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Jul 14, 2015 - ABSTRACT: Ampelopsis grossedentata, an indigenous plant in southern China, has been used for treating pharyngitis in traditional Chines...
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Suppression of Inflammatory Responses by Dihydromyricetin, a Flavonoid from Ampelopsis grossedentata, via Inhibiting the Activation of NF-κB and MAPK Signaling Pathways X. L. Hou, Q. Tong, W. Q. Wang, C. Y. Shi, W. Xiong, J. Chen, X. Liu, and J. G. Fang* Department of Pharmacy, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China ABSTRACT: Ampelopsis grossedentata, an indigenous plant in southern China, has been used for treating pharyngitis in traditional Chinese medicine for hundreds of years. In this study, we explored the anti-inflammatory activity of dihydromyricetin (1), its major bioactive component, and the underlying mechanism of this action. We demonstrated that 1 suppressed the levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) as well as increased the level of the anti-inflammatory cytokine interleukin-10 (IL-10) in lipopolysaccharide (LPS)-treated mice. Moreover, 1 was found to markedly inhibit the production of nitric oxide (NO) and the levels of TNF-α, IL-1β, and IL-6, whereas it increased the level of IL-10 in LPS-induced RAW 264.7 macrophage cells. Compound 1 also reduced the protein expression of inducible nitric oxide synthase (iNOS), TNF-α, and cyclooxygenase-2 (COX-2) in macrophage cells. Furthermore, 1 suppressed the phosphorylation of NF-kappa B (NF-κB) and IκBα as well as the phosphorylation of p38 and JNK but not ERK1/2 in LPS-stimulated macrophages. Taken together, the present results suggest that 1 exerts its topical anti-inflammatory action through suppressing the activation of NF-κB and the phosphorylation of p38 and JNK. Thus, 1 may be a potentially useful therapeutic agent for inflammatory-related diseases.

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kinase (ERK), and c-Jun NH2-terminal kinase (JNK), are also demonstrated to regulate the transcription of COX-2, iNOS, and inflammatory cytokines in inflammatory processes.10,11 Ampelopsis grossedentata (Hand.-Mazz.) W. T. Wang (Vitaceae), known as vine tea, is a medicinal and edible herb widely distributed in several provinces in the Yangtze River region of China (including Hubei, Fujian, Guangdong, Guangxi, Hunan, Guizhou, Yunnan, and Jiangxi). As recorded in the Chinese Materia Medica, vine tea carries out several functions such as clearing away heat, promoting diuresis and blood circulation, and removing channel obstructions (Editorial Committee of Chinese Materia Medica, 1999). Vine tea has been widely utilized not only as a healthy tea but also as a medicinal herb in traditional Chinese medicine to cure diseases including pharyngitis, sore throat, cold-related fever, and allergenic skin disease for hundreds of years, especially in the clinical treatment of pharyngitis.12,13 Several studies have demonstrated that vine tea possesses many beneficial pharmacological properties including anti-inflammatory, antioxidative, antihypertensive, hepatoprotective, and antiviral activities.14 As the most abundant (with a content of more than 30% in the tender stems and leaves of A. grossedentata) and bioactive constituent in vine tea,15 dihydromyricetin (1) was reported to

nflammation is a normal defensive reaction of the body to infection. However, an excessive inflammatory response can lead to negative effects on tissue function or can even result in overt tissue damage when allowed to continue unchecked.1 Further, numerous reports have demonstrated that inflammation can even be detrimental, as it is associated with many diseases such as atherosclerosis, cancer, asthma, and rheumatoid arthritis.2,3 Although a great deal of effective antiinflammatory agents are available, such as nonsteroidal antiinflammatory drugs, biological anti-inflammatory agents, and statins, it is still a challenge for pharmaceutical researchers to develop more effective and less toxic agents to treat the various symptoms of acute and chronic inflammatory diseases.4 Inflammation is usually mediated by immune cells, such as monocytes and macrophages.5 The excessive production of inflammatory substances such as nitric oxide (NO) and inflammatory cytokines and the excessive expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by macrophages in response to pro-inflammatory stimuli play key roles in the inflammatory response.6,7 Accumulating evidence has highlighted the crucial role of NF-kappa B (NF-κB) in the expression of those inflammatory mediators.8 As a transcription factor, NF-κB is activated by the phosphorylation of IκB and then translocates into the nucleus, where it transcribes various NF-κB-dependent genes in stimulated macrophages.9 Mitogen-activated protein kinases (MAPKs), such as p38 kinase, extracellular signal-regulated © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 20, 2015

