2018 Volume 41 Issue 8 Pages 1219-1227
The natural aporphine alkaloids including crebanine (CN), O-methylbulbocapnine (OMP), and dicentrine (DC), and protoberberine alkaloids, tetrahydropalmatine (THP) and N-methyl tetrahydropalmatine (NTHP), have been found in Stephania venosa. Previous reports demonstrated CN and THP exhibited anti-inflammatory properties. In this study, we investigated anti-inflammatory effect of CN analogs including OMP, DC, THP, and NTHP in RAW264.7 macrophages. The pre-treatment of macrophages with CN, OMP and DC suppressed lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and mediators including interleukin-6 (IL-6), tumor necrosis factor alpha, prostaglandin E2 and nitric oxide, in which the rank-order of inhibitory potency was DC>CN≥OMP. Whereas, high dose THP (30–40 µg/mL) reduced LPS-induced IL-6 production in RAW264.7 cells but NTHP did not effect. Moreover, CN, OMP and DC inhibited the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2. OMP and DC inhibited LPS-induced nuclear factor kappa B (NF-κB) activation by suppressing the phosphorylation of NF-κB at Ser536, but not the nucleus translocation and inhibitor of kappaB (IκB)-α degradation. In addition, OMP and DC also reduced the phosphorylation and nucleus translocation of activator protein-1 (AP-1). Furthermore, OMP and DC suppressed the LPS-activated myeloid differentiation factor 88 (MyD88), Akt and mitogen-activated protein kinases (MAPKs) signaling pathway, which were the upstream signaling regulators of AP-1 and NF-κB. Collectively, OMP and DC have an anti-inflammatory effect on RAW264.7 macrophages by the suppression of pro-inflammatory cytokines and mediators. The inhibitory property of OMP and DC is mediated by blockage the activation of MyD88, MAPKs, Akt, NF-κB and AP-1 signaling molecules.
Chronic inflammation has been known to play a role in many diseases including arthritis, cancer, cardiovascular diseases, diabetes and bronchitis. Macrophages are innate immune cells that are involved in the development of chronic inflammatory responses. The overproduction of inflammatory mediators from macrophage such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), nitric oxide (NO) and prostaglandin E2 (PGE2) eventually lead to deleterious consequences that are associated with the pathogenesis of inflammatory diseases.1,2) Inducible nitric oxide synthase (iNOS)-catalyzed NO is generally oxidized to free radicals and results in cell deaths in surrounding tissue.3) Cyclooxygenase-2 (COX-2) is an inducible enzyme that catalyzes the production of prostanoid biosynthesis including PGE2.4) Therefore, the inhibition of inflammatory mediators from macrophage can be used for the treatment of inflammatory diseases.5,6)
The lipopolysaccharide (LPS), an endotoxin of Gram-negative bacteria, is one of the most powerful inflammatory stimulants. LPS binds to the Toll-like receptor 4 (TLR4) and induces the release of inflammatory mediators from the macrophage via activated intracellular signaling cascades including mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K)/Akt, activator protein-1 (AP-1) and the nuclear factor kappa B (NF-κB) signaling pathway.7–9) NF-κB and AP-1 are critical transcription factor which promotes the expression of iNOS, COX-2, IL-6 and TNF-α.10) Thus, the regulation of these signaling pathways has been considered as an important target for the development of anti-inflammatory agents.
