Sinomenine Inhibits Inflammation Through α7nAChR And Downregulates α7nAChR Expression By ERK/Egr1 In Macrophages Stimulated By LPS

Objective:Sinomenine (SIN), an alkaloid derived from the plant Sinomenium acutum, has multiple functions, including antirheumatic, anti-inflammation and immune suppression, which is good efficacy and safety, fewer side effects on treatment of rheumatoid arthritis. Macrophages play an important role in the pathogenesis of RA and other autoimmune diseases. Even though pharmacological effect of SIN has been widely reported, the pharmacological mechanisms of SIN acting α7nAChR has not been reported. In our previous research, we found anti-inflammation effect of SIN in macrophages via α7nAChR. To analyze anti-inflammation effect and the signal mechanisms of SIN in macrophages via α7nAChR, Mouse macrophage cell strain Raw264.7 was stimulated by lipopolysaccharide(LPS) to induce cell inflammation model, and nAChR specific antagonist mecamylamine and alphα7nAChR specificity antagonist a-bungarotoxin were used as the blocker. Besides, we aslo found that SIN can inhibit expression of α7nAChR, the mechanism of SIN regulating α7nAChR expression was analyzed in this study.Methods:1. Sinomenine effect on the effector molecules by acting on α7nAChR in macrophages stimulated by LPSMouse macrophage cell strain Raw264.7 was stimulated by LPS to induce cell inflammation model, nicotine was chosen as the positive drug, and nAChR specific antagonist mecamylamine and alphα7nAChR specificity antagonist a-bungarotoxin as the blocker. TNF-α、IL-1β and MCP-1 secreted were measured by ELISA. The expression level of MIF and MMP-9 protein were detected by Western2. The effect of sinomenine on CD14, TLR4 expression and [Ca2+]i via α7nAChR in macrophagesRAW264.7 cells treated with LPS (1μg/ml) can up-regulate the expression of CD14 and TLR4, SIN or Nic-pretreated cells down-regulated the effect. In order to know the involvement of α7nAChR in the SIN effects, we examined the effects of nAChR specific antagonist mecamylamine and alphα7nAChR specificity antagonist α-bungarotoxin on CD14 and TLR4 expression. nAChR specific antagonist mecamylamine and alphα7nAChR specificity antagonist a-bungarotoxin reversed the effects of SIN and Nic on the expression of CD14 and TLR4. We found that theintracellular free calcium level([Ca2+]i)was enhanced in cells after 1 h stimulated by LPS compared with the control group, while [Ca2+]i was attenuatedin the SIN or Nic pre-treated group according to [Ca2+]i fluorescence detection. Further, to determine whether the effect of SIN or Nic on [Ca2+] i were mediated by α7nAChR, we pretreated cells with nAChR specific antagonist mecamylamine and alphα7nAChR specificity antagonist a-bungarotoxin. The result showed BTX prevented the effect of SIN or Nicin[Ca2+]i3. The effect of sinomenine on ERK1/2, p-ERK1/2 and Egr-1 expression via α7nAChR in macrophagesLPS induced a significant enhancement of ERK1/2 phosphorylation. This response was reduced by SIN or Nic. To ascertain whether SIN decrease ERK1/2 phosphorylation and Egr-1 expression through α7nAChR, RAW264.7 cells were pre-incubated with the α7nAChR selective antagonist BTX before treated with SIN or Nic. The results showed BTX could significantly abolished the effect of SIN on ERK1/2 phosphorylation and Egr-1 expression.4. The effect of sinomenine on α7nAChR regulation mechanismin in macrophagesWhen RAW264.7cells were treated with LPS, the expression of p-ERK1/2, and Egr-1 were up-regulated, after treated with U0126(MEK inhibitor), the effect of p-ERK1/2 and Egr-1 were attenuated, while the effect of ERK1/2 had no change. To clarify the molecular basis underlying LPS-induced α7nAChR expression, we examined the effect of U0126 on protein expression of α7nAChR. Immunoblotting analysis revealed that LPS-induced α7nAChR proteinexpression was significantly attenuated by treatment of U0126.Conclusion:1. Sinomenine through effect on α7nAChR to inhibit the effector moleculesthen exert an anti-inflammatory effect2. Sinomenine through effect on α7nAChR to inhibitthe expression of CD14 and TLR4, also reduce[Ca2+]i exert an anti-inflammatory effect3. Sinomenine through effect on α7nAChR to inhibit the ERK1/2 phosphorylation and Egr-1 expression exert an anti-inflammatory effect,4. Sinomenine downregulates α7nAChR expression by ERK/Egr-1 in macrophages stimulated by LPS.