Study On The Effect-mechanism Of Atractyloside A On Spleen Deficiency Diarrhea Mice Based On Intestinal Flora And TLR4/NF-κB Signal Pathway

1 PurposeSpleen deficiency diarrhea（SDD）is one of the most typical types of diarrhea,which is closely related to intestinal barrier dysfunction and intestinal flora disorder.Atractyloside A is one of the main components of Atractylodes lancea（Thumb.）DC and one of the components with obvious increment after frying or charring Atractylodes macrocephala bran,which can act on the gastrointestinal tract and treat diarrhea,the effect of strengthening intestines and relieving diarrhea was enhanced after frying Atractylodes rhizome,but the underlying mechanism of the enhancement was not clear.Therefore,this experiment selects the incremental component Atractyloside A as the research object,takes the SDD mice as the model,and takes the diarrhea index,pathological section and inflammatory factors as the indicators to investigate the improvement of Atractyloside A on the diarrhea symptoms of model mice,so as to clarify the therapeutic effect of Atractyloside A on SDD.On this basis,the regulation law of Atractyloside A was analyzed based on the regulation of intestinal flora,and the mechanism of Atractyloside A in the treatment of SDD was discussed from the inflammatory pathway.This study provides a theoretical reference for the anti diarrhea effect of Atractylodes A,and provides a basis for explaining the mechanism of the enhancement of intestinal consolidation and anti-diarrheal effect of processing Atractylodes.2 Method2.1 Pharmacodynamic study of Atractyloside A on spleen deficiency diarrheaICR mice were divided into control group（Ctrl）,the spleen deficiency diarrhea group（SDD）,Atractyloside A low-dose group（AAL）,Atractyloside A high-dose group（AAH）and loperamide group（LPM）,with 12 mice in each group.Except the Ctrl group,the spleen deficiency diarrhea was induced by Folium Sennae（SE）,and then treated with 5 mg·kg-1,10 mg·kg-1 Atractyloside A and 3 mg·kg-1 loperamide respectively for 7 days.The therapeutic effects of each drug on diarrhea were investigated by the body weight,fecal moisture content,diarrhea index and diarrhea score of mice as indicators.The macroscopic pathological state of colon and inflammatory were used as indicators to investigate the effects of drugs on the target organs—colon,in other words,the macroscopic changes of colon were observed based on HE staining,and the tumor necrosis factor（TNF-α）in mouse serum and colon was detected based on ELISA and q RT-PCR（TNF-α）,Interleukin-1β（IL-1β）,Interleukin-6（IL-6）,Interleukin-10（IL-10）,Diamine oxidase（DAO）,serotonin（5-HT）.The serotonin 3 receptor（5-HT3R）was evaluated based on immunohistochemical staining,and finally the pharmacodynamic effect of Atractyloside A on SDD was evaluated.2.2 Study on the interaction between Atractyloside A and intestinal floraThe cecal contents of experimental animals were collected with sterile EP tube,based on 16 Sr DNA sequencing technology,the intestinal flora structure of mice in each group was analyzed to investigate the effects of different drugs on the intestinal flora structure of mice.Then,the role of intestinal flora was verified by sterility verification experiment and intestinal flora transplantation experiment（FMT）.In addition,Atractyloside A was co-cultured with intestinal flora from healthy mice in vitro.The effect of intestinal flora on Atractyloside A was investigated based on HPLC,and the interaction between Atractyloside A and intestinal flora was preliminarily explored.2.3 Study on the mechanism of Atractyloside A against spleen deficiency diarrheaICR mice were divided into control group（Ctrl）,the spleen deficiency diarrhea group（SDD）,Atractyloside A low-dose group（AAL）,Atractyloside A high-dose group（AAH）and loperamide group（LPM）,with 12 mice in each group.After the pharmacodynamic study of each drug on SDD,the serum and colon of each mouse were taken and stained based on AB-PAS staining.Immunohistochemical staining and Western blot were used to detect the distribution and expression of goblet cells,MUC2,tight junction protein ZO-1and occludin in the colon of mice in each group.The expressions of aquaporins（AQPs）AQP3,AQP4 and AQP8 in serum and colon of mice in each group were detected based on ELISA and q RT-PCR.The different expressed genes（DEGs）were screened and enriched by RNA sequencing technology.The gene enrichment results were verified based on Western blot and immunofluorescence.3 Results3.1 Pharmacodynamic study of Atractyloside A on spleen deficiency diarrheaCompared with the SDD group,the treatment of Atractyloside A promoted the weight gain of experimental animals.Compared with SDD group,the fecal moisture