The traditional folk medicine of Stephania vernosa has been used for treatment of diabetes mellitus, nerve tonic, appetizer, asthma, and cancer.11) According to previous phytochemical investigations, several natural alkaloids, including crebanine (CN), O-methylbulbocapnine (OMP), dicentrine (DC), tetrahydropalmatine (THP), and N-methyl tetrahydropalmatine (NTHP) have been found in the tubes of S. vernosa.12–14) CN, OMP, and DC are aporphine alkaloids and they show the structural differences in the position of the dimethoxy group on the D-ring. Whereas, THP and NTHP are derivatives of protoberberine alkaloids and differ only the presence or absence of methyl group on the quaternary nitrogen atom in the position 7. In the recent years, the biological activities of alkaloids in S. vernosa have been reported to possess potential anti-cancer, antagonizes morphine abused, and inhibiting acetylcholinesterase.15–17) Furthermore, CN and THP exhibited anti-inflammation in LPS-stimulated macrophage cells. Our previous study demonstrated that CN reduced inflammatory mediators through modulation of AP-1 and NF-kB activity.18) On the other hand, THP inhibits LPS-induced IL-8 secretion via blocking MAPKs signaling pathway.19)
Based on the anti-inflammatory activity of CN and THP, it has led us to investigate the inhibitory activities of its analogues, OMP, DC, and NTHP, on LPS-induced inflammation responses in RAW264.7 macrophage cells, as well as to explore the relevant molecular mechanisms.
Dulbecco’s modified Eagle’s medium (DMEM) and penicillin–streptomycin were purchased from GIBCO-BRL (Grand Island, NY, U.S.A.). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, U.S.A.). Griess reagent, LPS, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). PGE2 enzymes-link immune assay (ELISA) kit was obtained from Abcam (Cambridge, MA, U.S.A.). Quantitation mouse ELISA kit of TNF-α and IL-6 were purchased from Biolegend Inc. (San Diego, CA, U.S.A.). Antibodies against COX-2, iNOS, phospho-extracellular signal-regulated kinase (ERK)1/2, ERK1/2, phospho-p38, p38, phospho-c-Jun, c-Jun, phospho-NF-κB p65, phosphor-Akt, Akt, inhibitor of kappa B-alpha (IκB-α), and myeloid differentiation factor 88 (MyD88) were ordered from Cell Signaling Technology Inc. (Beverly, MA, U.S.A.). Antibodies to NF-κB, poly(ADP-ribose) polymerase (PARP) and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). DC was ordered from Chengdu Biopurify Phytochemicals Ltd. (Sichuan, China). The alkaloids CN, OMP, THP, and NTHP isolated from the tubes of S. vernosa were kindly provided by Dr. Wilart Pompimon. The chemical structures of CN, OMP, DC, THP, and NTHP are shown in Fig. 1.
RAW264.7 macrophage cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were maintained in a humidified incubator with an atmosphere comprised of 95% air and 5% CO2 at 37°C.
Cell ViabilityThe cytotoxic effects of the alkaloids to RAW264.7 cells were determined using the MTT assay.20) RAW264.7 cells were seeded into a 96-well plate (1×104 cells/well) in DMEM with 10% FBS. The cells were treated with various concentrations of alkaloids and incubated for 24 h. Eventually, 15 µL of MTT solution (5 mg/mL) was added to each well. After incubation at 37°C for 4 h, the formazan crystals in each well were dissolved in 200 µL of dimethyl sulfoxide (DMSO). The absorbance of purple formazan was detected at 570 nm using a microplate reader. The percent of cell viability was calculated compared with control.
Measurement of Pro-inflammatory Cytokines and Mediators ProductionRAW264.7 cells were seeded into 12-well plates (5.0×105 cells/well) and pre-treated with various concentrations of alkaloids. After 4 h of treatment, the cells were incubated with or without LPS (1 µg/mL) for 24 h. The culture mediums were collected and analyzed for IL-6, TNF-α, and PGE2 production using ELISA kits according to the manufacturer’s protocol.21,22) To determine the total concentration of NO in the culture media, 150 µL of Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride and 2.5% phosphoric acid) was mixed with 150 µL of the supernatant and the absorbance at 540 nm was evaluated using a microplate reader.23)
Western Blot AnalysisPreparations of whole cell lysates, cytoplasmic and nuclear fractions were carried out according to the previously published procedures.24) The protein concentrations were quantified with Bradford assay kit. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to the nitrocellulose membrane. Then, the membranes were immediately blocked with non-fat milk in phosphate buffered saline (PBS) containing 2.5% (v/v) Tween 20. Thereafter, the membranes were further incubated with desired primary antibody at 4°C overnight followed by incubation with horseradish peroxidase conjugated secondary antibody. Bound antibodies were detected by using chemiluminescent detection system and then exposed to the X-ray film (GE Healthcare Ltd., U.K.).
