基于“肠-肝轴”探讨逍遥散加味方对代谢相关脂肪性肝病小鼠肠道屏障及肠道菌群的影响

Effect of Modified Xiaoyao Powder on intestinal barrier and intestinal flora in mice with metabolic associated fatty liver disease based on"gut-liver axis"

基于“肠-肝轴”研究逍遥散加味方对代谢相关性脂肪性肝病(MAFLD)小鼠肠黏膜屏障和肠道菌群的影响,探讨其作用。雄性C57BL/6小鼠随机分成正常组,模型组,双歧杆菌四联活菌片组,逍遥散加味方低、中、高剂量组,每组10只。除正常组外,其余组给予高脂饲料诱导MAFLD模型并予不同剂量药物干预,共12周。检测小鼠体质量、肝湿重、肝指数;生化试剂盒检测血清天门冬氨酸氨基转氨酶(AST)、丙氨酸氨基转氨酶(ALT)、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、脂多糖(LPS)的水平及肝脏肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的含量;苏木素-伊红(HE)染色和油红O染色观察肝脏、回肠及结肠组织形态;爱先蓝-糖原(AB-PAS)染色检测回肠组织杯状细胞数量;免疫组化和蛋白免疫印迹检测回肠、结肠组织紧密连接蛋白1(ZO-1)、闭合蛋白(occludin)、封密蛋白1(claudin-1)的表达;16S核糖体RNA(16S rRNA)基因测序分析小鼠肠道菌群变化。结果显示,与正常组相比,模型组小鼠体质量、肝湿重、肝指数均升高;血清TC、TG、ALT、AST、LDL-C和LPS含量均升高,HDL-C降低;肝组织IL-6、TNF-α含量均升高,肝脏脂质蓄积增多,表明模型诱导成功。与模型组相比,逍遥散加味方中、高剂量组和双歧杆菌四联活菌片组小鼠体质量、肝湿重、肝指数均降低;逍遥散加味方中剂量组和双歧杆菌四联活菌片组小鼠血清TC、TG、LDL-C和ALT、AST水平均显著下降,HDL-C水平显著升高;逍遥散加味方低、中剂量组和双歧杆菌四联活菌片组小鼠肝脏炎症因子IL-6、TNF-α和血清中LPS水平均显著降低,claudin-1、occludin和ZO-1蛋白表达均显著升高。肠道菌群分析显示,与模型组比较,逍遥散加味方中剂量能显著提高小鼠肠道菌群的丰富度和多样性;在门水平上,降低厚壁菌门/拟杆菌门比值;在属水平上,升高乳杆菌属、布劳特菌属、拟杆菌属和阿克曼菌属,降低普雷沃菌属、苏黎世杆菌属和脱硫弧菌属,差异物种以拟杆菌科Odorbacteraceaae和消化链球菌科Peptostreptococcaceae发挥主要作用。总之,逍遥散加味方可通过抑制炎症、调节肠道菌群稳态以及促进肠黏膜屏障的修复来治疗MAFLD。

This study explores the effects and mechanisms of Modified Xiaoyao Powder on the intestinal barrier and intestinal flora in mice with metabolic associated fatty liver disease(MAFLD)based on the"gut-liver axis".Sixty male C57BL/6 mice were randomly divided into the normal group,model group,bifidobacterium tetrad tablet group(SQ),and Modified Xiaoyao Powder groups with low,medium and high doses(XL,XM,XH),with 10 mice in each group.All the mice were administrated with a high-fat diet to build the MAFLD model except the normal group and then treated with related drugs for 12 weeks.Body mass,liver wet weight,and liver index were detected.Serum aspartate aminotransferase(AST),alanine aminotransferase(ALT),total cholesterol(TC),triacylglycerol(TG),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),and lipopolysaccharide(LPS)levels were detected using the biochemical kits.The contents of tumor necrosis factor-α(TNF-α)and interleukin(IL-6)in the liver were tested simultaneously.The morphological changes of the liver and intestine were observed using hematoxylin-eosin(HE)staining and oil red O staining.The goblet cells in the ileum were detected by periodic acid Schiff and alcian blue stain(AB-PAS)staining.The expression of zonula occludens-1(ZO-1),recombinant occludin(occludin),and recombinant claudin 1(claudin-1)in ileum and colon were detected by immunohistochemistry and Western blot.The changes of intestinal flora in mice were analyzed by 16S rRNA gene sequencing.The results showed that compared with the normal group,body weight,liver wet weight and liver index in the model group increased.The contents of TC,TG,ALT,AST,LDL-C,and LPS in the serum of the model group increased,while HDL-C decreased.Meanwhile,the contents of TNF-αand IL-6 in liver tissue increased and liver lipid accumulation increased,indicating successful model induction.Compared with the model group,body weight,liver wet weight,and liver index were decreased in XM,XH groups and SQ group.Serum levels of TC,TG,LDL-C,ALT and AST in XM group and SQ group were significantly decreased,and HDL-C levels were increased.The levels of IL-6,TNF-αin liver tissue and serum LPS in the XL,XM groups and SQ group were significantly decreased.The protein expression of claudin-1,occludin and ZO-1 in XL,XM groups and SQ group were increased.The analysis of intestinal flora showed that compared with the model group,Modified Xiaoyao Powder with a medium dose could significantly improve the richness and diversity of intestinal flora in mice.At the phylum level,the Firmicutes/Bacteroidetes(F/B)ratio decreased;at the genus level,Lactobacillus,Brautella,Bacteroides,and Ackermannia increased,while Prevotella,Desulfovibrio and Turicibacter decreased.The main differential species were Odorbacteraceaeae and Peptostreptococcaceae.In conclusion,Modified Xiaoyao Powder could inhibit inflammation,regulate intestinal flora homeostasis,and promote the repair of the intestinal mucosal barrier in mice with MAFLD.