Anti-Inflammatory Effects of Cajaninstilbene Acid and Its Derivatives

The effects of CSA and its derivatives of 5c, 5e, and 5h on cytokines (TNF-α ...... Wei, Z. F.; Jin, S.; Luo, M.; Pan, Y. Z.; Li, T. T.; Qi, X. L.; E...
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Anti-Inflammatory Effects of Cajaninstilbene Acid and Its Derivatives Mei-Yan Huang,† Jing Lin,† Kuo Lu, Hong-Gui Xu, Zhi-Zhong Geng, Ping-Hua Sun, and Wei-Min Chen* College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China ABSTRACT: Cajaninstilbene acid (CSA) is one of the active components isolated from pigeon pea leaves. In this study, antiinflammatory effects of CSA and its synthesized derivatives were fully valued with regard to their activities on the production of nitric oxide (NO) and pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in vitro cell model, as well as their impacts on the migration of neutrophils and macrophages in fluorescent protein labeled zebrafish larvae model by live image analysis. Furthermore, the anti-inflammatory mechanism of this type of compounds was clarified by westernblot and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that CSA, as well as its synthesized derivatives 5c, 5e and 5h, exhibited strong inhibition activity on the release of NO and inflammatory factor TNF-α and IL-6 in lipopolysaccharides (LPS)-stimulated murine macrophages. CSA and 5c greatly inhibited the migration of neutrophils and macrophages in injury zebrafish larvae. CSA and 5c treatment greatly inhibited the phosphorylation of proteins involved in nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Moreover, we found that peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor GW9662 could reverse partly the roles of CSA and 5c, and CSA and 5c treatment greatly resist the decrease of PPARγ mRNA and protein induced by LPS stimulation. Our results identified the promising anti-inflammatory effects of CSA and its derivatives, which may serve as valuable anti-inflammatory lead compound. Additionally, the mechanism studies demonstrated that the anti-inflammatory activity of CSA and its derivative is associated with the inhibition of NF-κB and MAPK pathways, relying partly on resisting the LPS-induced decrease of PPARγ through improving its expression. KEYWORDS: cajaninstilbene acid, anti-inflammation, PPARγ, NF-κB, MAPK

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INTRODUCTION Inflammation is known as an immune response initiated by pathogen invasion or tissue injury, and aggregation studies have realized that inflammation is always accompanied by various diseases, such as atherosclerosis,1,2 Alzheimer’s disease,3,4 diabetes,5,6 and cancer,7,8 and the treatment for inflammation is helpful to improve these diseases.9−13 Steroidal antiinflammatory drugs (SAIDs) and nonsteroidal anti-inflammatory drugs (NSAIDs) are the common drugs using in inflammation treatment nowadays, but more and more adverse side effects have been found in clinical use, which makes it urgent to find new drugs for inflammatory treatment. Food in traditional diet, especially that with pharmacological effects, are an important source of new drugs; thus, searching from food that with pharmacological effects may be an potential approach to the discovery of novel anti-inflammatory agents.14−16 Pigeon pea [Cajanus cajan (L.) Millsp.] is one of the most important grain legume crops in subtropical and tropical regions. Apart from its valuable applications in food and agricultural industry, pigeon pea can be also used for medicinal treatment. Traditionally, the seeds, leaves and roots of pigeon pea and its extract are used for curing diabetes,17−19 jaundice,17 measles,20 antifungals,21 ischemic necrosis of femoral head,22 osteoporosis23,24 as well as antioxidant activities.25−28 Particularly, cajaninstilbene acid (CSA) (Figure 1), one of the most important active components isolated from pigeon pea leaves,29,30 has attracted great interest due to its various pharmacological effects, such as antioxidation,31,32 deoxyribose nucleic acid (DNA) damage protection,33 relaxing renal artery,34 and antitumor activities.35−37 In addition to the above-mentioned active properties, ethanol extract from pigeon pea and the water extract © XXXX American Chemical Society

Figure 1. Chemical structure of CSA.

