肠道微生物来源石胆酸对脓毒症相关肝损伤的保护作用  

Protective effect of gut microbiota-derived lithocholic acid on sepsis-associated hepatic injury

作　　者：杨瑾 刘雪克 张媛媛[1] 金钧[1] 韦瑶 Jin Yang;Xueke Liu;Yuanyuan Zhang;Jun Jin;Yao Wei(Department of Intensive Care Unit,the First Affiliated Hospital of Soochow University,Suzhou 215006,China)

机构地区：[1]苏州大学附属第一医院重症医学科,江苏苏州215006

出　　处：《中华危重症医学杂志（电子版）》2024年第4期265-274,共10页Chinese Journal of Critical Care Medicine:Electronic Edition

基　　金：江苏省卫生健康委科研项目(M2020013)。

摘　　要：目的:探讨肠道微生物来源的石胆酸(LCA)对脓毒症小鼠肝损伤的保护作用。方法:将15只C57BL/6J小鼠分为假手术组(Sham组)、脓毒症组(Sep组)、粪菌移植组(Sep-fmt组),每组各5只。采用盲肠结扎穿孔术(CLP)构建脓毒症小鼠模型,Sham组仅行腹壁开关手术;Sep-fmt组于CLP后第2天给予粪菌液连续灌胃3 d;Sham组和Sep组根据体质量给予等剂量含10%甘油的无菌磷酸盐缓冲液连续灌胃3 d。采用粪便进行16S核糖体RNA(rRNA)测序分析并筛选出胆汁酸代谢差异代谢产物。然后将30只C57BL/6J小鼠分为假手术组(Sham组,6只)、脓毒症组(Sep组,12只)、LCA组(LCA-Sep组,12只),各组分别保证6只大鼠进行后续实验分析。LCA-Sep组在脓毒症造模之前给予100 mg/kg的LCA灌胃,连续5 d;Sham组、Sep组根据体质量给予等剂量溶媒连续灌胃5 d。采用粪便进行16S rRNA测序分析,观察各组小鼠死亡情况并进行临床严重程度评分(CSS)。检测血清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-10水平并评估各组小鼠肝脏组织病理损伤。结果:Sham组、Sep组和Sep-fmt组小鼠各次级胆汁酸比较,差异均有统计学意义(P均<0.001),且Sep-fmt组中LCA的增幅最大,故后续实验选取LCA对脓毒症小鼠进行灌胃。CLP术后24 h,Sham组小鼠无死亡,Sep组小鼠死亡4只,LCA-Sep组小鼠死亡2只;各组存活小鼠CSS比较,差异具有统计学意义[(1.0±0.0)、(3.5±0.5)、(2.5±0.5)分,F=47.500,P<0.001]。α多样性分析显示,3组小鼠肠道菌群丰富度[Chao1指数和基于丰度的覆盖估计值(ACE)指数]和多样性(Shannon指数和Simpson指数)比较,差异均有统计学意义(H=8.766、8.766、9.704、10.994,P均<0.05),且LCA-Sep组肠道菌群多样性高于Sep组(P均<0.05)。β多样性分析显示,3组小鼠的菌群结构存在显著差异(H=18.322,P=0.001)。3组小鼠拟杆菌门的平均相对丰度分别为62.17%、11.10%、34.47%,变形菌门的平均相对丰度分别为10.99%、62.81%、40.ObjectiveTo investigate the protective effect of lithocholic acid (LCA), originating from the gut microbiota, on liver damage in septic mice.MethodsA total of fifteen C57BL/6J mice were randomly divided into a sham surgery group (Sham group), a sepsis group (Sep group) and a fecal microbiota transplantation group (Sep-fmt group), five in each group. The sepsis model was induced using cecal ligation and puncture (CLP), while the Sham group underwent the abdominal wall incision and closure only. The Sep-fmt group received fecal microbiota solution for 3 d on the 2nd day after CLP, and the Sham group and Sep group were given an equal dose of sterile phosphate buffer containing 10% glycerol for 3 d according to body mass. Faecal samples were collected for 16S ribosomal RNA (rRNA) sequencing to analyze differences in bile acid metabolism. Then thirty C57BL/6J mice were assigned to a sham surgery group (Sham group, n = 6), a sepsis group (Sep group, n = 12) and a LCA group (LCA-Sep group, n = 12). Six mice were guaranteed in each group for subsequent experimental analysis. The LCA-Sep group received intragastric administration of 100 mg/kg LCA for five consecutive days before sepsis modeling. The Sham group and Sep group were administered an equal dose of solvent according to body weight for five consecutive days. Faecal samples were analyzed by 16S rRNA sequencing to assess changes in gut microbiota. The mortality, clinical severity score (CSS), serum tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-10 were measured, and the liver tissue pathology was evaluated.ResultsBile acid metabolism analysis showed that there were significant differences in secondary bile acids among the Sham group, Sep group and Sep-fmt group (all P < 0.001). The increase of LCA was the largest in the Sep-fmt group, so LCA was selected for subsequent experiments. At 24 h after CLP, no mice died in the Sham group, four mice died in the Sep group, and two mice died in the LCA-Sep group. The CSS of surviving mice among these three

关 键 词：脓毒症 石胆酸 肠道菌群 16S核糖体RNA 肝损伤 巨噬细胞 

分 类 号：R459.7[医药卫生—急诊医学] R575[医药卫生—治疗学]