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samples were collected. Our results collectively indicated that treatment with 1 significantly reduced the serum concentrations of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 (Figure 2A, B, and C), while the serum concentration of the anti-inflammatory cytokine IL-10 was significantly elevated (Figure 2D). Effect of Dihydromyricetin (1) on the Viability and Cytotoxicity of RAW 264.7 Cells. To select an optimal concentration in cell experiments, we investigated the effects of 1 on cell viability and cytotoxicity by an MTT reduction assay and LDH release assay, respectively. As seen in Figure 3A and B, exposure to 1 (37.5−600 μM) for 24 h did not influence the viability of RAW 264.7 macrophages. Further, pretreatment with 1 (37.5−600 μM) for 2 h followed by treatment with LPS (1 μg/mL) for 12 or 24 h did not significantly affect cell viability. However, a significant difference in cell viability was seen in cells treated with 600 μM for 48 h (Figure 3C). Therefore, we chose a concentration of 1 ranging from 0 to 300 μM and an incubation time of 24 h for our experiments. Additionally, selective inhibitors used in these studies (SB230580 for p38, SP600125 for JNK, U0126 and PD98059 for ERK1/2) showed no effect on the viability of macrophage cells in the presence or absence of 1 μg/mL LPS (Figure 3D). Inhibitory Effect of Dihydromyricetin (1) on NO Production and iNOS Protein Expression in RAW 264.7 Cells. To confirm the anti-inflammatory effects of 1 obtained in vivo, nitrite oxide in the culture media of LPS-activated RAW 264.7 cells was measured by the Griess reaction, whereas protein expression of iNOS was assayed by Western blotting. As seen in Figure 3E, 1 at 300 μM reduced LPS (1 μg/mL)stimulated NO production of macrophage cells by 17.4%, compared with the control group. Furthermore, treatment of cells with 1 (37.5−300 μM) for 2 h prior to incubation with LPS for 24 h significantly reduced LPS-induced iNOS protein expression, as illustrated in Figure 4B. Thus, the inhibitory effect of 1 on iNOS expression occurred in parallel with the comparable inhibition of NO production. Inhibitory Effect of Dihydromyricetin (1) on COX-2 Expression in LPS-Stimulated RAW 264.7 Cells. As COX-2 has been reported to take part in inflammatory responses,19 we explored the protein expression levels of COX-2 by Western blotting. As seen in Figure 4A and B, LPS (1 μg/mL) significantly increased COX-2 protein expression in RAW 264.7 cells. Cells preincubated with various concentrations of 1 (37.5, 75, 150, 300 μM) for 2 h prior to treatment with LPS significantly reduced the expression of COX-2, by 51.2%, 85.5%, 84.3%, and 86.4%, respectively. Effect of Dihydromyricetin (1) on Pro- and Antiinflammatory Cytokine Production in LPS-Stimulated RAW 264.7 Cells. To further evaluate the anti-inflammatory effect of 1 on LPS-induced RAW 264.7 cells, the secretion of pro- and anti-inflammatory cytokines in macrophage culture medium was measured by ELISA. Cells were pretreated with 1 (37.5, 75, 150, or 300 μM) for 2 h and stimulated with LPS (1 μg/mL) for 24 h. The results showed that 1 decreased LPSinduced secretion of TNF-α, IL-1β, and IL-6 in a concentration-dependent manner (Figure 5A, B, and C), whereas LPS-induced secretion of IL-10 was increased in a concentration-dependent manner (Figure 5D). Dihydromyricetin (1) Interferes with the Phosphorylation and Degradation of IκBα as Well as P65 Phosphorylation in LPS-Induced RAW 264.7 Cells. It is well known that the translocation of NF-κB from the cytoplasm

possess numerous pharmacological activities, including antioxidative, anticancer, antimicrobial, and antihypertensive activity as well as hepatoprotective effects.16−18 However, few reports have been issued on the anti-inflammatory activities of 1 or the molecular mechanisms involved. Therefore, we explored the anti-inflammatory properties of 1 in RAW 264.7 macrophages in vitro, in lipopolysaccharide (LPS)-challenged mice, and in 12-O-tetradecanoylphorbol-13-acetate (TPA)induced acute ear edema in mice, as well as the molecular mechanisms involved.

