肠道菌群异质性与脓毒症小鼠获得性肌无力的关系

Relationship between intestinal microbiota heterogeneity and acquired myasthenia in septic mice

目的评价肠道菌群异质性与脓毒症小鼠获得性肌无力的关系。方法SPF级健康雄性C57BL/6小鼠80只,体质量18~20 g,6~8周龄。取小鼠40只,采用腹腔注射LPS 10 mg/kg的方法制备脓毒症模型,筛选脓毒症敏感型小鼠(建模后24 h内出现濒死状态甚至死亡)和脓毒症抵抗型小鼠(生存7 d并恢复活跃状态),建模前收集小鼠粪便制备粪菌液。取小鼠40只,采用随机数字表法分为4组(n=10):对照组(C组)不予以任何处理;抗生素组(ABX组)灌胃复合抗生素1次/d,连续5 d;抵抗组(Res组)灌胃复合抗生素5 d后,灌胃脓毒症抵抗型小鼠粪菌液150μl,1次/d,连续3 d,随后腹腔注射LPS 15 mg/kg;敏感组(Sen组)灌胃复合抗生素5 d后,灌胃脓毒症敏感型小鼠粪菌液3 d(方法同Res组),随后腹腔注射LPS 15 mg/kg。脓毒症建模后24 h时进行脓毒症严重程度评分。于粪菌移植后以及脓毒症建模后24 h时测定四肢抓力。脓毒症建模后24 h时测定腓肠肌复合肌动作电位(CMAP),随后取血液标本,采用ELISA法测定血清IL-1、IL-6和TNF-α浓度,取胫骨前肌和腓肠肌组织,采用蛋白免疫印迹法测定胫骨前肌肌肉特异性环指蛋白1(MuRF-1)和肌肉萎缩盒F蛋白(MAFbx)表达,腓肠肌HE染色后评估测量肌纤维直径和横截面积。Res组和Sen组于粪菌移植3 d后收集小鼠粪便,提取DNA进行16S rDNA测序和非靶向代谢组学分析。结果与C组比较,Res组脓毒症严重程度评分升高,脓毒症建模后四肢抓力降低,血清IL-6浓度升高,腓肠肌肌纤维直径和横截面积减少(P<0.05),其余指标差异无统计学意义,ABX组上述指标差异无统计学意义(P>0.05)。与C组和Res组比较,Sen组脓毒症严重程度评分升高,脓毒症建模后四肢抓力降低,CMAP潜伏期延长、振幅降低,血清IL-1、IL-6、TNF-α浓度升高,腓肠肌肌纤维直径和横截面积减少,胫骨前肌MuRF-1和MAFbx表达上调(P<0.05)。肠道菌群16S rDNA测序:与Sen组比较,Res组Chao1指数、Good-coverage指数、Simpson指数差异无统计学意义(P>0.05),Shannon指数升高(P<0.05),β多样性差异无统计学意义(P>0.05);阿克曼氏菌科、疣微菌目、嗜黏蛋白阿克曼氏菌属、疣微菌纲、疣微菌门在Sen组显著富集,肠球菌属、肠球菌科在Res组显著富集(P<0.05)。非靶向代谢组学分析:Res组和Sen组代谢物图谱提示模型建立良好(R2Y>Q2Y);与Sen组比较,Res组显著上调的代谢物质90个,下调的代谢物质88个,显著上调的代谢物质包括维生素K1、γ生育酚、牛磺酸等(P<0.05);差异代谢物KEGG富集通路包括2-氧羧酸代谢和泛素酮等萜类醌的生物合成(P<0.05)。结论肠道菌群异质性可能参与脓毒症小鼠获得性肌无力的发生机制。