content,diarrhea index and diarrhea grade score in mice of AAH group were significantly lower,in other words,Atractyloside A effectively alleviated the diarrhea of SDD.All indexes in this part showed that the efficiency of high-dose Atractyloside A was better than the low-dose.In addition,Atractyloside A also significantly improved the symptoms of redness,swelling and bleeding in the colon of SDD mice.We also observed from HE section that Atractyloside A and loperamide inhibited the intestinal inflammatory invasion of SDD mice,reduced intestinal injury and protected the intestine.Compared with the SDD group,the expression of TNF-α,IL-1 β,IL-6,serum DAO and 5-HT were decreased in varying degrees,and the level of IL-10 increased significantly.Through the study of the pharmacodynamic effect of Atractyloside A on SDD mice,it was found that Atractyloside A can significantly improve the diarrhea,intestinal injury and inflammation caused by SE by reducing the fecal moisture content,inhibiting the storm of inflammatory factors in the body,and reducing the levels of neurotransmitters5-HT and DAO.3.2 Study on the interaction between Atractyloside A and intestinal floraThrough 16 Sr DNA sequencing analysis of the cecal contents of mice in each group,it was found that the composition of intestinal flora of mice in each group had changed greatly.Compared with the Ctrl group,the species richness of mice in the SDD group decreased（P<0.05）.At the genus level,compared with the Ctrl group,the abundance of Lactobacillus in the SDD group decreased significantly,and the abundance increased significantly after the treatment of Atractyloside A.After the intestinal flora in mice was eliminated with antibiotics,then Atractyloside A was used to treat germ-free SDD mice.It was found that the anti-diarrhea effect of Atractyloside A disappeared;however,when the intestinal flora from AAH mice were transplanted into germ-free SDD mice,it was found that the diarrhea of mice was alleviated.In addition,Atractyloside A was co-cultured with the intestinal flora of healthy mice in an anaerobic environment to simulate the metabolic process of Atractyloside A in the intestine.It was found that the content of Atractyloside A decreased by about 60 %.The above shows that Atractyloside A can treat SDD by inhibiting pathogenic bacteria and promoting the growth of beneficial bacteria,and the intestinal flora has biotransformation effect on Atractyloside A.3.3 Study on the mechanism of Atractyloside A against spleen deficiency diarrheaWhen further studying the anti-diarrhea mechanism of Atractyloside A,it was found that Atractyloside A could reverse the decrease of goblet cell number,secretion of Mucin 2（MUC2）,expression of AQP3,AQP4,AQP8 and tight junction protein ZO-1 and Occludin in the colon of SDD mice,which was conducive to maintaining the integrity of intestinal barrier.The DGEs between different groups were analyzed by RNA sequencing technology,and it was found that they were highly enriched in NF-κB and TNF pathways.Further validation of related genes in NF-κB and TNF pathway were conducted,and Atractyloside A was found to interfere with phosphorylation of NF-κB and nuclear translocation of p65,and it down regulated the expression of inflammatory genes,including p-65,IκB-α,IKK-α,My D88,TLR4,TNF-α etc.4.ConclusionIn this study,the mouse model of SDD was induced by Folium Sennae,and then treated with Atractyloside A to explore the therapeutic effect of Atractyloside A on SDD mice.Through the degree of diarrhea and intestinal injury in mice,the pharmacodynamic effect of Atractyloside A on SDD was clarified,and then the change of intestinal flora and the regulation of inflammation were deeply studied.The results showed that Atractyloside A could significantly improve the diarrhea symptoms of SDD mice,inhibit the release of inflammatory factors,reduce inflammation and improve the pathological changes of intestinal tissue.The mechanism is mainly through two aspects.On the one hand,Atractyloside A can protect the intestinal tract by increasing beneficial bacteria and reducing pathogenic bacteria in the intestinal tract,and the sterility verification and intestinal flora transplantation experiments made it clear that the intestinal flora is an indispensable link for Atractyloside A to play its therapeutic role.On the other hand,the expression of genes related to TLR4/NF-κB signaling pathway could be inhibited by Atractyloside A,which decreased the secretion of intestinal pro-inflammatory factors,and increased the synthesis of aquaporin and tight junction protein on cell membrane,and increased the number of goblet cells and mucin.Finally,Atractyloside A plays two roles in the treatment of diarrhea.