from pigeon pea leaves as well as active compounds that isolated from it also show anti-inflammatory activity. For instance, 50% ethanol extract of pigeon pea suppressed the production of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-6 (IL-6) in hydrogen peroxide (H2O2)-treated RAW264.7 macrophages.38 The cajanolactone isolated from pigeon pea leaves was reported to reduce the release of inflammatory factor TNF-α (IC50 < 22 μM) and IL-1β (IC50 < 40 μM) in lipopolysaccharides (LPS)stimulated RAW264.7 cells and J774A.1 cells.39 CSA also shows anti-inflammatory activity in inhibiting carrageenin induced murine paw edema.40 Thus, natural active compounds extracted from pigeon pea, such as CSA, and their derivatives are likely to be an excellent source for the discovery of novel antiinflammatory agents. However, the anti-inflammatory activity and the action mechanism of pigeon pea extracts still have not been fully understood yet. Received: January 17, 2016 Revised: March 20, 2016 Accepted: March 21, 2016

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DOI: 10.1021/acs.jafc.6b00227 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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After LPS-stimulation for 24 h, NO levels in cell supernatant were dectected using Griess reagent. Indometacin was used as a positive drug control. The experiment procedure and the calculation formula of NO inhibition rate are the same as previously described.43 ELISA. The effects of CSA and its derivatives of 5c, 5e, and 5h on cytokines (TNF-α and IL-6) release in RAW264.7 cells was evaluated using an enzyme-linked immunosorbent assay (ELISA).43 Briefly, RAW264.7 cells were seeded at a density of 3 × 104 cells per well in 96-well plates, and compounds were added to cells for 2 h prior to LPS-stimulation. When RAW264.7 cells were stimulated by LPS for 24 h, TNF-α and IL-6 levels in cell culture supernatant were measured as described in our previous work.43 Indometacin was used as a positive drug in this experiment. MTT Assay. The cell viability was measured using MTT assay44 as described in our previous work.43 Briefly, RAW264.7 cells were seeded at a density of 0.8 × 104 cells per well in 96-well plates and after treated with compounds for 48 h, the cell viability was measured and calculated as previously described.43 Zebrafish Larvae Live Image Analysis. Neutrophil/ macrophage double transgenic zebrafish (Coroninla-eGFP/LycdsRed) described in our previous work were used for the experiment.43 The preparing of 4 days post fertilization (dpf) zebrafish larvae, tail-cutting surgery, compound treatment, and image analysis were all performed as previously described.43 Western-Blot Analysis. RAW264.7 cells and PMA-pretreated U937 cells were seeded at a density of 2 × 106 cells per well in 6-well plates and were stimulated by LPS. When after LPS-stimulation for 1 h (RAW264.7) and 8 h (U937) respectively, the cells were collected for protein extraction and western-blot analysis. Protein extraction and western-blot analysis were performed as the protocol described previously.43 RT-PCR. RAW264.7 cells were seeded at a density of 2 × 106 cells per well in 6-well plates and were stimulated by LPS after pretreated by compounds for 2 h. When stimulated by LPS for 8 h, the cells were collected for extraction of total RNA. Total RNA was extracted by using RNA fast 200 (#220010, Fastagen Technology) according to the manufacture’s instruction, and the reverse transcription (RT) was using PrimeScript RT reagent kit (#RR047A, Takara). The relative mRNA levels of PPARγ were detected by quantitative real time PCR (qPCR). The primes used were listed as follows: GAPDH (forward: 5′AGGTCGGTGTGAACGGATTTG-3′; reverse: 5′-TGTAGACCATGTAGTTGAGGTCA-3′), PPARγ (forward: 5′CTGCATGTGATCAAGAAGAC-3′; reverse: 5′-AGTGCAATCAATAGAAGGAAC-3′). The reaction mixture was prepared using Premix Ex Tag II(#RR820A, Takara) according to the manufacture’s instruction. qPCR was performed 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, 58 °C for 60 s in Bio-Rad CFX96. Relative mRNA levels of PPARγ were obtained by normalized to control group and the revel of GADPH. Statistical analysis. Data were presented as means ± standard error (SD). Student’s t test was used for comparisons between two experiments. A value of p < 0.05 was considered statistically significant.