Statistical AnalysisThe results were expressed as represent mean±standard deviation (S.D.) of three independent experiments. All statistical differences of significances were observed via one-way ANOVA and followed by Dunnett’s test using SPSS software. p values <0.05 and <0.01 were considered statistically significant.
Initially, the effects of alkaloids on the cytotoxicity of RAW264.7 cells were determined by MTT assay. As is shown in Figs. 2A–E, no cytotoxicity was observed (% cell survival >80%) when RAW264.7 cells were treated with CN, OMP, DC, THP, and NTHP with concentrations of less than 14, 15, 3.5, 37.5, and 200 µg/mL, respectively. Next, we investigated whether CN, OMP, DC, THP, and NTHP modulated IL-6 and TNF-α production. Treatment of the cells with CN, OMP, DC, and THP decreased LPS-induced IL-6 production in a dose dependent manner, with IC50 at values of 8, 7, 2.5, and 32 µg/mL, respectively (Figs. 2F–I). However, treatment the cell with 100 µg/mL of NTHP did not affect IL-6 production (Fig. 2J). Moreover, CN and DC significantly inhibited the LPS-induced TNF-α production in a dose-dependent manner with IC50 at 12 and 3 µg/mL, respectively (Figs. 2K, M). Whereas, OMP, only at a level of 15 µg/mL, significantly reduced LPS-induced TNF-α to 42% when compared with the control (Fig. 2L). In contrast, THP and NTHP at 40 and 100 µg/mL had no effect on LPS-induced TNF-α production (Figs. 2N, O).
The cells were treated with indicated concentrations of alkaloids for 24 h and the cell viability was determined by MTT assay. The effect of the alkaloids on cytokine level in LPS-induced RAW264.7 cells is presented. The macrophages were pre-treated with CN (2.5–15 µg/mL), OMP (2.5–15 µg/mL), DC (1–4 µg/mL), THP (10–40 µg/mL), and NTHP (25–100 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 24 h. After incubation, the culture medium was used for determination of the level of IL-6 (F–J) and TNF-α (K–O) using an ELISA kit. Data are represented as the mean±S.D. of three independent experiments. Statistical differences between various concentration of alkaloids compared with LPS treatment only were considered significant at values of * p<0.05 and ** p<0.01.
The inhibitory effects of CN, OMP, DC, THP and NTHP on LPS-induced PGE2 production in macrophage cells were assessed. Pre-treatment with CN, OMP and DC significantly and dose-dependently inhibited PGE2 production when compared to LPS-treated only with IC50 at values of 8, 10, and 2.5 µg/mL, respectively (Figs. 3A–C). Whereas, THP and NTHP did not effect on LPS-induced PGE2 production (data did not show). COX-2 is known as the key regulatory enzyme in the production of PGE2. Therefore, we determined that the inhibitory effect of the alkaloids on PGE2 production was related to COX-2 expression. As is shown in Figs. 3D–F, pre-treatment with CN, OMP and DC suppressed the LPS-induced expression of COX-2 in a dose-dependent manner. In particular, treatment the cells with 5 µg/mL of CN, 10 µg/mL of OMP, and 2 µg/mL of DC decreased LPS-induced COX-2 expression more than 50% (Figs. 3G–I).
The cells were pre-treated with CN (2.5–15 µg/mL), OMP (2.5–15 µg/mL) and DC (1–4 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 24 h. PGE2 levels in the culture medium were determined by ELISA (A–C). The expression level of the COX-2 was detected in the whole-cell lysate by Western blot analysis (D–F). The intensity of COX-2 was quantified by using Image J, whereas the non-LPS-treated cells represented as 100% (G–I). Data are represented as the mean±S.D. of three independent experiments. Statistical differences between various concentration of alkaloids compared with LPS treatment only were considered significant at values of * p<0.05 and ** p<0.01.
