目的:探讨黄芪甲苷(AS-IV)对糖尿病肾病(DN)足细胞损伤的影响及其可能的作用机制。
方法:在体外实验中,足细胞分为4组,正常,高葡萄糖(HG),需要肌醇的酶1(IRE-1)α激活剂(HGthapsigargin1µmol/L),IRE-1α抑制剂(HG+STF-083010,20µmol/L)组。此外,足细胞分为4组,包括正常的,HG,AS-IV(HG+AS-IV20µmol/L),和IRE-1α抑制剂(HG+STF-083010,20µmol/L)组,分别。治疗24小时后,电镜观察足细胞和内质网的形态。细胞免疫荧光法检测葡萄糖调节蛋白78(GRP78)和IRE-1α的表达。在体内实验中,通过连续3天腹膜内注射链脲佐菌素(STZ)建立DN大鼠模型。共40只大鼠被分配到正常,DN,AS-IV[AS-IV40mg/(kg·d)],和IRE-1α抑制剂[STF-083010,10mg/(kg·d)]组(n=10),分别。一般情况,24小时尿量,随机血糖,尿蛋白排泄率(UAER),尿素氮(BUN),干预8周后测定大鼠血清肌酐(SCr)水平。通过苏木精和伊红(HE)染色观察肾组织的病理变化。定量逆转录-聚合酶链反应(RT-PCR)和Westernblot检测GRP78、IRE-1α、核因子κBp65(NF-κBp65),白细胞介素(IL)-1β,NLR家族pyrin结构域含3(NLRP3),caspase-1,gasderminD-N(GSDMD-N),和nephrin在体内和体外的mRNA和蛋白质水平,分别。
结果:在HG和IRE-1α激活剂组中观察到细胞质空泡化和ER肿胀。与HG组相比,AS-IV和IRE-1α抑制剂组的足细胞形态和ER扩张得到改善。细胞免疫荧光显示,与正常组相比,HG和IRE-1α激活剂组的GRP78和IRE-1α荧光强度明显升高,而AS-IV和IRE-1α抑制剂组降低(P<0.05)。与正常组相比,GRP78,IRE-1α,GRP78的mRNA和蛋白表达,NF-κBp65,IL-1β,HG组NLRP3、caspase-1和GSDMD-N增高(P<0.05)。与HG组相比,AS-IV和IRE-1α抑制剂组上述指标的表达降低,IRE-1α激活剂组的表达升高(P<0.05)。HG组nephrin的表达降低,AS-IV和IRE-1α抑制剂组升高(P<0.05)。体内实验结果表明,与正常组相比,血糖水平,甘油三酯,总胆固醇,BUN,DN组血肌酐和尿蛋白均较高(P<0.05)。与DN组相比,AS-IV和IRE-1α抑制剂组上述指标均降低(P<0.05)。HE染色显示肾小球肥大,DN组肾组织系膜增宽和系膜细胞增殖。与DN组相比,AS-IV和IRE-1α抑制剂组肾组织病理改变均有缓解。GRP78、IRE-1α的定量RT-PCR和Westernblot结果,NF-κBp65,IL-1β,NLRP3,caspase-1和GSDMD-N与免疫荧光分析一致。
结论:AS-IV可以减少ERS和炎症,改善足细胞焦亡,从而在DN中发挥足细胞保护作用,通过调节IRE-1α/NF-κB/NLRP3信号通路。
OBJECTIVE: To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism.
METHODS: In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1β, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively.
RESULTS: Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis.
CONCLUSIONS: AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.