RhoGTP酶对血吸虫病鼠肝窦毛细血管化的调控作用

目的探讨RhoGTP酶在肝窦内皮细胞转分化过程中的调控作用,及其对血吸虫病性鼠肝窦毛细血管化病变的影响和可能机制。方法采用腹部敷贴法感染血吸虫尾蚴建立血吸虫病性肝纤维化模型,于13周吡喹酮顿服杀虫,14周追加hydroxyfasudil。13周时正常对照组（A组,10只）与血吸虫病组（B组,6只）,16、19周和21周后（B组）、杀虫实验对照组（C组）、血吸虫病＋hydroxyfasudil组（D组）、杀虫＋hydroxyfasudil组（E组）各6只）剖腹取肝组织。分别作HE、Masson染色和透射电镜观察;免疫组化检测：p-膜联蛋白、CTGF、CD31、ColⅣ和LN;Western印迹检测：p-膜联蛋白、CTGF、RhoA、ColⅣ和LN蛋白表达;逆转录-聚合酶链反应（RT-PCR）检测：CTGF、RhoA、ROCKⅡ mRNA水平。结果与A组相比,动态观察B组肝组织RhoA、ROCKⅡ及CTGF mRNA表达水平明显上升,p-膜联蛋白、CTGF、RhoA、ColⅣ和LN蛋白表达量也增高。给予干预措施后,与C组及其它组相比,E组CTGF mRNA表达于16周显著下调,同时显示CTGF、LN、ColⅣ蛋白水平也降低。与B组相比,D组、E组p-膜联蛋白降低,16周时最为显著,但D组于19周开始p-膜联蛋白表达量渐恢复到原水平,其中E组明显低于D组。肝窦的超微结构观察,于21周时C组分别与B、D组相比,浸润的炎性细胞明显减少,Disse腔内胶原纤维有所减少,但肝窦内皮细胞窗孔、肝窦内皮下基底膜差异无统计学意义。E组与C组比较肝细胞器形态明显恢复,可见窗孔,未见基底膜。结论RhoGTP酶在调控血吸虫病性鼠肝窦内皮细胞转分化过程中,通过上调CTGF和p-膜联蛋白发挥作用,抑制RhoGTP酶信号通路有可能为防治肝窦毛细血管化提供新的有效靶点。

Regulatory effects of RhoGTPase on transition of liver sinusoidal capillarization: experiment with mice of schistosomal hepatic fibrosis

OBJECTIVE: To investigate the regulatory effects of RhoGTPase on the transition of liver sinusoidal endothelial cells and the potential mechanism thereof on the sinusoidal capillarization in schistosomal hepatic fibrosis. METHODS: Eight-eight mice underwent abdominal infection of schistosomal cercaria so as to establish liver fibrosis models. 13 weeks later the mice were divided into 5 groups: Group A (normal control group, n = 10), Group B (group of schistosomiasis, n = 24), Group C (anti-schistosoma control group, treated with biltricide), Group D (group of schistomiasis + hydroxyfudil, treated with hydroxyfudil since the week 14, n = 18), and Group E (biltricide + hydroxyfudil, treated with biltricide since week 13 and added with hydroxyfudil since week 14, n = 18). The mice in Group A and 6 mice of Group B were killed in week 13, and 6 mice of Groups B, C, D, and E were killed in weeks 16, 19, and 21 each. The livers were taken out to undergo electron microcopy. Immunohistochemistry was used to detect the expression of p-moesin, connective tissue growth factor (CTGF), RhoA, collagen IV (Col IV), and laminin (LN) protein expressions were assessed by Western blotting, and RT-PCR was used to detect the mRNA expression of CTGF, RhoA, and ROCK II. RESULTS: Compared with Group A, the mRNA levels of RhoA, ROCK II, and CTGF were significantly increased (all P < 0.05) and the protein expression levels of p-moesin, CTGF, RhoA, Col IV, and LN were significantly increased (all P < 0.05) in Group B. After intervention with biltricide and/or hydroxyfasudil, the CTGF mRNA expression was significantly decreased (P < 0.05) in Group E in week 16 and the protein expression levels of CTGF, Col IV, and LN were decreased (all P < 0.05) compared with other groups, and the expression of p-moesin of Group E was significantly lower than that of Group D (P < 0.05). Electron microcopy showed that the liver sinusoids of the mice in Group E was significantly better compared with the other groups, and there was no significant difference between Groups B and D. CONCLUSION: An upregulation of RhoGTPase that contributes to increased CTGF expression and phosphorylation of moesin may induce a transition of liver sinusoidal endothelial cells in schistosomiasis.