To assess the inhibitory effect of the alkaloids on the production of NO in LPS-induced RAW264.7 macrophages, the cells were pre-treated with different concentrations of alkaloids for 4 h following incubation with LPS for 24 h. After incubation, the level of NO production was determined using Griess reagent. As is shown in Figs. 4A–C, CN, OMP, and DC significantly exhibited the inhibitory effects on LPS-induced NO production in a dose-dependent manner with IC50 at 6, 7 and 3 µg/mL, respectively. On the other hand, THP at 40 µg/mL reduced LPS-induced NO production to 57%. Whereas, NTHP at 100 µg/mL had no effect on NO production (data did not show). Overproduction of NO is associated with the upregulation of iNOS expression. Therefore, we have determined the effects of alkaloids on the expression of iNOS in LPS-induced RAW264.7 cells using Western blot analysis. As is shown in Figs. 4D and E, pre-treatment of the cells with CN, OMP and DC decreased LPS-stimulated iNOS protein expression in a dose-dependent manner. Moreover, treatment the cells with 5 µg/mL of CN and OMP or 3 µg/mL of DC reduced LPS-induced iNOS expression more than 50% (Figs. 4G and H).
The cells were pre-treated with CN (2.5–15 µg/mL), OMP (2.5–15 µg/mL) and DC (1–4 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 24 h. NO production in the culture supernatant was determined using Griess reagent (A–C). The protein levels of iNOS were determined in whole-cell lysate using Western blot analysis (D–F). The intensity of iNOS was quantified by using Image J, whereas the non-LPS-treated cells represented as 100% (G–I). Data are represented as the mean±S.D. of three independent experiments. Values of * p<0.05 and ** p<0.01 indicate significant differences from the specimens that were only treated with LPS.
AP-1 and NF-κB are the major transcription factors in the inflammatory response to LPS. The expression of pro-inflammatory cytokines and mediators are controlled by NF-κB and AP-1 activities. Our previous study has demonstrated that CN inhibits LPS-induced NF-κB and AP-1 activities. Therefore, we evaluated the ability of OMP and DC to inhibit LPS-stimulated AP-1 and NF-κB activities by examining the translocation and phosphorylation of AP-1 and NF-κB. As is shown in Figs. 5A and B, The LPS-induced translocation of c-Jun (AP-1) was significantly reduced by OMP and DC with dose-dependent manner. Moreover, OMP and DC significantly inhibited LPS-induced phosphorylation of c-Jun (Figs. 5C and D). Next, we investigated the regulation of OMP and DC on the transcriptional activity of NF-κB by determine the nucleus translocation and phosphorylation of NF-κB. In resting cells, NF-κB is resides in the cytosol as an inactive complex with inhibitor of kappaB (IκB)-α. Once induced with LPS, IκB-α is phosphorylated and then degraded, resulting free NF-κB translocated to nucleus. As is shown in Figs. 6A and 6B, OMP and DC did not have an effect on LPS-induced IκB-α degradation. Moreover, treatment with OMP and DC did not affect the LPS-induced translocation of p65 (Figs. 6C and D). Since transactivation of NF-κB is regulated by phosphorylation, the effects of OMP and DC on the LPS-induced phosphorylation of p65 at Ser536 was determined. As is shown in Figs. 6E and F, treatment of the cells with OMP and DC significantly inhibited the LPS-induced phosphorylation of p65.
The cells were pretreated with OMP (2.5–15 µg/mL) and DC (1–4 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 30 min. The nucleus extracts were used to investigate the nucleus translocation of c-Jun (A and B). The phosphorylation levels of c-Jun at Ser63 in the whole-cell lysate were analyzed using Western blot analysis (C and D). The experiment was repeated three times and similar results were obtained.
