过表达SIRT1通过增强自噬水平调控巨噬细胞M2型极化减轻脓毒症诱导的急性肺损伤

Overexpression of SIRT1 attenuates sepsis induced acute lung injury by increasing autophagy level and regulating M2 macrophage polarization

目的:探究过表达沉默信息调节因子1(SIRT1)对脓毒症诱导的急性肺损伤(ALI)巨噬细胞表型转化的影响及调控机制。方法:体外培养小鼠巨噬细胞系RAW264.7,LPS+IFN-γ与IL-4定向诱导其M1与M2型极化,Western blot检测细胞SIRT1和M1型标志物iNOS、CD86与M2型标志物Arg1、Mcr1蛋白表达。转染过表达SIRT1的质粒,RT-PCR检测转染效率,LPS诱导脓毒症ALI体外细胞模型,并分为4组:LPS组、LPS+pcDNA3.1-SIRT1组、LPS+pcDNA3.1-NC组和Control组,Western blot检测各组细胞iNOS、CD86与Arg1、Mcr1和自噬标志物LC3B-Ⅱ/Ⅰ、Beclin1与p62及SIRT1/FOXO1信号通路SIRT1、FOXO1蛋白表达。免疫荧光检测各组细胞LC3B荧光表达。采用盲肠结扎穿孔(CLP)术构建脓毒症ALI小鼠模型,通过慢病毒在小鼠体内过表达SIRT1,并分为4组:假手术(Sham)组、CLP组、CLP+LV-NC组、CLP+LV-SIRT1组,RT-PCR、HE染色、ELISA检测各组小鼠肺组织SIRT1 mRNA表达、病理损伤及支气管肺泡灌洗液(BALF)中炎症因子TNF-α、IL-6与IL-10分泌,记录并分析造模72 h后各组小鼠存活率。结果:体外成功诱导了RAW264.7细胞M1、M2型极化,且M1型巨噬细胞SIRT1蛋白表达明显降低(P<0.05),而M2型巨噬细胞SIRT1表达显著升高(P<0.05)。转染pcDNA3.1-SIRT1后细胞SIRT1 mRNA表达明显升高(P<0.05)。与LPS组或LPS+pcDNA3.1-NC组相比,LPS+pcDNA3.1-SIRT1组iNOS、CD86、p62蛋白表达显著降低(P<0.05),而Arg1、Mcr1、LC3Ⅱ/Ⅰ和Beclin1、SIRT1、FOXO1表达明显升高(P<0.05),LC3B荧光斑点数显著增加(P<0.05)。小鼠体内实验显示,与CLP组或CLP+LV-NC组相比,CLP+LV-SIRIT1组SIRT1 mRNA表达、BALF中抗炎因子IL-10分泌和造模72 h后小鼠存活率均明显升高(P<0.05),而肺组织病理损伤、BALF中促炎因子TNF-α、IL-6水平显著降低(P<0.05)。结论:过表达SIRT1可在体内外改善脓毒症ALI,而通过SIRT1/FOXO1通路提高自噬水平促进巨噬细胞M2型极化可能是其发挥保护作用的机制之一。

Objective:To explore effect of SIRT1 over-expression on phenotype transformation of macrophages in sepsis induced acute lung injury(ALI)and its regulatory mechanism.Methods:RAW264.7 macrophages were cultured in vitro and treated with LPS+IFN-γand IL-4 to induce M1 and M2 polarization.Western blot was used to detect protein expressions of M1 markers iNOS,CD86 and M2 markers Arg1,Mcr1 and SIRT1;SIRT1 overexpression plasmid was transfected and transfection efficiency was detected by RT-PCR.Spesis ALI cells model in vitro induced by LPS were divided into four groups:LPS group,LPS+pcDNA3.1-SIRT11 group,LPS+pcDAN3.1-NC group and Control group.Western blot was used to detect protein expressions of iNOS,CD86,Arg1,Mcr1,autophagy markers LC3B-Ⅱ/Ⅰ,Beclin1 and p62,and SIRT1 and FOXO1 in SIRT1/FOXO1 signaling pathway.Immunofluo‐rescence was used to detect fluorescence expression of LC3B in cells;sepsis ALI mice model was established by cecal ligation and puncture(CLP),SIRT1 was overexpressed by lentivirus in mice,and were divided into four groups:Sham group,CLP group,CLP+LV-NC group,CLP+LV-SIRT1 group;RT-PCR,HE staining and ELISA were used to detect expressions of SIRT1 mRNA,pathological injury and expressions of inflammatory factors TNF-α,IL-6,IL-10 in BALF of each group;survival rate of mice in each group was observed and analyzed 72 h after modeling.Results:M1 and M2 polarization of RAW264.7 cells were successfully induced in vitro,and expression of SIRT1 protein was significantly decreased in M1 group(P<0.05),while significantly increased in M2 group(P<0.05);expression of SIRT1 mRNA in pcDNA3.1-SIRT1 transfected cells was significantly increased(P<0.05).Compared with LPS group or LPS+pcDNA3.1-NC group,protein expressions of iNOS,CD86 and p62 in LPS+pcDNA3.1-SIRT1 group were significantly decreased(P<0.05),while expressions of Arg1,Mcr1,LC3BⅡ/Ⅰ,Beclin1,SIRT1 and FOXO1 were significantly increased(P<0.05),fluorescent spot numbers of LC3B was increased(P<0.05).In vivo experiments showed that compared with CLP group or CLP+LV-NC group,expression of SIRT1 mRNA,secretions of anti-inflammatory factors IL-10 in BALF and survival rate of CLP+LV-SIRT1 group were significantly increased(P<0.05),while pathological injury,TNF-α,IL-6 levels in BALF were decreased(P<0.05).Conclusion:Overexpression of SIRT1 can protect sepsis ALI in vitro and in vivo,whose one of mechanisms may be that SIRT1/FOXO1 pathway enhances autophagy level to promote M2 macrophage polarization.