脂联素调控丝裂原活化蛋白激酶/细胞外信号调节激酶对大鼠滑膜细胞代谢能力的分子机制

The molecular mechanism of adiponectin regulating the metabolic ability of mitogen-activated protein kinase/extracellular signal-regulated kinase on rat synovial cells

目的探讨脂联素(AD)调控滑膜细胞产生炎性因子进而诱发骨关节炎(OA)。方法采用细胞计数试剂盒(CCK-8)检测刺激细胞的最适加药浓度;通过荧光定量聚合酶链反应(PCR)检测丝裂原活化蛋白激酶(MAPK)/细胞外信号调节激酶(ERK)通路标志基因如促分裂原活化的蛋白激酶(MEK)、丝裂原活化蛋白激酶(ERK1/2)的表达变化。蛋白质印迹法(Western blot)分析AD的刺激对ERK1/2、p38的磷酸化作用;酶联免疫吸附测定(ELISA)法测定AD作用后滑膜细胞炎性因子的产量。多个样本比较采用单因素方差分析。结果AD(1μg/ml)可显著对滑膜细胞产生增殖抑制作用(F=136.900、11.930,P<0.05);荧光定量PCR结果显示,与对照组比较,AD可上调MEK及ERK基因表达(1.026±0.072、2.926±0.158,t=21.910,P<0.05;0.960±0.194、5.433±0.532,t=15.810,P<0.05);Western blot结果表明,与对照组比较,AD可刺激滑膜细胞增加ERK1/2、p38的磷酸化(0.488±0.083、1.152±0.050,t=11.860,P<0.05;0.407±0.014、1.312±0.063,t=24.370,P<0.05);ELISA法结果显示,AD可显著促进由趋化蛋白引起的下游炎性因子如白细胞介素(IL)-1β、IL-6、基质金属蛋白酶(MMP)-3和MMP-13、肿瘤坏死因子-α(TNF-α)的产生[(15.077±0.884)、(69.947±2.950)pg/ml、(9.225±0.323)、(134.925±15.02)ng/ml、(93.055±6.073)pg/ml],与对照组比较,差异有统计学意义(t=3.239、4.838、5.312、4.366、3.292,P<0.05),与抑制剂组比较,差异有统计学意义(t=2.275、6.251、4.331、1.221、3.456,P<0.05)。结论AD可通过影响MAPK/ERK信号通路来增强滑膜细胞炎性因子的产生。

Objective Explore adiponectin(AD)to regulate synovial cells to produce inflammatory factors and induce osteoarthritis(OA).Methods Use cell counting kit-8(CCK-8)method to detect the optimal concentration of stimulating cells;detect mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK)pathway marker genes such as mitogen-activated protein by fluorescent quantitative polymerase chain reaction(PCR)The expression changes of kinase(MEK)and ERK1/2.Western blotting analysis of AD stimulation on the phosphorylation of ERK1/2,p38;enzyme linked immunosorbent assay(ELISA)method to determine the production of inflammatory factors in synovial cells after AD.Results AD(1μg/ml)can significantly inhibit the proliferation of synovial cells(F=136.900,11.930,P<0.05);fluorescence quantitative PCR results show that AD can upregulate MEK and ERK compared with the control group Gene expression(1.026±0.072,2.926±0.158,t=21.910,P<0.05;0.960±0.194,5.433±0.532,t=15.810,P<0.05);Western blotting results show that AD can stimulate synovial cells compared with the control group Increase the phosphorylation of ERK1/2 and p38(0.488±0.083,1.152±0.050,t=11.860,P<0.05;0.407±0.014,1.312±0.063,t=24.37,P<0.05);ELISA results show that AD can be significant Promote downstream inflammatory factors caused by chemotactic proteins,such as interleukin(IL)-1β,IL-6,matrix metalloproteinases(MMP)-3 and MMP-13,tumor necrosis factor-α(TNF-α)production[(15.077±0.884),(69.947±2.950)pg/ml,(9.225±0.323),(134.925±15.02)ng/ml,(93.055±6.073)pg/ml],compared with the control group The difference was statistically significant(t=3.239,4.838,5.312,4.366,3.292,P<0.05),and the difference was statistically significant compared with the inhibitor group(t=2.275,6.251,4.331,1.221,3.456,P<0.05).Conclusion AD can enhance the production of inflammatory factors in synovial cells by affecting the MAPK/ERK signaling pathway.