黄蜀葵花总黄酮抑制糖尿病肾脏疾病足细胞坏死性凋亡和肾脏纤维化的作用和机制

Effects and mechanisms of total flavones of Abelmoschus manihot in inhibiting podocyte necroptosis and renal fibrosis in diabetic kidney disease

研究表明,高血糖介导的“慢性微炎症”可以导致糖尿病肾脏疾病(diabetic kidney disease,DKD)患者出现“炎症性足细胞损伤”。其中,“坏死性凋亡”是新的足细胞死亡形式,与肾脏纤维化密切相关。为了探究传统治肾中药黄蜀葵花的提取物——黄蜀葵花总黄酮(total flavones of Abelmoschus manihot,TFA)在体内改善DKD足细胞坏死性凋亡和肾脏纤维化的作用和机制,并进一步揭示其“多途径、多靶点”的科学内涵,笔者将全部大鼠随机分为4组,即正常组、模型组、TFA组以及雷帕霉素(rapamycin,RAP)组。在改良型DKD大鼠模型成功建立后,每天经灌胃分别给予4组大鼠双蒸水、TFA混悬液以及RAP混悬液。在药物干预的第4周末,处死全部大鼠,收集尿液、血液以及肾组织等标本,进而针对模型鼠足细胞坏死性凋亡和肾脏纤维化各项指标进行观察、检测和分析。结果显示,对于改良型DKD模型鼠,TFA和RAP改善了一般情况、体质量、血清肌酐、尿白蛋白以及肾脏肥大指数;改善了肾脏纤维化指标,包括肾小球组织形态特征、肾小球内纤维连接蛋白(fibronectin,FN)、Ⅰ型胶原(collagen typeⅠ,collagenⅠ)染色程度以及肾组织FN、collagenⅠ、转化生长因子-β1(transforming growth factor-β1,TGF-β1)和Smad2/3蛋白表达水平;改善了足细胞损伤,包括足突形态以及肾组织中podocin、CD2AP蛋白表达水平;改善了肾脏中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)介导的足细胞坏死性凋亡,包括足细胞坏死性凋亡形态特征以及肾组织中TNF-α、磷酸化底物混合谱系激酶样蛋白(phosphorylated mixed lineage kinase domain like pseudokinase,p-MLKL)表达程度和蛋白表达水平,其中,对于p-MLKL,RAP的作用更强;改善了肾脏中受体相互作用蛋白激酶(receptor interacting serine/threonine protein kinase,RIPK)1/RIPK3/MLKL信号轴活性,包括其关键信号分子蛋白表达水平,如磷酸化受体相互作用蛋白激酶(phosphorylated receptor interacting serine/threonine protein kinase,p-RIPK)1、p-RIPK3、p-MLKL和半胱氨酸蛋白水解酶-8(cysteinyl aspartate specific proteinase-8,caspase-8),其中,对于p-RIPK1,TFA的作用更强。总之,该研究阐明:TFA通过抑制TNF-α介导的RIPK1/RIPK3/MLKL信号轴活性而减轻DKD足细胞坏死性凋亡和肾脏纤维化。笔者的发现为深入揭示TFA治疗DKD肾脏纤维化的科学内涵提供了新的药理学证据。

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD).Therein,necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF).To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA),an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases,on podocyte necroptosis and RF in DKD,and to further reveal its scientific connotation with multi-pathway and multi-target,the authors randomly divided all rats into four groups:a namely normal group,a model group,a TFA group and a rapamycin(RAP)group.After the modified DKD rat models were successfully established,four group rats were given double-distilled water,TFA suspension and RAP suspension,respectively by gavage every day.At the end of the 4th week of drug treatment,all rats were sacrificed,and the samples of their urine,blood and kidneys were collected.And then,the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed,detected and analyzed,respectively.The results indicated that,general condition,body weight(BW),serum creatinine(Scr),urinary albumin(UAlb),and kidney hypertrophy index(KHI)in these modified DKD model rats were both improved by TFA and RAP.Indicators of RF,including glomerular histomorphological characteristics,fibronectin(FN)and collagen typeⅠ(collagenⅠ)staining extent in glomeruli,as well as the protein expression levels of FN,collagenⅠ,transforming growth factor-β1(TGF-β1)and Smad2/3 in the kidneys were improved respectively by TFA and RAP.Podocyte damage,including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP.In addition,tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys,including the morphological characteristics of podocyte necroptosis,the extent and levels of the protein expression of TNF-αand phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL)was improved respectively by TFA and RAP.Among them,RAP had the better effect on p-MLKL.More importantly,the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys,including the expression levels of its key signaling molecules,such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1),p-RIPK3,p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8)was improved respectively by TFA and RAP.Among them,the effect of TFA on p-RIPK1 was superior.On the whole,in this study,the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys.The authors′findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.