益气养阴通络方通过lncRNA UCA1靶向调控miR-485-5p抑制糖尿病肾病大鼠肾小管上皮细胞凋亡及炎症反应作用机制

Mechanism of Yiqi Yangyin Tongluo formula targeting regulation of miR-485-5p on apoptosis and inflammatory response of renal tubular epithelial cells in DN rats by lncRNA UCA1

目的:探讨益气养阴通络方通过lncRNA UCA1靶向调控miR-485-5p抑制糖尿病肾病(DN)大鼠肾小管上皮细胞凋亡和炎症反应作用机制。方法:选取雄性Wistar大鼠50只,随机分为空白组、模型组和益气养阴通络方低、中、高剂量组,每组各10只;除空白组外,其余组大鼠采用高糖高脂胆固醇饮食造模。检测各组大鼠肾功能指标和炎症因子水平;采用流式细胞术检测各组大鼠细胞凋亡率;WB检测各组大鼠细胞凋亡蛋白表达;PCR检测大鼠UCA1、miR-485-5p mRNA水平;采用双荧光素酶报告基因实验检测UCA1与miR-485-5p的结合位点。结果:与模型组比较,益气养阴通络方不同剂量组24 h尿蛋白(24 hUTP)、血清肌酐(SCr)、白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)水平较低,且呈剂量依赖性(均P<0.05);与模型组比较,益气养阴通络方不同剂量组大鼠细胞凋亡率较低,且呈剂量依赖性(均P<0.05);与模型组比较,益气养阴通络方不同剂量组NLRP3、Caspase-1蛋白表达量较低,且呈剂量依赖性(均P<0.05);与模型组比较,益气养阴通络方不同剂量组UCA1 mRNA水平较高,miR-485-5p mRNA水平较低,且呈剂量依赖性(均P<0.05);UCA1与miR-485-5p存在结合位点,具有靶向调控关系。结论:益气养阴通络方可能通过lncRNA UCA1靶向调控miR-485-5p抑制DN大鼠肾小管上皮细胞凋亡和炎症反应。

Objective:To investigate the effect and mechanism of the regulation of miR-485-5p on the apoptosis and inflammatory response of renal tubular epithelial cells in rats with diabetic nephropathy(DN)by the targeting regulation of Yiqi Yangyin Tongluo formula by lncRNA UCA1.Methods:Fifty male Wistar rats were randomly divided into blank group,model group,low-dose,medium-dose and high-dose Yiqi Yangyin Tongluo formula groups,with 10 rats in each group.In addition to the blank group,the diabetic nephropathy rat model was established by high sugar and high fat cholesterol diet.The levels of renal function indexes and inflammatory factors were detected in each group.Flow cytometry was used to detect the number of apoptosis in each group.WB was used to detect the expression of apoptotic protein in rats.UCA1 and miR-485-5p mRNA levels in rats were detected by PCR.Dual luciferase reporter gene assay was used to detect the binding relationship between UCA1 and miR-485-5p.Results:Compared with model group,24 h urinary protein(24 hUTP),serum creatinine(SCr),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)levels in different doses of Yiqi Yangyin Tongluo formula groups were lower,and the levels were dose-dependent(all P<0.05).Compared with model group,the apoptosis rate of rats in different doses of Yiqi Yangyin Tongluo formula was lower,and the levels were dose-dependent(all P<0.05).Compared with model group,NLRP3 and Caspase-1 protein expressions in different dose of Yiqi Yangyin Tongluo formula groups were lower and the levels were dose-dependent(all P<0.05).Compared with model group,UCA1 mRNA level was higher and miR-485-5p mRNA level was lower in different dose of Yiqi Yangyin Tongluo formula groups,and the levels were dose-dependent(all P<0.05).There are binding sites between UCA1 and miR-485-5p,showing a targeted regulatory relationship.Conclusion:Yiqi Yangyin Tongluo formula can regulate miR-485-5p to inhibit the apoptosis and inflammatory response of renal tubular epithelial cells in DN rats through the targeting of lncRNA UCA1.