The cells were pretreated with OMP (2.5–15 µg/mL) and DC (1–4 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 30 min. The cytoplasmic extracts were prepared to analyze IκB-α degradation by using Western blot analysis (A and B). The nucleus extracts were prepared in order to analyze p65 nucleus translocation using Western blot analysis (C and D). The whole cell lysis was used to determine the phosphorylation of NF-κB p65 at Ser536 using Western blot analysis (E and F). The experiment was repeated three times and similar results were obtained.
LPS-activated MAPKs and Akt signaling pathways have been implicated in the transcriptional regulation of pro-inflammatory cytokine gene expression through AP-1 and NF-κB activation. Therefore, the effects of OMP and DC on LPS-induced activation of Akt and MAPKs, including ERK1/2, p38, and c-Jun N-terminal kinase (JNK), were investigated by Western blot analysis. OMP significantly inhibited the phosphorylation of ERK1/2 and p38 in a dose-dependent manner, whereas the phosphorylation of JNK was not affected when compared to the cells that were treated with LPS (Fig. 7A). On the other hand, DC significantly reduced the phosphorylation of ERK1/2 and JNK in a dose-dependent manner, but had no effect on the phosphorylation of p38 (Fig. 7B). Furthermore, OMP and DC significantly suppressed the LPS-induced phosphorylation of Akt in a concentration dependent manner, while no changes occurred in the total Akt (Figs. 7C and D). From the above results, OMP and DC suppressed LPS-induced NF-κB, AP-1, MAPKs, and PI3K/Akt signaling molecules. Those pathways are transduced by MyD88 adaptor protein. Therefore, we examined the effect of OMP and DC on LPS-induced MyD88 protein in Raw264.7 cells. As shown in Fig. 7E, LPS treatment stimulated the expression of MyD88. Incubation the cells with OMP and DC at 15 and 4 µg/mL decreased LPS-induced MyD88 level to 67±10 and 46±18%, respectively when compared to control (Fig. 7F).
The whole cell lysates were prepared from the cells that were pre-treated with or without OMP (2.5–15 µg/mL) and DC (1–4 µg/mL) for 4 h and stimulated with LPS (1 µg/mL) for 15 min. The levels of the phospho and nonphospho forms of the MAPKs signaling molecules, including ERK1/2, JNK, and p38 (A and B), the Akt signaling pathway (C and D) were detected by Western blot analysis. To study the expression of MyD88, the cells were pre-treated with OMP (5–15 µg/mL) and DC (2–4 µg/mL) for 4 h and stimulated with LPS (1 µg/mL) for 45 min. The whole cell lysates were collected for determine the level of MyD88 (E) by using Western blot analysis. The intensity of MyD88 was quantified by using Image J, whereas the non-LPS-treated represented as 100% (F). All experiments were repeated three times and the representative results are shown. Values of * p<0.05 indicate significant differences from the specimens that were only treated with LPS.
The way that the agents inhibited the level of the inflammatory mediators and cytokines is regarded as an effective therapeutic strategy for treatment inflammatory diseases. Our previous study demonstrated that CN, aporphine alkaloid from S. venosa, displays anti-inflammatory activities in RAW264.7 macrophages.18) In this study, we determined the anti-inflammatory effect of two alkaloids groups, aporphine (CN, OMP, and DC) and protoberberine (THP and NTHP), from S. venosa. Here, we have again shown the inhibition of CN on LPS-induced pro-inflammatory cytokine. Importantly, our results indicated for the first time that OMP and DC, which are CN derivatives, exhibit a significant level of suppression on the LPS-induced production of cytokines and mediators, including TNF-α, IL-6, PGE2, and NO, in macrophages. In addition, we have also demonstrated that OMP and DC exerted anti-inflammatory properties by reducing LPS-induced the expression of iNOS and COX-2. Interestingly, the effects of OMP on the reduction level of pro-inflammatory cytokines and mediators were found to be less than or equal to CN, while DC showed the highest level of potency when compared with experiments using IC50. On the other side, THP has been reported anti-inflammation properties via inhibited pro-inflammatory mediators in LPS-stimulated human monocyte cells.19) Our results confirm that THP reduced LPS-induced IL-6 and NO production in Raw264.7 macrophages whereas its analog, NTHP, did not effect. However, the effective concentration of THP on anti-inflammatory activity is greater than the aporphine derivatives. Thus, our results suggested that aporphine derivatives from S. venosa exerts more potent anti-inflammatory activity than protoberberine.
