新加肾元方调控RIPK1/RIPK3/MLKL通路对高糖诱导的糖尿病肾病肾小球足细胞损伤的影响

Effects of Xinjiashenyuan Formula Regulation of RIPK1/RIPK3/MLKL Pathway on Diabetic Nephropathy Glomerular Podocyte Injury Induced by High Glucose

目的:探讨新加肾元方对高糖诱导的糖尿病肾病(diabetic nephropathy,DN)大鼠肾小球足细胞损伤及RIPK1/RIPK3/MLKL通路的影响,并分析其可能的作用机制。方法:选取70只SD大鼠,随机取12只为正常对照组,另58只采用高糖高脂饲料喂养联合腹腔注射STZ法建立糖尿病肾病大鼠模型成功48只,随机分为模型组,阳性对照组,新加肾元方低、高剂量组,各12只。阳性对照组厄贝沙坦水溶液0.017g/kg灌胃,新加肾元方低、高剂量组0.72g/kg、1.44g/kg灌胃,正常对照组、模型组等体积生理盐水灌胃。检测肾功能:尿素氮、血清肌酐、蛋白尿;HE染色观察肾组织病理形态;透射电镜观察肾小球足细胞超微结构变化;TUNEL染色检测肾组织细胞凋亡情况;ELISA检测肾组织TNF-α、IL-6水平;WB检测肾组织RIPK1/RIPK3/MLKL通路相关蛋白表达;对新加肾元方中所含活性成分进行收集与筛选,并对“有效成分-靶点”拓扑分析所得度值较高的有效成分进行分子对接。结果:模型组大鼠蛋白尿、血清肌酐、尿素氮水平、凋亡指数、TNF-α、IL-6水平、RIPK1、RIPK3、MLKL蛋白表达增加较正常对照组增加,WT-1蛋白表达较正常对照组下降(P<0.05);阳性对照组、新加肾元方低、高剂量组大鼠蛋白尿、血清肌酐、尿素氮水平、凋亡指数、TNF-α、IL-6水平、RIPK1、RIPK3、MLKL蛋白表达较正常对照组下降,WT-1蛋白表达较正常对照组增加(P<0.05)。模型组大鼠肾小球基底膜变厚,系膜细胞增生,肾小管浊肿变性,肾间质纤维化;阳性对照组、新加肾元方低、高剂量组肾组织病理形态改变较模型组出现不同程度减轻。模型组大鼠足细胞足突融合或消失,基底膜增厚;阳性对照组、新加肾元方低、高剂量组大鼠肾组织超微结构病理形态改变较模型组出现不同程度改善。新加肾元方中人参主要活性成分22个,炙黄芪主要活性成分20个。分子对接结果显示与关键靶点对接较好的成分有人参中的Celabenzine、炙黄芪中的Mairin,与RIPK1、RIPK3、MLKL的结合能分别为-5.3、-6.42、-5.48和-5.39、-7.71、-5.41。结论:新加肾元方通过多成分、多靶点调控RIPK1/RIPK3/MLKL信号通路,降低炎症因子TNF-α、IL-6水平,缓解炎症反应,改善糖尿病肾病大鼠肾小球足细胞损伤。

Objective:To investigate the effects of Xinjiashenyuan formula on glomerular podocyte injury and RIPK1/RIPK3/MLKL pathway in diabetic nephropathy rats induced by high glucose,and to analyze its possible mechanism.Methods:A total of 70 SD rats were selected,and 12 were randomly selected as normal control group,and the remaining 58 rats were fed with high-sugar and high-fat diet for 8 weeks.Then,the DN rat model was established by intraderitoneal injection of 35 mg/kg streptozotocin,and 48 rats were successfully established.They were randomly divided into model group,positive control group,low-dose and high-dose Xinjiashenyuan Formula groups,with 12 rats in each group.Irbesartan solution was 0.017g/kg in positive control group,0.72g/kg and 1.44g/kg in Xinjiashenyuan formula group,and an equal volume of normal saline was the gavage for normal control group and model group.Renal function:urea nitrogen,serum creatinine,proteinuria;HE staining was used to observe the pathological morphology of renal tissue.Ultrastructural changes of glomerular podocyte were observed by transmission electron microscopy.TUNEL staining was used to detect the apoptosis of renal cells.The levels of TNF-αand IL-6 in renal tissue were detected by ELISA.The expression of RIPK1/RIPK3/MLKL pathway related proteins in renal tissues was detected by WB.The active ingredients contained in Xinjiashenyuan formula were collected and screened,and molecular docking was performed on the active ingredients with high degree values obtained from topological analysis of"active ingredient target".Results:Compared with normal control group,proteinuria,serum creatinine,urea nitrogen levels,apoptosis index,TNF-α,IL-6 levels,RIPK1,RIPK3 and MLKL protein expressions in model group were increased,while WT-1 protein expression was decreased(P<0.05).Proteinuria,serum creatinine,urea nitrogen levels,apoptosis index,TNF-α,IL-6 levels,RIPK1,RIPK3 and MLKL protein expressions in positive control group,low and high dose groups were decreased compared with normal control group,while WT-1 protein expression was increased compared with normal control group(P<0.05).In model group,the glomerular basement membrane was thickened,mesangial cells were hyperplasia,renal tubule turbidness and renal interstitial fibrosis were observed.The pathological morphological changes of renal tissues in positive control group,low dose group and high dose group were reduced in different degrees compared with model group.In model group,podocyte processes fused or disappeared and basement membrane was thickened.The pathological changes of renal ultrastructure in positive control group,low dose group and high dose group were improved in different degrees compared with model group.There are 22 active components of Panax ginseng in Xinjiashenyuanfang and 20 active components of zhihuangqi.Molecular docking results showed that Celabenzine in Ginseng and Mairin in prepared astragalus had better docking with key targets.The binding energies of Celabenzine in Ginseng and Mairin in prepared astragalus were-5.3,-6.42,-5.48 and-5.39,-7.71,-5.41 with RIPK1,RIPK3 and MLKL respectively.Conclusion:Xinjiashenyuan formula regulates RIPK1/RIPK3/MLKL signaling pathway through multiple components and multiple targets,reduces the levels of inflammatory factors TNF-αand IL-6,alleviates inflammatory response,and improves glomerular podocytic injury in diabetic nephropathy rats.