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Food Science and Human Wellness 6 (2017) 155–166
Comparison of chicoric acid, and its metabolites caffeic acid and caftaric
acid: In vitro protection of biological macromolecules and inflammatory
responses in BV2 microglial cells
∗
Qian Liu, Fan Liu, Liling Zhang, Yajie Niu, Zhigang Liu, Xuebo Liu
Laboratory of Functional Chemistry and Nutrition of Food, College of Food Science and Engineering, Northwest A&F University, Yangling, China
Received 11 October 2016; received in revised form 11 August 2017; accepted 11 September 2017
Available online 23 September 2017
Abstract
Chicoric acid (CA), a natural phenolic acid, has been used as a nutraceutical food ingredient due to its powerful antioxidant, anti-HIV and
anti-diabetic bioactivities. CA could be partly metabolized into caffeic acid (CFA) and caftaric acid (CTA) on cytochrome P450s in rat liver
microsomes. To compare the protective effects of CA and its metabolites on biomolecules and inflammatory responses, oxidative damage induced
by free radicals in vitro and microglial inflammation triggered by lipopolysaccharide in BV2 cells were constructed. Results showed that CA,
CTA and CFA all significantly inhibited protein degradation and carbonylation induced by hydroxyl radicals and alcoxyl radicals, and suppressed
hemin/nitrite/H2O2 triggered-nitration. Moreover, CA, CTA and CFA all exerted remarkable inhibition capacities on linoleic acid and soybean
lecithin liposomes peroxidation in a dose-dependent manner, and restrained the oxidation of herring sperm DNA, as well as the breakage of pBR322
plasmid DNA. Furthermore, CA and its metabolites suppressed lipopolysaccharide-induced decline of BV2 cell viability and the production of NO
and ROS. However, bioactivities of CA were significantly stronger than those of its metabolites within a certain concentration range. This study
provides scientific basis for the application of CA and its metabolites as nutrition and natural antioxidants.
© 2017 Beijing Academy of Food Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Chicoric acid and its metabolites; Oxidative damage; Biomolecules; Microglia; Inflammation
1. Introduction sive ROS/RNS will attack biological macromolecules such as
proteins, lipids and DNA, disrupt cell structure and function,
Oxidative stress plays a pivotal role in the pathophysiology and also cause inflammation responses [3,4]. Nicotinamide
of diseases including neurodegenerative diseases, aging cancer, adenine dinucleotide phosphate oxidase is a major source of
obesity and cardiovascular diseases [1,2]. Once being in a state of ROS production in many non-phagocytic cells. Microglial cells
␣
oxidative stress or disease, generation of reactive oxygen species produce inflammatory cytokines such as TNF- induced by acti-
(ROS)/reactive nitrogen species (RNS) would increase. Exces- vated nicotinamide adenine dinucleotide phosphate oxidase [5].
ROS/RNS can also act as a second messenger to activate NFB
signaling pathway in microglia cells, causing gene expression
related to inflammation and lead to the production of inflamma-
Abbreviations: CA, chicoric acid; CTA, caftaric acid; CFA, caffeic acid;
␣ β
ROS, reactive oxygen species; RNS, reactive nitrogen species; AAPH, 2,2 - tory mediators, including TNF- , IL-1 , NO and PGE2 [6].
Azobis (2-methylpropionamidine) dihydrochlorid; BSA, bovine serum albumin; Phytochemical antioxidants have been widely used due to
TBARS, thiobarbituric acid reactive substances; hsDNA, herring sperm DNA;
the higher natural and secure properties compared with syn-
DCFH-DA, 2 ,7 -dichlorofluorescin diacetate.
∗ thetic antioxidants. A large number of studies have confirmed
Corresponding author.
that phytochemicals, especially plant phenols could prevent or
E-mail address: [email protected] (X. Liu).
Peer review under responsibility of Beijing Academy of Food Sciences. alleviate oxidative stress. It has been reported that polyphenolic
extract of Geoffroea decorticans (chanar)˜ exhibited antioxidant
+• •
activities by scavenging ABTS , OH, hydrogen peroxide,
and protecting protein and lipid against oxidative damage [7].
