抑制组织蛋白酶B对脓毒症急性肺损伤小鼠肺组织细胞焦亡的影响

Effect of inhibition of cathepsin B on pyroptosis of lung tissue in mice with sepsis-induced acute lung injury

目的探讨组织蛋白酶B(CTSB)下调对脓毒症急性肺损伤(ALI)小鼠肺组织细胞焦亡的影响。方法SPF级健康雄性C57BL/6小鼠30只,随机分为假手术组(n=6)、脓毒症组(n=15)及脓毒症+组织蛋白酶B抑制剂CA-074组(脓毒症+CA-074组,n=9)。采用盲肠结扎穿孔法(CLP)建立脓毒症模型。假手术组除不行盲肠结扎及穿孔外,其他操作同脓毒症组。脓毒症+CA-074组于造模后15 min腹腔注射10 mg/kg CA-074。造模后24 h,收集肺泡灌洗液进行白细胞计数,并采用ELISA法检测白细胞介素(IL)-18及IL-1β的浓度。收集肺组织,行HE染色及肺组织损伤评分,测定肺组织湿/干重比;采用RT-PCR检测各组肺组织中小凹蛋白-1(caveolin-1)、单核细胞趋化蛋白诱导蛋白1(MCPIP1)、组织蛋白酶B(CTSB)、半胱天冬酶(caspase)-1及成孔效应蛋白D(GSDMD)的mRNA表达水平;采用Western blotting测定各组肺组织中CTSB、caspase-1及GSDMD蛋白的表达水平。结果假手术组小鼠观察期内无异常,术后24 h存活率100.0%。脓毒症组小鼠术后出现背部竖毛、行动迟缓、饮食不振及精神萎靡等症状,而这些症状在脓毒症+CA-074组小鼠中有所改善,两组术后24 h存活率分别为40.0%及66.7%。与假手术组比较,脓毒症组肺泡灌洗液中白细胞计数增加,炎性因子IL-18及IL-1β水平上升,肺组织湿/干重比及肺损伤评分增高,肺组织中caveolin-1 mRNA表达量上调,MCPIP1mRNA表达量下调,CTSB、caspase-1及GSDMD蛋白表达上调,差异均有统计学意义(P<0.05);与脓毒症组比较,脓毒症+CA-074组中白细胞计数减少,炎性因子水平降低,肺组织湿/干重比及肺损伤评分降低,caveolin-1 mRNA表达量下调,MCPIP1 mRNA表达量上调,焦亡相关蛋白表达明显下降,CTSB蛋白表达降低,差异有统计学意义(P<0.05),而CTSB mR NA表达无明显变化(P=0.973)。结论脓毒症肺损伤过程中肺组织CTSB表达增高,抑制CTSB的表达可通过减轻肺组织细胞焦亡而改善脓毒症急性肺损伤。

Objective To investigate the effects of cathepsin B(CTSB)down-regulation on pyroptosis of lung tissue in mice with sepsis-induced acute lung injury.Methods Thirty SPF healthy male C57BL/6 mice were randomly divided into three groups:sham group(n=6),sepsis group(n=15),and sepsis plus CTSB inhibitor CA-074 group(sepsis+CA-074 group,n=9).Sepsis was induced by cecal ligation and puncture(CLP).Except for cecal ligation and perforation,other operations in the sham group were the same as those in sepsis group.10 mg/kg CA-074 was intraperitoneally injected in sepsis+CA-074 group at 15 min after CLP.The mice were sacrificed 24 h after CLP.Bronchoalveolar lavage fluid(BALF)was collected for measurement of the white blood cell count and IL-18 and IL-1β concentrations with ELISA.Lung tissues were obtained for the determination of lung injury score,wet/dry lung weight ratio,the expressions of caveolin-1,MCPIP1,CTSB,caspase-1,and GSDMD mRNA(by RT-PCR),as well as the protein expressions of CTSB,caspase-1,and GSDMD(by Western blotting).Results The mice in sham group were normal throughout the observation period,and the 24 h survival rate after the operation was 100.0%.The sepsis mice showed obvious symptoms such as piloerection,dilatory,anorexia,and apathy,and these symptoms were improved in sepsis+CA-074 group.The 24 h survival rate after surgery in the two groups was 40.0% and 66.7%,respectively.Compared with sham group,the count of leukocytes and the expression of IL-18 and IL-1β in BALF were significantly increased,the wet/dry lung weight ratio and lung injury score were increased,the expression of caveolin-1 mRNA was up-regulated,the expression of MCPIP1 mRNA was downregulated,and the expressions of CTSB,caspase-1,and GSDMD were up-regulated in the sepsis group(P<0.05).Compared with sepsis group,the count of leukocytes and the expressions of inflammatory factors were decreased,the wet/dry lung weight ratio and lung injury score were decreased,the expression of caveolin-1 mRNA was down-regulated,the expression of MCPIP1 mRNA was up-regulated,proteins associated with pyroptosis were significantly decreased,and the protein expression of CTSB was decreased in sepsis+CA-074 group(P<0.05).However,there was no significant change in the parameter of CTSB mRNA between the sepsis and sepsis+CA-074 groups(P=0.973).Conclusion Increased CTSB expression occurs in lung tissue with sepsis-induced acute lung injury,and inhibition of CTSB may alleviate sepsis-induced acute lung injury by suppressing pyroptosis.