In the present work, the anti-inflammatory effects of CSA and its derivatives which were synthesized in our previous work41 were evaluated on both in vitro and in vivo models, and their antiinflammatory mechanism was deeply investigated by westernblot and reverse transcription-polymerase chain reaction (RTPCR) analysis as well. Toxicity of CSA and its derivatives on cells were also evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. The results showed the promising anti-inflammatory activity but low cytotoxicity of CSA and its derivatives, and revealed that their anti-inflammatory mechanism is via regulating peroxisome proliferator-activated receptor gamma (PPARγ) mediated nuclear factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signal pathways.

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MATERIALS AND METHODS Reagents. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco (Invitrogen, California, USA). The lipopolysaccharide (LPS, Escherichia coli 0127:B8) and MTT were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Indomethacin was purchased from national institutes for food and drug control (Beijing, China). N-(1-Naphthyl) ethylenediamine dihydrochloride and sulfanilamide were purchased from Sinopharm chemical reagent Co. Ltd. (Shanghai, China). TNF-α enzyme immunoassay kits (EK2822) and IL-6 enzyme immune assay kits (EK2062) were purchased from Multi Sciences Biotechnology (Hangzhou, China). Phorbol-12-myristate-13-acetate (PMA, S1819), IP cell lysis buffer (P0013), and phenylmethanesulfonyl fluoride (PMSF, ST506) were purchased from Beyotime Biotechnology (Shanghai, China). Antibeta actin monoclonal antibody (E021020−01) was purchased from EarthOx, LLC, CA. Primary antirabbit inducible NO synthase (iNOS, #2982), NF-κB pathway sampler Kit (#9936S), p-MAPK family antibody sampler kit (#9910S), MAPK family antibody sampler kit (#9926), and PPARγ (81B8) rabbit antibody (#2443) were purchased from Cell Signaling Technology. BCA protein assay kit (KGPBCA) was purchased from KeyGEN Biotechnology (Nanjing, China). The enhanced chemiluminescent (ECL) Detection Kit (WBKLS0050) was purchased from Merck Millipore Corporation (Merck KGaA, Darmstadt, Germany). CSA and its derivatives (purity >97%) were synthesized and the chemical structures were identified using 1H and 13C nuclear magnetic resonance (NMR) analysis in our laboratory as previously described.41 All compounds in stock including indomethacin, CSA and its derivatives were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 50 mM and the amount of DMSO was fixed at 0.1% (v/v) when added to cells. Cells and Treatment Protocol. RAW264.7 murine macrophages and U937 human monocyte cell line were purchased from American Type Culture Collection (ATCC). RAW264.7 cells were cultured in DMEM containing 10% FBS, penicillin/ streptomycin and U937 cells were cultured in 1640 containing 10% FBS, penicillin/streptomycin at 37 °C in a 5% CO2 humidified atmosphere. U937 cells were differentiated into macrophage-like cells by incubated with 20 ng/mL PMA for 24 h. For inflammatory experiments, RAW264.7 and PMA-pretreated U937 cells were prestimulated with the synthetic compounds for 2 h prior to LPS (100 ng/mL) stimulation. NO Production Assay. NO was detected by Griess assay42 as described in our previous work.43 Briefly, RAW264.7 cells were seeded at a density of 5 × 105 cells per well in 24-well plates, and compounds were added to cells for 2 h prior to LPS-stimulation.

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RESULTS AND DISCUSSION Effect of CSA on Inhibition of NO Release in LPSStimulated RAW264.7 Cells. Macrophages are the important immune cells in mammals and produce a great amount of NO after stimulated by LPS, which results in inflammation.45 To further study the activity of CSA against inflammation, we B

DOI: 10.1021/acs.jafc.6b00227 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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also designed and synthesized a series of CSA derivatives with different R group (Table 2) and evaluated their inhibition effects

incubated CSA with RAW264.7 cells to determine its dosageeffect and time-effect against NO production. In brief, RAW264.7 cells were first treated with CSA for 2 h and then 100 ng/mL LPS were added to the cells. After LPS treatment, the cell medium was collected and the amount of NO was determined using Griess reagent. The results (Table 1) were

Table 2. Chemical structures of CSA derivatives 5b−5k

Table 1. Dosage Effect of CSA on NO Release in LPSStimulated RAW264.7 Cells Compd. CSA

indometacin a

concentration (μM)

