基于肠道菌群研究枳术丸治疗脾虚证慢传输型便秘的作用机制

The mechanism of Zhi-zhu Wan in treatment of slow transit constipation with spleen deficiency syndrome based on intestinal flora

目的观察枳术丸对脾虚证慢传输型便秘(slow transit constipation,STC)小鼠肠道菌群、短链脂肪酸(SCFAs)、5-羟色胺(5-HT)和肠道传输功能的影响,探讨枳术丸治疗STC的机制。方法将50只健康的KM小鼠按随机数字表法分为正常组(15只)和造模组(35只),正常组小鼠基础饲料喂养,造模组小鼠以番泻叶灌胃和限制饮食饮水方法制作脾虚证STC小鼠模型。造模成功后,将造模组小鼠分为模型组、莫沙必利组和枳术丸组,每组10只,对应给药治疗7 d。治疗结束后分别采用16S rRNA基因测序技术分析小鼠粪便菌群多样性和菌群结构,气相色谱/质谱法检测粪便中SCFAs水平,HE染色观察各组小鼠结肠组织病理形态改变,使用ELISA法检测小鼠脑组织、结肠组织中5-HT水平。结果造模后,各造模组小鼠体质量显著低于正常组(均P<0.01);治疗结束后,模型组小鼠体质量和肠道推进率显著低于正常组(均P<0.01);与模型组比较,莫沙必利组和枳术丸组小鼠体质量和肠道推进率升高(均P<0.05)。小鼠结肠组织HE病理显示模型组腺体排列不齐,结肠黏膜轻微水肿、充血,轻度炎症浸润。与正常组比较,模型组小鼠肠道菌群丰富度和多样性皆降低,枳术丸组和莫沙必利组小鼠肠道菌群多样性和丰富度更趋近于正常组。在门分类水平上,与正常组比较,模型组小鼠厚壁菌门、疣微菌门相对丰度降低,拟杆菌门、变形菌门相对丰度增加;与模型组比较,枳术丸组小鼠疣微菌门相对丰度增加,拟杆菌门相对丰度降低。在属分类水平上,与正常组比较,模型组小鼠拟杆菌属、乳杆菌属相对丰度增加,异形藻属、梭菌属、另枝菌属相对丰度降低;与模型组比较,枳术丸组小鼠拟杆菌属相对丰度降低,异形藻属、梭菌属相对丰度增加。与正常组比较,模型组小鼠粪便乙酸、丙酸水平降低(均P<0.05),结肠组织、脑组织5-HT水平降低(均P<0.05);与模型组小鼠比较,枳术丸组小鼠粪便乙酸、丙酸水平显著升高(均P<0.01),结肠组织、脑组织5-HT水平升高(均P<0.05)。结论枳术丸可通过调节肠道菌群的平衡,促进SCFAs的分泌和5-HT的分泌释放,提高推进率,促进肠道蠕动治疗脾虚证STC。

Objective To observe the effect of Zhi-zhu Wan(ZZW)on intestinal flora,short-chain fatty acids(SCFAs),5-HT and intestinal transport function in mice with slow transit constipation(STC)with spleen deficiency syndrome,and explore the mechanism of ZZW in the treatment of STC.Methods A total of 50 healthy KM mice were randomly divided into normal group(n=15)and model group(n=35).The normal group was fed on the basic feeding.The model group was gavaged with senna leaf.After successful modeling,the mice in the model group were divided into model group,mosapride group,and ZZW group,and the corresponding medication was administered for 7 days.After the treatment,16S rRNA gene high-throughput sequencing was used to analyze the diversity and structure of intestinal flora in the feces of mice,gas chromatography/mass spectrometry was used to detect the content of SCFAs in feces,and HE staining was used to observe the pathological changes of colon tissue in each group of mice.The contents of 5-HT in mouse brain tissue and colon tissue were detected with ELISA methods.Results After modeling,the body weight in each modeling group was significantly lower than that in the normal group(all P<0.01);after the treatment,the body weight and intestinal propulsion rate of the mice in the model group were significantly lower than those in the normal group(all P<0.01).In comparison,the body weights and intestinal propulsion rates in the mosapride group and ZZW group increased(all P<0.05).HE pathology showed that the glands in the model group were irregularly arranged,the colonic mucosa had slightly edema,hyperemia,and mildly inflammatory infiltration.Compared with the normal group,the richness and diversity of intestinal flora in the model group were decreased,and those in the ZZW group and mosapride group were closer to the normal group.At the phylum classification level,compared with normal group,the relative abundance of Firmicutes and Verrucomicrobia were decreased in model group,while the relative abundance of Bacteroidota and Proteobacteria were increased.Compared with model group,the relative abundance of Verrucomicrobia in ZZW group was increased,while the relative abundance of Bacteroidota was decreased.At the genus classification level,compared with the normal group,the relative abundance of Bacteroides and Lactobacillus were increased in the model group,while the relative abundance of Heterophylla,Clostridium and Allocladomyces were decreased.Compared with model group,the relative abundance of Bacteroides in ZZW group was decreased,while the relative abundance of Heterophylla and Clostridium were increased.Compared with the normal group,the content of acetic acid and propionic acid in the feces of mice in the model group were decreased(all P<0.05),and those of 5-HT in the colon and brain tissue were decreased(all P<0.05).The contents of acetic acid and propionic acid in the feces of mice in the ZZW group were significantly increased(all P<0.01),and those of 5-HT in the colon tissue and brain tissue were increased(all P<0.05).Conclusion Zhi-zhu Wan can treat STC with spleen deficiency syndrome by regulating the homeostasis of intestinal flora,promoting the secretion of SCFAs,the secretion and release of 5-HT,increasing the propulsion rate,and promoting intestinal peristalsis.