目的 观察姜黄素对内毒素(LPS)诱导的血管平滑肌细胞(VSMCs)内Toll样受体4(TLR4)信号通路及炎症因子释放的影响.方法 原代培养VSMCs,分为5组:对照组、LPS组、LPS+姜黄素5μmol/L组、LPS+姜黄素10μmol/L和LPS+姜黄素30μmol/L组.MTT法测定不同浓度姜黄素对VSMCs细胞活性的影响;酶联免疫吸附法(ELISA)测定各组细胞上清液中单核细胞趋化因子-1(MCP-1)和白细胞介素(IL-6)蛋白的表达;Real-time PCR及Western-blot法检测各组细胞TLR4 mRNA和蛋白的表达;ELISA法测定丝裂原蛋白活化激酶(MAPKs)通路的磷酸化水平.结果 姜黄素在0～80μmol/L浓度范围内对细胞活性没有显著影响.姜黄素(5、10、30μmol/L)可以有效地抑制LPS诱导的MCP-1和IL-6过分泌和TLR4的高表达,同时降低MAPKs(ERK1/2、p38MAPK、JNK1/2)信号通路的磷酸化水平,并具有浓度依赖性.结论 姜黄素能够有效地抑制LPS诱导的VSMCs炎症因子的分泌、TLR4的过表达及下游MAPKs信号通路的磷酸化.%Objective To explore the inhibitory effect of curcumin on LPS-induced inflammation and the activation of Toll-like receptor 4 (TLR4)-dependent signaling pathway in vascular smooth muscle cells (VSMCs). Methods Primary VSMCs were cultured and divided into control group, LPS group, LPS +curcumin 5 μmol/L group, LPS+curcumin 10 μmol/L group, and LPS+curcumin 30 μmol/L group. Cell activity was observed by MTT assay. The secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) was measured by enzyme linked immunosorbent assay (ELISA) kits. The mRNA and protein expression of TLR4 were detected by real-time PCR and Western blot, respectively. The phosphorylation of mitogen-activated protein kinases (MAPKs) (ERK, p38MAPK and JNK) was analyzed by ELISA kits. Results VSMCs activities were not significantly affected by curcumin at the concentration between 0 and 80 μmol/L. Curcumin (5, 10 and 30 μmol/L) significantly inhibited LPS-induced oversecretion of MCP-1 and IL-6 as well as overexpression of TLR4 at the mRNA and protein levels, and suppressed phosphorylation of MAPKs (ERK1/2, p38MAPK, and JNK1/2) in VSMCs in a concentration-dependent manner. Conclusion Curcumin has a concentration-dependent inhibitory effect on the secretion of inflammatory cytokine, overexpression of TLR4, and phosphorylation of MAPKs in VSMCs stimulated by LPS.
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