NO inhibition rate (%) ± SD

10 20 50 20

21.16a ± 1.91 30.63a ± 2.21 40.92a ± 3.18 37.74a ± 3.74

p < 0.01, compared with cells treated by LPS alone.

reported as NO inhibition rate means ± standard error determined from at least three independent experiments. Table 1 shows a concentration-dependent NO release inhibition rates of CSA, where NO release inhibition rates were 40.92 ± 3.18%, 30.63 ± 2.21%, 21.16 ± 1.91%, corresponding to 50, 20, and 10 μM CSA. Interestingly, NO release inhibition rate at a CSA concentration of 20 μM was found close to that of 20 μM indometacin, where the NO release inhibition rate is 37.74 ± 3.74%. To determine the effect of CSA on NO release at different time in LPS-stimulated RAW264.7 cells, we collected cell medium at different intervals during LPS treatment. As shown in Figure 2, cells without CSA and LPS treatment show no changes

on NO release in LPS-stimulated RAW 264.7 cells. Protocols were the same as described above for CSA. Remarkably, as shown in Table 3, derivatives 5c, 5e, and 5h present stronger inhibition Table 3. Inhibitory Effects of 20 μM of CSA Derivatives on NO Release in LPS-Stimulated RAW264.7 Cells

Figure 2. Time effect of CSA on NO release in LPS-stimulated RAW264.7 cells. RAW264.7 cells were first treated with CSA for 2 h and then treated by 100 ng/mL LPS. After LPS treatment, the cell supernatant at different time points was collected and determined the amount of NO by Griess reagent. The data shown were averaged by at least three different experiments. ##P < 0.01, difference between the control group and LPS-treated alone group at the same time point, **P < 0.01, difference between the CSA-pretreated group in the present of LPS and LPS-treated alone group at the same time point.

in the NO release over time, while release of NO was found increased greatly after 6 h incubation for cells pretreated with LPS. However, when cells pretreated with 20 μM of CSA before LPS addition, release of NO was much lower than that in the cells with LPS treatment alone under the same time point after 12 h incubation, suggesting the inhibition effect of CSA on LPSstimulated NO release along with time. Taking together, we believe that CSA is a promising leading compound for antiinflammatory drug design. Effects of CSA Derivatives on Inhibition of NO Release in LPS-Stimulated RAW264.7 Cells. To fully investigate the anti-inflammatory effect of CSA skeleton, in addition to CSA, we

Compd.

NO release inhibition rate (%) ± SD

Compd.

NO release inhibition rate (%) ± SD

5b 5c 5d 5e 5f

36.51a ± 5.96 60.59a ± 13.47 39.73a ± 2.44 49.79a ± 4.92 28.88a ± 4.98

5g 5h 5i 5j 5k

21.09a ± 2.85 47.21a ± 10.74 26.89a ± 2.94 22.45a ± 5.07 27.89a ± 7.67

a

p < 0.01, compared with cells treated by LPS alone.

effect on NO release than that of CSA at the same concentration of 20 μM, where the corresponding inhibition rates are 60.59 ± 13.47%, 49.79 ± 4.92%, and 47.21 ± 10.74%, correspondingly. These results indicate that some of the CSA derivatives also exhibit the similar even improved anti-inflammatory activity, which confirmed the anti-inflammatory potential of this class of compounds that with CSA skeleton. Moreover, taken structure and bioactivity analysis together, compounds with halogen C

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Figure 3. Effects of CSA and its derivatives on TNF-α and IL-6 release in LPS-stimulated RAW264.7 cells. All data represent the mean ± SD of three different experiments. ##P < 0.01, different from the control group, **P < 0.01, *P < 0.05, different from the LPS-treated alone group, §§P < 0.01, different from the Indo-pretreated group in the presence of LPS.

Table 4. Effects of CSA and Its Derivatives on RAW264.7 Viability Compd.

50% cell viability concentration (μM) ± SD

20 μM cell viability rate (%) ± SD

CSA 5b 5c 5d 5e 5f

84.34 ± 7.18 62.95 ± 9.48 64.11 ± 4.69 84.76 ± 2.44 63.53 ± 13.18 76.04 ± 14.98

104.51 ± 11.37 100.60 ± 5.96 102.01 ± 13.47 103.88 ± 2.44 106.40 ± 4.92 96.59 ± 14.98

Compd.

