抑制MST1通路对糖尿病大鼠肾组织的保护作用及其机制研究

Protective effect and mechanism of MST1 inhibition on kidney tissue in diabetic rats induced by streptozotocin

目的探讨抑制哺乳动物不育系20样激酶1（MST1）通路对链脲佐菌素（STZ）诱导的糖尿病肾病大鼠的保护作用及其机制。方法54只雄性健康SD大鼠随机被分为健康对照组（A组，n=18）；MST1抑制组（B组，n=18）；糖尿病模型组（C组，n=18）。采用单侧腹腔注射链脲佐菌素（50mg／kg）方法制备糖尿病大鼠模型。除接受链脲佐菌素外，B组大鼠接受含MST1干扰RNA序列（shRNA）的慢病毒载体注射；C组大鼠接受空载慢病毒载体注射。造模后4、8、12周为观察时间点，自动生化仪上检测各时间点大鼠24h尿蛋白量、血糖及血肌酐；HE染色观察肾组织病理改变；透射电镜下观察足细胞及基底膜改变；免疫组织化学方法观察MSTl表达强度及定位；Western印迹法检测．肾组织MST1、磷酸化MST1、足细胞相关蛋白nephrin、凋亡相关蛋白半胱天冬蛋白酶（Caspase-3）、肾细胞凋亡相关基因（FasL）等蛋白的表达变化。结果（1）3组大鼠各观察时间点血肌酐比较差异无统计学意义（均P〉0．05）；与A组比较，B、C组大鼠造模后72h血糖明显升高（均P〈0．05）；B、C组间比较，各时间点血糖差异无统计学意义（均P〉0．05）；与C组比较，B组大鼠24h尿蛋白量在造模后4周明显减少（P〈0．05）。（2）A组大鼠肾组织未见明显病变；B、C组大鼠肾组织可见系膜增生性改变，未见明显K．w结节样改变；电镜下可见足细胞融合、基底膜增厚改变。系膜增生系数（MEI）比较结果示B组病变轻于C组（P〈0．05）。（3）Western印迹结果显示，与A组比较，B、C组大鼠各时间点肾组织MST1、磷酸化MST1、Caspase-3、FasL表达明显增高，nephrin表达明显降低（均P〈0．05）；与同时间点C组比较，B组大鼠肾组织MST1、磷酸化-MST1、Caspase-3、FasL表达降低，liephrin表达增高（均P〈0．05）。（4）免疫组化结果显示，A组大鼠肾小球及间质可见有MST1表达，以肾小管上皮细胞胞质多见；B、C组大鼠肾组织MST1表达均增多，变化趋势与Western印迹结果相似。结论MST1通路可能参与了糖尿病肾病的组织损伤，抑制MST1表达可减轻链脲佐菌素诱导的糖尿病模型的肾组织细胞损伤。

Objective To investigate the protective effect and mechanism of MST1 inhibition on kidney tissue in diabetic rats, and to find a new therapeutic target for diabetic nephropathy. Methods Total of 54 male SD rats enrolled in this study were divided into 3 groups including normal control （group A, n=18）, MST1 inhibition group （Group B, n=lS） and diabetes group （group C, n=18）.Diabetes was induced by a single streptozotocin （STZ, 50 mg/kg） injection in group B and group C. rats in group B received lentiviral vector contain Mstl interference RNA （shRNA） and the rats in group C received empty vector. The end of 4th, 8th and 12th week after modeling were considered as time points in this study. At each time point, the level of 24 hours urine protein （24-HUP）, blood glucose and serum creatinine were examined. Pathological changes were observed with HE stain; Injury of podocyte and glomerular basement membrane （GBM） were examined with transmission electron microscope （TEM）. The intensity and location of MST1 in kidney tissue were detected by immunohistochemistry. The level of MST1, Phosphorylated-MST1, nephrin, Caspase-3 and FasL were detected by western bloting. Results （1） At the starting point, there were no significant differences among groups in terms of weight, activity, eating and drinking. Since the end of 72nd hour after modeling, the levels of glucose in both group B and group C, compared to those in group A, significantly increased （P 〈 0.05）. There was no significant difference between group B and group C for glucose level at each time point （P 〉 0.05）; the level of 24-HUP increased significantly since the end of 4th week after modeling, and the level in group C was higher than its counterpart in group B at the same point （P 〈 0.05）; （2） There was no significant pathological lesion observed in group A. Without obvious K-W nodular changes, mesangial proliferation was observed in group B and group C. It was shown by TEM that podocyte fusion and thickening of the GBM could be found in group B and group C. The pathological change in group B was better than that in group C; （3） Compared to group A, it was shown by western blot that the levels of MST1, Phosphorylated-MST1, Caspase-3 and FasL in group B and group C were significantly higher （P 〈 0.05）, and the levels of nephrin in group B and group C were significantly lower （P 〈 0.05） since the end of 4th week after modeling. Meanwhile, the levels of MSTI, Phosphorylated-MST1, Caspase- 3 and FasL in group B were significantly lower than that in group C at each time point （P 〈 0.05）, the level of nephrin in group B was significantly higher than the one in group C; （4） It was shown by immunohistochemistry that there was low MST1 expression in normal condition, especially in cytoplasm of tubular epithelial ceils. The level of MST1 in group B and group C significantly increased after modeling, and the cbange could be the same as Western blot shown. Conclusions MST1 pathway could be involved in kidney injury induced by diabetes. MST1 inhibition could alleviate the kidney injury in STZ-induced diabetes animal model.