二甲双胍对单侧输尿管梗阻小鼠肾脏纤维化的影响及其作用机制

Effect of metformin on renal fibrosis in mice with unilateral ureteral obstruction and its mechanism

目的探讨二甲双胍对单侧输尿管梗阻(UUO)诱导的小鼠肾脏纤维化的影响及其作用机制。方法选取30只C57BL/6小鼠,分为假手术组(仅分离左侧输尿管但不结扎,n=10)、UUO组(单侧输尿管结扎术法建立UUO小鼠模型,n=10)、二甲双胍组[单侧输尿管结扎术法建立UUO小鼠模型,造模前1 d开始二甲双胍灌胃,200 mg/(kg·d),连续7 d;n=10]。HE染色检测3组肾组织形态变化;Masson染色测定3组肾间质纤维化情况;免疫组化检测3组肾组织中YAP1、TGF-β1和CD68的表达;免疫荧光检测3组肾组织中F4/80的表达;Western blotting检测YAP1、p-YAP、LATs1、TEAD的表达。结果与假手术组比较,UUO组尿N-乙酰-β-D-葡萄糖苷酶增加,肾间质纤维化程度增加,CD68、F4/80、TGF-β1、YAP1、TEAD表达增高,而p-YAP、LATs1的表达下降,差异均有统计学意义(均P<0.05)。与UUO组比较,二甲双胍组尿N-乙酰-β-D-葡萄糖苷酶下降,肾间质纤维化程度减轻,CD68、F4/80、TGF-β1、YAP1、TEAD表达减少,而p-YAP、LATs1的表达增加,差异均有统计学意义(均P<0.05)。结论UUO诱导的肾脏纤维化激活肾组织中Hippo/YAP通路,上调CD68、F4/80、TGF-β1蛋白表达水平促进肾纤维化,引起肾结构及功能异常;二甲双胍可能通过干预Hippo/YAP通路改善UUO诱导的小鼠肾脏结构及功能的损伤。

Objective To determine the effect of metformin on renal fibrosis induced by unilateral ureteral obstruction(UUO)and to clarify the role and mechanism of the Hippo/YAP pathway in renal fibrosis.Methods Thirty C57BL/6 mice were divided into three groups:a sham group(only the left ureter was isolated without ligation,n=10),a UUO group(unilateral ureteral ligation,n=10),and a metformin group[unilateral ureteral ligation,with metformin administered by gavage 1 day before modeling 200 mg/(kg·d),for 7 days,n=10].Hematoxylin and eosin staining was used to observe morphological changes in kidney injury in the three groups.Renal interstitial fibrosis was assessed by Masson staining.Immunohistochemistry was used to detect the expression of YAP1,TGF-β1,and CD68 in renal tissue,while the expression of F4/80 among the three groups was detected using immunofluorescence.Western blotting was used to detect the expression of YAP1,p-YAP,LATs1,and TEAD.Results Compared with the sham group,the urinary N-acetyl-β-D-glucosidase in the UUO group had significantly increased.Accompanied by an increase in renal interstitial fibrosis,the expressions of CD68,F4/80,TGF-β1,YAP1,and TEAD increased significantly,while the expressions of p-YAP and LATs1 decreased significantly(all P<0.05).Compared with that noted in the UUO group,urinary N-acetyl-β-D-glucosidase decreased;renal interstitial fibrosis alleviated;the expression levels of CD68,F4/80,TGF-β1,YAP1,and TEAD decreased;and the expression of p-YAP,and LATs1 increased in the metformin group(all P<0.05).Conclusion Renal fibrosis induced by UUO can significantly activate the Hippo/YAP pathway in renal tissue and further promote the progression of renal fibrosis by up-regulating the expressions of CD68,F4/80,and TGF-β1,which eventually leads to renal dysfunction and structural damage.Metformin may decrease renal structural damage and dysfunction by intervening the Hippo/YAP pathway.