehman † and Myeong Ok Kim * Division of Applied Life Science (BK 21), College of Natural Sciences, Gyeongsang National University, Jinju 52828, Korea; [email protected] (A.A.); [email protected] (T.A.); shafi[email protected] (S.U.R.) * Correspondence: [email protected]; Tel.: +82-55-772-1345; Fax: +82-55-772-2656 These authors contributed equally to this work. †


Received: 16 May 2019; Accepted: 11 June 2019; Published: 14 June 2019


Abstract: Phytomedicine based natural flavonoids have potent antioxidant, anti-inflammatory, and neuroprotective activities against neurodegenerative diseases. The aim of the present study is to investigate the potent neuroprotective and antioxidant potential effects of fisetin (natural flavonoid) against central nervous system (CNS)-insult, lipopolysaccharide (LPS)-induced reactive oxygen species (ROS), neuroinflammation, neurodegeneration, and synaptic/memory deficits in adult mice. The mice were injected intraperitoneally (i.p.) with LPS (250 µg/kg/day for 1 week) and a fisetin dosage regimen (20 mg/kg/day i.p. for 2 weeks, 1 week pre-treated to LPS and 1 week co-treated with LPS). Behavioral tests, and biochemical and immunofluorescence assays were applied. Our results revealed that fisetin markedly abrogated the LPS-induced elevated ROS/oxidative stress and activated phosphorylated c-JUN N-terminal Kinase (p-JNK) in the adult mouse hippocampus. Fisetin significantly alleviated LPS-induced activated gliosis. Moreover, fisetin treatment inhibited LPS-induced activation of the inflammatory Toll-like Receptors (TLR4)/cluster of differentiation 14 (CD14)/phospho-nuclear factor kappa (NF-κB) signaling and attenuated other inflammatory mediators (tumor necrosis factor-α (TNF-α), interleukin-1 β (IL1-β), and cyclooxygenase (COX-2). Furthermore, immunoblotting and immunohistochemical results revealed that fisetin significantly reversed LPS-induced apoptotic neurodegeneration. Fisetin improved the hippocampal-dependent synaptic and memory functions in LPS-treated adult mice. In summary, our results strongly recommend that fisetin, a natural potent antioxidant, and neuroprotective phytomedicine, represents a promising, valuable, and therapeutic candidate for the prevention and treatment of neurodegenerative diseases.

Keywords: phytomedicine; natural flavonoids; fisetin; central nervous system (CNS) insult; lipopolysaccharide (LPS); oxidative stress; neuroinflammation; neurodegeneration; synaptic and memory functions

1. Introduction Neuroinflammation is considered to be a key event in the neurodegeneration process inherent to various aging pathologies. Within the central nervous system (CNS), microglia are considered a key player in the immune system that trigger various neuroinflammatory responses [1]. Literature reviews suggest that microglial activation plays a pivotal role in various stress conditions including oxidation, neuroinflammation, and neurodegenerative diseases [2,3]. Under varying environmental conditions such as infections and the introduction of toxins and endogenous proteins, microglia

J. Clin. Med. 2019, 8, 850; doi:10.3390/jcm8060850 www.mdpi.com/journal/jcm J. Clin. Med. 2019, 8, x FOR PEER REVIEW 2 of 23 J. Clin. Med. 2019, 8, 850 2 of 23 become over-activated and release reactive oxygen species (ROS) that cause neurotoxicity and initiate an immune response via toll-like receptors (TLRs), whose activation communicates downstream becomeinflammatory over-activated mediators and [4,5]. release The reactiveelevated oxygen levels of species oxidative (ROS) stress that resulting cause neurotoxicity from an increased and initiate level anof immuneROS production, response a viadecrease toll-like in receptorsthe antioxidant (TLRs), system, whose or activation both, play communicates a critical role downstreamin the aging inflammatoryprocess and mediatorsin the development [4,5]. The elevated of levelsdegenerat of oxidativeive diseases stress resulting[6]. The from inflammatory an increased agent level oflipopolysaccharide ROS production, a (LPS) decrease is inan the endotoxin antioxidant derived system, from or both, the playouter a criticalmembrane role in of the Gram-negative aging process andbacteria in the and development is a strong activator of degenerative of host defense diseases responses. [6]. The Recent inflammatory studies suggested agent lipopolysaccharide that the systemic (LPS)administration is an endotoxin of LPS derivedcauses inflammatory from the outer processe membranes in the of body, Gram-negative which causes bacteria deleterious and is effects a strong in activatorthe brain of [7–13]. host defense LPS administration responses. Recent induces studies memory suggested impairments that the and systemic neuroinflammation administration via of LPSactivated causes TLR4/NF inflammatoryKB signaling processes and regulates in the body, various which downst causesream deleterious proinflammator effects iny themediators, brain [7 –such13]. LPSas nitric administration oxide (NO), induces prostaglandin memory E2 impairments (PGE2), and and cyclooxygenase neuroinflammation (COX)2, via activatedand proinflammatory TLR4/NFκB signalingcytokines, and including regulates interleukin-1 various downstream (IL-1), IL-6, proinflammatory and tumor necrosis mediators, factor- suchα (TNF- as nitricα) oxide[14]. Recent (NO), prostaglandinstudies have suggested E2 (PGE2), that and among cyclooxygenase the mammalian (COX)2, TLRs and families, proinflammatory TLR4 plays cytokines, an important including role in interleukin-1inflammation (IL-1), pathogenesis. IL-6, and tumor TLR4 necrosis acts as factor- a primaryα (TNF- initiatorα)[14]. Recentof innate studies immune have suggested responses that to amongpathogens the mammalianby activating TLRs a cascade families, of pro-inflamma TLR4 plays antory important events. The role LPS in inflammation administration pathogenesis. triggers the TLR4activation acts asof aTLR4 primary via downstream initiator of innate signaling immune factors, responses such as toadaptor pathogens myeloid by activating differentiation a cascade protein of pro-inflammatory88 (MyD88), leading events. to the The activation LPS administration of nuclear triggersfactor-KB the (NF- activationKB) and of ultimately TLR4 via downstreaminducing the signalingexpression factors, of inflammation-related such as adaptor myeloid genes. di Thefferentiation systematic protein LPS administration 88 (MyD88), leading impaired to the learning activation and ofmemory nuclear performance, factor-κB (NF- increasedκB) and ultimatelyamyloid beta inducing (Aβ)-burden, the expression and diminished of inflammation-related synaptic and memory genes. Thefunctions systematic [13,15,16]. LPS administration impaired learning and memory performance, increased amyloid beta (ATheβ)-burden, epidemiological and diminished comprehensive synaptic surveys and memory reported functions that over [13 the,15 past,16]. few years, it has become a primaryThe epidemiological focus of researchers comprehensive to search for surveys potential reported effective that therapeutic over the past agents few from years, natural it has becomesources ato primary combat focusinflammatory of researchers and neurodegenerative to search for potential diseases. effective Among therapeutic the natural agents sources, from naturalphytomedicine sources toderived combat flavonoids inflammatory are a and primary neurodegenerative focus because diseases. of their Amongpotent biological the natural and sources, therapeutic phytomedicine activities derivedwhich are flavonoids beneficial are for a primaryhealth improvements focus because of[17–20]. their potent Flavonoids biological are andpolyphenolic therapeutic compounds activities whichabundantly are beneficial found in for foods health and improvements vegetables and [17 widely–20]. Flavonoids used as nutritional are polyphenolic supplements compounds for the abundantlytreatment of founddiabetes, in obesity, foods and and vegetables cardiovascular and and widely neurological used as nutritionaldisorders [17,21–24]. supplements Fisetin for (3, the 7, treatment3,4-tetrahydroxyflavone) of diabetes, obesity, (Figure and 1), cardiovascular is a well-known and and neurological potent flavonoid, disorders which [17,21 is –commonly24]. Fisetin found (3, 7, 3,4-tetrahydroxyflavone)in many fruits and vegetables (Figure such1), is as a well-knownapples, grapes, and kiwis, potent strawberries, flavonoid, which onions, is commonlypersimmons, found and incucumbers. many fruits Several and vegetables studies have such suggested as apples, that grapes, fisetin kiwis, is a biochemically strawberries, and onions, physiologically persimmons, active and cucumbers.flavonoid based Several phytomedicine, studies have suggestedshowing various that fisetin strong is a pharmacological biochemically and activities physiologically against cancer, active flavonoidoxidative stress, based phytomedicine,inflammatory bowel showing disease, various and co stronggnitive/ pharmacological ynaptic dysfunctions activities [25–30]. against Similarly, cancer, oxidativewe also recently stress, inflammatoryreported the neuroprotective bowel disease, andeffect cognitive of fisetin/ynaptic against dysfunctions Aβ-induced [neurotoxicity25–30]. Similarly, and wecognitive/synaptic also recently reported dysfunctions the neuroprotective [31]. Therefore, effect the of present fisetin againststudy was Aβ -induceddesigned neurotoxicityto investigate and the cognitivepotential/ antioxidantsynaptic dysfunctions and neuroprotective [31]. Therefore, effects theof phytomedicine-based present study was designed small flavonoid to investigate molecules the potentialsuch as fisetin antioxidant against and the neuroprotective LPS-induced neurotoxicity effects of phytomedicine-based in the adult brain smallwith particular flavonoid moleculesand major suchfocus as to fisetin the hippocampus. against the LPS-induced neurotoxicity in the adult brain with particular and major focus to the hippocampus.

Figure 1. Chemical structure of phytomedicine-based fisetin.