http://dx.doi.org/10.1016/j.fshw.2017.09.001
2213-4530/© 2017 Beijing Academy of Food Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
156 Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166
2+
It has been proved that caffeic acid could improve cognitive Cu /H2O2 were determined according to the previous method
function through inhibition of lipid peroxidation and NO pro- with some modifications. CA, CFA, and CTA (10, 50, 100, 500,
duction [8]. Another study demonstrated that isoorientin could 1000 mol/L) were mixed with 0.8 mg/mL BSA solution. H2O2
inhibit inflammatory responses induced by lipopolysaccharide (25 mmol/L) and CuSO4 (0.1 mmol/L) were added to induce
through ROS-related MAPK/NF B signaling pathway in BV2 protein oxidative degradation followed by the pre-incubation at
◦
microglial cells [9]. 37 C for 30 min. And the control group used pH 7.4 phosphate
◦
Chicoric acid (CA), isolated from chicory [10], is widely buffer instead of CuSO4 and H2O2. After incubation at 37 C
distributed in the nature, which was also found in iceberg let- for 90 min, the protein content were measured by commasie bril-
tuce, dandelion, ocimum basilicum, cymodoceanodosa and other liant blue staining, and the optical densities were analyzed by
plants [11,12]. As a natural phenolic acid, CA has intense effects Quantity One 4.6.2.
in anti-oxidation, anti-obesity, antiviral, and hpyerglycemic Assays for the inhibition of CA and its metabolites on BSA
activities [13–15]. Our previous report demonstrated that CA oxidative degradation induced by AAPH were measured by the
was metabolized to caffeic acid (CFA) and caftaric acid (CTA) on method described by Mayo et al. [18]. The treatments were the
2+ ◦
cytochrome P450s in rat liver microsomes. CA and its metabo- similar with Cu /H2O2 reaction system. Incubation at 37 C for
• • +•
lites all could significantly scavenge DPPH , OH, ABTS 2 min made AAPH (50 mmol/L) thermal decomposition, pro-
◦
free radicals and had strong ferric reducing capacities in a dose- ducing alcoxyl radicals. After incubation at 37 C for 4 h, the
dependent manner, among which, CA has the highest functional protein content were measured by commasie brilliant blue stain-
activities [16]. However, comparisons on the protection abilities ing, and the optical densities were analyzed by Quantity One
of biological macromolecules and anti-inflammatory activities 4.6.2.
among CA and its metabolites have rarely been reported.
Therefore, this study is aimed to compare the inhibition of
2.3. BSA carbonylation
CA and its metabolites on the protein oxidation, carbonylation
and nitration, lipid peroxidation and DNA oxidation break-
DNPH method was used to detect the degree of BSA car-
age induced by hydroxyl radicals or alcoxyl radicals. Besides,
bonylation [19]. 400 L samples were added into 4 mL DNPH
in order to evaluate the effects of CA and its metabolites on
solution, and placed the solution in the dark at room tempera-
lipopolysaccharide induced inflammation in BV2 microglial
ture for 1 h. Then mixed with 4 mL 10% icy trichloroacetic acid,
◦
cells, cell viability and the production of NO, ROS were
and centrifugated at 4000 rmp at 4 C for 10 min, followed by
detected. This study will improve our understanding of the
washing precipitate three times with ethanol and ethyl acetate in
antioxidant and anti-inflammatory activities of CA and its
1:1 proportions. The absorbance of the precipitate dissolved in
metabolites, and provide a scientific basis for the application
4 mL guanidine hydrochloride was measured at 370 nm using a
of CA and its metabolites as nutrition and natural antioxidants.
UV–vis spectrophotometer. The carbonyl contents were calcu-
lated using formula (1):
2. Materials and methods
carbonyl contents (μmol/mg)
2.1. Chemicals and reagents
=
OD370/(22.0mmol/L · cm) × 1000 (1)
Chicoric acid, caffeic acid, and caftaric acid (purity ≥ 98%)
were purchased from Weikeqi Biological Technology Co.,
Ltd. (Sichuan, China). Bovine serum albumin (BSA), 2,2-
2.4. BSA nitration
azobis (2-amidinopropane) dihydrochloride (AAPH), herring
sperm DNA (hsDNA, D3159), linoleic acid (LA), Anti-DNP
Hemin/NaNO /H O reaction system was used to induce
antibody, lipopolysaccharide, and 3-(4,5-dimethyl-2-thiazolyl)- 2 2 2
protein nitration [20]. CA, CFA, and CTA (10, 50, 100, 500,
2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased
1000 mol/L) were mixed with 1.0 mg/mL BSA solution and
from Sigma Chemical Co. (St. Louis, MO, USA). Soybean
preincubating for 5 min. And then hemin (100 mol/L), NaNO
lecithin was obtained from Huamaike Biotechnology., Ltd. (Bei- 2
(10 mmol/L) and H O (1 mmol/L) were added. After incuba-
2 2
jing, China). PBR322 plasmid was purchased from Takara ◦
tion at 37 C for 30 min, the levels of nitration were measured
Shuzo Co., Ltd. (Kyoto, Japan). Hydrogen peroxide (H2O2)
by western blotting, and the optical densities were quantified by
was obtained from Sinopharm Group Chemical Reagent Co.,
Quantity One 4.6.2.
