基于Nrf2-NLRP3途径研究桃红四物汤干预糖尿病大鼠心肌损伤的作用机制

Mechanism of Action of Taohong Siwu Decoction in Intervention Against Myocardial Injury in Rats with Diabetes:A Study Based on the Nuclear Factor Erythroid 2-related Factor 2/NOD-like Receptor Protein 3 Pathway

目的观察桃红四物汤对糖尿病大鼠心脏的保护作用,同时基于核因子E2相关因子2(nuclear factor-E2-related factor 2,Nrf2)/血红素加氧酶-1(heme oxygenase-1,HO-1)和NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)通路探讨其作用机制。方法腹腔注射链脲佐菌素复制糖尿病心肌病大鼠模型,将模型复制成功的大鼠分为模型组,桃红四物汤低、中、高剂量组,每组10只,另设10只正常大鼠作为对照组。比较各组大鼠血糖、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)和B型利钠肽(B-type natriuretic peptide,BNP)、肌钙蛋白I(cardiac troponin I,cTnI)水平。采用苏木精—伊红染色和Masson染色观察大鼠心肌组织病理变化。制备心肌组织匀浆,使用流式细胞术进行线粒体膜电位检测。Western blot法检测Nrf2、HO-1、Kelch样环氧氯丙烷相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)、NLRP3、半胱氨酸天冬氨酸酶1剪切体(cleaved-caspase-1)、消皮素D(gasdermin D,GSDMD)和消皮素D蛋白N端结构域(N-terminal gasdermin D-N,GSDMD-N)蛋白表达水平。结果与对照组比较,模型组大鼠血糖升高,心肌纤维断裂、排列紊乱,炎症细胞浸润明显,胶原沉积明显;与模型组比较,桃红四物汤低、中、高剂量组大鼠心肌纤维化明显改善。与对照组比较,模型组SOD活性显著降低(P<0.05),MDA、BNP及cTnI水平显著升高(P<0.05);与模型组比较,桃红四物汤低、中、高剂量组SOD活性显著升高(P<0.05),MDA、BNP、cTnI水平显著降低(P<0.05)。与对照组比较,模型组心肌组织线粒体膜电位显著降低(P<0.05);与模型组比较,桃红四物汤低、中、高剂量组心肌组织线粒体膜电位显著升高(P<0.05)。与对照组比较,模型组心肌组织中Nrf2、HO-1、NLRP3、cleaved-caspase-1、GSDMD和GSDMD-N蛋白表达水平均显著升高(P<0.05);与模型组比较,桃红四物汤低、中、高剂量组心肌组织中Nrf2和HO-1蛋白表达水平均显著升高(P<0.05),Keap1、NLRP3、cleaved-caspase-1、GSDMD和GSDMD-N蛋白表达水平均显著降低(P<0.05)。结论桃红四物汤改善糖尿病心肌病的机制可能与调节Nrf2-NLRP3途径,抗氧化应激及抑制细胞焦亡相关。

Objective To investigate the protective effect of Taohong Siwu Decoction on the heart of rats with diabetes and its mechanism of action based on the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/NOD-like receptor protein 3(NLRP3)pathway.Methods Intraperitoneal injection of streptozotocin was performed to establish a rat model of diabetic cardiomyopathy,and then the rats were divided into model group and low-,middle-,and high-dose Taohong Siwu Decoction groups,with 10 rats in each group.Ten normal rats were selected as control group.The above groups were compared in terms of blood glucose,superoxide dismutase(SOD),malondialdehyde(MDA),B-type natriuretic peptide(BNP),and cardiac troponin I(cTnI).HE staining and Masson staining were used to observe the histopathological changes of the myocardium.Myocardial tissue homogenate was prepared,and flow cytometry was used to measure mitochondrial membrane potential.Western blot was used to measure the protein expression levels of Nrf2,HO-1,Kelch-like ECH-associated protein 1(Keap1),NLRP3,cleaved-caspase-1,gasdermin D(GSDMD),and N-terminal gasdermin D(GSDMD-N).Results Compared with the control group,the model group had an increase in blood glucose,rupture and disordered arrangement of myocardial fibers,significant inflammatory cell infiltration,and marked collagen deposition,and compared with the model group,the low-,middle-,and high-dose Taohong Siwu Decoction groups had improvements in myocardial histopathology.Compared with the control group,the model group had a significant reduction in the level of SOD(P<0.05)and significant increases in the levels of MDA,BNP,and cTnI(P<0.05),Compared with the model group,the Taohong Siwu Decoction groups had a significant increase in the level of SOD and significant reductions in the levels of MDA,BNP,and cTnI(P<0.05).Compared with the control group,the model group had a significant reduction in mitochondrial membrane potential in myocardial tissue(P<0.05),and the low-,middle-,and high-dose Taohong Siwu Decoction groups had a significant increase in mitochondrial membrane potential(P<0.05).Compared with the control group,the model group had significant increases in the protein expression levels of Nrf2,HO-1,NLRP3,cleaved-caspase-1,GSDMD,and GSDMD-N in myocardial tissue(P<0.05),and the low-,middle-,and high-dose Taohong Siwu Decoction groups had significant increases in the protein expression levels of Nrf2 and HO-1(P<0.05)and significant reductions in the protein expression levels of Keap1,NLRP3,cleaved-caspase-1,GSDMD,and GSDMD-N(P<0.05).Conclusion Taohong Siwu Decoction can improve diabetic cardiomyopathy possibly by regulating the Nrf2/NLRP3 pathway,intervening against oxidative stress,and inhibiting pyroptosis.