益气养阴活血方调控Nrf2/NLRP3信号通路对糖尿病肾病大鼠的肾脏保护作用研究

Protective Effect of Yiqi Yangyin Huoxue Recipe(益气养阴活血方)Regulating Nrf2/NLRP3 Signaling Pathway on Kidney of Diabetes Nephropathy Rats

目的探讨益气养阴活血方通过调控核因子E2相关因子2(Nrf2)/NOD样受体蛋白3(NLRP3)信号通路对糖尿病肾病(DN)大鼠的肾脏保护作用机制。方法清洁级雄性SD大鼠40只,适应性喂养1周后,随机分为:空白组、模型组、缬沙坦组、益气养阴活血方等剂量(等剂量)组及高剂量(高剂量)组。除空白组外各组均应用高脂高糖喂养,联合单次注射链脲佐菌素(STZ)建立DN大鼠模型。造模成功后中药两组均灌服益气养阴活血方颗粒剂,缬沙坦组灌服缬沙坦,空白组和模型组灌服生理盐水,6周后留取大鼠24 h尿液、血液和肾脏组织。生化检测各组大鼠24 h尿总蛋白定量(24 h-UTP)、血肌酐(SCr)、血尿素氮(BUN)、葡萄糖(GLU)、总胆固醇(CHO)、甘油三酯(TG);酶联免疫吸附测定法(ELISA)检测血清白介素1β(IL-1β)、白介素18(IL-18)水平;HE染色光镜观察肾组织病理改变;蛋白免疫印迹(Western Blot)检测肾组织中Nrf2、血红素加氧酶-1(HO-1)、NLRP3蛋白水平;实时荧光定量聚合酶链式反应(PCR)检测大鼠肾脏Nrf2、HO-1、NLRP3mRNA表达水平。结果生化:与空白组相比,模型组24 hUTP、Scr、BUN、GLU、CHO、TG均显著升高(P<0.01);与模型组对比,各干预组24 h-UTP、Scr、BUN、GLU、CHO、TG均显著降低(P<0.05或P<0.01)。ELISA:与空白组相比,模型组血清IL-1β、IL-18水平显著升高(P<0.01);与模型组比较,各干预组血清IL-1β、IL-18水平明显降低(P<0.05或P<0.01)。肾脏病理:与空白组对比,模型组大鼠肾脏组织病变严重;与模型组对比,各干预组肾脏病理均有改善。WB及PCR:与空白组对比,模型组肾脏Nrf2 mRNA、HO-1 mRNA及Nrf2、HO-1蛋白表达降低(P<0.05或P<0.01),NLRP3mRNA、NLRP3蛋白表达升高(P<0.05或P<0.01);与模型组对比,各干预组Nrf2 mRNA、HO-1 mRNA及Nrf2、HO-1蛋白表达升高(P<0.05或P<0.01),NLRP3mRNA、NLRP3蛋白表达降低(P<0.05或P<0.01)。结论益气养阴活血方可能通过调控Nrf2/NLRP3信号通路,减轻氧化应激,抑制DN大鼠炎症反应,从而改善上述生化指标,减轻肾脏病理损害。

Objective To explore the renal protective mechanism of Yiqi Yangyin Huoxue Recipe(益气养阴活血方)on diabetes nephropathy(DN)rats by regulating nuclear factor E2 related factor 2(Nrf2)/NOD like receptor protein 3(NLRP3)signal pathway.Methods Forty male SD rats of clean grade were randomly divided into blank group,model group,valsartan group,equal dose group of Yiqi Yangyin Huoxue Recipe and high dose group after adaptive feeding for one week.Except the blank group,the nts in each group were fed with high fat and high sugar,combined with a single injection of streptozotocin(STZ)to establish the DN rat model.After successful modeling,the two groups of Chinese medicine were given Yiqi Yangyin Huoxue Recipe by gavage.The valsartan group was given valsartan by gavage,the blank group and the model group were given normal saline by gavage,and 24 hours urine,blood and kidney tissue of rats were taken out after 6 weeks.Biochemical tests were carried out to determine 24 h urine total protein(24 h-UTP),blood creatinine(SCR),blood urea nitrogen(BUN),glucose(GLU),total cholesterol(CHO)and triglyceride(TG)of rats in each group.The senum levels of interleukin 1β(IL-1β)and interleukin 18(IL-18)were detected by enzyme-linked immunosorbent assay(ELISA).HE staining and light microscopy were used to observe the pathological changes of renal tissue.Protein levels of Nrf2,heme oxygenase-1(HO-1)and NLRP3 in renal tissues were detected by Western blat.The expressions of Nrf2,H0-1 and NLRP3 mRNA in the kidney of rats were detected by real-time fluorescence quantitative polymerase chain reaction(PCR).Results Biochemistry:Compared with those of the blank group,the levels of 24 h-UTP,Ser,BUN,GLU,CHOand TG in the model group were significantly increased(P<0.01).Compared with those of the model group,the levels of 24 h UTP,Ser,BUN,GLU,CHO and TG in each intervention group were significantly reduced(P<0.05 or P<0.01).ELISA:Compared with those of the blank group,the serum levels of IL-1β and IL-18 in the model group increased significantly(P<0.01).Compared with those of the model group,the serum levels of IL-1β and IL-18 of each intervention group decreased significantly(P<0.05 or P<0.01).Renal pathology:Compared with those of the blank,the pathological changes of renal tissue in the model group were serious.Compared with those of the model group,the renal pathology of each intervention group was improved.Western Blot and PCR:Compared with those of the blank group,the expressions of Nrf2 mRNA,HO-1 mRNA and Nrf2,HO-1 protein in the kidney of the model group decreased(P<0.05 or P<0.01),and the expressions of NLRP3 mRNAand NLRP3 protein increased(P<0.05 or P<0.01).Compared with those of the model group,the expressions of Nrf2 mRNA,HO-1 mRNA and Nrf2,HO-1 protein in each intervention group increased(P<0.05 orP<0.01),and the expressions of NLRP3 mRNA and NLRP3 protein decreased(P<0.05 or P<0.01).Conclusion Yiqi Yangyin Huoxue Recipe may improve the above biochemical indexes and alleviate the renal pathological damage by regulating Nr2/NLRP3 signal pathway,reducing oxidative stress and inhibiting the inflammatory reaction of DN rats.