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RESULTS AND DISCUSSION Effect of Dihydromyricetin (1) on TPA-Induced Ear Edema. Topical application of TPA (10 μL) induced acute inflammation when applied to the ears of KM mice. One hour later, 1 (2.3 and 4.6 mg per ear) was applied topically on the surface of the ear. Mice were sacrificed 5 h post-TPA application by cervical dislocation, and ear punch biopsies (8 mm in diameter) were collected. Inflammation was evaluated by measuring changes in ear weight as a surrogate for edema. As shown in Figure 1, treatment with 1 (2.3 and 4.6 mg

Figure 1. Inhibitory effects of 1 on TPA-induced mouse ear edema. After topical application of TPA (2.5 μg per ear) on the right ear of mice for 1 h, 1 (2.3 and 4.6 mg per ear) was applied on the surface of the ear. Edema was assayed 5 h after TPA application by measurement of the ear weight. Values are expressed as the increase in ear weight ± SD, n = 6. Statistical analyses were performed by one-way ANOVA test and Dunnett’s test. #p < 0.001 vs the control group; *p < 0.05, **p < 0.01 vs the TPA-stimulated group.

per ear) was found to significantly suppress TPA-induced increase in ear weight by 45.2% and 52.0%, respectively, compared to the control group. Effect of Dihydromyricetin (1) on Serum TNF-α, IL-1β, IL-6, and IL-10 Levels in LPS-Treated Balb/c Mice. To further determine the anti-inflammatory effects of 1, serum cytokine levels of LPS-stimulated Balb/c mice were measured by ELISA. Compound 1 (57.5, 115, 230, or 460 mg/kg) was administered as a single dose by intraperitoneal injection (ip). Two hours later, all mice received LPS (5 mg/kg) by ip, mice were sacrificed 6 h after LPS treatment, and then serum B

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Figure 2. Effect of 1 on plasma levels of TNF-α (A), IL-1β (B), IL-6 (C), and IL-10 (D) in LPS-treated mice. Compound 1 (57.5, 115, 230, or 460 mg/kg) was administrated (ip) 2 h prior to LPS challenge (5 mg/kg, i.p.), mice were sacrificed 6 h after LPS treatment, and then serum samples were collected for ELISA. Values are presented as mean ± SD, n = 5. Statistical analyses were performed by one-way ANOVA test and Dunnett’s test. #p < 0.001 vs the control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs the LPS-stimulated group.

To further confirm the signaling pathway involved in the anti-inflammatory effects of 1, selective inhibitors (SB230580 for p38, SP600125 for JNK, U0126, and PD98059 for ERK1/2, respectively) were employed. Cells were pretreated with inhibitor for 2 h and then were incubated with LPS (1 μg/mL) for 1 h. As seen in Figure 8A, the p38-specific inhibitor SB230580 markedly decreased the protein expression of TNF-α and COX-2 as well as the production of IL-6 (Figure 8B) in LPS-stimulated macrophages. The JNK-specific inhibitor SP600125 significantly decreased the protein expression of COX-2 and the production of IL-6, while the ERK1/2-specific inhibitors (U0126 and PD98059) showed no effects on the protein expression of either TNF-α or COX-2, nor did it influence the production of IL-6. The present study was undertaken to evaluate the antiinflammatory activity of 1, using established inflammatory models including a mouse model of LPS-induced acute inflammation in vivo, LPS-induced RAW 264.7 macrophage cells in vitro, and a TPA-induced mouse model of skin inflammation to confirm the above results. Our study clearly demonstrated that treatment of mice with 1 significantly reduced acute ear edema, as well as the plasma levels of inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and increased the level of plasma IL-10 in LPS-stimulated mice. These results have been further confirmed by examining the level of these cytokines in culture medium of LPS-induced macrophages, where cells were pretreated with 1 for 2 h followed by incubation with LPS (1 μg/mL) for 24 h. Macrophages, a prominent inflammatory cell type in the immune system, play a crucial role in many inflammatory processes.21,22 LPS, an essential component of the outer

