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Open Access Full Text Article ORIGINAL RESEARCH -Loaded Solid Lipid Nanoparticle Attenuates Diabetic Nephropathy Induced by Streptozotocin Nicotinamide Through Nrf2/ HO-1/NF-kB Signalling Pathway

This article was published in the following Dove Press journal: International Journal of Nanomedicine

Pingping Li1 Background: Apigenin is known to have a broad-spectrum efficacy in oxidative stress and Syed Nasir Abbas Bukhari 2 conditions due to inflammation, although weak absorption, fast metabolic rate and a fast Tahseen Khan 3 elimination (systemic) limit the pharmacological efficacy of this drug. Hence, we propose the Renukaradhya Chitti4 usage of highly bioavailable Apigenin-solid lipid nanoparticles (SLNPs) to recognize such Davan B Bevoor5 limitations. The defensive function of Apigenin-SLNPs on renal damage induced by strep­ 6 tozotocin (STZ) in animals was studied. Anand R Hiremath −1 7 Materials and Methods: We initially injected the rats with 35 mg kg streptozocin intraper­ Nagaraja SreeHarsha −1 8 itoneally, and after 7 days, the rats were then injected 150 mg kg of metformin intragastrically Yogendra Singh followed by a once-daily intragastric dose of Apigenin-SLNP (25 or 50 mg kg−1) for 9 Kumar Shiva Gubbiyappa a continuous period of 30 days. We then measured the level of insulin and blood glucose, 1Department of Nephrology, Zhengzhou superoxide dismutase, catalase and malondialdehyde in the tissues of the kidney. We also Central Hospital Affiliated to Zhengzhou observed messenger-RNA expression of Interleukin-1β, Interleukin-6 and Tumor Necrosis University, Zhengzhou City, Henan Province 450007, People’s Republic of Factor-alpha in renal tissue through RT-PCR technique. Moreover, H&E staining and Western China; 2Department of Pharmaceutical blotting observed the histopathological variations and protein expression of nuclear factor Chemistry, College of Pharmacy, Jouf erythroid 2-related factor 2/heme oxygenase/Nuclear Factor-κB signaling pathway, respectively. University, Sakaka, Aljouf 2014, Saudi Arabia; 3School of Pharmacy, Suresh Results: An enhancement in the expressing of nuclear factor erythroid 2-related factor 2 and Gyan Vihar University, Jaipur, India; heme oxygenase-1 and a suppression in the expression of Nuclear Factor-κB occurred due to 4Department of Pharmacy Practice, Sri Apigenin-SLNPs treatment, which was a result of the protective mechanism of Apigenin- Adichaunagiri College of Pharmacy, Adichunchanagiri University, SLNPs which is because of not only its anti-inflammatory function (by inhibition of release Adichunchanagiri, Mandya, India; of inflammatory factors) but also their anti-oxidant activity (through reduction of lipid 5Department of Pharmacy Practice, SCS College of Pharmacy, Harapanahalli, peroxidation production). Karnataka, India; 6Department of Conclusion: We found that a protective effect on diabetic nephropathy was shown due to Pharmacology, Bapuji College of Apigenin-SLNPs, in rats induced with streptozocin maybe through the pathway of nuclear Pharmacy, Davanagere, Karnataka, India; 7Department of Pharmaceutical Sciences, factor erythroid 2-related factor 2/heme oxygenase-1/Nuclear Factor-κB. College of Clinical Pharmacy, King Faisal Keywords: Apigenin-SLNPs, DN, Nrf2, HO-1, NF-κB University, Al-Ahsa, Saudi Arabia; 8Department of Pharmaceutical Sciences, Mahatma Gandhi College of Pharmaceutical Sciences, Jaipur, India; 9School of Pharmacy, GITAM University, Introduction Hyderabad, India Diabetes mellitus (DM) is a multi-dimensional metabolic disorder which is asso­ ciated with a deficit, wherein, the glucose utilization of the body and insulin homeostasis maintenance are affected. A chronic ruination, deterioration and an eventual breakdown of the organs mainly the eye, kidney, nerves and the cardio Correspondence: Yogendra Singh Email [email protected] vascular system is a result of a constant hyperglycaemia. Globally, there are submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2020:15 9115–9124 9115 DovePress © 2020 Li et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php http://doi.org/10.2147/IJN.S256494 and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Li et al Dovepress

