双链RNA依赖的蛋白激酶活性抑制剂对脓毒症小鼠器官损伤及炎症因子的影响

Effect of inhibiting the activity of double-stranded RNA-dependent protein kinase in sepsis mice

目的观察双链RNA依赖的蛋白激酶(PKR)抑制剂2-氨基嘌呤(2-AP)对盲肠结扎穿刺(CLP)的脓毒症模型小鼠器官损伤血浆炎症因子表达及死亡率的影响。方法无特异病原体(SPF)级C57BL/6小鼠40只,随机分为假手术(Sham)组、CLP组、2-AP组和CLP+2-AP组(n=10)。术后24 h收集外周血血清,进行丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、血肌酐(Cr)、血尿素氮(BUN)及炎症因子(IL-1β、IL-10和TNF-α)的检测;取肺组织进行病理检测;取外周血和腹腔灌洗液进行细菌清除率检测。另外取60只C57BL/6小鼠,按照上述分组(n=15)进行7 d生存率观察。组间计量资料比较采用独立样本t检验。结果CLP组和CLP+2-AP组小鼠肝损伤指标(ALT和AST水平)和肾损伤指标(Cr和BUN)均较Sham组显著升高(均P<0.001)。CLP+2-AP组ALT和AST水平均显著低于CLP组(t=27.88、11.33,均P<0.001);肾功能损伤指标方面,CLP+2-AP组Cr和BUN水平均较CLP组显著下降(t=11.02、7.15,均P<0.001)。与Sham组相比,CLP组血浆中促炎(IL-1β和TNF-α)及抑炎(IL-10)细胞因子水平均显著升高(均P<0.001);CLP+2-AP组小鼠血浆IL-1β和IL-10水平均显著降低(均P<0.001),而血浆TNF-α水平下降不明显(P=0.33)。Sham组小鼠7 d生存率为100%,CLP+2-AP组为13.3%,2-AP组为86.7%,CLP+2-AP组为20.0%。抑制PKR活化可轻微改善CLP模型小鼠7 d生存率趋势(Mantel-Cox检验分析,χ^2=0.0012,P=0.97)。结论在脓毒症小鼠模型中,抑制PKR活性可对降低血浆中炎症因子表达,减少血液和腹腔中细菌负荷,对器官损伤具有保护作用,提示抑制PKR活性在脓毒症治疗中具有应用潜力。

Objective To observe the effects of 2-aminopurine(2-AP),a double-stranded RNA-dependent protein kinase(PKR)inhibitor,on organ function,plasma inflammatory factor expression and 7 days mortality in sepsis mice induced by cecal ligation puncture(CLP).Methods Forty specific specific pathogen free C57BL/6 mice were randomly divided into sham group(n=10),CLP group(n=10),CLP+2-AP group(n=10)and 2-AP group(n=10).CLP was used to establish sepsis mice models.Peripheral blood serum was collected 24 hours after operation,alanine aminotransferase(ALT),aspartate aminotransferase(AST),serum creatinine(Cr),blood urea nitrogen(BUN)and inflammatory factors(IL-1β,IL-10 and TNF-α)were detected;peripheral blood and peritoneal lavage fluid were taken for bacterial clearance detection.Another 60 C57BL/6 mice were selected to observe the 7-day survival rate according to the above groups(n=15).Independent sample t test was used to compare the measurement data between groups.Results The levels of ALT,AST,Cr and BUN in CLP Group and CLP+2-AP group were significantly higher than those in sham group(all P<0.001).The levels of ALT and AST in CLP+2-AP group were significantly lower than those in CLP Group(t=27.88,11.33,both P<0.001);the levels of Cr and BUN in CLP+2-AP group were significantly lower than those in CLP Group(t=11.02,7.15,bothP<0.001).Compared with sham group,the levels of pro-inflammatory(IL-1βand TNF-α)and anti-inflammatory(IL-10)cytokines in CLP group were significantly higher(all P<0.001);the levels of IL-1βand IL-10 in CLP+2-AP group were significantly lower(all P<0.001),but the levels of TNF-αin CLP+2-AP group were not significantly lower(P=0.33).The 7-day survival rate was 100%in sham group,13.3%in CLP+2-AP group,86.7%in 2-AP group and 20.0%in CLP+2-AP group.Inhibition of PKR activation slightly improved the trend of 7-days survival rate of CLP model mice(analysis by mantel Cox test,χ^2=0.0012,P=0.97).Conclusion In sepsis mice model,inhibition of PKR activity can reduce the expression of inflammatory factors in plasma,decrease bacterial load in blood and abdominal cavity,and protect organ function,which could suggest that inhibition of PKR activity has potential application in sepsis treatment.