摘要
目的观察积雪草酸(asiatic acid,AA)能否抑制内毒素脂多糖(lipopolysaccharide,LPS)诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)炎症反应,并探讨其作用机制。方法原代培养SD大鼠主动脉VSMCs;不同浓度AA干预后,采用MTT法测定VSMCs的活性;分别采用酶联免疫吸附法(ELISA)和real-time PCR检测IL-6、MCP-1、TNF-α蛋白及mRNA水平;Western blot和real-time PCR法测定TLR4和PPAR-γ的蛋白及mRNA的表达水平。结果当AA浓度在0~30μmol·L^(-1)范围内时,对VSMCs细胞活性无明显影响,当500μg·L^(-1)LPS刺激VSMCs后,IL-6、MCP-1、TNF-α、TLR4蛋白及mRNA水平明显升高(P<0.05),而PPAR-γ的表达明显降低(P<0.05);AA(10、20、30μmol·L^(-1))对LPS诱导的VSMCs细胞IL-6、MCP-1和TNF-α蛋白及mRNA水平的降低作用呈浓度依赖性;此外,AA能浓度依赖性地抑制LPS诱导的VSMCs细胞TLR4 mR-NA和蛋白表达,且TLR4-siRNA能够起到与AA类似的抗炎作用。AA还可上调VSMCs细胞内PPAR-γmRNA和蛋白表达,而PPAR-γ拮抗剂GW9662则能够部分抵消AA的抗炎作用。结论AA能有效抑制LPS诱导的VSMCs细胞IL-6、MCP-1、TNF-αmRNA和蛋白表达,AA的抗炎作用可能与其下调TLR4表达和上调PPAR-γ活性作用相关。
AimTo observe whether asiatic acid(AA)can inhibit lipopolysaccharide(LPS)-induced inflammatory response in VSMCs,and explore its mechanism of action.MethodsThe VSMCs isolated from aorta of SD rats were primarily cultured.The effect of AA on the cell viability of VSMCs was measured by MTT assay.The protein and mRNA expression of IL-6,MCP-1,and TNF-α,were measured by ELISA assay and real-time PCR,respectively.The protein and mRNA of TLR4 and PPAR-γwere measured by Western blot and real-time PCR,respectively.ResultsAA exhibited no effect on cellular viability between the concentration from 0 to 30μmol·L^(-1).After treating VSMCs with LPS(500μg·L^(-1))for 6h or 24 h,the protein and mRNA expression of IL-6,MCP-1,TNF-α,and TLR4 significantly increased(P<0.05);and on the contrary,the activity of PPAR-γwas significantly reduced(P<0.05).Treatment with AA(10,20,30μmol·L^(-1))could concentration-dependently inhibit LPS-induced protein and mRNA expression of IL-6,MCP-1,TNF-α.AA could also reduce LPS-induced protein and mRNA expression of TLR4,and pretreatment of the cells with TLR4-siRNA could reduce LPS-induced inflammation.Moreover,treatment with AA could up-regulate the mRNA and protein expression of PPAR-γin VSMCs;however,GW9662,a PPAR-γantagonist,partially attenuated AA’s anti-inflammatory effect.ConclusionAA can significantly inhibit LPS-induced mRNA and protein expression of IL-6,MCP-1,TNF-α,in VSMCs,which is partially dependent on suppressing TLR4 and upregulating PPAR-γ.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2018年第1期60-67,共8页
Chinese Pharmacological Bulletin
基金
河南省卫生厅科技攻关项目(No 201403051)