366 million DM patients and the numbers of new cases are Materials and Methods 1 predicted to double up by the year 2030. Diabetic nephro­ Chemicals pathy (DN) is a common disorder that occurs because of We obtained Apigenin and STZ from Sigma. Glucose, UA, both type 1 and type 2 DM and affects DM-associated Creat. and commercial kits were provided by Jiancheng mortality drastically. It is also responsible for renal dys­ Bioengineering Institute. Rest of the chemicals and function in high-income countries. The clinical assessment reagents in use were of analytical grade. of DN is done through a five-stage criterion, wherein each stage features unique sets of functional and clinical altera­ tions along with alterations in the standard renal function Preparation of Apigenin-SLNPs markers.2 According to various studies, insulin resistance We used microemulsification method to prepare SLNPs. and the state of DM govern the production of oxidative We then briefly placed a mixture of 45.45% Tween 80, stress to a large extent. Recent studies have suggested DN 0.58% PLPC, and water in a beaker which was then to be an inflammatory process and the progression of subjected to heat to a temperature to attain the lipid- which can lead to implication of immune cells, even . We also melted Lipid (7.27%) at though the heamodynamic and metabolic factors are a temperature of 82°C to 85°C separately. We then added most common causative factors of DN.3–5 Mesangial Apigenin (25 mg) aqueou phase containing Tween 80, expansion, alterations of extracellular matrix, tubulointer­ later we dropped the hot aqueousemulsifier mix into the stitial renal fibrosis, and glomerulosclerosis are the leading lipid melt all at once. This was done under magnetic pathological conditions of DN. This is due to the diverse stirring for obtaining a clear micro-emulsion. We then adverse effects caused by chronic use of hyperglycemic transferred hot microemulsion in cold water (~2°C) of an drugs, and hence, plant sources with negligible adverse equal amount. This process was carried out under mechan­ effects are now being considered in the development of ical stirring at a speed of 5000 rpm for a time period of 1.5 hyperglycemic drugs.6 Phytoconstituents with a persuasive hrs. The SLNPs are formed in aqueous medium by crystal­ antioxidant property have proved to be a breakthrough lization of the hot droplets of lipid that are present in the remedy against DM. Also, they have been highly consid­ microemulsion. The aqueous SLNP dispersion that was 13 ered as source of biologically active substance (antioxi­ prepared was refrigerated until further analysis. dants, anti-hyperglycemics and anti-hyperlipidaemics).7 Apigenin is a naturally present in tea, berries, Characterization Studies of fruits, and vegetables. These have had various biological Apigenin-SLNPs functions, like antioxidant and anti-inflammatory activity.8 Nano-ZS ZEN 3600 measured the polydispersion (PDI), Apigenin was reported to be playing a protective role in zeta potential, mean size, and size distribution of oxidative-related disorders like CVD and neurology- Apigenin-SLNPs using the dynamic light scattering tech­ related diseases. Apigenin alleviates myocardial toxicity nique. We measured the scattering frequency at 90° and by modulating oxidative stress and inflammation in myo­ 25° angles. TEM investigated on the size of the Apigenin- cardial injury model reactive oxygen species (ROS) and SLNPs. We examined the morphology of the Apigenin- malondialdehyde (MDA) decreased significantly in rat SLNPs by using SEM. We then viewed SEM images of acrylonitrile-induced subchronic sperm injury.9–11 Apigenin-SLNPs by a Scanning Electron Microscope. Pharmacological studies have confirmed that Apigenin markedly inhibited glomerular mesangial cell prolifera­ tion, glomerulus hypertrophy, and extracellular matrix Evaluation of Apigenin-SLNPs development and aggregation in DN patients and mice. Encapsulation Efficiency Apigenin plays a multi-target role in DN treatment. Its We determined the nanoformulation encapsulation- low solubility, short half-life, low renal concentration, efficiencyas the percent of Apigenin trapped in the nanopar­ and minimal bioavailability hamper Apigenin uses.12 ticles. We then dissolved 25 mg of Apigenin nanoparticles Therefore, in laboratory animals, this research aimed to with 1 mL of 90% , further subjected to sonication examine the attenuating effects of Apigenin-solid lipid for 5 mins in order to cause disruption of the nanoparticles nanoparticles(SLNPs) in diabetic nephropathy caused by and release encapsulated Apigenin. The resulting solution at streptozotocin (STZ) nicotinamide. 10,000xg was centrifugated and we then gathered the