J. Clin. Med. 2019, 8, x FOR PEER REVIEW 3 of 23

Figure 1. Chemical structure of phytomedicine-based fisetin. J. Clin. Med. 2019, 8, 850 3 of 23 2. Materials and Methods 2. Materials and Methods 2.1. Chemicals 2.1. ChemicalsFisetin, LPS, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemical Co. (St. Louis,Fisetin, MO, LPS, USA). and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2.2. Animals Used in the Experiment 2.2. AnimalsAnimals Used used in for the the Experiment experimental purpose were wild-type C57BL/6N mice (28–32 g, 10 weeks old) Animalspurchased used from for Samtako the experimental Bio (Osan, purpose S. Korea). were The wild-type mice were C57BL kept/ 6Nfor mice1 week (28–32 in the g, university 10 weeks old)animal purchased housing from for acclimatization Samtako Bio (Osan, under S. a Korea).12-hr/12 The-hr light/dark mice were cycle kept forat a 1 controlled week in the temperature university animal(23 °C) housingwith 60 ± for 10% acclimatization humidity and under were provided a 12-hr/12-hr with light food/dark and cyclewater at ad a libitum. controlled For temperature animal care (23and C)treatment, with 60 we10% have humidity followed and the were guidelines provided (A withpproval food ID: and 125) water issued ad libitum.by the ethics For animal committee care ◦ ± and(IACUC), treatment, Division we haveof Applied followed Life the Sciences, guidelines Gyeo (Approvalngsang National ID: 125) University, issued by theSouth ethics Korea. committee Efforts (IACUC),were made Division to minimize of Applied the suffering Life Sciences, of the animals. Gyeongsang National University, South Korea. Efforts were made to minimize the suffering of the animals. 2.3. Study Designing, Animals Grouping, and Experimental Approach 2.3. Study Designing, Animals Grouping, and Experimental Approach In order to accomplish our hypothesis, we designed the following studies (Figure 2) as per our previousIn order reports to accomplish [9,12,31], and our designed hypothesis, fundamental we designed studies the followingparticularly studies for the (Figure optimization2) as per of our the previousfisetin dosage reports regimen. [9,12,31 To], and evade designed ambiguous fundamental sex-dependent studies differences, particularly we for involved the optimization only male of mice the fisetinin the dosagestudy. regimen.Adult male To evademice (13 ambiguous mice per sex-dependent group) were dirandomlyfferences, divided we involved into the only following male mice 3 ingroups: the study. (i) mice Adult treated male micewith (13saline mice as per a vehicle group) for were 14 randomlydays (control divided (C)); into (ii) themice following treated with 3 groups: LPS (i)injected mice treatedat a dose with of 250 saline μg/kg as a for vehicle 7 days; for and 14 days(iii) mice (control treated (C)); with (ii) miceLPS (250 treated μg/kg) with for LPS 7 days injected and atfisetin a dose (20 of mg/kg) 250 µg for/kg 14 for days 7 days; (7 days and prior (iii) mice to LP treatedS and with7 days LPS with (250 LPS;µg /LPS+Fis).kg) for 7 daysFisetin and was fisetin first (20dissolved mg/kg) in for 0.1% 14 days (v/v (7) DMSO days prior and to then LPS was and 7diluted days with with LPS; saline LPS such+Fis). that Fisetin the wastotal firstadministered dissolved involume 0.1% (vwas/v) DMSOin 0.9% and saline. then The was LPS, diluted fisetin, with and saline saline such were that administered the total administered intraperitoneally volume (i.p.) was into 0.9%the mice. saline. The LPS, fisetin, and saline were administered intraperitoneally (i.p.) to the mice.

Figure 2. Study design, animals grouping, dosage regimen for drug and behavioral analyses as well as the biochemical and morphological experimental approach for the whole study. J. Clin. Med. 2019, 8, 850 4 of 23

2.4. Behavioral Studies To know the beneficial effect of phytomedicine-based fisetin on behavioral performance, we designed a behavioral study (13 mice/group) through a Morris water maze (MWM) and a Y-maze test. The MWM test is a parameter task to evaluate memory functions. The experimental apparatus consisted of a circular water tank (100 cm in diameter, 40 cm in height) containing water (23 1 C) ± ◦ to a depth of 15.5 cm, which was rendered opaque by adding white paint. A transparent escape platform (10 cm in diameter, 20 cm in height) was hidden 1 cm below the water surface and placed at the midpoint of one quadrant. The MWM test was started on day 7 and completed on day 14 of the experimental schedule (Figure2). Each mouse received 3–4 training periods each day for 4 consecutive days using a single hidden platform in one quadrant with three rotating starting quadrants. Latency to escape from the water maze (finding the submerged escape platform) was calculated for each trial. The probe test was performed by removing the platform and allowing each mouse to swim freely for 60 s. The number of crossing and time spent in the target quadrant (where the platform was located during hidden platform training) was measured. The time spent in the target quadrant was considered to represent the degree of memory consolidation that has taken place after learning. All data were recorded using video-tracking software (SMART, Panlab Harvard Apparatus Bioscience Company, Holliston, MA, USA). The Y-maze was constructed from painted black wood. Each arm of the maze was 50 cm long, 20 cm high, and 10 cm wide at the bottom and top. The Y-maze was initiated on day 12 and completed on day 14 of the experimental schedule (Figure2). Each mouse was placed at the center of the apparatus and allowed to move freely through the maze for three 8-min sessions. The series of arm entries was visually observed. The spontaneous alternation was defined as the successive entry of the mice into the three arms in overlapping triplet sets. Alteration behavior (%) was calculated as follows: (successive triplet sets (entries into three different arms consecutively)/total number of arm entries-2) 100. × 2.5. Protein Extraction from Mice Brains for Biochemical Analyses At the completion of behavioral testing, mice brains were immediately removed, and hippocampal tissue was carefully separated, frozen on dry ice, and stored at 80 C until processing. Further, the − ◦ hippocampal tissue was homogenized in 0.2 M Phosphate Buffer saline (PBS) with a phosphatase inhibitor and protease inhibitor cocktail and then centrifuged at 10,000 g at 4 C for 25 min. × ◦ The supernatants were collected and stored at 80 C until processing for biochemical analyses. − ◦ 2.6. Western Blot Analysis The protein concentrations were measured through a BioRad protein assay kit (BioRad Laboratories, CA, USA). Equal amounts of protein (20–30 µg) underwent electrophoresis using 4%–12% BoltTM Mini Gels (Novex, Life Technologies, Kiryat Shmona, Israel). The membranes were blocked in 5% (w/v) skim milk to reduce non-specific binding and incubated with primary antibodies (Table1) overnight at 4 ◦C at a 1:1000 dilution. After undergoing a reaction with a horseradish peroxidase-conjugated secondary antibody, as appropriate, the proteins were detected using an Electrochemiluminescence (ECL) detection reagent according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). Then, X-ray films were scanned, and the optical densities of the bands were analyzed through densitometry using the computer-based Sigma Gel program, version 1.0 (SPSS, Chicago, IL, USA).

2.7. Antibodies Used in the Western Blotting The primary antibodies used in the Western blot analysis are explained in Table1. The secondary antibodies used in our experiments, goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG were purchased from Santa Cruz Biotechnology. J. Clin. Med. 2019, 8, 850 5 of 23

Table 1. Primary antibodies detail information.

Antibody Host Application Manufacturer Catalog Number Concentration Santa Cruz Iba-1 Rabbit WB/IF SC: 98468 1:1000/1:100 Biotechnology, USA GFAP Mouse WB = SC: 33673 1:1000 p-JNK Mouse WB/IF = SC: 6254 1:1000/1:100 TLR-4 Goat WB = SC: 16240 1:1000 p-NF-κB Mouse WB/IF = SC 8008 1:1000/1:100 CD14 Mouse WB = SC: 58951 1:1000 TNF-α Mouse WB/IF = SC: 8436 1:1000/1:100 COX-2 Rabbit WB = SC: 7951 1:1000 IL-1β Mouse IF = SC: 32294 1:100 Apaf-1 Mouse WB = SC: 65891 1:1000 Cyto. c Mouse WB = SC: 13156 1:1000 PARP-1 Mouse WB = SC: 8007 1:1000 Caspase-3 Mouse WB = SC: 7272 1:1000 PSD-95 Mouse WB/IF = SC: 71933 1:1000/1:100 Synaptophysin Rabbit WB = SC: 17750 1:1000 SNAP-23 Mouse WB = SC: 374215 1:100 Caspase-9 Rabbit WB Cell Signaling, USA 9508S 1:1000 p-CREB (Ser 133) Rabbit WB = 9198S 1:1000 p-GluR1 (Ser 845) Rabbit WB = 8084S 1:1000 p-IKKBβ/α Rabbit WB Abcam, USA Ab59195 1:1000 8-OxoG Mouse IF Millipore MAB3560 1:100 WB: Western blot; IF: immunofluorescence.

2.8. Tissue Sample Preparation for Morphological Analysis At the completion of behavioral testing, mice were perfused transcardially with 4% ice-cold paraformaldehyde, and brains were post-fixed for 72 h in 4% paraformaldehyde and transferred to 20% sucrose for 72 h. Then, brains were frozen in O.C.T. compound (A.O, USA), and 14-µm coronal sections were cut using a CM 3050C cryostat (Leica, Wetzlar, Germany). The sections were thaw-mounted on Probe-on Plus charged slides (Fisher, Rock-ford, IL, USA).

2.9. Immunofluorescence Staining The morphological evaluations were performed as previously described with some modifications [13,21]. The prepared tissue slides were washed twice for 10 min in 0.01 M PBS, followed by incubation for 1 h in a blocking solution containing 2% normal serum according to the antibody treatment and 0.3% Triton X-100 in PBS. After blocking, the slides were incubated overnight at 4 ◦C in the primary antibodies (mouse polyclonal phosphorylated c-JUN N-terminal Kinase (p-JNK), rabbit polyclonal anti-p-NFκB, mouse polyclonal TNF-α, rabbit polyclonal anti- ionized calcium-binding adaptor molecule 1 (Iba-1), mouse monoclonal post synaptic density-95 (PSD-95) from Santa Cruz Biotechnology and mouse monoclonal 8-Oxoguanine (8-OxoG) from Millipore) and diluted 1:100 in blocking solution. After incubation with primary antibodies, the sections were incubated for 2 h in the secondary tetramethylrhodamine (TRITC)/fluorescein isothiocyanate (FITC)-labeled antibodies (1:50) (Santa Cruz Biotechnology, Dallas, Texas, USA). After secondary antibody incubation, tissue slides were washed twice for 5 min. Slides were mounted with 40, 60-diamidino-2-phenylindole (DAPI) and Prolong Antifade Reagent (Molecular Probe, Eugene, OR, USA). Then, slides were examined using a confocal laser-scanning microscope (Flouview FV 1000, Olympus, Tokyo, Japan).