50% cell viability concentration (μM) ± SD

20 μM cell viability rate (%) ± SD

5g 5h 5i 5j 5k

54.30 ± 8.81 84.27 ± 10.35 88.35 ± 6.35 50.56 ± 3.47 88.80 ± 3.42

101.10 ± 10.74 103.09 ± 9.18 108.19 ± 8.94 94.67 ± 5.07 101.89 ± 7.67

Effects of CSA and Its Derivatives on the Migration of Neutrophils and Primitive Macrophages in Injury Transgenic Zebrafish Larvae. In vitro anti-inflammatory activity of CSA and its derivatives have been fully proved by above experiments, but are they also effective in vivo? To answer this question, next the injury transgenic zebrafish larvae was selected as the in vivo model for studying the anti-inflammatory effects of CSA and its derivatives. Neutrophil and macrophage are two important immune cells in living body, a large number of which will be produced and transported to the sites of inflammation when inflammation occurs. In this work, a double-transgenic line (Coroninla-eGFP/Lyc-dsRed) was used, in which neutrophils were double labeled by eGFP and dsRed presenting yellow color, and primitive macrophages were labeled by eGFP only presenting green color. The injury inflammation was induced by tail cutting; indometacin was used as the control drug. As shown in Figure 4, after tail cutting for 3 and 6 h, neutrophils and primitive macrophages in the control group obviously accumulated around the wound, which is consistent with what is described in other zebrafish inflammatory experiments.46−49 Compared to the control group, accumulation of neutrophils and primitive macrophages around the wound evidently reduced after treatment with 1.25 and 5 μM of CSA and its derivative of 5c in all the evaluated time, while the tendency is similar to that treated with indometacin. In conclusion, all the above results suggested the excellent in vitro and in vivo anti-inflammation activity of CSA and its derivative. Then, further efforts were performed to figure out their anti-inflammatory mechanism. Western Blot for Interpreting Possible Mechanism. Generally, two pathways can be activated in the LPS-induced inflammation. They are NF-κB signal pathway and MAPK pathway. Activation of NF-κB signal pathway leads to the phosphorylation of inhibitor kappa B alpha (IκBα) and NF-κB p65 proteins, which can then lead to the activation of NF-κB and the expression of inflammatory genes.50 While activation of MAPK pathways result in the increase in extracellular-signal-

substituents at phenyl B displayed better anti-inflammatory effect. Effects of CSA and Its Derivatives on TNF-α and IL-6 Production in LPS-Stimulated RAW264.7 Cells. To further investigate the anti-inflammatory activity of CSA and its derivatives, TNF-α and IL-6, two crucial pro-inflammatory mediators, were measured in LPS-stimulated RAW264.7 cells after treatment with different compounds. In this study, we first added CSA and derivatives of 5c, 5e and 5h to RAW264.7 cells for 2 h treatment, then 100 ng/mL LPS were added. After 24 h, cell medium was collected and the amount of TNF-α and IL-6 was quantified using Elisa kit, results are given in Figure 3. Generally, TNF-α and IL-6 detected in the cell medium after treatment with 20 μM CSA and its derivatives were lower than that only treated with LPS, notably, at the same concentration of 20 μM, the inhibitory effect of CSA on the production of IL-6 was even significant stronger than that of the positive control drug indomethacin, these results further suggest the excellent anti-inflammtory effects of CSA and its derivatives 5c, 5e, and 5h. Cytotoxicity of CSA and Its Derivatives. To examine the cytotoxicity of CSA and its derivatives and to investigate whether their effects on suppression of the production of NO, TNF-α, and IL-6 were related to cell viability, RAW264.7 cells were treated with this class of compounds for 48 h, and then cell viability was determined using MTT assay. As shown in Table 4, 50% cell viability concentrations of all the compounds are higher than 50 μM, which indicate the relative low toxicity of this class of compounds. As low toxicity is the basis for the development as drugs, this result suggests that CSA analogous are good antiinflammatory candidates. Notably, no obvious decrease in cell viability was observed when cells treated with 20 μM of those compounds, the relative cell viabilities of the treated cells were all almost 100%. This result suggests that CSA and its synthetic derivatives are all nontoxic at the effective concentration and the anti-inflammatory effects of them could probably be attributed to the interaction of them with their specific target. D