9116 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2020:15 DovePress Dovepress Li et al supernatant. Supernatant absorption was read to quantify SLNPs (25 mg kg−1) or Apigenin-SLNPs (50 mg kg−1) Apigenin at 425 nm in a spectrophotometer.14 intragastrically, once daily for 30 days consecutively. Control and Streptozocin group were simultaneously given In vitro Drug Release equal volume of distilled water. When the experiment period Apigenin-SLNPs were screened with a dialysis bag tech­ was completed, we sacrificed the rats after the rats were nique for the in vitro discharge profile. In short, a 25 mg given anesthesia (urethane 20%). We obtained blood sam­ Apigenin-SLNPs in PBS (2 mL) were suspended and ples, which were kept for 20 mins at ambient temperature, moved to a dialysis bag (molecular weight 12,000–­ meanwhile the clots formed. The samples were further sub­ 14,000Da). We secured the pocket with pins which were jected to centrifugation at a speed of 3000 rpm for a period of held in a bottle at 50 mL phosphate buffer saline (pH 7.4) 10 mins. For histopathological examination, we excised three with 1% of polysorbate 80 under steady agitation at renal tissues and then fixed in neutral buffered formalin a speed of 100 rpm. One millilitre PBS has been removed (10%). Remaining tissues of the kidney were preserved at at 1 hr intervals and spectrometric tests of Apigenin have −70°C till further usage.16,17 been conducted at 425 nm. After each removal, we intro­ duced 1 mL new phosphate buffer saline in the container Measurements of Blood Glucose and to preserve fluid circumstances.15 Insulin Experimental Design Determination of insulin and blood glucose in the serum Animals was done by the aid of glucose and ELISA kit as per the We acquired adult rats (male) weighing 180 to 200 gm protocol of the manufacturer. from the animal house of Zhengzhou Central Hospital, China. We then allowed them to adapt to a new environ­ Superoxide Dismutase (SOD), Catalase ment for 5 days. We maintained the animals in a 12 hr (CAT), and MDA Measurement in Renal light-dark natural cycle at an ambient temperature of 24 ± Tissue 1°C. The animals were given a standard diet and water ad We determined the renal tissues’ Catalase, superoxide dis­ lib. We obtained approval from Zhengzhou University mutase, and malondialdehyde levels with the help of pur­ Committee on Animal Care, also we followed all the chased test kits purchased. We carried out all the procedures as per the legislation of China on the use and procedures in accordance to the manual.18 care of experimental animals and the guidelines given by Institute for Experimental Animals of Zhengzhou Central Hospital. qRT-PCR Total RNA were subjected to isolation from kidney sam­ Induction of Diabetes ples we prepared with the use of guanidinium thiocyanate To prepare diabetic models, we injected the rats with 35 mg reagent. By RT, complementary DNA was synthesized in kg−1 of STZ intraperitoneally, which was made in 0.1 accordance to the protocol of the manufacturer. We per­ M citrate buffer with a pH of 4.4. Alongside, we adminis­ formed qRT-PCR with a std. Synergy Brands, Inc. green tered the same amount of citrate buffer to the control group Polymerase Chain Reaction kit, we also performed a PCR (n=10). We collected the blood sample from the orbital amplification which was gene specific by the use of ABI venous plexus. Post 7 days of streptozocin, we estimated 7300. We calculated levels of relative gene expression by their levels of blood glucose with the help of a kit. For further the use of 2-ΔΔCt methodology after normalizing the study, we only selected animals who had a blood glucose levels of messenger RNA of GAPDH. Primers used for level ≥11.1 mmol L−1. We randomly divided the rats with real-time PCRs are as follows: Tumor Necrosis Factor- DM into four groups having 10 rats in each group. The alpha, Fwd: 5ʹ-ACTTTGGAGTGATCGGCCCC-3ʹ; Rev: groups were: Streptozocin group, Streptozocin + Metformin 5ʹ-TTCTGTGTGCCAGACACCCTA-3ʹ, IL-6, Fwd: 5ʹ- (150 mg kg−1) group, Streptozocin + Apigenin-SLNPs CCTTCTCCACAATACCCCCAGG-3ʹ; Rev: 5ʹ- TGTGC (25 mg kg−1) group, and Streptozocin + Apigenin-SLNPs CCAGTGGACAGGTTT-3ʹ and IL-1β, Fwd: 5ʹ- ACCTG (50 mg kg−1) group. On confirmation of DM in the rats, we AGCTCGCCAGTGAAAT-3ʹ; Rev: 5ʹ-ACCCTAAGGC administered them with Metformin (150 mg kg−1), Apigenin- AGGCAGTTGG-3ʹ, GAPDH, Fwd: 5ʹ- TGGGGTGA

