黄芪甲苷调控TLR4/NF-κB通路对系膜增生性肾小球肾炎大鼠发挥保护作用

The protective effect of Astragaloside IV on mesangial proliferative glomerulonephritis rats by regulating the TLR4/NF-κB pathway

目的探究黄芪甲苷(AS-IV)调控Toll样受体4(TLR4)/核因子κB(NF-κB)通路对系膜增生性肾小球肾炎大鼠的保护作用。方法将Wistar大鼠分为假手术组、模型组、AS-IV低、中、高剂量组、NF-κB抑制剂组(BAY11-7082)组,每组16只。假手术组大鼠开腹后游离左肾后关腹。AS-IV低、中、高剂量组大鼠在系膜增生性肾小球肾炎模型制备结束后,分别灌胃2.5 mg/(kg·d)、5 mg/(kg·d)、10 mg/(kg·d)AS-IV,BAY11-7082组静脉注射5μmol/(kg·d)BAY11-7082,假手术组、模型组大鼠腹腔注射生理盐水,连续给药4周。给药2周、4周后测定尿液24 h蛋白量,酶联免疫法检测血清肌酐(Scr)、尿素氮(BUN)水平,HE、PSA染色观察肾脏组织形态学变化,TUNEL染色检测肾脏细胞凋亡情况;蛋白免疫印迹(WB)分析肾组织TLR4、NF-κB蛋白表达;免疫组化法检测肾脏中基质金属蛋白酶-9(MMP-9)、金属蛋白酶组织抑制因子-1(TIMP-1)蛋白阳性表达。结果与假手术组相比,模型组24 h尿蛋白、Scr、BUN升高,肾小球病理分级、肾小管间质损伤评分均升高,细胞凋亡率升高、TLR4、NF-κB蛋白表达升高、MMP-9蛋白阳性表达率降低,TIMP-1蛋白阳性表达率升高(P<0.05)。与模型组相比,AS-IV各组、BAY11-7082组24 h尿蛋白、Scr、BUN降低,肾小球病理分级、肾小管间质损伤评分降低,细胞凋亡率降低、TLR4、NF-κB蛋白表达降低、MMP-9蛋白阳性表达率升高、TIMP-1蛋白阳性表达率降低(P<0.05)。结论AS-IV通过抑制TLR4/NF-κB信号通路,影响MMP-9、TIMP-1蛋白表达进而发挥对系膜增生性肾小球肾炎大鼠的保护作用。

Objective To investigate the protective effect of astragaloside IV(AS-IV)on mesangial proliferative glomerulonephritis rats by regulating the Toll like receptor 4(TLR4)/nuclear factorκB(NF-κB)pathway.Methods Wistar rats were divided into sham operation group(Control),model group(Model),AS-IV low,middle and high dose groups(AS-IV-L,AS-IV-M,AS-IV-H),NF-κB inhibitor group(BAY11-7082),with 16 cases in each group.In the sham-operated group,the left kidney was closed and the abdomen was closed after laparotomy.Rats in the low,medium and high doses of AS-IV were given 2.5 mg/(kgod),5 mg/(kgod),10 mg/(kgod)AS-IV after the model preparation.BAY11-7082 group received intravenous injection of 5μmol/(kgod)BAY11-7082.The rats in the sham operation group and the model group were intraperitoneally injected with normal saline for 4 weeks.The amount of 24 h protein in urine was measured after2 weeks and 4 weeks of administration,serum creatinine(Scr)and urea nitrogen(BUN)levels were measured by enzyme-linked immunoassay,the changes of renal histomorphology were observed by HE and PSA staining,TUNEL staining was used to detect the apoptosis of renal cells;the expressions of TLR4 and NF-κB proteins in renal tissue were analyzed by Western blot(WB);the positive expressions of matrix metalloproteinase-9(MMP-9)and tissue inhibitor of metalloproteinase-1(TIMP-1)was detected by immunohistochemistry.Results Compared with group Control,the 24 h urine protein,Scr and BUN increased in group Model,glomerular pathological grading and renal tubulointerstitial injury score increased,the rate of apoptosis increased,the expressions of TLR4 and NF-κB increased,and the positive expression rate of MMP-9 protein decreased,the positive expression rate of TIMP-1 protein increased(P<0.05).Compared with group model,24 h urinary protein,Scr and BUN decreased in group AS-IV and group BAY11-7082,glomerular pathological grading and renal tubulointerstitial injury score decreased,the rate of apoptosis decreased,the expressions of TLR4 and NF-κB protein decreased,the positive expression rate of MMP-9 protein increased,the positive expression rate of TIMP-1 protein decreased(P<0.05).Conclusion AS-IV can affect the expressions of MMP-9 and TIMP-1 proteins by inhibiting the TLR4/NF-κB signaling pathway and play a protective role in mesangial proliferative glomerulonephritis rats.