2.10. Fluoro-Jade B (FJB) Staining FJB staining was performed as previously described [13] with some modifications. After air-drying the tissue slides overnight, the slides were immersed in a solution of 1% sodium hydroxide and 80% ethanol for 5 min. Then, slides were immersed in 70% alcohol and distilled water for 2 min each. Tissue slides were transferred to a solution of 0.06% potassium permanganate for 10 min, rinsed with distilled water and then immersed in a solution of 0.1% acetic acid and 0.01% FJB for 20 min. The slides J. Clin. Med. 2019, 8, 850 6 of 23 were washed with distilled water and allowed to dry for 10 min. Glass coverslips were mounted using Dibutylphthalate Polystyrene Xylene (DPX) non-fluorescent mounting medium, and images were assessed with a confocal laser scanning microscope (Flouview FV 1000 MPE, Olympus, Tokyo, Japan). The images were proceeded for quantification using the computer-based ImageJ program.

2.11. Cresyl Violet (Nissl) Staining Cresyl violet (Nissl) staining was performed to evaluate the histological examination and extent of neuronal cell death or neuronal survival. Slides containing 14-µm sections of tissue were washed twice for 15 min in 0.01 M PBS and stained with a 0.5% cresyl violet solution (containing a few drops of glacial acetic acid) for 10–15 min. Then, sections were washed with distilled water and dehydrated in a graded ethanol series (70%, 95%, and 100%), placed in xylene and coverslipped using mounting medium, and finally, slides were examined with fluorescent light microscopy. The results were analyzed using the computer-based ImageJ program.

2.12. ROS Assay in Mouse Hippocampus Homogenates

The ROS assay was based on the oxidation of 20 70-dichlorodihydrofluorescein diacetate (DCFH-DA) to 20 70-dichlorofluorescein (DCF). Subsequently, brain homogenates were diluted with ice-cold Lock’s buffer at a 1:20 ratio to make a final concentration of 2.5 mg tissue/500 mL. The reaction mixture of Lock’s buffer (1 mL, pH = 7.4), 0.2 mL homogenate, and 10 mL of the stock solution of DCFH-DA (5 mM) was incubated at room temperature for 15 min to convert DCFH-DA to the fluorescent product DCF. The conversion of DCFH-DA to DCF was performed using a spectrofluorimeter with excitation at 484 nm and emission at 530 nm. For background fluorescence assessment (conversion of DCFH-DA in the absence of homogenate), we ran parallel blanks. The quantification analysis of ROS is expressed as pmol DCF formed/mg protein.

2.13. Lipid Peroxidation (LPO) Analysis in Mouse Hippocampus Homogenates The LPO levels were determined in the hippocampus (n = 8 mice per group) homogenates through analyzing the malondialdehyde (MDA) level, a biomarker of LPO, by using the commercial lipid peroxidation kit (catalog # K739-100) from Biovision Incorporated, A 95035 USA. The assay was performed according to the provided protocol.

2.14. Glutathione (GSH) Analysis in Mouse Hippocampus Homogenates The GSH levels in the hippocampus (n = 8 mice per group) homogenates were assessed by using the commercially available glutathione assay kit (BioVision’s catalog #K264-100) according to the provided protocol.

2.15. Statistical Analysis Western blot bands were scanned and analyzed through densitometry using the Sigma Gel System (SPSS Inc., Chicago, IL). Density values are expressed as the mean standard error of the mean (SEM). ± ImageJ software was used for immunohistological quantitative analysis. The data are the mean SEM. ± Statistical analysis was performed through one-way ANOVA followed by post-hoc analysis. Statistical calculations and graphs were made through Prism 5 software (Graph-Pad Prism 5 Software, San Diego, USA). P-values less than 0.05 were considered to be statistically significant. * p < 0.05 control versus LPS, # p < 0.05, LPS versus LPS + fisetin.

3. Results

3.1. Effect of Fisetin Dosage Regimen on LPS-Induced Oxidative Stress in the Mouse Brain A recently reported study showed that LPS exposure mediates ROS accumulation, which in turn enhances the expression level of pro-inflammatory mediators in addition to playing the main J. Clin. Med. 2019, 8, 850 7 of 23 role in various neurological disorders [29]. Herein, consistently, we also observed that systemic LPS administration enhanced the production and accumulation of ROS and oxidative stress (p < 0.05). Further, we found that fisetin treatments significantly (p < 0.05) attenuated the upregulated levels of ROS and LPO compared to the LPS-only treated group (Figure3A,B). We next performed the GSH assay to study alterations in oxidative stress levels. We observed reduced levels of GSH (p < 0.05) in the LPS-injected mice brain than that of the control saline-injected mice. Fisetin treatment to the LPS-treated group escalated the GSH expression level as compared to the LPS-only treated group (p < 0.05) (Figure3C). We also performed immunofluorescence analysis to evaluate the expression of 8-OxoG, a predominant parameter of oxidative stress and potentially expressed in the degenerated brain [27]. Interestingly, our immunofluorescence results indicated that LPS significantly enhances the immunofluorescence reactivity of 8-OxoG in the Cornu Ammonis 1 (CA1) (molecular layer and pyramidal cells) and Cornu Ammonis 3 (CA3) (molecular layer and pyramidal cells) regions as well as in the Dentate gyrus (DG) (hilum and granular cells) region of the hippocampus (p < 0.05). Fisetin dosage significantly reversed the increased expression levels of 8-OxoG in the fisetin-treated group relative to LPS-only treated group (p < 0.05) (Figure3D). These results suggest that the natural-based phytomedicine flavonoid, fisetin, has the potential effect to mitigate the systemic LPS-induced accumulatedJ. Clin. Med. ROS 2019 and, 8, x FOR LPO PEER in REVIEW the hippocampus of adult mice brains. 8 of 23

FigureFigure 3. Effect 3. Effect of fisetin of fisetin treatments treatments on on lipopolysaccharide lipopolysaccharide (LPS)-induced (LPS)-induced oxidative oxidative stress stress in a mouse in a mouse brain. (A) The graph represents the levels of reactive oxygen species (ROS) accumulation in the brain. (A) The graph represents the levels of reactive oxygen species (ROS) accumulation in the hippocampi of adult mice. The results were shown as the means ± SEM (number = 8 mice/group) for hippocampi of adult mice. The results were shown as the means SEM (number = 8 mice/group) three repeated and reproducible independent experiments. (B) The ±graph represents the levels of for threemalondialdehyde repeated and reproducible(MDA) in the hippocampi independent of adult experiments. mice. The (resultsB) The were graph shown represents as the means the levels ± of malondialdehydeSEM (number (MDA) = 8 mice/group) in the hippocampi for three repeated of adult and mice. reproducible The results independent were shown experiments. as the means (C) The SEM ± (numbergraph= 8 micerepresents/group) the forlevels three of glutathione repeated and (GSH) reproducible in the hippocampi independent of adult experiments. mice. The results (C) were The graph representsshown the as levels the means of glutathione ± SEM (number (GSH) = 8 mice/group) in the hippocampi for three repeated of adult and mice. reproducible The results independent were shown as theexperiments. means SEM (D) Representative (number = 8 images mice/group) of immunofluore for threescence repeated of 8-OxoG and (green, reproducible FITC; blue, independent 4′, 6′- diamidino-2-phenylindole± (DAPI)) in the CA1 (molecular layer and pyramidal cells), CA3 (molecular experiments. (D) Representative images of immunofluorescence of 8-OxoG (green, FITC; blue, 40, layer and pyramidal cells) and DG (hilum and granular cells) regions of the hippocampi of adult mice. 6 -diamidino-2-phenylindole (DAPI)) in the CA1 (molecular layer and pyramidal cells), CA3 (molecular 0 The results were shown as the means ± SEM (number = 5 mice/group) for three repeated and layer andreproducible pyramidal independent cells) and experiments. DG (hilum Magnified and granular 10×. Scale cells) bar regions = 50 μm. of * the shows hippocampi a significant of adult mice. The results were shown as the means SEM (number = 5 mice/group) for three repeated and difference between control and LPS-treated± groups; # shows a significant difference between LPS- reproducibletreated and independent LPS+Fis-treate experiments.d groups. Significance Magnified = p < 10 0.05.. Scale bar = 50 µm. * shows a significant × difference between control and LPS-treated groups; # shows a significant difference between LPS-treated and3.2. LPS Effect+Fis-treated of Fisetin Dosage groups. on Significance LPS-Induced =Activap < 0.05.tion of p-JNK Expressions in the Mouse Hippocampus Various studies have demonstrated that LPS treatment exaggerates the expression of the stress associated kinases. Among them, p-JNK is the main marker which promotes neuroinflammation and neurodegeneration [9,31]. Recently, a well-known study demonstrated that LPS induced the activation of p-JNK, which led to neuroinflammation and neuronal cell death [31]. In our study, we also investigated the LPS-induced activation of p-JNK expression through Western blot analysis. Our results revealed that fisetin treatments significantly reversed the upregulated expression level of p- JNK proteins in the LPS-treated group (p < 0.05) relative to the LPS-only treated group (p < 0.05) (Figure 4A). To confirm the Western blot results, we performed immunofluorescence staining of p- JNK. Our immunofluorescence results confirmed that LPS-treatment increased levels of p-JNK (p < 0.05) in the DG (hilum and granular cells) and CA3 (molecular layer and pyramidal cells) regions of the hippocampus compared to saline-treated controls (Figure 4B), while fisetin supplementation for two weeks significantly (p < 0.05) inhibited the higher expression and immunofluorescence reactivity of p-JNK in the DG (hilum and granular cells) and CA3 (molecular layer and pyramidal cells) of the LPS-treated group compared to the LPS-only treated group (Figure 4B).

