法舒地尔对软骨细胞基质降解的影响及其机制

The effect of Fasudil on degradation of chondrocyte matrix and its mechanism

目的:研究法舒地尔对大鼠软骨细胞的基质降解的影响,并探讨其可能的作用机制。方法:用CCK-8检测法舒地尔是否具有毒性并确定合适的药物浓度。分别按空白组、法舒地尔组(10μmol/L)、IL-1β组(10ng/mL)和IL-1β(10ng/mL)+法舒地尔组(10μmol/L)对细胞进行分组处理。使用硝酸还原酶法测定NO水平;Western blot检测COX-2、iNOS、MMP-13、p-MYPT、ROCK1、ROCK2、p-Erk/Erk、p-JNK/JNK与p-p38/p38水平;qRT-PCR测定MMP-13和二型胶原(Collagen-II)mRNA表达;细胞免疫荧光检测MMP-13的表达水平。结果:与IL-1β组比,IL-1β+法舒地尔组ROCK1、ROCK2和p-MYPT受到抑制(P<0.05);与IL-1β组比,IL-1β+法舒地尔组的COX-2、NO、iNOS和MMP-13的表达受到显著抑制(P<0.05);IL-1β组比,IL-1β+法舒地尔组的p-Erk、p-JNK、p-p38表达降低,并且Erk、JNK与p38磷酸化水平降低(P<0.05);与IL-1β组比,IL-1β+法舒地尔组二型胶原(Collagen-II)表达得到上调(P<0.05)。结论:法舒地尔可抑制IL-1β诱导的软骨细胞基质降解,该作用可能通过抑制MAPK信号通路来实现。

Objective:To study the effect of Fasudil on matrix degradation of humolan chondrocytes and to explore its possible mechanism.Methods:CCK-8 was used to determine whether Fasudil was toxic and to determine the appropriate drug concentration.The cells were grouped as control group,Fasudil group,IL-1βgroup and Fasudil+IL-1βgroup.NO levels were determined by nitrate reductase assay;Western blot was used to detect COX-2,iNOS,MMP-13,p-MYPT,ROCK1,ROCK2,p-Erk/Erk,p-JNK/JNK and p-p38/p38 levels;qRT-MMP-13 and collagen-II expressions were determined by PCR;cell immunofluorescence was used to detect the expressions of MMP-13.Results:Compared with the IL-1βgroup,ROCK1,ROCK2 and p-MYPT were inhibited in the Fasudil+IL-1βgroup;the expression of COX-2,NO,iNOS and MMP-13 was inhibited,so did the expression of p-Erk,p-JNK and p-p38;the phosphorylation levels of Erk,JNK and p38 were decreased,and the expression of collagen-II was up-regulated.The differences were statistically significant(P<0.05).Conclusion:Fasudil inhibited IL-1β-induced degradation of chondrocyte matrix,which is probably achieved by inhibiting MAPK signaling pathway.