to the nucleus is preceded by the phosphorylation and proteolytic degradation of IκBα. To investigate whether the decrease of iNOS and COX-2 protein expression as well as the regulation of the levels of inflammatory cytokines by 1 is associated with the suppression of the activity of NF-κB, we analyzed the phosphorylation and protein level of p65 and IκBα by Western blotting. As seen in Figure 6A, the levels of p-NFκB p65, indicating NF-κB activation, were increased after LPS stimulation, but were down-regulated in cells treated with 1. Furthermore, LPS was found to cause phosphorylation and degradation of IκBα in the cytosol, whereas pretreatment with 1 effectively blocked the increased phosphorylation of IκBα, as well as increased IκBα protein content. Dihydromyricetin (1) Affects MAP Kinase Activation in LPS-Stimulated RAW 264.7 Cells. Previous studies have reported that the three MAP kinases, ERK1/2, p38, and JNK, are involved in LPS-mediated inflammatory responses.20 In the present experiment, Western blot analysis was used to assess the effect of 1 on the induction of these kinases. Anti-p-ERK1/2, anti-p-p38, and anti-p-JNK antibodies were applied to detect the phosphorylation status of these MAPKs. As seen in Figure 7, pretreatment with 1, followed by LPS stimulation, did not modify the phosphorylation level of ERK1/2, whereas 1 significantly decreased the phosphorylation level of p38 and JNK. However, we observed that the total protein levels of p38 MAPK and JNK in macrophages remained unchanged by treatment with either LPS alone or cotreated with 1. These results suggest that 1 strongly inhibited the activation of p38 and JNK but not that of ERK1/2 in LPS-induced macrophages. These results indicated that the anti-inflammatory activities of 1 are partly due to suppression of the MAPK pathway. C

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Figure 4. Effect of 1 treatment on protein levels of iNOS and COX-2 in LPS-stimulated RAW 264.7 macrophages. The data are presented as mean ± SD, n = 3. Statistical analyses were performed by a one-way ANOVA test and Dunnett’s test. #p < 0.001 vs the control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs the LPS-stimulated group.

demonstrated that treatment with 1 (2.3, 4.6 mg per ear) significantly inhibited TPA-induced acute inflammation in mice (Figure 1). Although 1 was reported to have anti-inflammatory effects in vitro,26 our animal experimental results show a clearer picture of anti-inflammatory properties of 1 in vivo. As one of the key steps during inflammation, leukocyte infiltration is regulated chiefly by chemokines from neutrophils and monocytes. It was previously shown that production of these chemokines is controlled by iNOS-derived NO from inflammatory cells, such as macrophages.27,28 Moreover, iNOS is expressed, especially in inflammatory cells, in response to diverse pro-inflammatory stimuli. Similarly, COX-2 is significantly upregulated by inflammatory stimuli and contributes to pain and swelling in inflammatory diseases.29 Thus, we examined the production of NO and the expression of iNOS and COX-2 in RAW 264.7 macrophage cells. It was found that 1 treatment, at a concentration of 300 μM, on LPSinduced RAW 264.7 cells could reduce the production of nitrite and significantly decrease the protein expression of iNOS and COX-2 (Figure 3E, Figure 4). The nitrite inhibition by 1 was not due to cell damage since 1 was not cytotoxic at a dose of 300 μM (Figure 3A, B, and C). In macrophage cells stimulated with LPS, the expression of iNOS as well as COX-2 and the production of various inflammatory mediators such as cytokines, growth factors, NO, and enzymes are promoted by the activation of NF-κB. The activation of NF-κB is a multistep process induced via various upstream signal transduction pathways. By and large, upon cellular stimulation by LPS the IKK complex is activated, phosphorylating IκBα at specific serine residues and subsequently undergoing proteasomal degradation, which results in the release of p65 from the IκB-p65 complex, allowing it to translocate to the nucleus, where it transactivates target genes.30 To further explore the mechanism underlying the antiinflammatory effects of 1, we examined the effect of 1 on the proteolysis of IκBα and the nuclear translocation of NF-κB in

