抑制p38MAPK信号通路对尿源性脓毒血症兔肾组织细胞凋亡的影响及其作用机制

Effect of inhibiting p38MAPK signaling pathway on apoptosis of renal tissue in rabbits with urosepsis and its mechanism

目的探讨抑制p38MAPK信号通路对尿源性脓毒血症兔肾组织细胞凋亡的影响及其作用机制。方法将60只新西兰兔按随机数字表法分为4组:假手术组(sham)、模型组(Model)、p38MAPK抑制剂SB203580组(SB203580)、SB203580+p53激活剂Nutlin-3组(SB203580+Nutlin-3),每组15只。采用输尿管近端注入大肠埃希菌菌液法制作尿源性脓毒血症模型,术前30 min静脉注射30μg/kg SB203580或腹腔注射128 mg/kg Nutlin-3进行干预,1次/d,共3 d。记录术后24、48、72 h时各组兔肛温、呼吸频率和心率;全自动分析仪检测白细胞计数以及血清肌酐、尿素氮含量;ELISA检测血清白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)含量;TUNEL染色观察肾组织细胞凋亡情况;qRT-PCR法检测肾组织MDM2和p53 mRNA表达水平;Western blot法检测肾组织Cleaved caspase-3、Bax、Bcl-2、p-p38MAPK、MDM2、p53和PUMA等蛋白表达水平。结果与Model组比较,SB203580组兔肛温、呼吸频率、心率、白细胞计数、血清IL-1β、IL-6和TNF-α水平以及血清肌酐和尿素氮含量降低(P<0.05),同时肾组织细胞凋亡率、p53 mRNA以及p53、Cleaved caspase-3、Bax、PUMA、p-p38MAPK等蛋白水平降低(P<0.05),而MDM2 mRNA以及MDM2、Bcl-2等蛋白水平增加(P<0.05)。与SB203580组比较,SB203580+Nutlin-3组兔肛温、呼吸频率、心率、白细胞计数、血清IL-1β、IL-6和TNF-α水平以及血清肌酐和尿素氮含量升高(P<0.05),同时肾组织细胞凋亡率、p53 mRNA以及p53、Cleaved caspase-3、Bax、PUMA等蛋白水平增加(P<0.05),Bcl-2蛋白水平降低(P<0.05),而MDM2 mRNA以及MDM2、p-p38MAPK等蛋白水平无显著变化(P>0.05)。结论抑制p38 MAPK信号通路通过促进MDM2表达而抑制p53介导的细胞凋亡途径,从而减少尿源性脓毒血症兔肾组织细胞凋亡。

Objective To investigate the effect of inhibition of p38MAPK signaling pathway on renal cell apoptosis in rabbits with urosepsis and its mechanism.Methods Sixty New Zealand rabbits were randomly divided into 4 groups:sham operation group(sham),model group(model),p38MAPK inhibitor SB203580 group(SB203580)and SB203580+p53 activator Nutlin-3 group(SB203580+Nutlin-3),with 15 rabbits in each group.The urosepsis model was made by injecting Escherichia coli into the proximal end of the ureter.Intravenous injection of 30μg/kg SB203580 or intraperitoneal injection of 128 mg/kg Nutlin-3 was used for intervention at 30 minutes before the operation,once daily,for a total of 3 d.The rectal temperature,respiratory rate and heart rate of the 4 groups of New Zealand rabbits were recorded at 24 h,48 h and 72 h after operation.The automatic analyzer detected the white blood cell count,serum creatinine and urea nitrogen content.ELISA was used to detect the levels of serum Interleukin-1β(IL-1β),Interleukin-6(IL-6)and Tumor necrosis factor-α(TNF-α).Renal tissue was taken for TUNEL staining to observe the apoptosis;qRT-PCR method was used to detect the expression of MDM2 and p53 mRNA in renal tissue;Western blot method was used to detect the expression of Cleaved caspase-3,Bax,Bcl-2,p38MAPK,p-p38MAPK,MDM2,p53 and PUMA protein levels in renal tissue.Results Compared with the Model group,rabbits rectal temperature,respiratory rate,heart rate,white blood cell count,serum IL-1β,IL-6 and TNF-αlevels,and serum creatinine and urea nitrogen levels reduced in the SB203580 group(P<0.05),and cell apoptosis rate of renal tissue,the mRNA expression of p53 and the proteins expression of p53,Cleaved caspase-3,Bax,PUMA and p-p38MAPK decreased(P<0.05),while the expression levels of MDM2 mRNA and MDM2,Bcl-2 proteins increased(P<0.05).Compared with the SB203580 group,the rabbits rectal temperature,respiratory rate,heart rate,white blood cell count,serum IL-1β,IL-6 and TNF-αlevels,and serum creatinine and urea nitrogen levels increased in the SB203580+Nutlin-3 group(P<0.05),and renal cell apoptosis rate,the mRNA expression of p53 and the proteins expression of p53,Cleaved caspase-3,Bax and PUMA increased(P<0.05),the protein expression of Bcl-2 significantly decreased(P<0.05),while the expression of MDM2 mRNA and MDM2,p-p38MAPK proteins had no changes(P>0.05).Conclusion Inhibition of p38MAPK signaling pathway inhibits the p53-mediated apoptosis pathway by promoting the expression of MDM2,thereby reducing renal cell apoptosis in rabbits with urosepsis.