The structural differences of CN, OMP, and DC involve the position of the dimethoxy group between C-8 to C-11 on ring D of the aporphine alkaloid. The dislocation of dimethoxy in CN from C-8 and C-9 to C-10 and C-11 of OMP revealed less of an effect on the anti-inflammatory activity. However, the presence of the dimethoxy group in C-9 and C-10 enhanced the anti-inflammatory effect of DC. These results suggest that the steric interaction between the C-11 methoxy group and the methylenedioxy ring may be involved in a reduction of the anti-inflammatory effect of OMP. Therefore, the location of the dimethoxy group on ring D plays an important role in inhibiting pro-inflammatory cytokines and mediators from LPS-induced macrophages. In addition, several studies indicated that DC inhibits topoisomerases activity.25) Our result shown that the cytotoxicity on macrophage of DC was higher than CN and OMP. This finding suggest that the location of dimethoxy group in C-9 and C-10 can enhance cellular toxicity.
Woo et al. indicated that DC inhibited topoisomerases II activity and led to inhibit cell proliferation better than bulbocapnine which present hydroxyl group at C-11.26) Unlike the case of aporphine alkaloids, only THP reduced LPS-induced IL-6 and NO production but not NTHP. These observation suggest that the presence of methyl group on the quaternary nitrogen atom in the position 7 of NTHP may cause a loss of anti-inflammatory activity of protoberberine.
The activation of the NF-κB and AP-1 transcription factors in macrophages is strongly associated with the activation of the downstream pro-inflammatory cytokines and mediators. The promoted region of the COX-2, NO, TNF-α, and IL-6 gene contains the AP-1 and NF-κB binding site, which indicates the association of AP-1 and NF-κB in the regulation of these genes.27–29) Our previous report has shown that CN reduced the level of AP-1 and NF-κB transcription activity.18) Therefore, we have investigated whether the inhibitory effects of OMP and DC on inflammatory mediators were related to the suppression of the NF-κB and AP-1 transcription activity. AP-1 is a dimeric complex protein consisting of different sub-families of Jun and Fos. The activity of AP-1 was regulated at the expression level and post-translation modification via phosphorylation and dephosphorylation.30,31) The activated AP-1 was translocated to the nucleus and bound to a DNA consensus sequence. Many agents that can reduce AP-1 activity have been shown to suppress inflammation.32) Here, we have investigated the activation of AP-1 by observing the phosphorylation and translocation of c-Jun in LPS-induced macrophage. In this study, OMP and DC were demonstrated to inhibit LPS-induced c-Jun phosphorylation and translocation to the nucleus of macrophages, which is similar to the mode of action of CN as has been previously reported.
A common form of NF-κB is a heterodimer consisting of p50/p65. In un-stimulated cells, NF-κB is present in cytosol and interacts with IκB-α. Upon LPS stimulation, IκB-α is catalyzed phosphorylation by IκB kinase (IKK) leading to IκB-α degradation. Free NF-κB is then get into a nucleus, which promotes the transcription of the corresponding pro-inflammatory genes.33) Therefore, we determined the activity of OMP and DC on LPS-induced the degradation of IκB-α and the translocation of p65 into the nucleus. Our results demonstrated that OMP and DC did not affect IκB-α degradation and p65 translocation. Based on this finding indicate that OMP and DC did not effect on signaling cascade of LPS induced IKK-IκB-α pathway. Moreover, a good deal of evidence has indicated the phosphorylation of the NF-κB subunit p65 modulates the NF-κB transcription activity.34) The phosphorylation of p65 on Ser536 residue modulated the DNA-binding and trans-activating activity of NF-κB, which is independent of the NF-κB translocation to the nucleus.35) In this study, we have investigated the effects of OMP and DC on the activation of NF-κB by determining the translocation and phosphorylation of NF-κB p65 in LPS-induced RAW264.7 cells. OMP and DC reduced the LPS-induced phosphorylation of p65 at Ser536. This result was similar to that of our previous findings, which have stated that CN inhibited LPS-induced NF-κB transcription activity by inhibiting the phosphorylation of p65, but not the translocation.18) Based on the above-mentioned results, we suggest that OMP and DC could decrease the expression of pro-inflammatory cytokines and mediators by the inhibition of AP-1 and NF-κB activation, in a similar manner to the action of CN.