Ltd. (Shanghai, China). The BCA protein assay kit was obtained
from Thermo Scientific (Rockford, IL, USA). All other reagents
were made in China and were of HPLC-grade or the highest 2.5. Western blotting
commercially available grade.
After the reaction, add 25 L 5 × SDS loading buffer to
◦
2.2. BSA oxidative degradation 100 L reaction solution, and then immediately heated at 95 C
for 10 min. The proteins were separated by SDS-PAGE and
2+
Hydroxyl free radicals generated by Cu react with H2O2 transferred onto PVDF membranes (0.45 m, Millipore). Block-
will cause protein oxidation [17]. The inhibition of CA and ing was carried out for 1.5 h in 5% nonfat dry milk in TBST
its metabolites on BSA oxidative degradation induced by buffer (20 mmol/L Tris, 166 mmol/L NaCl, and 0.05% Tween
Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166 157
20, pH 7.5). Then, the membrane was washed 3 times each for 2.10. pBR322 plasmid DNA oxidative cleavage
5 min using TBST buffer. The primary antibody 3-NT (sc-32731,
Santa Cruz, 1:300) was added overnight at 4C. Then, after three By adopting DNA oxidative cleavage method, the inhibitions
washes in TBST, secondary antibodies (sc-2005, Santa Cruz) of CA and its metabolites were studied [24]. Mix 100 ng pBR322
were added for 1.5 h. After another three washes with TBST, plasmid DNA with CA, CFA, and CTA (10, 50, 100, 500,
◦
the immunoreactive bands were visualized with an enhanced 1000 mol/L), 37 C preincubate for 10 min, and add 10 mmol/L
◦
chemiluminescence reagent (Thermo Fisher, China). AAPH to starting reactions. After 37 C for 4 h, the levels
of DNA oxidative cleavage were tested by agarose gel elec-
trophoresis, and the optical densities were quantified by Quantity
2.6. Lipidosome peroxidation
One 4.6.2.
Lipidosome peroxidation was determined by previous meth-
2.11. Cell culture and treatment
ods [21]. Mix CA, CFA, and CTA (10, 50, 100, 500,
1000 mol/L) with 20 L lipidosome (20 mg lecithin dissolved
Mouse microglial BV2 cells were obtained from the China
in 5 mL 0.05 mol/L pH 7.4 phosphate buffer filled with nitrogen)
Center for Type Culture Collection (Wuhan, China). BV2 cells
and preincubate for 10 min. Then 0.8 mmol/L FeSO4 was added
◦ were grown in RPMI 1640 medium with 10% fetal bovine serum,
to induce peroxidation. After 37 C for 40 min, the reaction fin- ◦
100 g/mL of streptomycin, and 100 U/mL of penicillin at 37 C
ished and the amount of thiobarbituric acid reactive substances
under a 5% CO2 atmosphere. The medium was replaced every
(TBARS) induced by lipid peroxidation was observed by col-
2 days. BV2 cells were pretreated with different concentrations
orimetry.
of CA, CFA, and CTA dissolved in dimethyl sulfoxide for 4 h,
and then treated with lipopolysaccharide (1 g/mL) for 12 h.
2.7. Linoleic acid peroxidation
2.12. Measurement of cell viability
2+
Fe /Vc reaction system was adopted to induce linoleic acid
peroxidation [22]. Linoleic acid (1 mmol/L) was mixed with The cell vitality was determined using MTT assay [25]. BV2
6
×
CA, CFA, and CTA (10, 50, 100, 500, 1000 mol/L). Reaction cells were seeded in 96-well plates at a density of 1 10
◦
solution was vortexed with 50 mol/L FeSO4 and 1 mmol/L Vc, cells/well and incubated over night at 37 C with 5% (v/v) CO2.
◦
follow by 37 C water bath for 24 h away from light. The degree After various treatments, the medium was removed carefully and
◦
of lipid peroxidation was assessed by TBARS. the cells were incubated with MTT (0.5 mg/mL) for 4 h at 37 C.
Formazan crystals formed by live cells were solubilized with
100 L DMSO and absorbance at 490 nm was measured with a
2.8. TBARS method microplate reader (Bio-Rad Laboratories Ltd., China). Cell via-
bility was expressed as a percentage of the control (untreated
TBA method was used to measure the lipid peroxidation level cells). The relative cell viability was calculated according to
[23]. Reacting 10% trichloroacetic acid 100 L and 0.8% thio- formula (3):
◦
barbituric acid 100 L with the samples in 100 C for 15 min.
