基于16S rDNA技术和TLRs/MyD88/NF-κB信号通路探讨参苓白术散抗腹泻型肠易激综合征大鼠的分子机制OA

Objective:Based on 16S rDNA technology and molecular biology methods,the molecular mechanism of Shenling Baizhusan in the treatment of diarrhea-predominant irritable bowel syndrome(IBS-D)was investigated.Methods:The 42 SD rats with SPF were randomly divided into no control group,SLBZS-H,medium(SLBZS-M),low(SLBZS-L)dose group,positive control group and model group,with 7 rats in each group.The rat model of IBS-D was prepared by ice-cold senna(0.45 g·mL-1)gavage(10 mL·kg-1)combined with restraint stress for 14 consecutive days.After successful modeling,the corresponding drugs were given to each group with a gavage volume of 10 mL·kg-1:The positive group was administered with 2.36,1.18,0.59 g·mL-1 of Shenling Baizhusan in the Positive group and the Model group with the same volume of normal saline for 14 d.The general condition of the rats:Weight,feces,mental state and death were observed and recorded.The body weight,abdominal wall retraction reflex score(AWR)and loose stool rate of rats in each group were measured before(the first day),after the model(day 14)and after treatment(day 28).Hematoxylin-eosin staining was used to observe the morphological characteristics of colon tissues of experimental animals.Enzyme-linked immunosorbent assay was used to quantitatively analyze the concentration of inflammatory mediators in the peripheral blood of experimental animals.Western blotting was used to detect the expression levels of key proteins of Toll-like receptor 4(TLR4),Toll-like receptor 2(TLR2),myeloid differentiation factor 88(MyD88)and nuclear factor-κB(NF-κB)signaling pathway in rat colon tissue.16S rDNA technology was used to detect the structural changes of intestinal microbiota in rats.Results:Compared with Control,the colon of the Model group showed partial mucosal epithelial shedding and inflammatory cell infiltration.The contents of TNF-α,IL-1β,IL-6 and 5-HT in serum increased(P＜0.05),the protein expressions of TLR2,TLR4,MyD88 and NF-κB in colon tissue increased(P＜0.05),the diversity indices of Richness,Chao1,abundance-based coverage estimator(ACE)and Shannon decreased(P＜0.05),and the phylum Firmicutes,Actinobacteria,The relative richness of Bacteroides-H,Lactobacillus and Ligilactobacillus decreased(P＜0.05),while the relative richness of Bacteroidetes,Proteobacteria and Prevotella increased(P＜0.05).Compared with the model group,the colonic structure and organization of the SLBZS-H group,SLBZS-M group,SLBZS-L group and Positive group were clearer,and only a small number of inflammatory cells were present in some areas,and the serum contents of TNF-α,IL-1β,IL-6 and 5-HT were decreased(P＜0.05),TLR2,TLR4,The protein expressions of MyD88 and NF-κB decreased(P＜0.05),and compared with the model group,the diversity indices of Richness,Chao1,ACE and Shannon in the SLBZS-H,SLBZS-M and SLBZS-L groups increased(P＜0.05),and the richness of Firmicutes and Actinobacteria increased(P＜0.05).The richness of Proteobacteria and Prevotella decreased(P＜0.05),and the abundance of Prevotella decreased(P＜0.05),Bacteroides-H,Muribaculum,Lactobacillus and salivarius in the Positive group salivarius(P＜0.05).Conclusion:Shenling Baizhusan can effectively treat IBS-D,and its molecular mechanism may be to play a therapeutic role by improving intestinal flora and inhibiting the TLRS/MyD88/NF-κB signaling pathway to reduce inflammatory response.