Figure 3. Effect of 1 on cell viability, cytotoxicity, and NO production, as well as the effects of selective inhibitors on RAW 264.7 macrophage cells in culture. (A) Cell viability was measured by an MTT assay. (B) Cell cytotoxicity was assessed by an LDH release assay. (C) Cells were treated with LPS (1 μg/mL) alone or with 1 as indicated for 12, 24, and 48 h, and cell viability was evaluated. (D) Cells were treated with selective inhibitors (SB230580, PD98059, U0126, and SP600125) alone or with LPS (1 μg/mL) for 24 h, and cell viability was evaluated. (E) Cell culture medium was collected for the nitrite assay using the Griess reagent. Statistical analyses were performed by a one-way ANOVA test and Dunnett’s test. #p < 0.001 vs the control group; *p < 0.05, **p < 0.01 vs the LPS-stimulated group.

membrane of Gram-negative bacteria, is considered as one of the most potent innate immune-activating stimuli known.23 It has been reported that LPS can induce macrophages to generate inflammatory cytokines (such as IL-1β, TNF-α, and IL-6), NO, and free radicals.24 Here, LPS-stimulated RAW 264.7 macrophage cells were selected to evaluate the antiinflammatory properties of 1. In addition, we found that LPS at a dose of 5 mg/kg by ip for 6 h exhibits similar proinflammatory effects to that of a 20 mg/kg dose. For this reason, we chose 5 mg/kg to perform our LPS-stimulated experiments in vivo. Given that cytokines mediate mechanisms of inflammation that impair organ integrity,25 we explored the effect of 1 on the production of cytokines in LPS-induced acute inflammation in vivo. Our results showed that 1 reduced the amount of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) that are significantly increased by LPS. In contrast, 1 markedly increased the amount of the anti-inflammatory cytokine IL-10 (Figure 2A, B, C, and D). Accordingly, pretreatment with 1 in vivo produced similar changes in the production of cytokines in LPS-induced RAW 264.7 macrophage cells (Figure 5A, B, C, and D). In our study, we also evaluated the anti-inflammatory effect of 1 against TPA-stimulated acute ear edema in mice. We have D

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Figure 5. Effect of 1 on LPS-induced production of TNF-α (A), IL-1β (B), IL-6 (C), and IL-10 (D) in RAW 264.7 macrophage cells. After pretreatment with or without 1 (37.5−300 μM) for 2 h, cells were treated with LPS (1 μg/mL) for 24 h followed by analysis of cytokine levels by ELISA. Experiments were performed in triplicate, and the data are presented as mean ± SD. Statistical analyses were performed by a one-way ANOVA test and Dunnett’s test. #p < 0.001 vs the control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs the LPS-stimulated group.

Figure 7. Effect of 1 on LPS-induced phosphorylation of MAP kinases. After pretreatment with or without 1 (37.5, 75, 150, 300 μM) for 2 h, cells were treated with LPS (1 μg/mL) and harvested for protein measurements after 1 h. Experiments were performed in triplicate, and the data are presented as mean ± SD. Statistical analyses were performed by one-way ANOVA and Dunnett’s test. #p < 0.001 vs the control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs the LPSstimulated group.

Figure 6. Effect of 1 on NF-κB in RAW 264.7 macrophage cells. Cells were treated with LPS (1 μg/mL) and 1 (37.5, 75, 150, 300 μM). After a 24 h incubation, IκBα and NF-κB p65 were evaluated by Western blot. Experiments were performed in triplicate, and the data are presented as mean ± SD. Statistical analyses were performed by a one-way ANOVA test and Dunnett’s test. #p < 0.001 vs the control group; *p < 0.05, ***p < 0.001 vs the LPS-stimulated group.

LPS-induced RAW 264.7 macrophage cells. Cells were treated with various concentrations of 1 for 2 h prior to incubation with LPS (1 μg/mL). As shown in Figure 6, the levels of p-NF-kB E

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Figure 8. Effect of inhibitors on LPS-induced alteration of different inflammatory mediators in RAW 264.7 cells. (A) TNF-α and COX-2 protein expression. (B) Release of IL-6 in macrophage cells. Cells were pretreated with SB203580 (10 μM), SP600125 (20 μM), U0126 (30 μM), or PD98059 (10 μM) for 2 h, then were incubated with LPS (1 μg/mL) for 1 h. Whole cell lysates were used in Western blots to evaluate the expression of iNOS and COX-2, and the secretion of IL-6 in macrophage culture medium was measured by ELISA. Statistical analyses were performed by a one-way ANOVA test and Dunnett’s test. #p < 0.001 vs the control group; **p < 0.01, ***p < 0.001 vs the LPS-stimulated group.