J. Clin. Med. 2019, 8, 850 8 of 23

3.2. Effect of Fisetin Dosage on LPS-Induced Activation of p-JNK Expressions in the Mouse Hippocampus Various studies have demonstrated that LPS treatment exaggerates the expression of the stress associated kinases. Among them, p-JNK is the main marker which promotes neuroinflammation and neurodegeneration [9,31]. Recently, a well-known study demonstrated that LPS induced the activation of p-JNK, which led to neuroinflammation and neuronal cell death [31]. In our study, we also investigated the LPS-induced activation of p-JNK expression through Western blot analysis. Our results revealed that fisetin treatments significantly reversed the upregulated expression level of p-JNK proteins in the LPS-treated group (p < 0.05) relative to the LPS-only treated group (p < 0.05) (Figure4A). To confirm the Western blot results, we performed immunofluorescence staining of p-JNK. Our immunofluorescence results confirmed that LPS-treatment increased levels of p-JNK (p < 0.05) in the DG (hilum and granular cells) and CA3 (molecular layer and pyramidal cells) regions of the hippocampus compared to saline-treated controls (Figure4B), while fisetin supplementation for two weeks significantly (p < 0.05) inhibited the higher expression and immunofluorescence reactivity of p-JNK in the DG (hilum and granular cells) and CA3 (molecular layer and pyramidal cells) of the J.LPS-treated Clin. Med. 2019 group, 8, x FOR compared PEER REVIEW to the LPS-only treated group (Figure4B). 9 of 23

Figure 4. EffectEffect of of fisetin fisetin on the LPS-induced activati activationon of p-JNK expression levels in the mouse hippocampus. (A) (A) The The Western Western blot band of p-JNK p-JNK was was quan quantifiedtified using Sigma Gel software, software, and the differencesdifferences areare representedrepresented by by a a histogram. histogram.β -actinβ-actin was was used used as as a loadinga loading control. control. The The density density values values are areexpressed expressed in arbitrary in arbitrary units units (A.U.) (A.U.) as the as meansthe meansSEM ± SEM (number (number= 8 mice= 8 mice/group)/group) for threefor three repeated repeated and ± andreproducible reproducible independent independent experiments. experiments. (B) A representative(B) A representative image of immunofluorescenceimage of immunofluorescence staining of stainingp-JNK (green, of p-JNK FITC; (green, blue, FITC; DAPI) blue, in the DAPI) DG (hilumin the DG and (hilum granular and cells) granular and CA3cells) (molecularand CA3 (molecular layer and layerpyramidal and pyramidal cells) regions cells) of theregions hippocampi of the hippocamp of adult mice.i of Theadult results mice. were The shownresults aswere the meansshown asSEM the ± means(number ± SEM= 5 mice (number/group) = 5 for mice/group) three repeated for three and reproducible repeated and independent reproducible experiments. independent Magnified experiments. 10 . × MagnifiedScale bar = 1050×µ. m.Scale * shows bar = a50 significant μm. * shows diff erencea significant between difference controland between LPS-treated control groups; and LPS-treated # shows a groups;significant # dishowsfference a betweensignificant LPS-treated difference and betw LPS+eenFis-treated LPS-treated groups. and Significance LPS+Fis-treated= p < 0.05. groups. Significance = p < 0.05.

3.3. Effect of Fisetin on LPS-Induced Activation of Microglia and Astrocytes in the Adult Mouse Hippocampus. Prominent in vitro and in vivo studies have demonstrated that LPS exposure induces glial activation, neuroinflammation, and neurodegeneration. Activated gliosis and neuroinflammation are crucial factors involved in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). The LPS-injection significantly induces astrocyte activation in an inflammatory rodent model [7,12,13,32]. Therefore, in our study, we also investigated the LPS-induced glial activation in the adult mouse hippocampus. Our Western blot results showed that LPS injection significantly induced the activation of both astrocytes and microglia in the LPS-treated group compared to the saline-treated control group (p < 0.05) (Figure 5A). Interestingly, fisetin dosage (20 mg/kg/day for 2 weeks, 1 week prior LPS and 1 week co-treated with LPS) suppressed the activation of both Iba-1 and GFAP protein expression levels compared to the LPS-only treated group (p < 0.05). Further, we also performed immunofluorescence analysis for Iba- 1 reactivity in the adult mouse hippocampus. Our morphological results demonstrated that LPS treatment significantly increases the immunofluorescence reactivity of activated Iba-1 in the CA1 (molecular layer and pyramidal cells) and CA3 (molecular layer and pyramidal cells) regions as well as in the DG (hilum and granular cells) regions of the hippocampus (p < 0.05). Fisetin treatment significantly reduced the amount of activated Iba-1 in LPS-treated mice compared to mice treated with LPS alone (p < 0.05) (Figure 5B). Therefore, these results suggest that fisetin acts as a potent

J. Clin. Med. 2019, 8, 850 9 of 23

3.3. Effect of Fisetin on LPS-Induced Activation of Microglia and Astrocytes in the Adult Mouse Hippocampus Prominent in vitro and in vivo studies have demonstrated that LPS exposure induces glial activation, neuroinflammation, and neurodegeneration. Activated gliosis and neuroinflammation are crucial factors involved in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). The LPS-injection significantly induces astrocyte activation in an inflammatory rodent model [7,12,13,32]. Therefore, in our study, we also investigated the LPS-induced glial activation in the adult mouse hippocampus. Our Western blot results showed that LPS injection significantly induced the activation of both astrocytes and microglia in the LPS-treated group compared to the saline-treated control group (p < 0.05) (Figure5A). Interestingly, fisetin dosage (20 mg/kg/day for 2 weeks, 1 week prior LPS and 1 week co-treated with LPS) suppressed the activation of both Iba-1 and GFAP protein expression levels compared to the LPS-only treated group (p < 0.05). Further, we also performed immunofluorescence analysis for Iba-1 reactivity in the adult mouse hippocampus. Our morphological results demonstrated that LPS treatment significantly increases the immunofluorescence reactivity of activated Iba-1 in the CA1 (molecular layer and pyramidal cells) and CA3 (molecular layer and pyramidal cells) regions as well as in the DG (hilum and granular cells) regions of the hippocampus (p < 0.05). Fisetin treatment significantly reduced the amount of

J.activated Clin. Med. 2019 Iba-1, 8, inx FOR LPS-treated PEER REVIEW mice compared to mice treated with LPS alone (p < 0.05) (Figure10 of5 B).23 Therefore, these results suggest that fisetin acts as a potent antioxidant and might possess potent antioxidantanti-inflammatory and might activity possess through potent the suppression anti-inflammatory of activated activity astrocytes through and microgliathe suppression in the adult of activatedmouse hippocampus. astrocytes and microglia in the adult mouse hippocampus.

Figure 5. Cont.

Figure 5. Effect of fisetin on LPS-induced activation of microglia and astrocytes in the adult mouse hippocampus. (A) The Western blot analysis of microglia (Iba-1) and astrocyte (GFAP) antibodies were quantified using Sigma Gel software, and the differences are represented by a histogram. β-actin was used as a loading control. The density values are expressed in arbitrary units (A.U.) as the means ± SEM (number = 8 mice/group) for three repeated and reproducible independent experiments. (B) The representative image of immunofluorescent staining of Iba-1 in the CA1 (molecular layer and pyramidal cells), CA3 (molecular layer and pyramidal cells), and DG (hilum and granular cells) regions of the hippocampi of adult mice. The results were shown as the means ± SEM (number = 5 mice/group) for three repeated and reproducible independent experiments. Magnified 10×. Scale bar = 50 μm. * shows a significant difference between control and LPS-treated groups; # shows a significant difference between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05.

3.4. Effect of Fisetin on LPS-Induced Activation of TLR4/ NFKB Signaling and Inflammatory Mediators in the Adult Mouse Hippocampus

J. Clin. Med. 2019, 8, x FOR PEER REVIEW 10 of 23 antioxidant and might possess potent anti-inflammatory activity through the suppression of activated astrocytes and microglia in the adult mouse hippocampus.

J. Clin. Med. 2019, 8, 850 10 of 23

Figure 5. Effect of fisetin on LPS-induced activation of microglia and astrocytes in the adult mouse Figurehippocampus. 5. Effect of fisetin (A) The on Western LPS-induced blot analysis activation of of microglia microglia (Iba-1) and astrocytes and astrocyte in the (GFAP) adult antibodiesmouse hippocampus.were quantified (A) The using Western Sigma Gelblot software, analysis andof microglia the differences (Iba-1) are and represented astrocyte by(GFAP) a histogram. antibodiesβ-actin werewas quantified used as using a loading Sigma control. Gel software, The density and the valuesdifferences are expressedare represented in arbitrary by a histogram. units (A.U.) β-actin as the wasmeans used as aSEM loading (number control.= 8 The mice density/group) values for three are repeated expressed and in reproduciblearbitrary units independent (A.U.) as the experiments. means ± ± SEM(B) The(number representative = 8 mice/group) image offor immunofluorescent three repeated and staining reproducible of Iba-1 independent in the CA1 (molecularexperiments. layer (B) and Thepyramidal representative cells), image CA3 (molecular of immunofluorescent layer and pyramidal staining cells), of Iba-1 and DGin the (hilum CA1 and(molecular granular layer cells) and regions pyramidalof the hippocampi cells), CA3 of (molecular adult mice. layer The resultsand pyrami were showndal cells), as the and means DG (hilumSEM (numberand granular= 5 mice cells)/group) ± regionsfor threeof the repeated hippocampi and reproducibleof adult mice. independent The results were experiments. shown as Magnified the means 10± SEM. Scale (number bar = =50 5 µm. × mice/group)* shows a for significant three repeated difference and between reproduc controlible independent and LPS-treated experiments. groups; # showsMagnified a significant 10×. Scale diff barerence = 50between μm. * LPS-treatedshows a significant and LPS +differenceFis-treated between groups. Significancecontrol and= LPS-treatedp < 0.05. groups; # shows a significant difference between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05. 3.4. Effect of Fisetin on LPS-Induced Activation of TLR4/NFκB Signaling and Inflammatory Mediators in the Adult Mouse Hippocampus 3.4. Effect of Fisetin on LPS-Induced Activation of TLR4/ NFKB Signaling and Inflammatory Mediators in the AdultIt Mouse has recently Hippocampus been reported that peripheral systemic LPS administration significantly mediates the activation of the TLR4/NFκB pathway in LPS-induced neuroinflammation and developed AD-like pathologies [9]. Therefore, in our study, we also investigated the anti-inflammatory activity of fisetin against LPS-induced activation of inflammatory signaling (TLR4/p-NFκB). Interestingly, our results showed that LPS treatment for seven days increased the TLR4, CD14, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB-β), and p-NFκB protein expression levels in the LPS-treated group (p < 0.05), while fisetin (20 mg/kg) treatment, a natural polyphenol, could significantly reverse their higher expression levels in the LPS-treated group compared to the LPS-only treated group (p < 0.05) (Figure6A). Recently, Badshah et al. 2015 reported that LPS administration significantly activated NFκB signaling, ultimately enhancing neuroinflammation [10]. Therefore, we further performed an immunofluorescence assay to determine the immunoreactivity of p-NFκB protein in the hippocampal regions of adult mice. The immunofluorescence results showed increased reactivity of p-NFκB in LPS-treated mice compared to the control group (p < 0.05), while fisetin (20 mg/kg) treatments for 2 weeks (1 week prior to LPS and 1 week co-treated with LPS) significantly reversed the LPS-induced enhancements of immunofluorescence reactivity in the CA1 (molecular layer and pyramidal cells) and CA3 (molecular layer and pyramidal cells) regions of the hippocampus compared to the LPS-only treated group (p < 0.05) (Figure6B). These results suggest that fisetin has the ability to suppress the LPS-induced activation of inflammatory signaling cascades and shows effective anti-inflammatory activity in the adult mouse brain. J. Clin. Med. 2019, 8, x FOR PEER REVIEW 11 of 23