= A /A ×
The reaction mixture was centrifuged for 10 min at 5000 r/min Relative cell viability (%) 1 0 100 (3)
and the absorbance of the supernatant was measured at 532 nm
where A1 is the absorbance of the experimental group, and A0
using a UV–vis spectrophotometer. The inhibition of CA and its
is the absorbance of the control group.
metabolites on lipid peroxidation induced by free radicals were
calculated according to formula (2):
2.13. Measurement of NO production
inhibition rate(%) = (1 − (A1 − A2) /A 0) × 100 (2)
The production of NO was determined with Griess reagent
[26]. 50 L samples or standards were added into 96-well plates,
where A1 is the absorbance of the experimental group, A2 is the
which were incubated with the same volume of the Griess
absorbance of the induced group (using phosphate buffer instead
reagent [0.1% (w/v) N-(1-naphathyl)-ethylenediamine and 1%
of FeSO4 solution), and A0 is the absorbance of the control group ◦
(w/v) sulfanilamide in 5% (v/v) phosphoric acid] at 37 C. Ten
(using phosphate buffer instead of the sample).
minutes later, the absorbance at 540 nm was measured using a
microplate reader. The amount of nitrite was calculated by using
2.9. hsDNA oxidative damage a sodium nitrite standard curve.
CA, CFA, and CTA (10, 50, 100, 500, 1000 mol/L) were 2.14. Measurement of ROS
mixed with 2.0 mg/mL hsDNA. 40 mmol/L AAPH was added
◦
to induce DNA oxidative damage. After incubation at 37 C for DCFH-DA fluorescence dye itself has no fluorescence, and
12 h, the degree of DNA oxidative damage was measured by can go through the cell membrane freely. However, it can be oxi-
TBA method. dized into DCF which has fluorescence by intracellular reactive
158 Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166
Fig. 1. Effects of CA and its metabolites on protein oxidative damage. (a–d) Effects of CA, CTA and CFA on protein oxidative degradation induced by hydroxyl
2+
radicals. BSA was incubated with Cu /H2O2 in the presence or absence of CA, CTA and CFA, as described in the Materials and Methods section; (e–h) Effects
of CA, CTA and CFA on protein oxidative degradation induced by alcoxyl radicals. BSA was incubated with AAPH in the presence or absence of CA, CTA and
CFA, as described in the Materials and Methods section. The protein contents were measured by commasie brilliant blue staining; (i–l) Effects of CA, CTA and
2+
CFA on protein carbonylation induced by hydroxyl radicals. BSA was incubated with Cu /H2O2 in the presence or absence of CA, CTA and CFA, as described
in the Materials and Methods section; (m–p) Effects of CA, CTA and CFA on protein carbonylation induced by alcoxyl radicals. BSA was incubated with AAPH
in the presence or absence of CA, CTA and CFA, as described in the Materials and Methods section. The carbonyl content was determined using a colorimetric
method. (q–t) Effects of CA and its metabolites on protein nitration. BSA was incubated with hemin/nitrite/H2O2 in the presence or absence of CA, CTA and CFA,
##
as described in the Materials and Methods section; The levels of nitration were measured by western blotting. Each value represents the mean ± SD (n ≥ 3),
**
represents significant differences compared to the control group (p < 0.01); * represents significant differences compared to the induced group (p < 0.05); represents
significant differences compared to the induced group (p < 0.01); represents significant differences compared to the same concentration of CA (p < 0.05);
represents significant differences compared to the same concentration of CA (p < 0.01).
oxygen species. Therefore, the fluorescence intensity can reflect 3. Results
the level of ROS [27].
BV2 microglial cells were washed with PBS 2–3 times after 3.1. Effects of CA, CTA and CFA on protein oxidative
treated, then DCFH-DA dyes diluted by serum free medium degradation
◦
were added, after 37 C incubation for 30 min, inverted fluores-
cence microscope was used to observe the generation of ROS Excessive generation of ROS will induce protein oxidative
and Multiscan Spectrum to make quantitative determinations of damage. And degradation and carbonylation are the main char-
2+
intracellular ROS. acteristics of protein oxidation [28]. Cu /H2O2 and AAPH
reaction systems were adopted to detect the effects of CA and
its metabolites on BSA oxidative damage induced by these two
different kinds of free radicals.