cytokines such as TNF-α, IL-1β, and IL-6, inhibiting the protein expression of iNOS and COX-2, and increasing the production of anti-inflammatory cytokine IL-10 in inflammatory responses. These anti-inflammatory effects, at least in part, might be mediated via suppressing the activation of NF-κB and the phosphorylation of p38 and JNK, thereby regulating the transactivation of target genes. Therefore, our findings provide useful mechanistic explanations for the anti-inflammatory effect of 1 and highlight its potential pharmaceutical importance. In the present study, we assessed the anti-inflammatory potential of 1 against TPA-induced skin inflammation in mice, and the topical application of 1 was found to significantly inhibit TPA-induced acute mouse ear edema. However, whether the protein expression of iNOS and COX-2 in the mouse ear is comparable to that in the RAW 264.7 macrophage cell-based study under the same dose of 1 for animals requires further elucidation. Additionally, as pharmacokinetic properties such as bioavailability play an important role in the development of a new drug, further pharmacological studies are needed to provide more detailed information on the absorption, metabolism, and distribution as well as possible biotransformation of 1 in vivo. Nonsteroidal anti-inflammatory drugs (NSAIDs) are often the first choice of anti-inflammatory agents worldwide. However, NSAIDs are associated with potentially harmful side effects, such as gastrointestinal damage, which has become an important public health problem due to the morbidity and elevated mortality rate.34,35 Thus, new and safe immunosuppressive agents with less side effects are needed to combat inflammatory diseases. Our findings suggest that 1, a flavonoid from A. grossedentata, showed significant anti-inflammatory properties both in vitro and in vivo. In particular, 1 showed no cytotoxicity on RAW 264.7 macrophage cells even at a dose of 600 μM for 24 h, and no mice died at a dose of 920 mg/kg by

p65 were increased after LPS stimulation, but were downregulated in cells treated with 1. In addition, 1 blocked LPS-stimulated NF-κB translocation by inhibiting the phosphorylation of IκBα in the cytosol. There is compelling evidence to support that MAPKs play an important role in regulating a wide range of physiological processes.31 Multiple lines of evidence have implicated MAPKs in the expression of LPS-induced inflammatory mediators, such as iNOS and COX-2, as well as the production of proinflammatory cytokines.32 MAPKs, including at least three families of MAPK (p38 MAPK, ERK, and JNK), can be activated by LPS and undergo phosphorylation, activating transcription factors during inflammatory processes.33 Indeed, MAPKs are considered targets for novel anti-inflammatory drugs.20 Therefore, we hypothesized that the p38 MAPK signaling pathway might be involved in the anti-inflammatory effect of 1. Our results show that incubation of macrophages with LPS led to the activation of p38 and JNK, but not ERK1/2. Treatment with 1 markedly suppressed the phosphorylation, but not total protein levels, of p38 MAPK and JNK (Figure 7). Our experiments with p38- and JNKspecific inhibitors (SB230580 and SP600125, respectively) confirm that the increase in protein expression of TNF-α and COX-2 (Figure 8A) and the release of IL-6 (Figure 8B) were due to the activation of p38 and JNK. These results indicate that p38 and JNK are involved in the anti-inflammatory effects of 1. However, we do not have a good explanation for the results that using JNK inhibitor SP600125 significantly inhibited the expression of COX-2 as well as the production of IL-6, but did not inhibit the expression of TNF-α. We consider that as another goal for future study. In conclusion, the present study demonstrated that 1, a flavonoid extracted from A. grossedentata, acts as an antiinflammatory agent. Compound 1 exerted anti-inflammatory effects partly by suppressing the production of pro-inflammatory F