It has recently been reported that peripheral systemic LPS administration significantly mediates the activation of the TLR4/NFKB pathway in LPS-induced neuroinflammation and developed AD- like pathologies [9]. Therefore, in our study, we also investigated the anti-inflammatory activity of fisetin against LPS-induced activation of inflammatory signaling (TLR4/p-NFKB). Interestingly, our results showed that LPS treatment for seven days increased the TLR4, CD14, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB-β), and p-NFKB protein expression levels in the LPS-treated group (p < 0.05), while fisetin (20 mg/kg) treatment, a natural polyphenol, could significantly reverse their higher expression levels in the LPS-treated group compared to the LPS-only treated group (p < 0.05) (Figure 6A). Recently, Badshah et al. 2015 reported that LPS administration significantly activated NFKB signaling, ultimately enhancing neuroinflammation [10]. Therefore, we further performed an immunofluorescence assay to determine the immunoreactivity of p-NFKB protein in the hippocampal regions of adult mice. The immunofluorescence results showed increased reactivity of p-NFKB in LPS-treated mice compared to the control group (p < 0.05), while fisetin (20 mg/kg) treatments for 2 weeks (1 week prior to LPS and 1 week co-treated with LPS) significantly reversed the LPS-induced enhancements of immunofluorescence reactivity in the CA1 (molecular layer and pyramidal cells) and CA3 (molecular layer and pyramidal cells) regions of the hippocampus compared to the LPS-only treated group (p < 0.05) (Figure 6B). These results suggest that fisetin has

J.the Clin. ability Med. 2019 to ,suppress8, 850 the LPS-induced activation of inflammatory signaling cascades and shows11 of 23 effective anti-inflammatory activity in the adult mouse brain.

Figure 6. Effect of fisetin on the LPS-induced activation of inflammatory signaling (TLR4/NFκB) in the Figure 6. Effect of fisetin on the LPS-induced activation of inflammatory signaling (TLR4/ NFKB) in β κ adultthe adult mouse mouse hippocampus. hippocampus. (A) The (A) WesternThe Western blot analysis blot analysis of CD14, of CD14, TLR4, TLR4, p-IKK p-IKK, p-NFβ, p-NFB, andKB, tumor and α α necrosistumor necrosis factor- factor-(TNF-α (TNF-) in theα) hippocampusin the hippocampus of mice. of mice. The bands The bands were were quantified quantified using using Sigma Sigma Gel software, and the differences are represented by a histogram. β-actin was used as a loading control. Gel software, and the differences are represented by a histogram. β-actin was used as a loading The density values are expressed in arbitrary units (A.U.) as the means SEM (number = 8 mice/group) control. The density values are expressed in arbitrary units (A.U.) as± the means ± SEM (number = 8 for three repeated and reproducible independent experiments. (B) Representative images of mice/group) for three repeated and reproducible independent experiments. (B) Representative immunofluorescence staining of p-NFκB in the CA1 (molecular layer and pyramidal cells) and images of immunofluorescence staining of p-NFKB in the CA1 (molecular layer and pyramidal cells) CA3 (molecular layer and pyramidal cells) regions of the hippocampus. The results were shown and CA3 (molecular layer and pyramidal cells) regions of the hippocampus. The results were shown as the means SEM (number = 5 mice/group) for three repeated and reproducible independent as the means ±± SEM (number = 5 mice/group) for three repeated and reproducible independent experiments. Magnified 10 . Scale bar = 50 µm. * shows a significant difference between control and × LPS-treated groups; # shows a significant difference between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05.

3.5. Effect of Fisetin on the LPS-Induced Upregulation of Inflammatory Mediators in the Adult Mouse Hippocampus Several studies suggest that the activation of gliosis, the elevation in pro-inflammatory cytokines and chemokines and the generation of ROS are responsible for immune response dysregulation, which contributes to neurodegeneration, neuroinflammation, and AD pathogenesis [7–13]. Therefore, in this study, we also investigated the LPS-induced activation of inflammatory mediators in the hippocampal region of adult mice brains. The Western blot results showed that there was a significant increase in the protein expression levels of the inflammatory markers TNF-α (p < 0.05), COX2 (p < 0.05), and IL1-β (p < 0.05) in LPS-treated group compared to the saline-treated control group. Interestingly, fisetin (20 mg/kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) supplementation reversed the elevated expression levels in the LPS-treated group compared to LPS-only treated group (p < 0.05) (Figure7A). Further, to verify the anti-inflammatory e ffect of fisetin, we performed an immunofluorescence assay of the key cytokine TNF-α. Our morphological analyses of immunofluorescence results demonstrated that fisetin supplementation significantly attenuated the increased immunoreactivity of TNF-α protein in the CA3 (molecular layer and pyramidal cells) and DG J. Clin. Med. 2019, 8, x FOR PEER REVIEW 12 of 23

experiments. Magnified 10×. Scale bar = 50 μm. * shows a significant difference between control and LPS-treated groups; # shows a significant difference between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05.

3.5. Effect of Fisetin on the LPS-Induced Upregulation of Inflammatory Mediators in the Adult Mouse Hippocampus Several studies suggest that the activation of gliosis, the elevation in pro-inflammatory cytokines and chemokines and the generation of ROS are responsible for immune response dysregulation, which contributes to neurodegeneration, neuroinflammation, and AD pathogenesis [7–13]. Therefore, in this study, we also investigated the LPS-induced activation of inflammatory mediators in the hippocampal region of adult mice brains. The Western blot results showed that there was a significant increase in the protein expression levels of the inflammatory markers TNF-α (p < 0.05), COX2 (p < 0.05), and IL1-β (p < 0.05) in LPS-treated group compared to the saline-treated control group. Interestingly, fisetin (20 mg/kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) supplementation reversed the elevated expression levels in the LPS-treated group compared to LPS-only treated group (p < 0.05) (Figure 7A). Further, to verify the anti-inflammatory effect of fisetin, we performed an immunofluorescence assay of the key cytokine TNF-α. Our

J.morphological Clin. Med. 2019, 8 ,analyses 850 of immunofluorescence results demonstrated that fisetin supplementation12 of 23 significantly attenuated the increased immunoreactivity of TNF-α protein in the CA3 (molecular layer and pyramidal cells) and DG (hilum and granular cells) regions of hippocampus in the LPS- (hilumtreated andgroup granular (p < 0.05) cells) relative regions to ofthe hippocampus LPS-only treated in the group LPS-treated (p < 0.05) group (Figure (p < 7B).0.05) These relative findings to the LPS-onlysuggest that treated fisetin group treatment (p < 0.05) (20 (Figuremg/kg, 7i.p.B). for These 2 weeks; findings 1 week suggest prior that to fisetin LPS and treatment 1 week (20co-treated mg /kg, i.p.with for LPS) 2 weeks; significantly 1 week suppressed prior to LPS the and LPS-induce 1 week co-treatedd activation with of LPS) inflammatory significantly mediators suppressed in the LPS-inducedadult mouse hippocampus. activation of inflammatory mediators in the adult mouse hippocampus.

Figure 7. Effect of fisetin on the LPS-induced upregulation of inflammatory mediators in the Figure 7. Effect of fisetin on the LPS-induced upregulation of inflammatory mediators in the hippocampus of the adult mouse. (A) Western blot analysis of inflammatory mediators using hippocampus of the adult mouse. (A) Western blot analysis of inflammatory mediators using the the antibodies to TNFα, cyclooxygenase (COX)2, and interleukin-1 (IL1)-β in the mouse hippocampus. antibodies to TNFα, cyclooxygenase (COX)2, and interleukin-1 (IL1)-β in the mouse hippocampus. The bands were quantified using Sigma Gel software, and the differences are represented by a histogram. The bands were quantified using Sigma Gel software, and the differences are represented by a β-actin was used as a loading control. The density values are expressed in arbitrary units (A.U.)

as the means SEM (number = 8 mice/group) for three repeated and reproducible independent ± experiments. (B) Representative images of immunofluorescent staining of TNFα in the CA3 (molecular layer and pyramidal cells) and DG (hilum and granular cells) regions of the hippocampus of the mouse brain. The results were shown as the means SEM (number = 5 mice/group) for three repeated and ± reproducible independent experiments. Magnified 10 . Scale bar = 50 µm. * shows a significant × difference between control and LPS-treated groups; # shows a significant difference between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05.

