Investigation Of Therapeutic Mechanism Of Tongfu Jiangzhuo Decoction On Rats With Constipation Predominant Irritable Bowel Syndrome Based On The Regulation Of Intestinal Microflora To Neuroendocrine Factors

Objective: To observe the effect of Tongfu Jiangzhuo decoction（TF）on the general condition,fecal character score,fecal water content and visceral sensitivity of rats with constipation predominant irritable bowel syndrome（IBS-C）,and to detection the change of intestinal microflora and substance P（SP）,and vasoactive intestinal peptide（VIP）,levels of the gut,discuss the therapeutic mechanism of TF and then provide experimental basis for the clinical application.Methods: 49 male SD rats were randomly divided into the normal control group（N）,the model group（N）,the western medicine group（X）,the prebiotics group（Y）,the high dose TF group（ZH）,the medium dose TF group（ZM）,the low dose TF group（ZL）,7 in each group.IBS-C rat models were established by ice water by gavage,except of the normal control group.After successfully modeling,corresponding medication was administered to each treatment group.Equal volume of saline solution was administered to rats in the normol control group and the model group,all for 14 days.The general condition,fecal character score,fecal water content were observed.The visceral sensitivity were determined by abdominal withdrawal reflex（AWR）.Immunohistochemical assay was used to detect expression levels of SP and VIP in colon tissue.The changes of intestinal microflora and the relevance with SP and VIP were analyed through Illumina high-throughput sequencing technology.Results: 1.The general condition Compared with N group,the rats appeared loss of appetite and energy, irritability,lower intake after modeling.Meanwhile,the fecal showed stem ball.After treatment,the general condition of each group improved.Expect of X group,the other rats of the treatment groups tended to be normal in regard to fecal character.2.The fecal character score and fecal water content Compared with N group,the fecal character score significantly reduced in the modeling groups（P<0.05）.After treatment,compared with M group,the fecal character score in Y group,ZH group,ZM group and ZL group all significantly increased（P<0.05）.Compared with N group,the fecal water content significantly reduced in the modeling groups（P<0.05）.After treatment,compared with M group,the fecal water content in X group,Y group,ZH group,ZM group and ZL group all significantly increased（P<0.05）.Compared with X group,the fecal water content in Y group,ZH group,ZM group and ZL group all significantly increased（P<0.05）.3.The visceral sensitivity Compared with N group,the visceral sensitivity significantly increased in the modeling groups（P<0.05）.After treatment,compared with M group,the visceral sensitivity in X group,Y group,ZH group,ZM group and ZL group all significantly reduced（P<0.05）.Compared with X group,the visceral sensitivity in Y group,ZH group,ZM group and ZL group all significantly reduced（P<0.05）.Compared with Y group,the visceral sensitivity in ZH group,ZM group and ZL group all significantly reduced（P<0.05）.4.The changes of intestinal microflora 49 samples were analyzed and tags were 4143321.The species accumulation boxplot showed that the sample content was adequate.Analysis of alpha diversity indicated that,the richness of intestinal microflora reduced in M group.After treatment,that of Y group and ZH group approached to normal.Analysis of beta diversity indicated that,there was significant difference in M group and N group for the structure of intestinal microflora.After treatment,that of ZH group was most closed to that of N group. The statistics analysis of intestinal microflora showed that,three bacterial phylum,namely firmicute,acidobacteria and virrucomicrobia,six bacterial class,namely bacilli,alphaproteobacteria et al,four bacterial family,namely lactobacillus,bifidobacterium et al all significantly reduced in IBS-C rats.Meanwhile,one bacterial phylum,namely bacteroidetes,one bacterial class,namely bacteroidia,16 bacterial family,namely revotellaceae_NK3B31_group,prevotella₉ et al all significantly increased in IBS-C rats.Tongfu Jiangzhuo detection can improve the balance of the intestinal microflora of IBS-C rats.5.The expression of SP and VIP in colon tissue Compared with N group,the expression of SP in colon tissue significantly reduced and that of VIP significantly increased in M group（P<0.05）.Compared with M group,the expression of SP in colon tissue significantly increased and that of VIP significantly reduced in X group,Y group,ZH group and ZM group（P<0.05）.6.Correlative analysis:the expression of SP and VIP in colonic tissue and the intestinal microflora Correlative analysis showed that,there was a positive correlation between verrucomicrobia,acidobacteria,bacilli,erysipelotrichia,lactobacillus,allobaculum et al and the expression of SP in colonic tissue,meanwhile,that bacteria exhibit a negative correlation with the expression of VIP in colonic tissue（P<0.05）.there was a negative correlation between bacteroidetes,bacteroidia,prevotellaceae_NK3B31_group,alloprevotella et al and the expression of SP in colonic tissue,meanwhile,that bacteria exhibit a positive correlation with the expression of VIP in colonic tissue（P<0.05）.Conclusions: 1.The expression of SP in colonic tissue is reduced and that of VIP is increased in the IBS-C rats.Intestinal microflora is of imbalance in the IBS-C rats.2.Tongfu Jiangzhuo decoction can improve the IBS-C rats fecal character score,increase the fecal water content and reduce the visceral sensitivity,which is an effective method for the treatment of IBS-C.3.Tongfu Jiangzhuo decoction can regulate the intestinal microflora,so as to up-regulate the expression of SP,down-regulate the expression of VIP in colonic tissue,finally balance the neuroendocrine factors,which may be the therapeutic mechanism.