2.15. Statistical analysis
2+
Fig. 1(a–d) showed that after Cu /H2O2 treatment, protein
bands were notably weakened compared to the control group,
All the experiments were performed with at least three repli-
2+
indicating hydroxyl free radicals produced by Cu /H O sys-
cations, and all the data were presented as the mean ± standard 2 2
tem substantially destroyed the structure of BSA. Pretreatment
errors (SD). Significant differences were analyzed using the
of CA and CTA increased the content of residual BSA in a con-
one-way analysis of variance (ANOVA) followed by Tukey’s
centration dependent manner, which demonstrated that CA and
multiple-range test with the SPSS 18.0 system.
Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166 159
Fig. 2. Effects of CA and its metabolites on lipid peroxidation. (a-d) Effects of CA, CTA and CFA on liposomes peroxidation induced by hydroxyl radicals. Lecithin
2+
liposomes were incubated with Fe in the absence or presence of CA, CTA and CFA, as described in the Materials and Methods section; (e–h) Effects of CA, CTA
2+
and CFA on linoleic acid peroxidation induced by hydroxyl radicals. Linoleic acid was incubated with Fe in the absence or presence of CA, CTA and CFA, as
##
described in the Materials and Methods section. The amount of TBARS formed was determined by colorimetry. Each value represents the mean ± SD (n ≥ 3),
**
represents significant differences compared to the control group (p < 0.01); * represents significant differences compared to the induced group (p < 0.05); represents
significant differences compared to the induced group (p < 0.01); represents significant differences compared to the same concentration of CA (p < 0.01).
160 Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166
CTA had strong abilities to protect BSA protein from hydroxyl dation concentration dependently. The ability of 100 mol/L CA
radicals induced degradation. However, the optical density of to inhibit liposome peroxidation was 55.60%, which was 2.81
protein bands in CFA groups decreased. The degradation inhi- times than that of CFA. However, it had no significant difference
bition rate of 1 mmol/L CA was 60.95%, which was 1.78 times compared with CTA (p < 0.05).
than that of CTA. Moreover, Fig. 2(e–h) exhibited a concentration-dependent
AAPH system producing alcoxyl radicals was also employed inhibition in linoleic acid peroxidation when treated with CA
to investigate the effects of CA and its metabolites on BSA and its metabolites, which were consistent with liposome per-
oxidative degradation (Fig. 1(e–h)). CA and its metabolites sup- oxidation system. The inhibition rates of CA at 1 mmol/L was
pressed BSA degradation in concentration-dependent manners. 1.54 and 0.78 times than those of CTA and CFA, respec-
The degradation inhibition rates of CA, CTA and CFA were tively.
79.74%, 55.00% and 66.88% at 100 mol/L, respectively (Sup-
plementary Fig. 1). 3.5. Effects of CA, CTA and CFA on DNA oxidative
damages
3.2. Effects of CA, CTA and CFA on protein carbonylation
DNA is the main attack target of free radicals. Oxidative
2+
As illustrated in Fig. 1(i–l), Cu /H2O2 dramatically stress leads to DNA damages such as DNA base modifica-
increased BSA carbonylation. Within the concentration range tions, double chain rupture, and base mutation. AAPH thermal
of 10–1000 mol/L, CA and its metabolites all exerted remark- decomposition generates alcoxyl radicals, which attack DNA
able inhibition capacities. Besides, the effects were strengthened and the degraded product of DNA (reactive carbonyl com-
along with the increase of the concentrations of CA and CTA. pounds) will react with thiobarbituric acid under acidic condition
However, CFA promoted carbonylation in 10–1000 mol/L, to produce TBARS, which has a significant absorption peak at
which is consistent with the protein degradation at same con- 532 nm. As demonstrated in Fig. 3(a–d), the absorbance of DNA
centration. The carbonylation suppression rate of 1 mmol/L CA reaction solution at 532 nm significantly (p < 0.01) increased
◦
was 37.36% higher than CFA, which had statistically significant after treatment with 40 mmol/L AAPH for 12 h at 37 C. The
difference (p < 0.01). absorbance of CA and its metabolites treatment group were
Fig. 1(m–p) showed that the level of BSA carbonylation obvi- significantly (p < 0.01) lower than AAPH-induced group, indi-
ously increased when treated with AAPH, while CA, CTA and cating that CA and its metabolites all had protective effects on
CFA all could reverse this trend. The results were consistent DNA. The degradation suppression rate of 100 mol/L CA was
with those obtained in BSA degradation system. Carbonylation 72.45%, which was 22.32% and 16.50% statistically signifi-
inhibition rates of CA at 100 mol/L was 51.46%, dramatically cant (p < 0.01) higher than those of CTA and CFA, respectively.
stronger than its metabolites. These results showed that the inhibiting effect of CA on her-
ring sperm DNA induced by alcoxyl radicals was significantly
3.3. Effects of CA, CTA and CFA on protein nitration (p < 0.01) stronger than its metabolites at same experimental
concentrations.
