ROCK信号通路对高糖诱导血管平滑肌细胞炎症反应的调控作用及其与Nur77表达的关系

Regulation of ROCK signaling pathway on inflammatory response of vascular smooth muscle cells induced by high glucose and its relationship with Nur77 expression

目的 观察阻断ROCK信号通路对高糖诱导的血管平滑肌细胞(VSMC)炎症反应的调控作用,并探讨其与孤儿核受体Nur77表达的关系。方法 分离提取大鼠主动脉VSMC,将细胞随机分为对照组、法舒地尔组、高糖组、高糖+法舒地尔组,分别加入葡萄糖5.5 mmol/L、葡萄糖5.5 mmol/L+ROCK信号通路抑制剂法舒地尔50μmol/L、葡萄糖30 mmol/L、葡萄糖30 mmol/L+法舒地尔50μmol/L,作用12 h;采用Western blotting法检测细胞ROCK1、ROCK2、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)、Nur77蛋白表达,实时荧光定量PCR法检测Nur77 mRNA表达。将VSMC随机分为Nur77高表达组和空白组,两组均加入葡萄糖5.5 mmol/L,Nur77高表达组同时加入Nur77激动剂Cytosporone B(CsnB)10μg/mL,作用12 h后比较其Nur77 mRNA及蛋白表达;更换培养基,加入葡萄糖30 mmol/L,作用12 h后比较其Nur77及IL-1β、IL-6蛋白表达。结果 高糖组细胞ROCK1、IL-1β、IL-6蛋白相对表达量均高于对照组(P均<0.05)。对照组、高糖+法舒地尔组、法舒地尔组细胞ROCK1、IL-1β、IL-6蛋白相对表达量比较,各组细胞ROCK2蛋白相对表达量比较差异均无统计学意义(P均>0.05)。高糖组细胞Nur77 mRNA及蛋白相对表达量均高于高糖+法舒地尔组、对照组、法舒地尔组(P均<0.05),高糖+法舒地尔组、对照组、法舒地尔组比较差异均无统计学意义(P均>0.05)。低糖环境下Nur77高表达组Nur77 mRNA及蛋白相对表达量均高于空白组,加入高糖后Nur77高表达组Nur77蛋白相对表达量高于空白组,IL-1β、IL-6蛋白相对表达量均低于空白组(P均<0.01)。结论 阻断ROCK信号通路和上调Nur77表达均可抑制高糖诱导的VSMC炎症反应,ROCK信号通路与Nur77表达之间可能存在一定的调控关系。

Objective To observe the inhibitory effect of ROCK signaling pathway blockade on inflammatory response of vascular smooth muscle cells(VSMC) induced by high glucose,and to explore its relationship with the expression of orphan nuclear receptor Nur77.Methods The rat aortic VSMC were randomly divided into the control group,fasudil group,high glucose group,and high glucose+fasudil group,which were added with 5.5 mmol/L glucose,5.5 mmol/L glucose+50 μmol/L ROCK signal pathway inhibitor fasudil,30 mmol/L glucose,30 mmol/L glucose+50 μmol/L fasudil,and were incubated for 12 h.The expression levels of ROCK1,ROCK2,interleukin(IL)-6,IL-1β and Nur77 protein was detected by Western blotting,and the Nur77 mRNA expression was detected by real-time fluorescence quantitative PCR.VSMC were randomly divided into the high Nur77 expression group and blank group;both groups were treated with glucose 5.5 mmol/L,and at the same time,the high Nur77 expression group was added with 10 μg/mL Nur77 agonist Cytosporone B(CsnB).After 12 hours of treatment,the expression levels of Nur77 mRNA and protein were compared;then,we changed the culture medium and added 30 mmol/L glucose,and the protein expression levels of Nur77,IL-1β and IL-6 were compared after 12 hours of treatment.Results The relative expression levels of ROCK1,IL-1βand IL-6 proteins in the high glucose group were higher than those in the control group(all P0.05).There was no significant difference in the relative expression of ROCK2 protein among all groups(P>0.05).The relative expression levels of Nur77 mRNA and protein in the high glucose group were higher than those in the high glucose+fasudil group,control group,and fasudil group(all P0.05).The relative expression levels of Nur77 mRNA and protein in the high Nur77 expression group under low glucose environment were higher than those in the blank group;after adding high glucose,the relative expression of Nur77 protein in the high Nur77 expression group was higher than that in the blank group,and the IL-1β and IL-6 protein expression levels were lower than those in the blank group(all P<0 01).Conclusion Blocking ROCK signaling pathway and up-regulating Nur77 expression can inhibit VSMC inflammatory response induced by high glucose,and there may be a certain regulatory relationship between ROCK signaling pathway and Nur77 expression.