三步止泻治疗小儿腹泻调控肠微生态的机制研究

Study on the Mechanism of Three-Step Antidiarrheal Therapy in the Treatment of Infantile Diarrhea by Regulating Intestinal Microecology

目的 通过本项目研究,了解三步止泻法治疗腹泻乳兔的可能作用机制,明确三步止泻法治疗小儿腹泻与肠微生态作用机制的强相关性。方法 将乳兔制作脾虚证模型后,分为六组即:补脾组、摩腹组、推上七节骨组、三步止泻手法组、益生菌组、模型组,经过治疗周期后,处死乳兔后,将胃黏膜、肠黏膜组织切片进行HE染色,观察乳兔胃、肠黏膜的组织学变化;检测腹泻兔血浆内毒素的表达水平;并收集各组大鼠粪便排泄物,进行微生物组总DNA提取,目标片段PCR扩增,扩增产物回收纯化,扩增产物荧光定量,测序文库制备,上机进行高通量测序,然后多元化对比,后统计分析。结果 兔胃肠组织HE染色提示治疗后三步止泻手法组、补脾组、摩腹组及推上七节骨组、益生菌组能有效的改善兔胃肠组织水肿及炎症情况;血浆内毒素的表达水平表明补脾组在降低兔血浆内毒素有较好的效果(P<0.05),三步止泻手法组、摩腹组、推上七节骨组及益生菌组对降低兔血浆内毒素也有一定的作用;OTU/ASV分析、Alpha多样性分析表明摩腹组及三步止泻手法组可纠正菌群紊乱,提高菌群的丰富度及均匀性,使肠道内环境恢复稳态。结论 三步止泻手法组具有刺激宿主产生免疫及清除功能、减少内毒素的吸收、调整肠道菌群间及其与人体生理机能的平衡状态,改善腹泻症状,且研究表明,各手法组在治疗中,针对性各有不同。

Objective:Through this study,to understand the possible mechanism of action of three-step antidiarrheal therapy in the treatment of diarrhea in suckling rabbits,and to clarify the strong correlation between the three-step antidiarrheal therapy in the treatment of diarrhea in children and the intestinal microecological mechanism of action.Methods:After the spleen deficiency syndrome model of the suckling rabbits was made,the rabbits were divided into six groups:spleen tonifying group,abdominal massage group,seven-segment bone pushing group,three-step antidiarrheal technique group,probiotics group and model group.After the treatment cycle and the suckling rabbits were sacrificed,the gastric mucosa and intestinal mucosa tissues were sliced for HE staining,and the histological changes of the gastric mucosa and intestinal mucosa were observed.The expression level of plasma endotoxin of the diarrhea rabbits was detected.The feces and excrement of the rabbits each group were collected for total DNA extraction of microbiome,PCR amplification of target fragments,recycling and purification of amplification products,fluorescence quantification of amplification products,preparation of sequencing library,high-throughput sequencing on the computer,and then diversified comparison,and statistical analysis.Results:The HE staining of the rabbit gastrointestinal tissue suggested that after treatment,the three-step antidiarrhea manipulation group,the spleen tonifying group,the abdominal massage group,the seven-segment bone pushing group and the probiotics group could effectively improve the edema and inflammation of the rabbit gastrointestinal tissue.The expression level of plasma endotoxin showed that the spleen tonifying group had a good effect on the reduction of the rabbit plasma endotoxin(P<0.05),and the three-step antidiarrhea manipulation group,the abdominal massage group,the seven-segment bone pushing group and the probiotics group also had a certain effect on the reduction of the rabbit plasma endotoxin.OTU/ASV analysis and Alpha diversity analysis showed that the abdominal massage group and the three-step antidiarrhea manipulation group could correct the flora disorder,improve the richness and uniformity of the flora,and restore the intestinal environment to a stable state.Conclusion:The three-step antidiarrheal manipulation group can stimulate the host to produce immunity and clearance function,reduce the absorption of endotoxin,adjust the balance between intestinal flora and human physiological function,and improve the symptoms of diarrhea.The study has shown that each manipulation group has different pertinence in the treatment.