DOI: 10.1021/acs.jafc.6b00227 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 6, similar trends were found as p-IκBα and p-p65, treating with CSA and 5c significantly decreased the production of pERK(1/2), p-JNK(1/2) and p-p38 in comparison with those treated only with LPS. Our results suggest that anti-inflammatory activity of CSA and 5c is due at least in part to suppressing the activation of NF-κB and MAPK pathways in LPS-stimulated macrophages. Additionally, considering that NF-κB and MAPK pathways activation have been proved to be modulated by PPARγ,53 the activation of these two pathways could be inhibited by activating PPARγ using PPARγ synthetic agonist, such as rosiglitazone.54,55 In order to investigate whether the inhibitory effects of CSA and 5c on NF-κB and MAPK pathways activation is mediated by PPARγ, an irreversible PPARγ inhibitor GW9662 was used to treat the cells before addition of CSA and 5c. As a parallel experiment, 5 μM of GW9662 was added for treating RAW264.7 cells for 30 min prior to the addition of CSA or 5c, while the following steps kept unchanged. As shown in Figure 5 and Figure 6, pretreatment with GW9662 obviously weaken the inhibitory ability of CSA and 5c on the expression of the NF-κB pathway associated proteins p-IκBα and p-p65 (Figure 5) as well as the MAPK pathways associated proteins p-ERK (1/2), p-JNK (1/2) and p-p38 (Figure 6). These results demonstrate that the roles of CSA and 5c could be reversed partly by PPARγ inhibitor GW9662, which suggests that the suppression effects of CSA and 5c on the activation of NF-κB and MAPK pathways are most likely through enhancing the activity of PPARγ, which could relate to improving the protein level or protein function of PPARγ. In order to clarify how does CSA and 5c regulate PPARγ, we further detected the protein and mRNA levels of PPARγ in macrophage cell models that with or without pretreated by CSA and 5c. As shown in Figure 7, both the protein and mRNA levels of PPARγ were greatly reduced by LPS treatment compared to that of control in macrophage cell models. These results are in line with the fact that PPARγ protein can be degraded in LPSstimulated inflammation.56 Notably, CSA and 5c pretreatment dramatically increase the protein as well as mRNA levels of PPARγ in LPS-stimulated macrophage cells. These results suggest that CSA and 5c is relying on resisting the LPS-induced decrease of PPARγ through improving its expression to enhance its activity. In summary, the anti-inflammatory mechanism of CSA and its derivatives is to suppress the activation of NF-κB and MAPK pathways by resisting the LPS-induced decrease of

Figure 4. Effects of CSA and 5c on migration of neutrophils and macrophages in injury transgenic zebrafish larvae. After tail cutting for 30 min, 4 dpf transgenic zebrafish larvae were treated by CSA and 5c (1.25 and 5 μM). Indometacin (Indo) was used as control drug. The images of neutrophils (yellow) and macrophages (green) in 3 and 6 h were recorded by fluorescent microscope. The blank was healthy larvae. The control was tail-cutting larvae but without compound treatment. 6− 8 larvae in each group were recorded. Scale bar = 400 μm.

regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 phosphorylation, which will promote the development of inflammation.51,52 To know exactly whether CSA and its derivatives realize their anti-inflammatory activity through these two pathways, expression of the above-mentioned proteins was further identified by performing Western blot analysis. In a typical experiment, 20 μM of CSA or 5c was added to treat RAW264.7 cells, then 100 ng/mL LPS was added to stimulate cells for 1 h. After that, for NF-κB pathway, the expression of phosphorylated IκBα (p-IκBα) and phosphorylated p65 (p-p65) was analyzed using Western blot analysis. The data (Figure 5A− C) showed that expression of p-IκBα and p-p65 were remarkably increased when cells were treated with LPS only, while addition of CSA and 5c significantly inhibited the expression of those proteins. Simultaneously, the expression of phosphorylated ERK, JNK, and p38 involved in MAPK pathways were also monitored after the same treatment as described above. As it is shown in

