基于YKL-40和Wnt/β-catenin通路探讨山茱萸颗粒对糖尿病肾病的保护作用

Study on the Protective Effect of Corni Fructus Granules in Diabetic Nephropathy based on YKL-40 and Wnt/β-catenin Signaling Pathways

目的:基于YKL-40和Wnt/β-catenin通路探讨山茱萸颗粒对糖尿病肾病(DN)的保护作用。方法:采用腹腔注射链脲佐菌素构建大鼠糖尿病肾病模型,实验分正常对照组、模型组、山茱萸颗粒(300 mg/kg)组、Wnt-C59(Wnt/β-catenin信号通路抑制剂,30 mg/kg)组,给药并观察记录各组大鼠的一般体征变化,连续2 w,造模第14天处死大鼠,检测大鼠血肌酐(Scr)、尿素氮(BUN);HE染色、PAS染色、Masson染色观察各组大鼠肾组织的形态变化,电镜观察各组大鼠肾组织的超微结构,免疫组化检测肾组织Ⅳ型胶原蛋白、转化生长因子(TGF-β1)的表达,Western blot和RT-PCR法检测肾组织YKL-40、Wnt4、β-catenin mRNA及蛋白表达。结果:与正常对照组大鼠比较,模型组大鼠Scr、BUN显著升高(P<0.05),组织病理学改变明显,肾小球系膜区的PAS阳性表达及肾小球和肾小管中纤维性物质沉积明显增多,肾小球和肾小管结构改变明显,肾组织Ⅳ型胶原蛋白、TGF-β1表达及YKL-40、Wnt4、β-catenin mRNA及蛋白表达显著升高(P<0.05)。与模型组比较,山茱萸颗粒组、Wnt-C59组大鼠Scr、BUN显著降低(P<0.05),组织病理学明显改善,肾小球系膜区PAS阳性表达及纤维性物质沉积在肾小球和肾小管中表达明显降低,肾小球和肾小管结构改变有所恢复,肾组织Ⅳ型胶原蛋白、TGF-β1表达及YKL-40、Wnt4、β-catenin mRNA及蛋白表达显著降低(P<0.05);山茱萸颗粒组与Wnt-C59组各指标差异无统计学意义(P>0.05)。结论:山茱萸颗粒能改善DN大鼠的肾脏损伤,其作用机制可能与抑制YKL-40表达,从而抑制Wnt/β-catenin信号通路有关。

Objective:To investigate the protective effect of Corni Fructus granules in diabetic nephropathy(DN)based on YKL-40 and Wnt/β-catenin signaling pathways.Methods:A rat model of diabetic nephropathy was established by intraperitoneal injection of streptozotocin.The experiment was divided into normal control group,model group,Corni Fructus granules group(300 mg/kg)and Wnt-C59 group(Wnt/β-catenin signaling pathway inhibitor group,30 mg/kg).The drugs were administered,the general signs of the rats in each group were observed and recorded for 2 w,the rats were sacrificed on the 14 th day of modeling.The serum creatinine(Scr)and urea nitrogen(BUN)were detected.HE staining,PAS staining and Masson staining were used to observe the morphological changes of renal tissues in each group of rats.The renal tissues of rats in each group were observed by electron microscope.Immunohistochemistry was used to detect the expressions of typeⅣcollagen and transforming growth factor(TGF-β1)in renal tissues,the expressions of protein and mRNA of YKL-40,Wnt4 andβ-catenin in renal tissues were detected by Western blot and RT-PCR.Results:Compared with the normal control group,the Scr and BUN in the model group were significantly increased(P<0.05),the histopathological symptoms changed significantly,and the PAS positive expression in the mesangial area and the expression of fibrous material deposition in glomeruli and renal tubules increased significantly.The structures of glomeruli and tubules were significantly changed.The expressions of typeⅣcollagen and TGF-β1,the protein and mRNA expressions of YKL-40,Wnt4 andβ-catenin were significantly increased(P<0.05).Compared with the model group,the Scr and BUN of the Corni Fructus granules group and the Wnt-C59 group were significantly decreased(P<0.05),the histopathological symptoms improved significantly,the PAS positive expression in the mesangial area and the expression of fibrous substances deposition in glomeruli and renal tubules was significantly decreased,the structural changes of glomeruli and tubules were restored,the expressions of typeⅣcollagen and TGF-β1 in renal tissues and the mRNA and protein expressions of YKL-40,Wnt4 andβ-catenin were significantly decreased(P<0.05).There was no significant difference between the Corni Fructus granules group and the Wnt-C59 group(P>0.05).Conclusion:Corni Fructus granules can improve kidney damage in DN rats.The mechanism may be related to the inhibition of YKL-40 expression and the inhibition of Wnt/β-catenin signaling pathway.