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TGCAGGTGCTAC-3ʹ; Rev: 5ʹ- GGACACGGAAGGCC Statistical Analysis 19 ATACCA-3ʹ. We expressed the data as mean ± SEM. We used ANOVA to evaluate the difference between the groups by using Histopathological Changes Tukey’s multiple comparison test (significance p value We harvested the kidney tissue longitudinally and was fixedin < 0.05). paraformaldehyde (4%), embedded with paraffin and sliced into sections (4-µm) and then stained with H&E staining, we Results then observed this under an optical microscope. We examined Characterization of Prepared deep coronal section with the help of a microscope, which was Apigenin-SLNPs then graded as per the magnitude of damage which was based SEM, TEM, and light dynamics were used to execute the on the % involvement of the kidneys. We graded the damage Morphology and Distribution of the Apigenin-SLNPs. The quantification from ten areas corresponding to the renal PT by prepared Apigenin-SLNPs are small, compact spheres use of following parameters: tubular cell necrosis, cytoplasmic with an average size of 152.7 ± 7.04 nm of particles as 20 vacuole formation, hemorrhage, and tubular dilatation. shown in Figures 1 and 2 and have a zeta potential of 52.18±3.9 mV and 0.692 PDI. Apigenin-SLNP’s encapsu­ Western Blot lation performance was 78.90%. Figure 2B shows the Western Blot quantity of the released drug against time for Apigenin- We performed western blot as mentioned previously. SLNPs. The average release rate of a medication up to 24 Shortly, 150 μg protein aliquots from the supernatant of hrs is 71.52% with a rapid release of 34.28% in 5 hrs. the tissue of the kidney were run on SDS-PAGE (10%). Thereafter, the percentage release over 24 hrs was steadily We blocked bull serum albumin (3%) in 0.2% to 0.4% increased. TBST for 1 hr. After doing so, we incubated the mem­ branes at 4°C overnight with primary Ab against active Apigenin-SLNPs Effect on Insulin and TAT-14 Peptide, Heme Oxygenase-1, and NFKB and later Blood Glucose with alkaline phosphatase-conjugated secondary Ab. We Table 1 shows, insulin and blood glucose levels in developed the membranes by 5-b-romo-4-chloro-3-indolyl diabetic animals induced by STZ were suggestively phosphate/nitroblue tetrazolium. We stained the blots with greater as compared to controls (p value<0.001). an anti-β-actin Ab. The protein levels were then normal­ Comparatively, Apigenin-SLNP (25 and 50 mg kg−1) ized concerning β-actin band density. We measured the and metformin therapies effectively reduced level of Ag-Ab products by Thermo Scientific Super Signal West glucose in the blood and increased insulin levels in Pico Chemiluminescent Substrate. We analyzed the result rats given Streptozocin (significance p<0.05, <0.01, with a Fluor Chem system.21 and <0.001). Such findings show that diabetic rats are

Figure 1 (A) SEM micrographs of the Apigenin-SLNPs, (B) TEM image of Apigenin-SLNPs. Abbreviations: SEM, scanning electron microscope; TEM, transmission electron microscope; SLNPs, solid lipid nanoparticles.

