基于NLRP3通路研究金樱子抑制肾小管上皮细胞间质化治疗糖尿病肾病的作用机制

Study on the mechanism of Rosa laevigata Michx inhibiting epithelial mesenchymal transformation in the treatment of diabetes nephropathy based on NLRP3 Pathway

[目的]研究金樱子对糖尿病肾病(DN)模型小鼠的治疗效果及基于核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)信号通路抑制肾小管上皮细胞间质化(EMT)的作用机制。[方法]通过链脲佐菌素(STZ)联合高脂饮食诱导建立DN小鼠模型,并灌胃不同剂量的金樱子。通过检测各组小鼠体肌酐(Cr)、尿素氮(BUN)、24 h尿蛋白,肾石蜡切片苏木精-伊红(HE)染色及过碘酸-Schiff(PAS)染色评估金樱子对DN模型小鼠的治疗作用;通过检测上皮性钙黏附蛋白(E-CDH)、波形蛋白(VIM)、α-平滑肌肌动蛋白(α-SMΑ)和转化生长因子β(TGF-β)mRNA和蛋白表达水平研究金樱子对DN小鼠EMT的影响;通过检测NLRP3、凋亡相关斑点样蛋白(ASC)、活化的半胱氨酸天冬氨酸蛋白酶1(cleaved Caspase-1)、成熟的白细胞介素-1β(mature IL-1β)、成熟的白细胞介素-18(mature IL-18)的蛋白水平评估金樱子对DN小鼠NLRP3信号通路的影响。[结果]金樱子可明显降低DN小鼠Cr、BUN及24 h尿蛋白水平;同时改善DN模型小鼠肾组织病理学变化,提示金樱子对DN模型小鼠具有治疗作用;进一步研究发现金樱子提升了E-CDH,降低了VIM、α-SMΑ和TGF-βmRNA和蛋白水平,改善了DN小鼠的EMT;降低了NLRP3、ASC、cleaved-Caspase-1、mature IL-1β、mature IL-18蛋白水平,提示金樱子干预降低了DN小鼠NLRP3信号通路的活化程度。[结论]金樱子对DN模型小鼠具有治疗作用,其作用机制可能与通过降低NLRP3信号通路活化抑制EMT有关。

[Objective]To investigate the therapeutic effect of Rosa laevigata Michx on diabetic nephropathy(DN)model mice and the mechanism of inhibiting epithelial mesenchymal transformation(EMT)based on NOD-like receptor protein 3(NLRP3)signaling pathway.[Methods]The DN mouse model was established by Streptozotocin(STZ)combined with high-fat diet,and different doses of Rosa laevigata Michx were intragastrically administered.Creatinine(Cr),Blood urea nitrogen(BUN),24 h urinary protein of mice in each group were detected.Hematoxylin-Eosin(HE)and Periodic acid-Schiff(Pas)staining evaluated the therapeutic effect of Rosa laevigata Michx on DN model mice.Epithelial cadherin(E-CDH),vimentin(VIM),alpha-smooth muscle actin(alpha-SMA)and the expression levels of TNF-βmRNA and protein were studied to investigate the effect of Rosa laevigata Michx on EMT in DN mice.The protein levels of NLRP3,apoptosis-associated speck-like protein(ASC),cleaved cysteinyl aspartate specific proteinase-1(cleaved Caspase-1),mature interleukin-1β(mature IL-1β),and mature IL-18 were used to evaluate the effects of Rosa laevigata Michx on the NLRP3 signaling pathway in DN mice.[Results]The levels of Cr,BUN and 24 h urinary protein in DN mice were decreased.At the same time,the renal histopathological changes of DN model mice were improved,suggesting that Rosa laevigata Michx has therapeutic effect on DN model mice.Further studies showed that Rosa laevigata Michx increased E-CDH,decreased VIM,α-SMA and TGF-βmRNA and protein levels,and improved EMT in DN mice.The levels of NLRP3,ASC,cleaved Caspase-1,mature IL-1β,and mature IL-18 proteins were decreased,suggesting that the NLRP3 signaling pathway in DN mice was reduced by the intervention of Rosa laevigata Michx.[Conclusion]Rosa laevigata Michx has therapeutic effect on DN model mice,and its mechanism may be related to the inhibition of EMT by decreasing the activation of NLRP3 signaling pathway.