As shown in Fig. 1(q–t), treatment with hemin/nitrite/H2O2 Fig. 3(e–h) showed that supercoiled form of DNA unwound
reaction system for 60 min significantly induced the formation of and gradually disappeared after the treatment of 10 mmol/L
3-nitrotyrosine in BSA, which suggested obvious BSA protein AAPH, while control group was almost all consisted of
nitration. In a certain concentration range (10–1000 mol/L), suprahelical helixes. Pretreatment with 5–100 mol/L CA and
CA and its metabolites had significant dose-dependent inhibition its metabolites increased superhelical structure concentration
abilities on nitration induced by hemin/nitrite/H2O2. In addi- dependently, implying that CA and its metabolites all remark-
tion, 500 mol/L CA and its metabolites pretreatment almost ably inhibited DNA oxidative cleavage caused by alcoxyl
completely restrained the formation of 3-nitrotyrosine. The sup- radicals. The content of supercoiled DNA was 82.73% when
pression rates of CA, CTA and CFA at 100 mol/L were 83.51%, treated with 100 mol/L CA, which was significantly (p < 0.01)
36.09% and 43.99%, respectively (Supplementary Fig. 2). higher than its metabolites CTA and CFA. The result demon-
Results indicated the stronger inhibiting capacity of CA on strated that the inhibiting effect of CA on pBR322 plasmid DNA
hemin/nitrite/H2O2-induced BSA nitration (p < 0.01) (Supple- breakage induced by alcoxyl radicals was significantly (p < 0.01)
mentary Fig. 2). stronger than its metabolites.
3.4. Effects of CA, CTA and CFA on lipid peroxidation 3.6. Effects of CA, CTA and CFA on BV2 cell viability
induced by lipopolysaccharide
Lipid peroxidation product such as malondialdehyde can
react with thiobarbituric acid to generate TBARS, which is an Chronic inflammation plays a pivotal role in pathogenesis of
important index in measuring the degree of lipid peroxidation. neurodegenerative diseases such as Alzheimer’s disease, Parkin-
Lipid had a slow process of auto-oxidation in the absence of son’s disease and multiple sclerosis. As illustrated in Fig. 4, CA
2+
Fe , and the content of TBARS in induced group increased sig- and its metabolites themselves had no influence on cell viability.
nificantly (p < 0.01) compared with control group (Fig. 2(a–d)). After treated with lipopolysaccharide (1 g/mL) for 12 h, via-
CA and its metabolites all inhibited the lecithin liposome peroxi- bility of BV2 cells significantly decreased to 81.58% compared
Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166 161
Fig. 3. Effects of CA and its metabolites on DNA oxidative damage. (a–d) Effects of CA, CTA and CFA on hsDNA oxidation induced by alcoxyl radicals. hsDNA
was incubated with AAPH in the presence or absence of CA, CTA and CFA, as described in the Materials and Methods section. The degree of DNA oxidative damage
was measured by TBA method; (e–h) Effects of CA, CTA and CFA on pBR322 plasmid DNA oxidative cleavage induced by alcoxyl radicals. pBR322 plasmid
DNA was incubated with AAPH in the presence or absence of CA, CTA and CFA, as described in the Materials and Methods section. The levels of DNA oxidative
##
cleavage were tested by agarose gel electrophoresis, and the optical densities were quantified by Quantity One 4.6.2. Each value represents the mean ± SD (n ≥ 3),
**
represents significant differences compared to the control group (p < 0.01); * represents significant differences compared to the induced group (p < 0.05); represents
significant differences compared to the induced group (p < 0.01); represents significant differences compared to the same concentration of CA (p < 0.01).
162 Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166
Fig. 4. Effect of CA and its metabolites on lipopolysaccharide −induced BV2 cell viability. BV2 cells were treated with lipopolysaccharide (1 g/mL) 12 h with
##
or without of pretreatment of CA, CTA and CFA for 4 h. Cell viability was measured by MTT assay. Each value represents the mean ± SD (n ≥ 8), represents
**
significant differences compared to the control group (p < 0.01); * represents significant differences compared to the induced group (p < 0.05); represents significant
differences compared to the induced group (p < 0.01).
to control group. However, pretreatment of various concentra- 4. Discussion
tion of CA and its metabolites improved cell viability compared
to lipopolysaccharide treatment group. After pretreatment with Various studies have confirmed that phytochemicals, espe-
80 mol/L CA, CTA and CFA, cell viability elevated back to cially plant phenols could prevent or alleviate oxidative stress
88.84%, 83.64%, and 84.24%, respectively. in diseases [29]. In this study, antioxidant effects of CA and
its metabolites on the protection of biological macromolecules
in vitro and inflammatory responses in microglial cells were
3.7. Effects of CA, CTA and CFA on the production of NO investigated. Excessive ROS/RNS will attack biological macro-
induced by lipopolysaccharide molecules such as proteins, lipids and DNA, disrupt cell
structure and functions.