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Figure 2 (A) DLS analysis of the Apigenin-SLNPs, and (B) in vitro release profile of Apigenin-SLNPs. Abbreviations: DLS, dynamic light scattering; SLNPs, solid lipid nanoparticles; STZ, streptozotocin. induced by STZ, and Apigenin-SLNPs exhibited anti- Effect of Apigenin-SLNPs on Interleukin- diabetic properties. 6, Interleukin-1β, and Tumor Necrosis Factor-Alpha mRNA Expression Apigenin-SLNPs Effect on Oxidative For STZ-administered rats, Interleukin-6, Tumor Necrosis Stress Factor-alpha and Interleukin-1β mRNA expression were Lipid peroxidation was measured by measuring SOD, expressively up-regulated (p<0.001) in contrast with con­ −1 catalase as well as MDA. Table 2 shows the stimulation trol rats. Thus, Apigenin-SLNPs (25 and 50 mg kg ) and of streptozocin markedly reduced superoxide dismutase metformin therapies are significantly down-regulated (p and catalase activities (p value <0.001). Thus, Apigenin- value< 0.05, < 0.01, and < 0.001) Interleukin-6, Tumor SLNPs (25 and 50 mg kg−1) as well as metformin thera­ Necrosis Factor-alpha and Interleukin-1β mRNA expres­ pies were successful in restoring superoxide dismutase and sion relative to diabetic rats induced by STZ (Figure 3). catalase levels (significant p<0.05, <0.01, and <0.001). Furthermore, STZ treatment revealed a remarkably strong Apigenin-SLNPs Effect on Renal Tissue MDA amount (p<0.01). On the opposite, prescribing Histopathology Apigenin-SLNPs (25 and 50 mg kg−1) and metformin Apigenin-SLNPs defensive function in physiological dys­ dramatically reduced MDA quality (p value <0.05, function, H&E staining was assessed. Histological analysis <0.01, and <0.001). Our findings have shown these of control-group renal tissue revealed normal cell architec­ Apigenin-SLNPs could decrease oxidative-stress STZ- ture. Conversely, diabetic rat kidneys showed extreme tub­ induced diabetic rats. ular necrosis, mild glomerular dilation, and interstitial inflammation.Nonetheless, Apigenin-SLNPs and metformin Table 1 Effects of Apigenin-SLNPs on Blood Glucose and Insulin attenuated the extent of renal damage. Analytical results showed that Apigenin-SLNPs strengthened the histopatho­ Treatment Blood Glucose Serum Insulin Groups (mmol/L) (ng/mL) logical status of streptozocin-induced DN (Figure 4).

Control 06.83±1.93 09.04±1.73 STZ 13.84±1.82z 4.92±2.83z Apigenin-SLNPs Effect on Proteins Metformin 07.02±1.03c 10.92±1.04c Expression of TAT-14 Peptide, NRKB and STZ+Apigenin- 10.55±1.78a 10.05±1.92b SLNPs (25) Heme Oxygenase-1 STZ+Apigenin- 07.14±1.21c 7.60±1.21c In the antioxidant reaction, TAT-14 Peptide/heme oxyge­ SLNPs (50) nase-1 has a crucial role. To examine if TAT-14 Peptide

Notes: Values are expressed as means ± SEM. Compared with control: zP < 0.001; and heme oxygenase-1 expression are intertwined with the compared with STZ: aP < 0.05, bP < 0.01 and cP < 0.001. defensive effects of Apigenin-SLNPs on STZ-induced oxi­ Abbreviations: Apigenin-SLNPs, Apigenin-solid lipid nanoparticles; STZ, streptozotocin. dative damage to the kidneys. As predicted, NF-kB protein

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Table 2 Effects of Apigenin-SLNPs on MDA, SOD and CAT

Treatment Groups MDA (nmol/mg prot) SOD (U/mg prot) CAT (U/mg prot)

Control 1.93±0.04 79.61±3.28 17.27±1.72 STZ 4.89±0.18z 20.88±1.04z 6.03±1.00z Metformin 1.98±0.49c 75.81±3.05c 16.77±1.32c STZ+Apigenin-SLNPs (25) 3.05±0.08a 58.26±3.29b 10.04±1.72a STZ+Apigenin-SLNPs (50) 2.02±0.50c 72.90±3.81c 14.28±1.04b

Notes: Values are expressed as means ± SEM. Compared with control: zP < 0.001; compared with STZ: aP < 0.05, bP < 0.01 and cP < 0.001. Abbreviations: Apigenin-SLNPs, Apigenin-solid lipid nanoparticles; CAT, catalase; MDA, malondialdehyde; SOD, superoxide dismutase; STZ, streptozotocin.