3.6. Effect of Fisetin on LPS-Induced Apoptotic Neurodegeneration in the Adult Mouse Brain To investigate the neuroprotective property of fisetin against LPS-mediated apoptotic neurodegeneration in the hippocampus of the adult mouse, we examined several apoptotic markers. Recently, mounting studies have shown that LPS administration significantly activates apoptotic neurodegeneration in adult mice. Badshah et al. recently showed that LPS-induced activation of neuroinflammatory and mitochondrial apoptotic pathways through the upregulation of several apoptotic markers such as Bax, cytochrome C, caspase-9, and caspase-3 in an adult mouse model [7–12]. Therefore, we examined the protein expression levels of cytochrome C, Apoptotic protease activating factor 1 (Apaf-1), caspase-9, caspase-3, and Poly (ADP-ribose) polymerase-1 (PARP-1) through Western blot analysis in the hippocampi of adult mice. Our Western blot results demonstrated that LPS J. Clin. Med. 2019, 8, 850 13 of 23

administration upregulated the expression levels of cytochrome C (p < 0.05), Apaf-1 (p < 0.05), caspase-9 (p < 0.05), caspase-3 (p < 0.05), and PARP-1 (p < 0.05) in the LPS-treated group compared to the control group (Figure8A). However, fisetin (20 mg /kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) treatment significantly reversed the enhanced expression levels of cytochrome C (p < 0.05), Apaf-1 (p < 0.05), caspase-9 (p < 0.05), caspase-3 (p < 0.05) and PARP-1 (p < 0.05) in the LPS-treated group relative to the LPS-only treated group (Figure8A). Furthermore, we investigated the neuroprotective effect of fisetin through morphological analysis using FJB and Nissl staining in the cortex and hippocampus of adult mice. FJB is known to be used to evaluate the extent of neuronal cell death, and, herein, we also used it to investigate the level of apoptotic neurodegeneration in LPS and fisetin-treated mice. Our FJB results demonstrated that LPS treatment significantly increased the number of degenerative neuronal cells in the cortex (p < 0.05), CA1 (molecular layer and pyramidal cells) (p < 0.05), CA3 (molecular layer and pyramidal cells) (p < 0.05), and DG (hilum and granular cells) (p < 0.05) regions of the hippocampus, while fisetin treatment markedly decreased the LPS-induced neurodegeneration, which was evident from the reduced number of FJB-positive cells compared to the LPS-only treated group (p < 0.05) (Figure8B). Furthermore, Nissl staining was carried out to evaluate the extent of neuronal damage in the LPS-treated and fisetin-treated adult mouse hippocampus and cortex. Our Nissl staining results demonstrated that LPS treatment significantly enhanced the number of damaged, shrunken, and fragmented neurons in the CA1 (molecular layer and pyramidal cells) (p < 0.05), CA3 (molecular layer and pyramidal cells) (p < 0.05), and DG (hilum and granular cells) (p < 0.05) regions of the hippocampus relative to the control group. Hence, fisetin treatment significantly reversed the LPS-induced neuronal damage in the LPS and fisetin-treated group (p < 0.05) and enhanced the viability of neurons relative to the LPS-only treated group (p < 0.05) (Figure8C). These results suggest that fisetin supplementation significantly inhibited LPS-induced neurodegeneration in the J. Clin.adult Med. mouse 2019, 8, brain. x FOR PEER REVIEW 14 of 23

Figure 8. Cont.

Figure 8. Effect of fisetin on the LPS-induced apoptotic neurodegeneration in adult mice brain. (A) Western blots analysis of apoptotic markers using antibodies Cyt.C, Apaf-1, caspase-9, cleaved caspase-3, and cleaved PARP-1 in the mice hippocampus. The bands were quantified using Sigma Gel software, and the differences are represented by a histogram. β-actin was used as a loading control. The density values are expressed in arbitrary units (A.U) as the means ± SEM (number = 8 mice/group) for three repeated and reproducible independent experiments. (B) Representative images of (fluoro-jade B) FJB staining in the cortex, CA1 (molecular layer and pyramidal cells), CA3 (molecular layer and pyramidal cells), and DG (hilum and granular cells) hippocampus of the mouse brain. Magnified 10×. Scale bar = 50 μm (C) Representative images of cresyl violet staining in the CA1 (molecular layer and pyramidal cells), CA3 (molecular layer and pyramidal cells), and DG (hilum and granular cells) hippocampus of the mouse brain. The presented data is relative to the control. Magnification 20×, Scale bar = 50 μm. * shows a significant difference between the control and LPS- treated groups; # shows a significant difference between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05.

3.7. Effect of Fisetin on LPS-Induced Disruption of Pre- and Post-Synaptic and Memory Function in the Adult Mouse Hippocampus It has been recently reported that LPS administration resulted in the disruption of synaptic and cognitive functions in mice [13]. Therefore, we also investigated the LPS-induced disruption of pre-

J. Clin. Med. 2019, 8, x FOR PEER REVIEW 14 of 23

J. Clin. Med. 2019, 8, 850 14 of 23

FigureFigure 8. 8.E Effectffect of offisetin fisetin onon thethe LPS-induced apopto apoptotictic neurodegeneration neurodegeneration in in adult adult mice mice brain. brain. (A) (AWestern) Western blots blots analysis analysis of apoptoticapoptotic markersmarkers usingusing antibodiesantibodies Cyt.C, Cyt.C, Apaf-1, Apaf-1, caspase-9, caspase-9, cleaved cleaved caspase-3,caspase-3, and and cleaved cleaved PARP-1 PARP-1 in the in micethe mice hippocampus. hippocampus. The bandsThe bands were were quantified quantified using Sigmausing Sigma Gel software,Gel software, and the and differences the differences are represented are represented by a histogram. by a βhistogram.-actin was usedβ-actin as awas loading used control. as a loading The densitycontrol. values The aredensity expressed values in are arbitrary expressed units in (A.U) arbitrary as the units means (A.U)SEM as the (number means= ±8 SEM mice /group)(number for = 8 ± threemice/group) repeated andfor reproduciblethree repeated independent and reproducible experiments. independent (B) Representative experiments. images (B) of Representative (fluoro-jade B)images FJB staining of (fluoro-jade in the cortex, B) FJB CA1 staining (molecular in thelayer cortex, and CA1 pyramidal (molecular cells), layer CA3 and (molecular pyramidal layer cells), and CA3 pyramidal(molecular cells), layer and and DG pyramidal (hilum andcells), granular and DG cells) (hilum hippocampus and granular of cells) the mousehippocampus brain. Magnifiedof the mouse 10 brain.. Scale Magnified bar = 50 µ10m×.( CScale) Representative bar = 50 μm ( imagesC) Representative of cresyl violet images staining of cresyl in the violet CA1 staining (molecular in the layer CA1 × and(molecular pyramidal layer cells), and CA3 pyramidal (molecular cells), layer CA3 and (molecul pyramidalar layer cells), and andpyramidal DG (hilum cells), and and granular DG (hilum cells) and hippocampusgranular cells) of thehippocampus mouse brain. of Thethe presentedmouse brain. data Th ise relative presented to the data control. is relative Magnification to the control. 20 , × ScaleMagnification bar = 50 µm. 20*× shows, Scalea bar significant = 50 μm. di *ff erenceshows betweena significant the control difference and LPS-treatedbetween the groups; control # and shows LPS- a significanttreated groups; difference # shows between a significant LPS-treated difference and LPS between+Fis-treated LPS-treated groups. Significanceand LPS+Fis-treated= p < 0.05. groups. Significance = p < 0.05. 3.7. Effect of Fisetin on LPS-Induced Disruption of Pre- and Post-Synaptic and Memory Function in the Adult Mouse Hippocampus 3.7. Effect of Fisetin on LPS-Induced Disruption of Pre- and Post-Synaptic and Memory Function in the AdultIt has Mouse been Hippocampus recently reported that LPS administration resulted in the disruption of synaptic and cognitive functions in mice [13]. Therefore, we also investigated the LPS-induced disruption It has been recently reported that LPS administration resulted in the disruption of synaptic and of pre-synaptic and post-synaptic proteins in the adult mouse hippocampus. Our Western blot cognitive functions in mice [13]. Therefore, we also investigated the LPS-induced disruption of pre- results showed that LPS administration significantly reduced the expression level of pre-synaptic synaptosomal-associated protein 23 (SNAP-23) (p < 0.05), SYN (p < 0.05)) and post-synaptic (PSD-95 (p < 0.05), phospho-glutamate receptor (p-GluR1) (p < 0.05), phospho- cAMP response element-binding protein (p-CREB) (p < 0.05)) proteins in the hippocampi of LPS-treated mice compared to the control group (Figure9A). However, fisetin treatment (20 mg /kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) significantly reversed the LPS effect on the pre-synaptic (SNAP-23, SYN) (p < 0.05) and post-synaptic (PSD-95, p-GluR1, p-CREB) (p < 0.05) protein expression levels compared to LPS-only treated group (Figure9A). Next, we performed immunofluorescence analysis of post-synaptic protein markers in the hippocampi of adult mice. Our morphological results showed reduce expression of post-synaptic (PSD-95) immunofluorescence reactivity in the LPS-treated group (p < 0.05) compared to the control group (Figure9B). Interestingly, fisetin treatment significantly enhanced the expression levels of PSD-95 in the CA1 (molecular layer and pyramidal cells) (p < 0.05) and CA3 (molecular layer and pyramidal cells) (p < 0.05) regions of the hippocampus, compared to the LPS-only treated group (Figure9B). These results suggest that fisetin treatment (20 mg /kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) significantly improved synaptic functions associated with the pre- and post-synaptic proteins in the hippocampi of adult mice. J. Clin. Med. 2019, 8, x FOR PEER REVIEW 15 of 23 synaptic and post-synaptic proteins in the adult mouse hippocampus. Our Western blot results showed that LPS administration significantly reduced the expression level of pre-synaptic synaptosomal-associated protein 23 (SNAP-23) (p < 0.05), SYN (p < 0.05)) and post-synaptic (PSD-95 (p < 0.05), phospho-glutamate receptor (p-GluR1) (p < 0.05), phospho- cAMP response element- binding protein (p-CREB) (p < 0.05)) proteins in the hippocampi of LPS-treated mice compared to the control group (Figure 9A). However, fisetin treatment (20 mg/kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) significantly reversed the LPS effect on the pre-synaptic (SNAP-23, SYN) (p < 0.05) and post-synaptic (PSD-95, p-GluR1, p-CREB) (p < 0.05) protein expression levels compared to LPS-only treated group (Figure 9A). Next, we performed immunofluorescence analysis of post-synaptic protein markers in the hippocampi of adult mice. Our morphological results showed reduce expression of post-synaptic (PSD-95) immunofluorescence reactivity in the LPS-treated group (p < 0.05) compared to the control group (Figure 9B). Interestingly, fisetin treatment significantly enhanced the expression levels of PSD-95 in the CA1 (molecular layer and pyramidal cells) (p < 0.05) and CA3 (molecular layer and pyramidal cells) (p < 0.05) regions of the hippocampus, compared to the LPS-only treated group (Figure 9B). These results suggest that fisetin treatment (20 mg/kg, i.p. for

J.2 Clin.weeks; Med. 20191 week, 8, 850 prior to LPS and 1 week co-treated with LPS) significantly improved synaptic15 of 23 functions associated with the pre- and post-synaptic proteins in the hippocampi of adult mice.

