黄芩汤对糖尿病肾病大鼠肾脏NF-κB/NLRP3/Caspase-1细胞焦亡通路的影响

Effect of Huangqin Decoction on NF-κB/NLRP3/Caspase-1 Signaling Pathway and Pyroptosis in Kidney of Diabetic Nephropathy Rats

目的:研究黄芩汤对糖尿病肾病(diabetic nephropathy,DN)大鼠肾组织核因子κB(nuclear factor kappa-B,NF-κB)/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)/胱天蛋白酶-1(cysteinyl aspartate specific proteinase-1,Caspase-1)细胞焦亡通路的影响。方法:将SD大鼠随机分为空白组、模型组、厄贝沙坦组(27 mg/kg)和黄芩汤低、高剂量组(5 g/kg和20 g/kg),高脂饲料喂养6周联合一次性腹腔注射链脲佐菌素(35 mg/kg)诱导DN大鼠模型,每组9只。灌胃给药6周后检测大鼠血清空腹血糖(fasting blood glucose,FBG)、总胆固醇(total cholesterol,TC)、甘油三酯(triacylglycerol,TG)、尿蛋白(urine protein,UP)、尿素氮(blood urea nitrogen,BUN)、血肌酐(serum creatinine,Scr)、白介素-1β(interleukin 1β,IL-1β)和IL-18水平;HE染色和Masson染色观察大鼠肾脏病理变化;Western blot和免疫组化检测肾脏NF-κB/NLRP3/Caspase-1细胞焦亡通路相关蛋白及阳性细胞表达。结果:与空白组比较,模型组大鼠FBG、TC、TG、UP、BUN、Scr、IL-1β和IL-18水平明显升高(P<0.01);肾脏出现肾小球体积增大及基底膜增厚,肾小管管腔扩张,炎性浸润及纤维化明显等病理变化;肾脏组织NF-κB的磷酸化水平,以及NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、Caspase-1、IL-1β和消皮素D(gasdermin D,GSDMD)的蛋白质表达明显升高(P<0.01);肾脏组织NLRP3和GSDMD阳性细胞表达水平明显升高(P<0.01)。与模型组比较,黄芩汤组大鼠上述血糖、血脂、肾功能及炎性因子水平均得到明显改善(P<0.05,P<0.01);肾脏肾小球及肾小管结构趋于正常,炎性浸润及纤维化程度得到改善;肾脏组织NF-κB的磷酸化水平,以及NLRP3、ASC、Caspase-1、IL-1β和GSDMD的蛋白质表达水平明显降低(P<0.05,P<0.01);肾脏组织NLRP3和GSDMD阳性细胞表达明显降低(P<0.05,P<0.01)。结论:黄芩汤对DN大鼠具有确切的疗效,机制可能与抑制NF-κB/NLRP3/Caspase-1细胞焦亡通路有关。

To study the effect of Huangqin Decoction on nuclear factor kappa B(NF-κB)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(Caspase-1)-dependent pyroptosis pathway in diabetic nephropathy(DN)rat model.Methods:Rats were randomly divided into control group,model group,irbesartan group(27 mg/kg),and Huangqin Decoction low-and high-dose groups(5 g/kg and 20 g/kg),and the DN rat model was induced by high-fat diet for 6 weeks combined with a single intraperitoneal injection of streptozotocin(35 mg/kg).Serum fasting blood glucose(FBG),total cholesterol(TC),triacylglycerol(TG),urine protein(UP),blood urea nitrogen(BUN),serum creatinine(Scr),interleukin 1β(IL-1β)and IL-18 levels were measured after 6 weeks of gavage administration;renal pathological changes were observed by HE and Masson staining;renal NF-κB/NLRP3/Caspase-1/pyroptosis pathway-related proteins and positive cell expression were detected by Western blot and immunohistochemistry.Results:Compared with the control group,the levels of FBG,TC,TG,UP,BUN,Scr,IL-1β and IL-18 were significantly increased in the model group(P<0.01);the kidneys showed pathological changes such as increased glomerular volume and basement membrane thickening,tubular lumen expansion,inflammatory infiltration and fibrosis;the phosphorylation levels of NF-κB in kidney tissues and NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),Caspase-1,IL-1β and gasdermin D(GSDMD)protein expression were significantly increasedc(P<0.01);NLRP3 and GSDMD positive cells expression in renal tissue were significantly increased(P<0.01).Compared with the model group,the above-mentioned blood glucose,lipids,renal function and inflammatory factor levels were significantly improved in the Huangqin Decoction groups(P<0.05,P<0.01);renal glomerular and tubular structures were normalized,and the degree of inflammatory infiltration and fibrosis was improved;the phosphorylation levels of NF-κB in renal tissue as well as NLRP3,ASC,Caspase-1,IL-1β and GSDMD protein expressions were significantly reduced(P<0.05,P<0.01);renal tissue NLRP3 and GSDMD positive cells expression were significantly reduced(P<0.05,P<0.01).Conclusion:Huangqin Decoction exerts a definite effect on DN rats,at least partly,through inhibition of the NF-κB/NLRP3/Caspase-1-dependent pyroptosis signaling pathway.