宣白承气汤对脓毒症小鼠肺-肠损伤及肠道菌群的调节作用

Regulation of Xuanbai Chengqi Decoction on lung-gut injury and intestinal microflora in sepsis mice

目的明确宣白承气汤对脓毒症小鼠肺和肠道损伤以及肠道功能的作用,分析其对脓毒症小鼠肠道菌群的影响。方法健康清洁级C57雄性小鼠36只,随机(随机数字法)分为三组,每组12只,分别为对照组、模型组和宣白承气汤治疗组。通过腹腔注射细菌脂多糖(lipopolysaccharide,LPS)5 mg/kg制备脓毒症模型,造模前24 h、造模后0.5 h及造模后12 h宣白承气汤灌胃给药,造模后24 h采集肺脏、肠道以及血液标本。采用实时荧光定量PCR测定肺部和肠道组织炎症因子白介素1β(interleukin-1β,IL-1β)、IL-6、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)含量,通过组织学染色评估肺部和肠道结构损伤和改变,通过特殊染色评价肠道黏蛋白表达,分别采用免疫组化和Western Blot分析肠道上皮紧密连接蛋白的表达,最后使用鲎试剂盒检测各组小鼠血浆中内毒素含量,并提取小鼠粪便DNA,通过菌群16S rDNA荧光定量PCR方法检测脓毒症小鼠肠道菌群变化。三组间的计量资料比较,采用单因素方差分析。结果(1)宣白承气汤能够降低脓毒症小鼠肺部炎症因子IL-1β、IL-6、TNF-α和MCP-1的表达(均P<0.05),减轻肺部炎症损伤;(2)与模型组相比,宣白承气汤治疗组小鼠肠道炎性损伤有所缓解,肠道炎症因子表达明显下调(均P<0.05),肠道黏蛋白和上皮紧密连接蛋白表达增加,肠道屏障功能改善;(3)宣白承气汤减轻脓毒症小鼠循环血液中内毒素的含量(P<0.05),并促进脓毒症小鼠肠道中有益菌阿克曼菌的生长,减少肠球菌属的表达(均P<0.05)。结论宣白承气汤可显著改善脓毒症小鼠肺部和肠道炎症损伤,调节肠道上皮屏障和肠道菌群,改善肠道功能。

Objective To investigate the effect of Xuanbai Chengqi Decoction(XBCQT)on lung-gut injury and intestinal function,and analyze its effect on intestinal flora in sepsis mice.Methods C57 male mice were randomly divided into three groups with 12 mice in each group:control group,model group and treatment group.The sepsis model was prepared by intra-peritoneal injection of lipopolysaccharide(LPS)5 mg/kg.XBCQT was administered by gavage 24 h before,0.5 h after and 12 h after modeling.The lung,colon and blood samples were collected at 24 h after modeling.The pulmonary and intestinal inflammatory cytokine content of interleukin-1β(IL-1β),IL-6,tumor necrosis factorα(TNF-α)and monocyte chemoattractant protein(MCP-1)was measured by real-time fluorescence quantitative PCR.HE staining was used to evaluate the structural damage and changes of lung and gut,and Western blot and Immunohistochemistry methods were used to analyze the expression of occludin and claudin-1 in intestinal epithelium.Finally,the plasma endotoxin content of each group was tested by Limulus test kit.Fecal DNA of mice was extracted and the changes of intestinal flora in sepsis mice were detected by 16S rDNA quantitative PCR.The measurement data among the three groups were compared by one-way analysis of variance.Results(1)XBCQT significantly reduced the pulmonary inflammatory cytokine IL-1β,IL-6,TNF-αand MCP-1 expression(all P<0.05),and attenuated lung injury.(2)Compared to the model group,the treatment group exhibited a reduction in intestinal damage and a decrease in the intestinal inflammatory cytokines(all P<0.05).XBCQT increased the expression of epithelial tight junction and mucin of colon,and improved the intestinal epithelium barrier function.(3)XBCQT treatment decreased the content of endotoxin in plasma of sepsis mice(P<0.05),promoted the growth of beneficial bacteria Akkermansia muciniphila and reduced the expression of Enterococcus in the intestine of sepsis mice(all P<0.05).Conclusions XBCQT can significantly improve the intestinal inflammatory injury,regulate the intestine epithelium barrier and improve the intestinal function in sepsis mice.