NO, an inflammatory product, results in biomacromolecule Recent studies have found that many diseases such as
damage and aggravates inflammatory process. Fig. 5 showed that Alzheimer’s, diabetes, cardiovascular disease are associated
CA and its metabolites had no effects on the production of NO with elevated levels of protein carbonylation [30–32]. Protein
in BV2 cells. However, 20–80 mol/L CA and its metabolites tyrosine nitration has been identified as an important selective
remarkably reduced the increased production of NO induced post-translational modification, and is vital in the initiation and
by lipopolysaccharide in concentration-depended manners. The aggravation in cardiovascular diseases, ischemia/reperfusion
NO inhibition rates of CA, CTA and CFA at 80 mol/L were injury, inflammation, and neurodegenerative disorder. CA, CFA
11.14%, 10.25% and 9.60%, respectively. and CTA all significantly inhibited protein degradation and car-
bonylation induced by hydroxyl radicals and alcoxyl radicals,
suppressed the RNS-induced nitration in the experimental con-
3.8. Effects of CA, CTA and CFA on the production of ROS centrations (10–1000 mol/L). And the protective effects of
induced by lipopolysaccharide CA were markedly stronger than its metabolites. It should be
noted that CFA promoted oxidation in high concentration range
3+
ROS is mainly produced by mitochondria and NADPH oxi- induced by hydroxyl radicals, may due to the reduction of Fe to
2+ 2+
dase located on the cell membrane. Excessive ROS can trigger a Fe , and the reaction between Fe and H2O2 forming hydroxyl
variety of signaling pathways such as NFB and MAPK, lead- radicals [33].
ing to cell proliferation, differentiate, or death. As demonstrated Lipid peroxidation readily occurs in mitochondria, cell mem-
in Fig. 6, lipopolysaccharide treatment induced elevated ROS brane, endoplasmic reticulum, lysosomes, perioxisomes and
levels in BV2 cells. As expected, CA significantly (p < 0.01) other membrane structures that contain rich polyunsaturated
reduced the ROS levels induced by lipopolysaccharide, which fatty acids. In the process of lipid peroxidation, a large num-
was 7.93 and 2.25 times than the effects of CTA and CFA. ber of toxicities including malondialdehyde, acraldehyde and
Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166 163
Fig. 5. Effect of CA and its metabolites on lipopolysaccharide-induced NO production. BV2 cells were treated with lipopolysaccharide (1 g/mL) 12 h with or
without of pretreatment of CA, CTA and CFA for 4 h. The accumulation of nitrite was measured to assess the NO production in BV2 cells. Each value represents the
##
mean ± SD (n ≥ 8), represents significant differences compared to the control group (p < 0.01); * represents significant differences compared to the induced group
**
(p < 0.05); represents significant differences compared to the induced group (p < 0.01).