Figure 9. Effect of fisetin on LPS-induced disruption of synaptic and memory function in the adult mice. (FigureA) Western 9. Effect blot of analysis fisetin ofon presynaptic LPS-induced (SNAP-23, disruption SYN) of andsynaptic postsynaptic and memory (PSD-95, function p-GluR1, in the p-CREB) adult proteinsmice. (A markers) Western in blot the mouseanalysis hippocampus. of presynaptic Bands (SNAP- were23, quantified SYN) and using postsynaptic Sigma Gel (PSD-95, software, p-GluR1, and the β dip-CREB)fferences proteins are represented markers byin athe histogram. mouse hippoca-actin wasmpus. used Bands as a loadingwere quanti control.fied The using density Sigma values Gel are expressed in arbitrary units (A.U) as the means SEM (number = 8 mice/group) for three repeated software, and the differences are represented by a± histogram. β-actin was used as a loading control. and reproducible independent experiments. (B) Representative image of immunofluorescence images The density values are expressed in arbitrary units (A.U) as the means ± SEM (number = 8 mice/group) of PSD-95 in the CA1 (molecular layer and pyramidal cells) and CA3 (molecular layer and pyramidal for three repeated and reproducible independent experiments. (B) Representative image of cells) of the hippocampus of mice brains (number = 5 mice/group). Magnified 10 . Scale bar = 50 µm. immunofluorescence images of PSD-95 in the CA1 (molecular layer and pyramidal× cells) and CA3 * shows a significant difference between control and LPS-treated groups; # shows a significant difference (molecular layer and pyramidal cells) of the hippocampus of mice brains (number = 5 mice/group). between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05. Magnified 10×. Scale bar = 50 μm. * shows a significant difference between control and LPS-treated 3.8. Effect of Fisetin on CNS-Insult, LPS-Induced Memory Dysfunction After one week of fisetin dosage (20 mg/kg, i.p.) and the start of LPS on day eight, the behavioral study via MWM test was started. Initially, for four days, we trained all animals in the MWM task where they were required to find a submerged hidden platform and we analyzed the time required to reach the hidden platform. We observed that LPS-injected animals took a long time to find the hidden platform compared to the control mice (p < 0.05). However, the fisetin treatment reversed the LPS effect and significantly improved the performance of the mice, as revealed by the mice taking less time to reach the hidden platform compared to the LPS-injected mice (p < 0.05) (Figure 10A). After a training session and training latency with a one-day interval, we performed a probe test. The probe test results also revealed that fisetin reversed the LPS effect, showing a decrease in the number of mice that crossed over the hidden platform (p < 0.05) and spent less time in the target quadrant (p < 0.05). However, the results revealed a significant increase in the number of platform crossings (p < 0.05) and an increase in the time spent in the target quadrant (p < 0.05) in which the hidden platform was previously located in the training session (Figure 10B,C). Next, we examined the mean swim speeds during training days to observe the motor ability among the saline, LPS alone, and LPS+Fisetin-treated group. We found a J. Clin. Med. 2019, 8, x FOR PEER REVIEW 16 of 23

groups; # shows a significant difference between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05.

3.8. Effect of Fisetin on CNS-Insult, LPS-Induced Memory Dysfunction. After one week of fisetin dosage (20 mg/kg, i.p.) and the start of LPS on day eight, the behavioral study via MWM test was started. Initially, for four days, we trained all animals in the MWM task where they were required to find a submerged hidden platform and we analyzed the time required to reach the hidden platform. We observed that LPS-injected animals took a long time to find the hidden platform compared to the control mice (p < 0.05). However, the fisetin treatment reversed the LPS effect and significantly improved the performance of the mice, as revealed by the mice taking less time to reach the hidden platform compared to the LPS-injected mice (p < 0.05) (Figure 10A). After a training session and training latency with a one-day interval, we performed a probe test. The probe test results also revealed that fisetin reversed the LPS effect, showing a decrease in the number of mice that crossed over the hidden platform (p < 0.05) and spent less time in the target quadrant (p < 0.05). However, the results revealed a significant increase in the number of platform crossings (p < 0.05) and an increase in the time spent in the target quadrant (p < 0.05) in which the hidden platform was previously located in the training session (Figure 10B,C). Next, we examined the mean swim speeds during training days to observe the motor ability among the saline, LPS alone, and LPS+Fisetin-treated group. We found a significantly (p < 0.05) lower swimming speed in the LPS- treated group compared to the saline-treated group. This represented the motor problem in animals which may add to the latency differences. However, the Fisetin treatment regulated the mean swim speeds in the LPS+Fisetin-treated mice group (p < 0.05) (Figure 10E). Next, in order to analyze the short term spatial working memory, we designed and evaluated spontaneous alternation behavior percentage (%) through the Y-maze task. On day 12, we trained the J. Clin. Med. 2019, 8, 850 16 of 23 mice until day 13. Following training, we observed the final performance of the mice on day 14. Our results revealed that LPS-injected mice showed a less spontaneous alternation behavior percentage (%)significantly than that of ( pthe< 0.05)control lower saline-treated swimming ( speedp < 0.05) in mice. the LPS-treated Fisetin treatment group compared improved to and the restored saline-treated the specialgroup. memory This represented (increased the the spontaneous motor problem alternation in animals behavior which percentage may add (%)), to the of latency the LPS-injected differences. miceHowever, (p < 0.05) the (Figure Fisetin treatment10D). All regulatedof these behavioral the mean swim results speeds showed in the that LPS fisetin+Fisetin-treated treatment mice to LPS- group injected(p < 0.05) mice (Figure reversed 10E). the LPS effect and significantly improved memory (Figure 10A–D).

Figure 10. Effect of fisetin on LPS-induced memory dysfunction. The behavioral studies were performed through the Morris water maze (MWM) and the Y-maze test. The mice (13 mice per group) were used for the behavioral analysis. (A) The time taken (escape latency (s)) to reach the submerged hidden platform during training. (B) The number of platform crossings during the probe test. (C) The graphs represent the time spent in the target quadrant (where the platform was located during the hidden platform training session) during the probe test. (D) The graphs represent the % of spontaneous alternation behavior in the Y-maze test. (E) Represents the mean swim speeds during training days. The graphs express the means SEM (n = 13 mice/group). * shows a significant difference between control and ± LPS-treated groups; # shows a significant difference between LPS-treated and LPS+Fis-treated groups. Significance = p < 0.05.

Next, in order to analyze the short term spatial working memory, we designed and evaluated spontaneous alternation behavior percentage (%) through the Y-maze task. On day 12, we trained the mice until day 13. Following training, we observed the final performance of the mice on day 14. Our results revealed that LPS-injected mice showed a less spontaneous alternation behavior percentage (%) than that of the control saline-treated (p < 0.05) mice. Fisetin treatment improved and restored the special memory (increased the spontaneous alternation behavior percentage (%)), of the LPS-injected mice (p < 0.05) (Figure 10D). All of these behavioral results showed that fisetin treatment to LPS-injected mice reversed the LPS effect and significantly improved memory (Figure 10A–D).