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Cells were allowed to grow until reaching 90−95% confluence and then were washed with phosphate-buffered saline (PBS), and the culture medium was replaced.40 To evaluate the anti-inflammation effects of 1, RAW 264.7 cells were pretreated with 1 (37.5, 75, 150, 300 μM) for 2 h followed by 1 or 24 h of stimulation with LPS (1 μg/mL). Cell Viability Assay. The effect of 1 on RAW 264.7 murine macrophage viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded in 96-well plates and grown overnight. Cells were treated with selective inhibitors (SB230580, SP600125, PD98059 and U0126 for p38, ERK, JNK, respectively) at separate concentrations (SB230580 at 10 μM, SP600125 at 20 μM, PD98059 at 10 μM, and U0126 at 30 μM) for 1 h and then cotreated with or without LPS (1 μg/mL) for 24 h and 1.41 Cells were then treated with MTT for 4 h at a final concentration of 5 mg/mL. The medium was subsequently replaced with 150 μL of DMSO.42 Finally, the absorbance at 490 nm was monitored using a universal microplate reader (Elx 800, Bio-TEK Instruments Inc., USA). Cytotoxicity Assay. The RAW 264.7 cells were seeded at 2 × 106 cells/well in a 96-well microplate for 12 h before 1 treatment. Cells were preincubated with 1 (37.5, 75, 150, and 300 μM) for 2 h and then stimulated with LPS (1 μg/mL) for another 24 h. Cytotoxicity was assessed by testing intracellular lactate dehydrogenase (LDH) leakage, using a cytotoxicity assay kit (Nanjing Jiangcheng Bioengineering Institute, Nanjing, People’s Republic of China) according to the manufacturer’s instructions. The release of LDH into the cell culture supernatant was quantified using a microplate reader at a wavelength of 450 nm.43 Assay of Nitrite Activity. The nitrite concentration in the culture media was used as an indicator of NO production and was measured by the Griess reaction. RAW 264.7 cells were cultured in 96-well plates for 12 h, followed by treated with or without 1 (37.5, 75, 150, and 300 μM) for 2 h, and then were stimulated with LPS (1 μg/mL) for another 24 h. Nitrite production in the supernatant was assayed using the Griess reagent system kit (Beyotime, Jiangsu, People’s Republic of China) according to the manufacturer’s protocols. A serial dilution standard curve was generated with NaNO2.41 Determination of TNF-α, IL-1β, IL-6, and IL-10 Production in LPS-Stimulated RAW 264.7 Macrophages. The effects of 1 on TNF-α, IL-1β, IL-6, and IL-10 production of macrophage cells were assayed by ELISA. In brief, RAW 264.7 cells cultured in six-well plates were preincubated for 2 h with 1 (37.5, 75, 150, and 300 μM) and then stimulated with LPS (1 μg/mL) for another 24 h. The concentration of TNF-α, IL-1β, IL-6, and IL-10 in supernatants of culture medium was assayed by using ELISA kits according to the manufacturer’s instructions.44 Western Blot Analysis. Cells were incubated with various concentrations of 1 (37.5, 75, 150, and 300 μM) for 2 h and then were stimulated with LPS (1 μg/mL) for another 1 or 24 h. After experimental treatments, cells were harvested and lysed in an ice-cold lysis buffer. Protein concentration in the supernatants was measured with BCA protein assay reagent (Beyotime, Jiangsu, People’s Republic of China). Equal amounts of protein (30 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride membranes at 120 mA for 2 h.45 The membranes were then blocked in PBS-Tween 20 containing 3% w/v defatted milk for 1 h and incubated with antibodies against GADPH, β-actin, iNOS, COX-2, TNF-α, phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, TLR4, phospho-IκBα, IκBα, and phospho-NFκB p65 (Ser536) (1:1000) in PBS containing 0.1% Tween 20 overnight. These antibodies were obtained from Cell Signaling Technology. Then, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. The intensities of the protein bands were visualized and were standardized with the density of the β-actin band.46,47 Statistical Evaluation. All data are given as the mean ± standard deviation of at least three experiments. Values were compared using a one-way analysis of variance (ANOVA) and Dunnett’s test. P values less than 0.05 was accepted as statistically significant.

ip within 6 h in vivo (data not shown). Therefore, 1 may be a potentially useful therapeutic agent for inflammatory diseases.