MAPKs are known to be serine/threonine kinase and are composed of three major sub-families including ERK1/2, JNK, and p38. LPS activated the MAPK signaling pathway in macrophages by binding to the TLR4.36) A large number of evidences have demonstrated that the activation of MAPKs by LPS is involved in increasing the level of inflammatory mediators via the stimulation of AP-1 and NF-κB.37) The present results showed that OMP reduced the level of phosphorylation of ERK1/2 and p-38, but not that of JNK. On the other hand, DC effectively suppressed the level of activation of ERK1/2 and JNK in LPS-activated RAW264.7 cells. This finding is in contrast with those of our previous reports, which have stated that CN reduced the level of phosphorylation of ERK1/2, p38, and JNK.18) Our results implied that small structural differences between CN, OMP, and DC produced differences in the specific recognition and inhibition of the MAPKs pathway. In addition to the MAPK pathway, the phosphatidylinositol 3′-kinase/Akt (PI3K/Akt) signaling pathway has been reported to play an important role in LPS-induced inflammatory responses. The PI3K/Akt signaling pathway is critical not only in NF-κB activation, but also for the modulation of the AP-1 activity.38) Here, OMP and DC were found to inhibit the LPS-induced phosphorylation of Akt. Thereby, we have suggested that the inhibition of LPS-induced NF-κB and AP-1 activation by OMP and DC resulted in an interruption of the downstream signaling molecules that are involved in the TLR-induced MAPKs and PI3K/Akt signaling pathways.
The binding of LPS to TLR4 triggers MyD88-dependent and independent signaling. In MyD88 dependent pathway, MyD88 recruits and activates IL-1 receptor-associated kinases (IRAKs). IRAKs are subsequently phosphorylated, lead to dissociate from MyD88, which activates the TNF receptor-associated factor 6 (TRAF6). TRAF6 subsequently activates transforming growth factor-β-activated kinase 1 (TAK1). TAK1, in turn, activates downstream IKK and MAPK signaling pathway.39) In addition, MyD88 have been involved on the activation of PI3K/Akt signaling pathway.40) Thus, the augmented level of MyD88 in LPS-induced macrophage leading to activation of several signaling transductions includes NF-κB, PI3K/Akt, and MAPK pathways, which contribute to the release of numerous inflammatory mediators. We hypothesized that MyD88 dependent TLR4 signaling could be the pharmacological target of OMP and DC. Our result demonstrated that OMP and DC attenuated the expression of MyD88 in LPS-induced macrophage or it might be because the OMP and DC induced the degradation of MyD88. Therefore, OMP and DC might inhibit LPS-induced MAPKs, PI3K/Akt, AP-1 and NF-κB activation in the MyD88-dependent pathway.
In conclusion, the results of the present study indicate for the first time that CN analogs, including OMP and DC, exert anti-inflammatory properties by suppressing iNOS, COX-2, TNF-α, and IL-6 expression in LPS-induced macrophages. These inhibitory effects are mediated by a coorperative inactivation of the NF-κB and AP-1 transcription factors via a blockade of the MAPKs, Akt, and MyD88 signaling pathways.
This work was supported by the Faculty of Medicine Research Fund (No. 074/2559), Faculty of Medicine, Chiang Mai University, Thailand and Center for Research and Development of Natural Products for Health, Chiang Mai University, Chiang Mai, Thailand.
The authors declare no conflict of interest.