4-hydroxynonenal are produced [34]. Linoleic acid, an essential nervous system, can secrete cytokines such as NO, TNF-␣,
fatty acid containing two easily oxidized double bonds, is an and IL-1β [40]. Lipopolysaccharide is an efficient activator
ideal material for studying lipid peroxidation [35]. The polyun- of microglial cells and it can increase the production of ROS
saturated fatty acids of very low density lipoprotein and low and pro-inflammatory cytokines, which damage the surrounding
density lipoprotein located in the C-2 of lecithin produce perox- nerve cells [41]. Lipopolysaccharide activates receptor ser-
2+
idation under the catalysis of Fe . So lecithin is usually used as a ine/threonine kinases to trigger the phosphorylation of a series
cell model in vitro. This study indicated that the inhibition of CA protein kinase through specific receptor CD14 and TLR4 on the
2+
on Fe -induced linoleic acid and soybean lecithin liposomes cell membrane, and it also activates p38MAPK and NFB sig-
peroxidation were remarkably stronger than its metabolites. It naling pathways to participate in the process of iNOS expression,
is also worthy of noting that CFA had a better protective effect which promotes the production of NO [42]. NO, as an intra-
of linoleic acid in comparison with CA. cellular messenger molecule, takes part in vascular regulation,
DNA oxidative damage caused by radicals attack is closely neurotransmitter, immune responses and other physiological
related to a variety of diseases such as inflammation, tumor, processes [43]. However, excess NO will react with superox-
•− −
cardiovascular disease and aging. In this work, CA and its ide anion (O2 ) to generate peroxynitrite (ONOO ), which
•
metabolites showed a concentration-dependent inhibition on the decomposes to generate a potent oxidant similar to HO in
oxidation of hsDNA and protection for pBR322 plasmid DNA reactivity, and it may cross cell membranes through anion
−
from breakage induced by alcoxyl radicals. Protective effects of channels [44]. ONOO induces protein nitration, lipid peroxida-
−
CA were statistically significant better than its metabolites. It has tion, and DNA breakage [45]. Increased formation of ONOO
been demonstrated that polyphenols in mango wine could pro- has been linked to Alzheimer’s disease, rheumatoid arthritis,
tect DNA against UV + H2O2 and ␥-irradiation induced DNA atherosclerosis, lung injury, amyotrophic lateral sclerosis, and
damage [36]. (+)-catechin hydrate, (−)-epigallocatechin gallate other diseases [46,47]. This study demonstrated that both CA
hydrate, morin hydrate, quercetin hydrate and resveratrol have and its metabolites significantly increased BV2 microglial cell
potential for protecting both the viability of cells and ability viability and inhibited the production of NO and ROS induced by
to proliferate from damage caused by UV-A-irradiation [37]. lipopolysaccharide. As noted, the inhibition of CA on the ROS
Green tea (Camellia sinensis) strongly reversed apoptotic hep- levels induced by lipopolysaccharide was stronger than CTA and
atic degeneration and prevented micronecrosis and mutagenic CFA, which was consistent with the scavenging of free radicals
• • +•
DNA damage induced by arsenic [38]. including DPPH , OH, ABTS and biological macromolecu-
Chronic inflammation in the brain is one of the most impor- lar protection effects in vitro. Whereas CA, CTA and CFA had
tant pathological features of neural degenerative diseases such no significant difference in NO levels and cell viability. Since
as Alzheimer’s, Parkinson’s disease and multiple sclerosis [39]. DCFH-DA assay was used to detect overall intracellular redox,
Microglia, the most representative immune cells in the central cells incubation with compounds with more hydroxyl groups is
164 Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166
Fig. 6. Effect of CA and its metabolites on lipopolysaccharide −induced ROS production. BV2 cells were treated with lipopolysaccharide (1 g/mL) 12 h with or
without of pretreatment of CA, CTA and CFA for 4 h. The production of ROS was monitored with DCFH-DA fluorescence dye. (a) using inverted fluorescence
microscope to observe the generation of ROS; (b) using multiscan spectrum to make quantitative determinations of intracellular ROS. Each value represents the
## **
mean ± SD (n ≥ 8), represents significant differences compared to the control group (p < 0.01); represents significant differences compared to the induced group
(p < 0.01); represents significant differences compared to the same concentration of CA (p < 0.01).
likely to have a lower DCF fluorescence, which may partly con- ings will provide scientific bases for the application of CA and
tributed to the more powerful inhibitory effects of CA. There its metabolites as nutrition and natural antioxidants.
are structure-activity relationships of polyphenols to inhibit-
ing lipopolysaccharide-induced inflammation [48]. However, Conflict of interest
the molecular mechanism remains to be further investigated.
The authors declare that there are no conflicts of interest.
5. Conclusion
Acknowledgement
In this study, effects of CA and its metabolites on in vitro
This work was supported by the National Key Research and
protection of biological macromolecules and inflammatory
Development Program of China (No. 2016YFD0400601) and
responses in microglial cell were investigated. Results demon-
the Science and Technology Coordination Project of Innovation
strated that CA and its metabolites all exerted remarkable
in Shaanxi province (NO. 2014KTCL02-07).
inhibition capacities to biological macromolecules damages
including protein, lipid, and DNA induced by different kinds
Appendix A. Supplementary data
of free radicals. In addition, CA and its metabolites suppressed
the decline of BV2 microglia cell viability and the production
of NO and ROS induced by lipopolysaccharide. Furthermore, Supplementary data associated with this article can be
found, bioactivities of CA were significantly stronger than those of its in the online version, at http://dx.doi.org/10.1016/
j.fshw.2017.09.001 metabolites within a certain concentration range. These find- .
Q. Liu et al. / Food Science and Human Wellness 6 (2017) 155–166 165
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