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EXPERIMENTAL SECTION

General Experimental Procedures. LPS, DMSO, and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-GADPH, anti-β-actin, anti-iNOS, anti-COX-2, anti-TNF-α, anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-JNK, anti-JNK, anti-phospho-IκBα, anti-IκBα, and antiphospho-NFκB p65 (Ser536) primary antibodies and U0126 were acquired from Cell Signaling Technology (Danvers, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific HyClone, Beijing, People’s Republic of China) was supplemented with fetal bovine serum (FBS, Kang Yuan biology, Shandong, People’s Republic of China). TPA and Griess reagent were purchased from Beyotime Institute of Biotechnology (Beyotime, Jiangsu, People’s Republic of China). ELISA for mouse TNF-α, IL-1β, IL-6, and IL-10 were purchased from eBioscience (San Diego, CA, USA). SP600125, SB230580, and PD98059 were obtained from Selleckchem (Houston, TX, USA). All other chemicals and reagents in this work were analytical grade. Isolation and Characterization of 1 from Ampelopsis grossedentata. Dihydromyricetin (1) was isolated from the leaves of A. grossedentata (Hand.-Mazz.) W. T. Wang as reported previously by Bai36 and characterized using IR, HPLC, and NMR. HPLC: column, Apollo C18 250 × 4.6 mm; particle size, 5 μm; eluent, methanol/0.1% phosphoric acid (30:70); flow rate, 0.8 mL/min; peak detection at 290 nm. IR: νmax3476, 1642, 1156 cm−1. The purity of the obtained DMY sample was over 99.5%, and this sample was used for the subsequent experiments in our studies. Animal Experiments. All animal care and experimental procedures were approved by the Institutional Ethics Committee of the Huazhong University of Science and Technology, Wuhan, People’s Republic of China (permission number: SYXK [Hubei]2010-0057, SYXK [Hubei]2014-0049). Six-week-old male Balb/c mice weighing 20−25 g and eight-week-old KM mice weighing 25−30 g (both from Hubei Provincial Center for Disease Control and Prevention, Wuhan, People’s Republic of China) were used for in vivo anti-inflammatory experiments. All animals were housed in a temperature (22 ± 2 °C)and humidity (40−60%)-controlled room under a 12 h light/dark cycle with a commercial standard diet (water, 88; protein, 216; fat, 45; fiber, 42 g/kg diet) and tap water ad libitum.37 To investigate the effects of 1 in vivo, mice were randomly assigned to six groups: a control group (water), an LPS-treated group (5 mg/kg), and four LPS- and 1-treated groups. Estimation of Serum TNF-α, IL-1β, IL-6, and IL-10 Levels in LPS-Challenged Mice. The mice were treated with LPS (5 mg/kg, ip) or 1 (57.5, 115, 230, 460 mg/kg, ip) followed by LPS (5 mg/kg, ip) after 1 h. Blood samples were collected after 6 h, separated by centrifugation, and stored at −80 °C. The concentration of inflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-10 in each sample was measured using ELISA kits according to the manufacturer’s protocol. TPA-Induced Acute Ear Edema. Skin inflammation was induced in the right ears of the mice by the application of TPA dissolved in 10 μL of acetone. Control mice received the same volume of acetone applied to the left ear. After topical application of TPA (2.5 μg per ear) for 1 h, 1 (2.3 and 4.6 mg per ear) was dissolved in the same volume of acetone and applied topically on the surface of the right ear of mice. Five hours after TPA treatment, mice were sacrificed by cervical dislocation.38,39 Left and right ear punches 8 mm in diameter were taken from each mouse. The increase in edema was directly proportional to the degree of inflammation. Tissues were frozen and stored at −80 °C until analyses were performed. Cell Culture Experiments: Cell Culture. RAW 264.7 murine macrophages were obtained from Wuhan Boster Biological Technology, Ltd. (Wuhan, People’s Republic of China). Cells were maintained in DMEM supplemented with 10% FBS and kept at 37 °C in a humidified 5% CO2 atmosphere. The medium was replaced every 2 days. G

DOI: 10.1021/acs.jnatprod.5b00275 J. Nat. Prod. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Author

*E-mail (J. G. Fang): [email protected]. Tel: +8602783649095. Fax: +8602783624090. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding for this work was partly provided by Technology Innovation Foundation, Innovation Institute, Huazhong University of Science and Technology (CXY13Q059).

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on July 14, 2015, with an error in the caption for Figure 2. The corrected version was reposted on July 16, 2015.

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DOI: 10.1021/acs.jnatprod.5b00275 J. Nat. Prod. XXXX, XXX, XXX−XXX