expressions have increased substantially, whereas diabetic subjected to heat till it attained lipid melting temperature. rats caused by STZ expressions of Nrf2 and HO-1 have We also melted 7.27% lipid at a temperature of 82°C to been substantially reduced relative to control (P<0.001). 85°C separately. We then added Apigenin to the aqueous Thus, Apigenin-SLNPs (25 and 50 mg kg−1) and metfor­ phase that contained Tween 80, after which we dropped min therapies effectively reduce controlled protein expres­ the hot aqueous emulsifier mixture into the lipid melt sion of NF-πB and TAT-14 Peptide and heme oxygenase-1 under continuous magnetic stirring in order to get regulated expression in support of diabetic rats caused by a microemulsion which is visibly clear and is more advan­ STZ (p value <0.05, <0.01, and <0.001), as seen in tageous than using Apigenin alone. An improvement in the Figure 5. biodistribution, elevated sensitivity and specificity, and reduced pharmacological toxicity can be attained by mod­ Discussion ification to nanoparticle. The obtained nanoparticles in our Apigenin was encapsulated into SLNP through the micro­ study had a particle size of 152.7 ± 7.04 nm, the small emulsification method. We placed 45.45% Tween 80, particle size may increase its antioxidant and anti- 0.58% PLPC, and water together in a beaker which was inflammatory properties.

Figure 3 Effect of Apigenin-SLNPs on the expression of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the kidney of STZ treated rats. Notes: (A) The mRNA expression of IL-1β, IL-6 and TNF-α in the kidney tissue of each group was measured by RT PCR. (B) Bands of each group were scanned, and the data are expressed as the fold change vs the control group. Values are expressed as means ± SEM. Compared with control: zP < 0.001; compared with STZ: aP < 0.05, bP < 0.01 and cP < 0.001. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-6, Interleukin-6; IL-1β, Interleukin-1β and TNF-α, Tumor Necrosis Factor-α; RT PCR; reverse transcription polymerase chain reaction; SLNPs, solid lipid nanoparticles; STZ, streptozotocin.

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Figure 4 Apigenin-SLNPs effect on histopathological changes of renal tissue. Notes: H&E staining of kidney sections. The kidney from control (A), STZ (B) showed extreme tubular necrosis, mild glomerular dilation, and interstitial inflammation, Metformin (C), and Apigenin-SLNPs 25 and 50 mg/kg (D and E) revealed normal cell architecture. Blue arrow mark showing a infiltration of cells. (F) Histopathological scoring. Values are expressed as means ± SEM. Compared with control: zP < 0.001; compared with STZ: aP < 0.05, and cP < 0.001. Abbreviations: H&E; hematoxylin and eosin; SLNPs, solid lipid nanoparticles; STZ, streptozotocin.

Figure 5 Effect of Apigenin-SLNPs on the protein’s expression of Nrf2, Nf-kB and HO-1 in the kidney of STZ treated rats. Notes: (A) The protein’s expression of Nrf2, Nf-kB and HO-1 in the kidney tissue of each group were measured by Western blot technique. (B) Bands of each group were scanned, and the data are expressed as the fold change vs the control group. Values are expressed as means ± SEM. Compared with control: zP < 0.001; compared with STZ: aP < 0.05, bP < 0.01 and cP < 0.001. Abbreviations: HO-1, heme oxygenase-1; NF-κB, nuclear factor-κB; and Nrf2, nuclear factor erythroid 2-related factor 2; SLNPs, solid lipid nanoparticles; STZ, streptozotocin.

DM is a common disorder related to metabolism and has diabetic rats is by its anti-oxidative and anti-inflammatory a partial relation to the lipid peroxidation derangements. properties.24 Streptozocin-induced diabetic model was con­ Pancreatic cell apoptosis is caused due to streptozocin, and firmedto be a well-established model due to elevated levels hence makes it an experimental model for inducing of blood glucose and insulin. Also, the levels of blood T1DM.22,23 In our study, the protective effect of Apigenin- glucose and insulin were decreased by Apigenin-SLNPs SLNPs on the kidney injury of streptozocin-induced treatment which indicated it to possess a protective shield

