SGK1抑制剂EMD638683对膀胱出口梗阻小鼠肾组织细胞焦亡的作用及机制研究

目的:确定小鼠膀胱出口梗阻(bladder outlet obstruction,BOO)模型引起下尿路梗阻介导的肾脏炎性损伤;研究血清和糖皮质激素调节激酶1(serum and glucocorticoid-regulated kinase 1,SGK1)抑制剂EMD638683抗梗阻性肾病炎症的作用机制,探讨SGK1与NLRP3-caspase-1-IL-1β/细胞焦亡(pyroptosis)通路诱导细胞损伤的关系。方法:构建小鼠BOO模型,HE染色观察肾组织病理变化及炎症细胞浸润,PAS染色观察肾脏的组织病变,Masson染色法检测肾小管的胶原沉积,免疫组化法和Western blot法检测NLRP3、caspase-1、F4/80、gasdermin D-N末端片段(GSDMD-N)、IL-1β和SGK1的表达。醛固酮(aldosterone,ALD)处理小鼠肾小管上皮细胞(mouse renal tubular epithelial cells,mRTECs),EMD638683进行干预,Western blot法检测NLRP3、caspase-1、IL-1β、SGK1及GSDMD-N的表达水平;ELISA法检测各组细胞上清液IL-1β含量。结果:梗阻3周的小鼠肾脏中HE染色观察到炎症细胞浸润,PAS染色出现肾脏组织病变,Masson染色检测到肾小管间质有胶原沉积。与正常组小鼠比较,梗阻3周小鼠组NLRP3、caspase-1、F4/80、GSDMD-N、IL-1β和SGK1含量显著升高(P<0.05或P<0.01)。细胞实验中,与正常组比较,ALD组NLRP3、caspase-1、IL-1β、SGK1及GSDMD-N含量显著升高(P<0.05或P<0.01);与ALD组相比,ALD加EMD638683组NLRP3、caspase-1、IL-1β、SGK1及GSDMD-N含量显著减少(P<0.05或P<0.01)。结论:成功构建小鼠BOO模型引起下尿路梗阻介导的肾脏炎性损伤;EMD638683通过抑制NLRP3-caspase-1-pyroptosis通路阻断细胞焦亡,从而发挥抗炎作用。

Role of SGK1 inhibitor EMD638683 in preventing renal tissue pyroptosis induced by bladder outlet obstruction in mice

AIM:To study the mechanism of serum and glucocorticoid-regulated kinase 1(SGK1)inhibitor EMD638683 against inflammation of obstructive nephropathy,and to explore the relationship between SGK1 and NLRP3-caspase1-IL-1β/pyroptosis pathway-induced cell damage in a mouse model of lower urinary tract obstruction-induced bladder outlet obstruction(BOO).METHODS:After the BOO model was constructed,the inflammatory cell infiltration was observed by HE staining,the tissue lesions of the kidney were observed by PAS staining,the collagen deposition of renal tubules was detected by Masson staining,and the expression levels of NLRP3,caspase-1,F4/80,gasdermin D N-terminal fragment(GSDMD-N),IL-1βand SGK1 were determined by immunohistochemistry and Western blot.The mouse renal tubular epithelial cells(mRTECs)were used,and treated with aldosterone(ALD)and EMD638683.The expression levels of NLRP3,caspase-1,IL-1β,SGK1 and GSDMD-N were detected by Western blot.The IL-1βcontent in the cell supernatant was determined by ELISA.RESULTS:After obstruction for 3 weeks in the mice,the infiltration of inflammatory cells was observed in the kidney by HE staining,renal tissue lesions were observed by PAS staining,and collagen deposition was detected in tubulointerstitium by Masson staining.Compared with normal group,the levels of NLRP3,caspase-1,F4/80,GSDMD-N,IL-1βand SGK1 were significantly increased in the 3-week obstructed mice(P<0.05 or P<0.01).The expression levels of NLRP3,caspase-1,IL-1β,SGK1 and GSDMD-N in mRTECs were increased significantly in ALD group(P<0.05 or P<0.01).Compared with ALD group,the expression of NLRP3,caspase-1,IL-1β,SGK1 and GSDMD-N were significantly reduced in ALD+EMD638683 group(P<0.05 or P<0.01).CONCLUSION:We successfully constructed renal inflammatory injury mediated by BOO-induced lower urinary tract obstruction in mice.EMD638683 blocked pyroptosis and exerted its anti-inflammatory effect by inhibiting NLRP3-caspase-1-pyroptosis pathway.