摘要
目的:观察当归补血汤对糖尿病肾病(DKD)大鼠足细胞损伤及受体相互作用蛋白激酶1/受体相互作用蛋白激酶3/混合系激酶区域样蛋白(RIPK1/RIPK3/MLKL)信号通路的影响,探讨其治疗DKD的可能作用机制。方法:50只SD大鼠随机选取8只设为正常组,其余大鼠6周高糖高脂饮食结合一次性腹腔注射链脲佐菌素(STZ)0.035 g·kg^(-1)制备2型糖尿病大鼠模型。模型制备成功后随机分为模型组,当归补血汤高、低剂量(1.44,0.72 g·kg^(-1))组,厄贝沙坦(0.017 g·kg^(-1))组。药物干预20周后检测各组大鼠空腹血糖(FBG),肾重指数(KI),尿微量白蛋白与尿肌酐比值(UACR);苏木素-伊红(HE)染色观察肾组织病理形态学变化;透射电镜观察足细胞超微结构变化;酶联免疫吸附测定法(ELISA)检测大鼠肾组织肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6)水平;免疫组化法检测大鼠肾组织RIPK1,RIPK3,MLKL蛋白表达;原位末端标记法(TUNEL)检测大鼠肾脏足细胞凋亡率;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠肾组织RIPK1,RIPK3,MLKL mRNA表达水平;蛋白免疫印迹法(Western blot)检测大鼠肾组织RIPK1,RIPK3,MLKL蛋白及足细胞标志蛋白肿瘤蛋白-1(WT-1)表达水平。结果:与正常组比较,模型组大鼠FBG,UACR,KI显著升高(P<0.01),肾小球肥大,基底膜增厚,系膜外基质增多,系膜增生,足突融合或丢失,肾组织凋亡细胞明显增多,TNF-α及IL-6水平显著升高(P<0.01),WT-1蛋白表达水平显著降低,RIPK1/RIPK3/MLKL mRNA及蛋白表达水平显著升高(P<0.01);与模型组比较,当归补血汤高剂量组明显降低FBG,UACR,KI水平,明显改善肾组织病理学,减少足细胞的丢失,减少肾组织细胞凋亡,降低TNF-α及IL-6水平,升高WT-1蛋白表达水平,降低RIPK1/RIPK3/MLKL mRNA及蛋白表达水平(P<0.05,P<0.01)。结论:当归补血汤可能通过调控RIPK1/RIPK3/MLKL信号通路,改善足细胞损伤,从而延缓DKD的发展进程。
Objective:To observe the effect of Danggui Buxuetang on the podocyte injury and receptorinteracting protein kinase 1/receptor-interacting protein kinase3/mixed lineage kinase domain-like protein(RIPK1/RIPK3/MLKL)signaling pathway in diabetic kidney disease(DKD)ratsand to explore its possible mechanism against DKD.Method:Eight of the 50 SD rats were randomly classified intoa normal group,and the remaining were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 0.035 g·kg^(-1)streptozotocin(STZ)for inducing type 2 diabetes.After successful modeling,they were randomized into the model group,high-and low-dose(1.44,0.72 g·kg^(-1))Danggui Buxuetang groups,and irbesartan(0.017 g·kg^(-1))group.After 20 weeks of drug intervention,the fasting blood glucose(FBG),kidney index(KI),and urinary microalbumin-to-urine creatinine ratio(UACR)were detected in each group.The pathological changes in renal tissue were observed by hematoxylin-eosin(HE)staining,followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope.The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in renal tissue of rats were determined by enzyme-linked immunosorbent assay(ELISA),and the protein expression levels of RIPK1,RIPK3,and MLKL in rat kidney tissue by immunohistochemistry.The apoptosis rate of podocytes was detected by in situ nick end-labeling(TUNEL)assay.The mRNA expression levels of RIPK1,RIPK3,and MLKL in kidney tissue of rats were measured by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and the protein expression levels of RIPK,RIPK3,and MLKL and podocyte marker Wilms tumor protein-1(WT-1)in rat kidney tissue were assayed by Western blot.Result:Compared with the normal group,the model group exhibited elevated FBG,UACR,and KI(P<0.01),glomerular hypertrophy,thickened basement membrane,increased extracellular matrix,mesangial hyperplasia,foot process fusion or loss,enhanced apoptosis in renal tissue,upregulated TNF-αand IL-6 levels(P<0.01)and RIPK1/RIPK3/MLKL mRNA and protein expression(P<0.01),and down-regulated WT-1 protein expression.Compared with the model group,Danggui Buxuetang high-dose group significantly reduced the levels of FBG,UACR,and KI,improved renal histopathology,podocyte loss,and apoptosis in renal tissue,down-regulated TNF-αand IL-6 levels and RIPK1/RIPK3/MLKL mRNA and protein expression(P<0.05,P<0.01),and up-regulated WT-1 protein expression.Conclusion:Danggui Buxuetang alleviates podocyte injury and delays the development of DKD possibly by regulating the RIPK1/RIPK3/MLKL signaling pathway.
作者
靳贺超
张冠文
梁胜然
强家维
郭登洲
JIN He-chao;ZHANG Guan-wen;LIANG Sheng-ran;QIANG Jia-wei;GUO Deng-zhou(Hebei University of Chinese Medicine,Shijiazhuang 050200,China;Hebei Provincial Hospital of Chinese Medicine,Shijiazhuang 050011,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第3期41-48,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81373804)
河北省博士研究生创新项目(CXZZBS2021087)。
关键词
当归补血汤
糖尿病肾病
足细胞
受体相互作用蛋白激酶
混合系激酶区域样蛋白
Danggui Buxuetang
diabetic kidney disease(DKD)
podocytes
receptor-interacting protein kinase(RIPK)
mixed lineage kinase domain-like protein(MLKL)