International Journal of Nanomedicine 2020:15 submit your manuscript | www.dovepress.com 9121 DovePress Li et al Dovepress against DM.25,26 At the same time, the study of histopathol­ Interleukin-1β, and Interleukin-6 release may decrease ogy of streptozocin-stimulated renal sections showed grave inflammation. Nuclear Factor-κB has a major role in the degeneration of the tubules (necrosis), glomerular dilatation nephropathy (pathophysiologically) in rats with DM with (moderate), thickening of the vascular wall and inflamma­ inflammation and oxidative stress. Activation of Nuclear tion of the interstitium. These alterations were reduced by Factor-κB expression is positively related to the expression Apigenin-SLNPs. The results on the whole suggested the of Tumor Necrosis Factor-alpha, Interleukin-1β, and efficacy of Apigenin-SLNPs over streptozocin-challenged Interleukin-6. The property of heme oxygenase-1 to diabetic rats. catabolize free heme and also to produce CO is responsible An imbalance between oxidants and antioxidants is for its inherent anti-inflammatory property by up-regulation known to cause Oxidative stress. The ROS which was of Interleukin-10. Nuclear factor erythroid 2-related factor 2 accumulated could have an interaction with fatty acids is an important regulator of antioxidative defence pathway (polyunsaturated) leading to lipid peroxidation formation which can be activated by diabetes and DN.35,36 in the renal tissues. This could ultimately result in damage An earlier study has shown that the enhancement of and toxicity.27 The fact that oxidative stress is the major Nuclear factor erythroid 2-related factor 2/heme oxygenase- factor for DM-related complications including DN is 1 pathway has shown an anti-apoptosis activity and also that acknowledged widely.28,29 The membrane fatty acid (poly­ activating Nuclear factor erythroid 2-related factor 2 could unsaturated) is degraded by ROS and leads to production lead to induction of heme oxygenase-1 expression.37 From of 4-HNE and MDA, which is an unstable aldehyde that the current study, we inferred that there was a down- could form a covalent protein adduct which is a trait of regulation in the expression of Nuclear factor erythroid oxidative stress in tissue injuries. Superoxide dismutase 2-related factor 2 and heme oxygenase-1 whereas, there and catalase are free radical scavenging enzymes, which was an up-regulation in the expression of Nuclear Factor- act as the first defense line against oxidative damage in 10 mammals. The oxidation/reduction/conversion of SOD κB in rats with DM. Treatment with Apigenin-SLNPs radicals (O2−) to molecular O2 and hydrogen peroxide is improved Nrf2 and HO-1 expression and reduced NF-kB catalyzed by superoxide dismutase. There is evidence of activity, suggesting that Apigenin-SLNP protection is based existence of a close association of superoxide dismutase on its anti-inflammatory activity by decreasing the develop­ and malondialdehyde with DN. Catalase is considered to ment of LPO by preventing the pro-inflammatory factors be a prominent antioxidant in the kidneys that are respon­ release and its anti-oxidant activity, which is likely dependent sible for the elimination of hydrogen peroxide and also on controlling activated IL-6, IL-1β, TNF-α and NF-κB guards the tissues from the reactive OH− radicals.30,31 through Nrf2/HO-1 signaling pathway on DN induced Studies in the past have shown an accelerated kidney by STZ. injury in DM due to catalase deficiency due to deficiency of the peroxisome. The result of our study confirms the Conclusion role of Apigenin-SLNPs in an increase in activity of super­ Our results showed that Apigenin-SLNPs possessed oxide dismutase and catalase and also reduced levels of a protective effect by reducing the OS and inflammation malondialdehyde in the renal tissues of rates stimulated which might be through the Nuclear factor erythroid with streptozocin. 2-related factor 2/heme oxygenase-1/Nuclear Factor-κB Besides, the anti-inflammatoryeffect of Apigenin-SLNPs signaling pathway. More studies are needed for the was validated from the mRNA expression of cytokines from exploration of clinical application of Apigenin-SLNPs. renal tissues. mRNA level of Interleukin-6, Tumor Necrosis Factor, and Interleukin-1β were highly stimulated in renal tissues of diabetic rats, whereas Apigenin-SLNPs treatment Ethical Statement decreased the level as that of normal rats. Studies have shown All the procedures carried out were as per the legislation that Interleukin-6 has a major function in leukocyte recruit­ of China on the use and care of lab animals and the ment, apoptosis, and activation of T-lymphocyte.32,33 Also, established guidelines by the Institute for Experimental Tumor Necrosis Factor is known to stimulate neutrocytes for Animals of Zhengzhou Central Hospital. Approval was transcribing and releasing of cytokines and chemokines also taken from Zhengzhou University Committee on biosynthesis.34 Inhibiting Tumor Necrosis Factor-alpha, care and use of experimental animals.

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