Figure 5. Inhibitory effects of CSA and its derivative on expression of proteins of NF-κB pathway in LPS-stimulated RAW264.7 cells. (A) Western blot analysis of p-p65 and p-IκBα in cytoplasm fractions of RAW264.7 cells treated with LPS (100 ng/mL) in the presence or absence of CSA and derivative 5c or PPARγ inhibitor GW9662 for 1 h. Equal amounts of the cytoplasm fractions were loaded into each lane. (B, C) Graphs showing changes in the levels of p-p65 and p-IκBα; densitometry values were normalized using β-actin for cytoplasm fractions as internal controls. All data represent the mean ± SD of three different experiments. ##P < 0.01, #P < 0.05, different from the control group, **P < 0.01, *P < 0.05, different from the LPS-treated alone groups, ★★P < 0.01, ★P < 0.05, different from the LPS and compounds-treated groups in the absence of GW9662. E

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Figure 6. Effects of CSA and its derivative on expression of proteins of MAPK pathways in LPS-stimulated RAW264.7 cells. (A) Western blot analysis of ERK(1/2), JNK(1/2) and p38 of MAPK pathways in cytoplasm fractions of RAW264.7 cells treated with LPS (100 ng/mL) in the presence or absence of CSA and derivative 5c or PPARγ inhibitor GW9662 for 1 h. Equal amounts of the cytoplasm fractions were loaded into each lane. (B−D) Graphs showed changes in the ratios of p-ERK/ERK, p-JNK/JNK and p-p38/p38. All data represent the mean ± SD of three different experiments. ##P < 0.01, different from the control group, **P < 0.01, *P < 0.05, different from the LPS-treated alone groups, ★★P < 0.01, ★P < 0.05, different from the LPS and compounds-treated groups in the absence of GW9662.

Figure 7. Effects of CSA and its derivative 5c on the protein and mRNA levels of PPARγ in LPS-stimulated macrophage cell models. (A) Western blot analysis of the protein levels of PPARγ in cytoplasm fractions of PMA-pretreated U937 macrophage cells treated with LPS (100 ng/mL) in the presence or absence of CSA and derivative 5c for 8 h. (B) Quantitative RT-PCR analysis of the mRNA levels of PPARγ in RAW264.7 cells treated with LPS (100 ng/mL) in the presence or absence of CSA and derivative 5c for 8 h. All data represent the mean ± SD of three different experiments. ##P < 0.01, different from the control group, **P < 0.01, *P < 0.05, different from the LPS-treated alone groups.

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PPARγ, an identified negative regulatory factor of inflammation

CONCLUSION

In conclusion, the anti-inflammatory activity of CSA (isolated from pigeon pea leaves) and its synthetic derivatives were investigated both in in vitro macrophage cell models and in in vivo transgenic zebrafish larvae. For the in vitro anti-

with the inhibitory effect on the activation of NF-κB and MAPK pathways,57−60 through improving its expression. F

DOI: 10.1021/acs.jafc.6b00227 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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inflammatory experiments, we found that those compounds presented the similar even better inhibitory effects on the release of inflammatory mediator and cytokines (NO, TNF-α, and IL-6) than indometacin, and compounds with halogen substituents at phenyl B displayed better anti-inflammatory effect. For the in vivo evaluation, CSA and its derivative 5c greatly reduced the production and migration of neutrophils and primitive macrophages in the injury zebrafish larvae. Moreover, we demonstrated that anti-inflammatory activity of CSA and its derivative 5c probably realized by suppressing the activation of NF-κB and MAPK pathways, most likely through resisting the LPS-induced decrease of PPARγ by improving its expression. In all, our work demonstrated that CSA and its derivatives could be potential candidates for the treatment of inflammation. CSA and its derivatives can be developed either as functional foods or as complementary medicines or original material for new drugs.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 20 8522 4497. Fax: +86 20 8522 4766. E-mail: [email protected]. Author Contributions †

These authors contributed equally to this paper.

Funding

We thank the Natural Science Foundation of Guangdong Province (2014A030311021) for financial support to this study. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jafc.6b00227 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

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DOI: 10.1021/acs.jafc.6b00227 J. Agric. Food Chem. XXXX, XXX, XXX−XXX