4. Discussion This study was designed to investigate the therapeutic efficacy of the natural based flavonoid fisetin against LPS-induced oxidative stress, neuroinflammation, synaptic loss, and neurodegeneration in the adult mouse brain. We have shown in our study that fisetin treatment for two weeks markedly inhibited the LPS-induced increased levels of in vivo ROS and LPO, and activation of an inflammatory cascade through the suppression of activated gliosis and TLR4/CD14/NFκB signaling pathways and J. Clin. Med. 2019, 8, 850 17 of 23 apoptotic neurodegeneration in the hippocampus of adult mice. Recently reported literature supports the notion that LPS induces oxidative stress, neuroinflammation, neurodegeneration, and synaptic dysfunction [7,13]. It has been evident from well-reported studies that bacterial LPS exacerbates chronic inflammation, beta-amyloid accumulation, memory defects, and neurodegeneration leading to early stages of AD [33,34]. Several in vitro and in vivo studies have demonstrated that the systemic administration of LPS causes the activation of ROS and activated glial cells (astrocytes and microglial cells), the elevation of cytokines including TNF-α, intracellular adhesion molecule-1, and IL-6, as well as the increase of inflammatory proteins, such as inducible nitric oxide synthase and cyclooxygenase-2, which are factors known to be responsible for neuroinflammatory disorders and lead to memory impairments [35,36]. It has been recently demonstrated that elevated levels of thiobarbituric acid-reactive substance (TBARS) as well as oxidative DNA damage, measured by 8-OxoG, and reduced levels of the protein responsible for removing damaged DNA, 8-OxoG DNA glycosylase 1 (OGG1), were detected in the brains of patients with AD [7,37]. LPS, a major constituent of Gram-negative bacteria, can trigger a variety of inflammatory reactions, including the release of proinflammatory cytokines, which in turn causes the release of ROS from mitochondria as well as NO and other cell mediators from monocytes and macrophages. The increased level of ROS is responsible for mediating various pathological events such as the peroxidation of lipids, DNA, and various proteins [38]. Similarly, our current study demonstrated that fisetin dosage (20 mg/kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) markedly overcomes the LPS-induced upregulation of ROS, lipid peroxidation, and 8-OxoG, a key biomarker of oxidative stress detected in the brains of AD patients [13]. Well-reported in vitro and in vivo evidences suggest that fisetin treatments show potent antioxidant activity and attenuate Aβ1-42-induced cognitive and synaptic dysfunction, neuroinflammation, and neurodegeneration [28,31]. Increased production of inflammatory mediators can induce severe neurodegenerative diseases, such as AD, PD, cerebral ischemia, multiple sclerosis, and trauma [34]. The stress kinase JNK is an important mediator of activated gliosis and is responsible for the release of pro-inflammatory cytokines such as IL-1β and TNF-α. Previously, it has been reported that LPS and oxidative stress together enhance apoptotic neurodegeneration and inflammatory responses [36,39]. Furthermore, recent reports have revealed that LPS treatment increases the phosphorylation of JNK, and the excessive production of proinflammatory cytokines related to neuroinflammation and apoptotic neurodegeneration [40,41]. In this study, we also found that LPS (250 µg/kg) exposure enhanced the phosphorylation of p-JNK, and their downstream pro-inflammatory cytokines (i.e., TNF-α, IL-1β, and COX2), whereas fisetin (20 mg/kg) supplementation for 2 weeks could inhibit their expression. Additionally, we hypothesized that fisetin treatment would significantly inhibit TLR4 downstream signaling through the TLR4/CD14 receptor complex and inhibit LPS-induced neuroinflammation and apoptotic neurodegeneration. The TLR4 signaling plays an important role in the brain and mediates autoimmune responses, inducing neuroinflammation and neurodegeneration diseases, such as AD [42]. Well-reported in vitro and in vivo studies have revealed that LPS-stimulated inflammatory mediator production, release of inflammatory cytokines, and suppression of NF-κB and TLR4 signaling pathways ultimately results in chronic inflammation and neurodegeneration [43]. Recently, evidence has suggested that the activation of TLR4 evokes proinflammatory cytokines through the signaling of different adaptor proteins, such as TIRAP and MyD88, and activates NFκB and activator protein (AP)-1, which mediate the activation of other inflammatory genes [44,45]. Interestingly, our data showed that dietary flavonoid fisetin dosage (20 mg/kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) significantly protected and inhibited the LPS-induced suppression of the TLR4/CD14 signaling pathway and its downstream inflammatory mediators in the mouse hippocampus. Previously, studies have shown that the interaction of CD14, a glycosylphosphatidylinositol-anchored monocytic antigen, with TLR4 is an early event of neuroinflammation signaling activation [46]. Additionally, upon LPS binding, CD14 associates with the extracellular domain of TLR4, which in turn activates the TLR4 intracellular J. Clin. Med. 2019, 8, 850 18 of 23 domain-mediated signaling complex including MyD88, IRAK, and TRAF, leading to downstream signal transduction through NF-κB-mediated inflammatory cascades [47,48]. In the progression of AD pathogenesis, neuroinflammation plays a key role and has been considered a main pathological event of numerous neurological disorders. Activation of microglia and astrocytes and increased levels of inflammatory mediators such as IL1-β, TNF-α, and TGFβ have been found in aged subjects [49,50]. Previously reported studies have shown that activated microglia are involved in all degenerative conditions in the CNS [51,52]. Therefore, the suppression of microglia activation may contribute to neuronal cell survival. Our results showed that LPS exposure markedly induced the activation of gliosis, as indicated by the increased expression of both Iba-1 (activated microglia) and GFAP (astrocytes) compared to that of control saline-treated adult mice. Recently, we reported that fisetin treatments attenuated the overactivation of glial cells in adult mice [25]. It has been previously reported that microglial overactivation is considered an early pathogenic event that precedes neutrophil destruction in patients with AD [53,54]. Yang et al. recently reported that LPS exposure mediates neuroinflammation and neurodegeneration through the activation of microglia, NF-κB, and the p38/JNK pathway in both in vitro and in vivo models [55]. Well-established studies have indicated that even a single systemic injection of LPS can impair spatial memory and long-term potentiation (LTP) and decrease neurogenesis in the hippocampus [56]. Recently, reported evidence indicates that a disruption of synaptic function is a primary feature of AD and causes cognitive dysfunction and memory impairments with or without the induction of neurodegeneration [57]. Our results indicate that fisetin dosage (20 mg/kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) restored the memory and synaptic dysfunctions via elevation of the expression of pre- and post-synaptic proteins, such as Synaptophysin (SYN), SNAP-23, PSD-95, p-GluR1, and p-CREB in the hippocampus of adult mice brains. Several well-published articles have revealed that LPS exposure mediates the activation of mitochondrial apoptotic pathways and enhances apoptotic cell death [58]. In recent years, LPS-induced apoptotic neurodegeneration has been widely investigated in the brain and multiple organs, including the lungs, liver, heart, etc. [59–62]. The apoptotic pathway involves a diverse array of stimuli that disrupts the mitochondrial membrane and causes the release of cytochrome C from the intermembrane space into the cytosol, followed by the activation of caspase-9, which in turn, cleaves caspase-3, leading to apoptotic cell death [55]. In the present study, LPS exposure markedly enhanced the release of cytochrome C from the mitochondria as well as Apaf-1 expression levels, while fisetin dosage (20 mg/kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) significantly inhibited the release of cytochrome C and Apaf-1 expression in the hippocampus of adult mice. The activation of caspase-3 plays a crucial role during the execution phase of apoptosis, which involves nuclear condensation, DNA degradation, and membrane blebbing [63]. In the present study, the decrement in the levels of cleaved caspase-3 and cleaved PARP-1 in the hippocampus of fisetin-treated mice indicates that fisetin treatments attenuated the LPS-induced neuronal apoptosis. Furthermore, the FJB and Nissl staining results revealed that fisetin treatments significantly overcame the LPS-induced apoptotic neurodegeneration in the adult mice brain. Interestingly, FJB and Nissl staining results further suggested the possible beneficial effect of fisetin on neurogenesis in the DG region of hippocampus, particularly LPS associated anxiety and depression models, where DG neurogenesis is required to overcome the detrimental effects of LPS. Previous studies [64–66], also reported that fisetin enhanced neurogenesis, which might be associated with the memory improving and anti-depressant effect of fisetin.

5. Conclusions In conclusion, the results of the current study suggest that a fisetin dosage (20 mg/kg, i.p. for 2 weeks; 1 week prior to LPS and 1 week co-treated with LPS) markedly reduced oxidative stress, systemic inflammation, neurodegeneration, and synaptic and memory dysfunction induced by LPS in the adult mouse brain. Interestingly, fisetin mitigated neuronal apoptosis by regulating J. Clin. Med. 2019, 8, x FOR PEER REVIEW 17 of 23

Figure 10. Effect of fisetin on LPS-induced memory dysfunction. The behavioral studies were performed through the Morris water maze (MWM) and the Y-maze test. The mice (13 mice per group) J. Clin. Med.were2019 used, 8 ,for 850 the behavioral analysis. (A) The time taken (escape latency (s)) to reach the submerged19 of 23 hidden platform during training. (B) The number of platform crossings during the probe test. (C) The graphs represent the time spent in the target quadrant (where the platform was located during the the intrinsic apoptotic cascades and provided potent neuroprotection in LPS-induced mouse model hidden platform training session) during the probe test. (D) The graphs represent the % of of neurodegeneration. The underling potent neuroprotective and anti-oxidant mechanism has been spontaneous alternation behavior in the Y-maze test. (E) Represents the mean swim speeds during described in the schematic diagram (Figure 11). Taken together, these results suggested that fisetin is a training days. The graphs express the means ± SEM (n = 13 mice/group). * shows a significant promisingdifference neuroprotective between control and and potent LPS-treated antioxidant groups agent; # shows that deservesa significant further difference exploration betweenas LPS- a safe, effective,treated and and therapeutic LPS+Fis-treate toold against groups. neurodegeneration Significance = p < 0.05. and age-related complications such as AD.

Figure 11. This representative schematic diagram predicts and highlights the proposed potent Figure 11. This representative schematic diagram predicts and highlights the proposed potent neuroprotective and antioxidant therapeutic effect of fisetin, a natural dietary flavonoid which protects neuroprotective and antioxidant therapeutic effect of fisetin, a natural dietary flavonoid which LPS-induced neurotoxicity in adult mice. protects LPS-induced neurotoxicity in adult mice.

Author4. Discussion Contributions: A.A. designed, executed the experimental work, and drafted the manuscript. T.A. analyzed the data and equally contributed to the manuscript writing. S.U.R. performed Western blot and morphological experiments.This study M.O.K. was is designed a corresponding to investigate author, whothe ther reviewedapeutic and efficacy approved of the manuscriptnatural based and flavonoid holds all responsibilities related to this manuscript. All authors reviewed the manuscript. fisetin against LPS-induced oxidative stress, neuroinflammation, synaptic loss, and Funding:neurodegenerationThis research in was the supported adult mouse by the brain. Brain Research We have Program shown through in our thestudy National that fisetin Research treatment Foundation for of Korea (NRF) funded by the Ministry of Science, ICT (2016M3C7A1904391). two weeks markedly inhibited the LPS-induced increased levels of in vivo ROS and LPO, and Acknowledgments: We are thankful to Sohail Khan for the assistance of animal handling and drug administration. activation of an inflammatory cascade through the suppression of activated gliosis and ConflictsTLR4/CD14/NF of Interest:KB Thesignaling authors pathways declare no and competing apoptotic financial neurodegeneration interests. in the hippocampus of adult mice. Recently reported literature supports the notion that LPS induces oxidative stress, Referencesneuroinflammation, neurodegeneration, and synaptic dysfunction [7,13]. It has been evident from 1.well-reportedBrown, G.C.; Neher,studies J.J. that Inflammatory bacterial neurodegeneration LPS exacerbates and mechanismschronic inflammation, of microglial killing beta-amyloid of neurons. accumulation,Mol. Neurobiol. memory2010, 41 defects,, 242–247. and [CrossRef neurodegeneration][PubMed] leading to early stages of AD [33,34]. Several 2.in vitroVon-Bernhardi, and in vivo R.; studies Eugenín-von have Bernhardi, demonstrated L.; Eugen thatín, the J. Microglial systemic cell administration dysregulation of in brainLPS causes aging and the neurodegeneration. Front. Aging Neurosci. 2015, 7, 124. [CrossRef][PubMed] 3. Skaper, S.D. The brain as a target for inflammatory processes and neuroprotective strategies. Ann. N. Y. Acad. Sci. 2007, 1122, 23–34. [CrossRef][PubMed] J. Clin. Med. 2019, 8, 850 20 of 23

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