Therapeutic Effect And Mechanism Of Yujin Powder Alcoholic Extracts On Ulcerative Colitis In Mice

Ulcerative colitis（UC）is a chronic non-specific inflammatory bowel disease involving the mucosa of colon and rectal.Due to the high incidence,lingering disease,treatment difficulty and poor prognosis,UC seriously affects the life quality and economic state of patients.Yujin Powder（YJP）is a classic prescription for the treatment of large intestine damp-heat diarrhea.Its active ingredients have the effects on anti-inflammation,repairing intestinal mucosa and anti-ulcer.In this study,we first evaluated therapeutic effect of Yujin Powder alcoholic extracts（YJP-A）on UC mice.And then,its therapeutic mechanism was explored through ILC3s-Tfh-TD IgA axis;on this basis,the effect of YJP-A on TD IgA-targeted colonic mucosal flora in UC mice was detected.Finally,the mechanism of YJP-A on UC mice was further explored through interaction of enteric nerve-immune system.The main methods and results were as follows:1.Evaluation of therapeutic effect of YJP-A on UC mice.Forty-eight BABL/c mice were randomly divided into normal control（NC）,Model,Sulfasalazine（SASP）,YJP-A high dose（YJP-A-H,14.56 g/kg）,YJP-A middle dose（YJP-A-M,7.28 g/kg）and YJP-A low dose（YJP-A-L,3.64 g/kg）groups.Except NC group was given distilled water,other groups were given 3.5%dextran sodium sulfate（DSS）drinking water for7 consecutive days.At the same time,the treatment groups were orally given different dose of YJP-A and SASP for treatment.During the experiment,the clinical symptoms,the body weight,stool consistency,and occult blood/thick blood of stool of mice in each group were observed and recorded,and the disease activity index（DAI）was monitored.After the experiment,the colorectum of mice in each group were collected and the colon length from the anus to ileocecal junction were measured and recorded.And then the colon tissue was detected for histopathology,some indicators of inflammatory cytokines and intestinal mucosal barrier.The results showed that different doses of YJP-A could significantly alleviate the weight loss and DAI increase of UC mice（P<0.05）;significantly inhibit the colon shortening and inflammatory response（P<0.05）;reduce pathological damage of colon and restore the expression of tight junction proteins（ZO-1 and Occludin）in intestinal mucosa（P<0.05）.The above treatment results showed an obvious dose-effect relationship,and the YJP-A-H had the best treatment effect.2.Exploration of the therapeutic mechanisms of YJP-A on UC mice by ILC3s-Tfh-TD IgA axis.For NC,Model,SASP and YJP-A-H groups,the number of ILC3s,Tfh and B cells,the expression of MHC II,Bcl6,IL-4,Aicda in the mesenteric lymph nodes and the expression of IgA in the colon were detected by flow cytometry,WB,ELISA or immunofluorescence.Finally,molecular docking technology was used to analyze the molecular regulatory mechanism of 13 active components in YJP-A on ILC3s-Tfh-TD IgA axis.The results showed that YJP-A significantly increased the ILC3s proportion and the expression level of MHC II（P<0.05）;significantly decreased the proportion of Tfh and B cells and the expression of Bcl6,IL-4,Aicda in mesenteric lymph nodes of colon in UC mice and IgA in colon（P<0.05）.In addition,molecular docking analysis showed that the 13 active components of YJP-A had strong binding to key proteins（MHC II,Bcl6,IL-4 and Aicda）in the ILC3s-Tfh-TD IgA axis,which suggested that these active components could activate ILC3s-Tfh-TD IgA axis through the above proteins.3.Effects of YJP-A on colonic mucosal flora in UC mice.For NC,Model,SASP and YJP-A-H groups,the colonic mucus was scraped with sterile slides,and the changes of colonic mucosal flora were analyzed by 16S r RNA high-throughput sequencing technology.The results showed that YJP-A could significantly increase alpha diversity,abundance and evenness of colonic mucosal flora in UC mice.In addition,YJP-A also significantly improved the structure of colonic mucosal flora,reduced the relative abundance of pathogens associated with inflammatory bowel disease（Mucispirillum and Lachnospiraceae）and increased the relative abundance of short chain fatty acids-producing bacteria（Allprevotella,Alistipes,Desulfovibrio,Ruminococcaceae）.4.Regulation of YJP-A on myenteric nervous-immune system in UC mice.The colonic muscularis of mice were collected among the NC,Model,SASP and YJP-A-H groups.The expression of NE,β₂-AR,Nlrp6 and Arg-1 in colonic muscularis were detected by WB,ELISA or immunofluorescence;the number of muscularis macrophages（MMs）and the damage of the myenteric plexus were detected by immunofluorescence.The results showed that YJP-A could significantly alleviate the damage of the myenteric plexus,decrease the number of MMs and expression of Arg-1（P<0.05）.In addition,YJP-A significantly increased the expression of NE,β₂-AR and decreased the expression of Nlrp6 in colonic muscularis of UC mice（P<0.05）.Conclusion:（1）YJP-A could exert therapeutic effect on UC in mice through alleviating weight loss and increasing DAI,inhibiting inflammatory response,repairing colon tissue damage and enhancing intestinal mucosal barrier function.（2）YJP-A could regulate ILC3s-Tfh-TD IgA axis in UC mice.（3）YJP-A could maintain the homeostasis of colonic mucosal flora in UC mice through regulating the abundance,composition and structural changes.（4）YJP-A could regulate the expression of Nlrp6,NE andβ₂-AR in colonic muscularis of UC mice,further protect the damage of the myenteric plexus and restore its interaction with MMs to normal.In conclusion,this study revealed part of the mechanism of YJP-A on the treatment of UC in mice,which broadened the strategy of treatment of UC,provided theoretical basis and foundation for YJP-A treating UC,and provided a new evidence for exploring new drug development.