蛋白激酶C和活化态C激酶1受体蛋白在血管平滑肌细胞泡沫化变中的作用

Roles of protein kinase C and receptor for activated C kinase 1 in foam cell transformation of vascular smooth muscle cells

目的探索血管平滑肌细胞泡沫化过程中活化态C激酶1受体蛋白(RACK1)、蛋白激酶C(PKC)通道蛋白的相关性,抑制PKC的作用后对动脉硬化的效果,为临床上的进一步治疗提供坚实的研究基础和新的治疗靶点。方法使用组织贴块法从SD大鼠的主动脉中提取原代血管平滑肌细胞并进行鉴定;取第3~9代的原代平滑肌细胞分为对照组和不同浓度脂氧化实验组(氧化型低密度脂蛋白ox-LDL处理组),采用高效液相色谱法(HPLC法)测定不同组细胞内胆固醇含量;通过油红O染色测定各组平滑肌细胞泡沫化程度;通过免疫印迹(Western blot)技术检测各组细胞中PKC、磷酸化蛋白激酶C(Cp-PKC)、RACK1、应激活化蛋白激酶(JNK)、磷酸化应激活化蛋白激酶(p-JNK)表达量的变化;加入不同浓度的PKC抑制剂(Calphostin C)处理细胞后检测抑制剂对PKC/RACK1通道及其下游相关蛋白的影响。用SPSS 17.0软件对数据进行统计学分析,多组间比较用单因素方差分析。结果与对照组相比,不同浓度ox-LDL刺激下平滑肌细胞内总胆固醇(TC)和胆固醇酯(CE)含量均明显增加,且随着ox-LDL浓度的增加,TC和CE含量逐渐增加,泡沫细胞内的脂滴含量也增加。Western blot检测发现,与对照组相比,实验组中加入ox-LDL刺激后,PKC的活性增强,其活化型受体RACK1和p-JNK的表达量也升高。加入PKC的抑制剂Calphostin C后会抑制平滑肌细胞脂质的蓄积,抑制泡沫细胞的形成,同样也会降低p-PKC、RACK1的表达。结论ox-LDL可以促进平滑肌细胞的泡沫化变,其作用机制可能与PKC/RACK1的作用有相关性。抑制PKC的作用后细胞内脂质蓄积降低,可以延缓动脉硬化的发生,为临床上治疗动脉硬化提供新的治疗方案。

Objective To explore the correlation between receptor for activated C kinase 1(RACK1)and protein kinase C(PKC)pathway proteins and the foaming process of vascular smooth muscle cells(VSMCs),and the effects of inhibiting PKC on atherosclerosis,to provide a solid research fundation and a new therapeutic target for clinical treatment.Methods The tissue patch method was used to isolate the primary VSMCs from the aorta of SD rats,and the primary VSMCs were identified.The primary smooth muscle cells of the third and ninth generations were divided into control group and lipoxygenation group(oxidized low density lipoprotein ox-LDL treatment group),the content of intracellular cholesterol in different groups was determined by high performance liquid chromatography(HPLC);and the foaming degree of smooth muscle cells in each group was determined by oil red O staining.The expression levels of PKC,p-PKC,RACK1,JNK and p-JNK in each group were detected by Western blot;after treatment of PKC inhibitor(Calphostin C),the effects of PKC inhibitor on PKC/RACK1 pathway and downstream related proteins were detected.The difference among more groups was compared by ANOVA.The used software was SPSS 17.0.Results As compared with the control group,the total cholesterol(TC)and cholesterol ester(CE)levels in VSMCs treated by different concentrations of ox-LDL increased significantly and with ox-LDL concentrations,also the lipid droplet level increased.Western blot assay showed that as compared with the control group,in ox-LDL treatment group,PKC activity increased,also the expression levels of RACK1 and p-JNK increased.After adding PKC inhibitor(Calphostin C),the intracellular lipid accumulation and foaming cells formation were inhibited,and the expression levels of p-PKC and RACK1 decreased.Conclusion ox-LDL could promote the foaming of smooth muscle cells,and the mechanism might be related to the effects of PKC/RACK1.The intracellular lipid accumulation decreased and the occurrence of atherosclerosis could delay after inhibiting PKC,which provided a new therapy protocol for atherosclerosis.