热休克蛋白27调控RhoA在大鼠膀胱出口梗阻致平滑肌重塑中的作用

Role of HSP27 regulating RhoA in bladder smooth muscle remodeling in rats after bladder outlet obstruction

目的探讨热休克蛋白27(heat shock protein 27,HSP27)调控RhoA在大鼠膀胱出口梗阻(bladder outlet obstruction,BOO)致平滑肌重塑中的作用及其机制。方法采用透明质酸尿道口注射法制作大鼠膀胱出口梗阻模型(BOO组),对照组大鼠注射相同剂量生理盐水。4周后行膀胱组织HE染色,Western blot检测大鼠膀胱组织中HSP27及RhoA表达水平;建立人膀胱平滑肌细胞HSP27过表达(过表达组)及敲低(敲低组)模型,以转染相应空载体细胞作为对照组,Western blot检测各组HSP27、pHSP27、RhoA及G-actin、F-actin蛋白水平。F-actin鬼笔环肽荧光染色观察各组细胞中肌动蛋白结构改变。结果成功建立BOO大鼠模型,4周后HE染色可见BOO组大鼠膀胱平滑肌明显肥厚;BOO组大鼠膀胱组织中HSP27及RhoA表达较对照组明显上调(P<0.05)。成功建立HSP27过表达及敲低模型,Western blot结果显示,与对照组比较,HSP27过表达组HSP27、p-HSP27及RhoA表达水平明显上调(P<0.05);HSP27敲低组HSP27、p-HSP27及RhoA表达水平均明显下调(P<0.05);HSP27过表达组F-actin/G-actin(F/G)比值明显上升(P<0.05),而敲低组F/G比值明显降低(P<0.05);鬼笔环肽染色结果显示,HSP27敲低组平滑肌细胞内肌动蛋白丝含量较对照组和过表达组明显减少,HSP27过表达组平滑肌细胞内肌动蛋白丝含量较对照组明显增多,排列紊乱。结论HSP27可通过调控RhoA,影响膀胱平滑肌细胞内肌动蛋白结构,可能在大鼠膀胱出口梗阻后膀胱平滑肌重塑中发挥重要作用。

Objective To explore the role and underlying mechanism of heat shock protein 27(HSP27)in bladder smooth muscle remodeling after bladder outlet obstruction(BOO).Methods Rat BOO model was established by urethral orifice hyaluronic acid injection,and the rats from the control group were given the injection of same amount of normal saline.In 4 weeks after model establishment,the bladder wall tissues of BOO and control group were observed by HE staining.The expression of HSP27 and RhoA was detected by Western blotting in both groups.Cultured human bladder smooth muscle cells(BSMCs)were transfected with lentiviral vectors expressing either HSP27 or HSP27-shRNA(shHSP27).Western blotting was used to detect the expression of HSP27,phosphorylated HSP27,RhoA,G-actin and F-actin.Phalloidin fluorescence staining of F-actin was performed in overexpression and control group to observe actin filaments in BSMCs.Results HE staining showed evident bladder smooth muscle hypertrophy in BOO group in 4 weeks after hyaluronic acid injection,indicating the successful establishment of rat BOO model.The expression levels of HSP27 and RhoA were increased significantly compared to those in the control group(P<0.05).BMSC cell models of HSP27 overexpression and knockdown were successfully established.The Results of Western blotting showed increased expression of HSP27,phosphorylated HSP27 and RhoA in HSP27 overexpression cells(P<0.05),and decreased expression of these protein in shHSP27 cells(P<0.05).Also,the F-actin/G-actin ratio was elevated in HSP27 overexpression cells(P<0.05)and an opposite result was seen in the shHSP27 cells(P<0.05).Phalloidin fluorescence staining showed there were less actin filaments in shHSP27 cells than the control and HSP27 overexpression cells,and more actin filaments in abnormal arrangement were observed in the overexpression cells than the control cells.Conclusion HSP27 can affect the structure of actin by regulating RhoA,and may play a critical role in bladder smooth muscle remodeling after BOO in rats.