Objective To evaluate the relationship between intestinal microbiota heterogeneity and acquired myasthenia in septic mice.Methods Eighty SPF healthy male C57BL/6 mice,weighing 18-20 g,aged 6-8 weeks,were included.Forty mice were selected to prepare a sepsis model by intraperitoneal injection of lipopolysaccharide(LPS)10 mg/kg.Sepsis-sensitive mice(state of dying or even death within 24 h after developing the model)and sepsis-resistant mice(survival for 7 days and recovery)were screened.The feces from donor mice were collected to make fecal bacteria fluid.Forty mice were selected and divided into 4 groups(n=10 each)by the random number table method:control group(C group),antibiotic group(ABX group),resistant group(Res group),and sensitive group(Sen group).Group C received no treatment.In ABX group,compound antibiotics were given by intragastric gavage once a day for 5 consecutive days.In Res group,the fecal solution from sepsis-resistant mice 150μl was given by gavage once a day for 3 consecutive days starting from 5 days after gavage administration of compound antibiotics,and then LPS 15 mg/kg was intraperitoneally injected.In Sen group,the fecal solution from sepsis-sensitive mice was given by gavage for 3 days(using the same method as previously described in Res group)starting from 5 days after gavage administration of compound antibiotics,and then LPS 15 mg/kg was intraperitoneally injected.The severity of sepsis was assessed and scored at 24 h after developing the model.The four limb grip strength was measured after fecal bacteria transplantation and at 24 h after developing the sepsis model.The Compound Muscle Action Potential(CMAP)of the gastrocnemius was measured at 24 h after developing the sepsis model.Then blood samples were collected for determination of the levels of interleukin-1(IL-1),IL-6 and tumor necrosis factorα(TNF-α)in serum by enzyme-linked immunosorbent assay.The tissues of anterior tibial muscle and gastrocnemius muscle were obtained for determination of the expression of muscle-specific ring finger protein 1(MuRF-1)and muscle atrophy box F protein(MAFbx)by Western blot.The diameter and cross-sectional area of gastrocnemius muscle fibers were measured after HE staining.The feces of mice were collected at 3 days after fecal bacteria transplantation,and DNA was extracted from mouse fecal samples for 16S rDNA sequencing and untargeted metabolomics analysis.Results Compared with C group,the score for severity of sepsis was significantly increased,the four limb grip strength was decreased after developing the sepsis model,the serum IL-6 concentration was increased,and the diameter and cross-sectional area of gastnemius muscle fibers were decreased(P<0.05),and no statistically significant changes were found in the other parameters in Res group,and no statistically significant changes were found in the indexes mentioned above in ABX group(P>0.05).Compared with C group and Res group,the score for severity of sepsis was significantly increased,the four limb grip strength was decreased,the latency of CMAP was prolonged,and the amplitude of CMAP was decreased,the serum concentrations of IL-1,IL-6 and TNF-αwere increased,the diameter and cross-sectional area of gastrocnemius muscle fibers were decreased,and the expression of MuRF-1 and MAFbx in anterior tibial muscle was up-regulated after developing the sepsis model in Sen group(P<0.05).16S rDNA sequencing of intestinal flora:Compared with Sen group,no significant change was found in Chao1 index,Good-coverage index and Simpson index in Res group(P>0.05),Shannon index was increased(P<0.05),and no significant change was found inβdiversity in Res group(P>0.05).LDA analysis showed that f_Akkermarisiaceae,o_Verrucomicrobiales,g_Akmansia,c_Verrucomicrobiae and p_Verrucomicrobiota were significantly enriched in Sen group,and g_Enterococcus and f_Enterococcaceae were significantly enriched in Res group(P<0.05).Nontargeted metabolic analysis:The metabolite profiles in Res group and Sen group suggested that the model was well developed(R2Y>Q2Y).Compared with Sen group,90 metabolites was significantly up-regulated and 88 down-regulated,significantly up-regulated metabolic substances including vitamin K1,gamma-tocopherol,and taurine in Res group(P<0.05).The differential metabolite KEGG enrichment pathway included 2-oxocarboxylic acid metabolism and ubiquinone and other terpenoid-quinone biosynthesis(P<0.05).Conclusions Intestinal flora heterogeneity may be involved in the pathogenesis of acquired myasthenia in septic mice.