矢志方抑制NLRP3减轻高尿酸血症小鼠肾小管上皮细胞焦亡的研究

Study on Shizhifang ameliorating pyroptosis of renal tubular epithelial cells induced by hyperuricemia through inhibiting NLRP3 inflammasome

目的:探讨矢志方抑制高尿酸血症(H U A)小鼠肾小管上皮细胞焦亡和抗炎的作用机制。方法:40只BALB/c小鼠随机分成正常组、模型组、别嘌醇组、矢志方组。除正常组外,其余组采用腹腔注射氧嗪酸钾(P O)诱导H U A,分别给予矢志方、别嘌醇治疗,观察血清尿酸(S U A)、血清肌酐(S C r)、血尿素氮(BUN);HE染色观察病理改变;Western Blot和免疫荧光技术检测焦亡相关蛋白表达;ELISA检测IL-1β、IL-18。培养人肾小管上皮细胞(HK-2),采用尿酸(UA)干预,分别给予矢志方、炎性小体3(NLRP3)抑制剂(MCC950)处理48 h,CCK-8法检测细胞活力;Western Blot检测焦亡相关蛋白表达;ELISA检测细胞上清IL-1β、IL-18含量。结果:与正常组小鼠比较,模型组SCr、BUN、SUA显著升高(P<0.01),肾组织NLRP3、Caspase-1、Cleaved-caspase-1、GSDMD、pro-IL-1β、IL-1β、IL-18表达升高(P<0.01),血清IL-1β和IL-18水平升高(P<0.01);与模型组比较,矢志方可有效降低HUA小鼠的SUA、SCr、BUN水平(P<0.01)和血清IL-1β和IL-18分泌水平(P<0.01,P<0.05)。与空白组细胞比较,UA诱导细胞NLRP3、cleaved-Caspase-1、Caspase-1、GSDMD、N-GSDMD表达升高(P<0.01);矢志方和MCC950可显著抑制UA诱导下细胞NLRP3、Caspase-1、cleaved-Caspase-1、GSDMD、N-GSDMD的表达(P<0.01,P<0.05),减少IL-1β、IL-18产生(P<0.01,P<0.05)。结论:矢志方靶向抑制NLRP3,抑制HUA小鼠肾小管上皮细胞焦亡,减轻肾脏炎性损伤。

Objective:To explore the mechanism of Shizhifang(SZF)ameliorates pyroptosis of renal tubular epithelial cells and anti-inflammatory in hyperuricemia(HUA)mice.Methods:Forty BALB/c mice were randomly divided into normal group,model group,allopurinol group and SZF group.HUA mice models were induced by intraperitoneal injection of potassium oxonate(PO).Mice were treated with SZF or allopurinol.Serum uric acid(SUA),serum creatinine(SCr)and blood urea nitrogen(BUN)were observed;HE staining to observe pathological changes;Western Blot and immunofluorescence techniques to detect pyroptosis protein expression;ELISA to detect IL-1β,IL-18.Human renal tubular epithelial cells(HK-2)stimulated by UA were treated with SZF or NLRP3 inhibitor(MCC950)for 48 h.The cell viability was detected by CCK-8 method.The expressions of pyroptosis protein were detected by Western Blot.IL-1βand IL-18 in cell supernatant were detected by ELISA.Results:Compared with the normal group,the SCr,BUN and SUA of the model group were significantly increased(P<0.01).The expressions of NLRP3,Caspase-1,pro-caspase-1,GSDMD,pro-IL-1β,IL-1βand IL-18 in renal tissue were increased(P<0.01),and the levels of IL-1βand IL-18 in serum were increased(P<0.01).Compared with model group,SZF effectively decreased the levels of SUA,SCr,BUN(P<0.01)and serum IL-1βand IL-18 secretion levels(P<0.01,P<0.05).Compared with the normal group,the expressions of NLRP3,pro-caspase-1,Caspase-1,GSDMD and N-GSDMD were increased in UA induced cells(P<0.01).SZF and MCC950 significantly inhibited the expression of NLRP3,Caspase-1,GSDMD and N-GSDMD in UA-induced cells(P<0.05,P<0.01),and decreased the production of IL-1βand IL-18(P<0.01,P<0.05).Conclusions:SZF can inhibit the pyroptosis of renal tubular epithelial cells and reduce the inflammatory injury of renal by targeting NLRP3.