A Pilot Study On The Treatment Effect Of Wumei Pills Of Interventions On Chemotherapy-induced Intestinal Mucositis

Objective&Significance:Background:Chemotherapy-induced intestinal mucositis(CIM)is a common dose-limiting toxic effect of drugs with diarrhea and inflammation as the main manifestations during anti-tumor treatment.There is a lack of effective methods in clinical practice.Traditional Chinese medicine can increase efficacy and reduce toxicity in the process of anti-tumor.The previous meta-analysis indicated that the use of traditional Chinese medicine during chemotherapy can effectively improve the treatment effect and KPS score of chemotherapy-induced diarrhea,and its safety is good.Wumei Pills(WMP)is a classic prescription in Treatise on Exogenous Febrile Disease for the treatment of syndrome of intermingled heat and cold.It has significant effect on inflammatory diarrhea caused by ulcerative colitis and irritable bowel syndrome.The literature research found that Wumei Pills has significant clinical relieving efficacy in treating CIM,but the specific mechanism of action is still unclear.Therefore,clarify the mechanism and links of Wumei Pills on CIM,and provide new targets for the prevention and treatment of CIM.More and more evidences indicate that the intestinal flora and its metabolites play an important role in the pathogenesis of CIM.Meta-analysis suggested that the application of probiotics before or during chemotherapy can effectively prevent cancer patients from developing CID.In addition,the combination of probiotics to treat CID can also improve the therapeutic effect of CID,and the safety is good.Therefore,the purpose of this topic is to:1.Use network pharmacology to determine the potential targets and mechanism of Wumei Pills to provide theoretical basis for following experimental research.2.From the overall animal experiment level,explore the specific mechanism of Wumei Pills's intervention on CIM.On the one hand,it provides theoretical basis for the clinical application and secondary development of Wumei Pills,and on the other hand,it lays the experimental foundation for the discovery of new drugs in clinical CIM and the development of microecological preparations,which has considerable clinical application prospects and scientific significance.Methods:1.Based on network pharmacology to explore the mechanism of Wumei pills in the treatment of chemotherapy-induced intestinal mucositisThe related active compounds of Wumei Pills were obtained from TCMSP database,TCM database@Taiwan and BATMAN database,and the potential targets of these compounds were collected through Uniprot.Then construct the CIM target gene database through GeneCards and OMIM database.Common targets were screened out,a protein interaction network was constructed,and select hub targets through topological parameters.Finally,perform GO function and KEGG pathway enrichment analysis.2.Research on the alleviating effect and mechanism of Wumei Pills on chemotherapy-induced intestinal mucositisIn this study,Balb/C mice were selected and 5-Fu(40mg/kg)was injected intraperitoneally for 5 consecutive days to establish a CIM mouse model.Wumei Pills were intragastrically administered for 10 consecutive days.The body weight,food intake,score of diarrhea,colon length and HE staining were observed.Detect the level of inflammatory cytokines(IL-1?,MPO,IL-6,TNF-?)and the expression of protein of TLR4/Myd88/NF-?B signal pathway to evaluate the efficacy of Wumei Pills on CIM.Further detect intestinal leakage,mucus layer thickness and the expression of mucin Mucin-2 and tight junction proteins(ZO-1,Claudin-1,E-cadherin)to evaluate the protective effect of Wumei Pill on the intestinal mucosal barrier.Next 16s rDNA sequencing was performed to detect the influence of Wumei Pills on the composition and abundance of intestinal flora;and use mass spectrometer(MS)to detect the content of short-chain fatty acids in mouse feces to evaluate the effect of Wumei Pills on the metabolites of intestinal flora.Finally,through fecal microbiota transplantation experiment,the fecal flora of the mice in the 22650 mg/kg Wumei pill group was given to the 5-Fu group mice,and the effects of fecal microbiota transplantation on the intestinal mucosal inflammation,intestinal mucosal barrier and short chain fatty acids were observed.Results:1.Based on network pharmacology to explore the mechanism of Wumei pills in the treatment of chemotherapy-induced intestinal mucositis97 active ingredients and 205 targets of Wumei pills were screened out.The results of DAVID enrichment analysis showed that there were 51 GO items(FDR<0.01)and 20 pathways(FDR<0.01)in Wumei Pills.The GO enrichment analysis indicated that in terms of biological functions,it mainly involves apoptosis,immune response,proliferation and inflammation.In terms of cell composition(CC),including the extracellular space,the outer plasma membrane and the extracellular area;molecular function(MF),it mainly involves cytokine activity,oxygen binding,growth factor activity and glutathione binding.KEGG enrichment analysis indicated that the main signaling pathways include endocrine resistance signaling pathway(hsa01522),HIF-1 signaling pathway(hsa04066),chemokine signaling pathway(hsa04062),Ras signaling pathway(hsa04014),MAPK signaling pathway(hsa04010),EGFR tyrosine the amino acid receptor signaling pathway(hsa01521),NF-?B signaling pathway(hsa04064).They are all related to inflammation,cell proliferation,and apoptosis,which are in line with GO enrichment analysis.In addition,the 19 hub nodes of Wumei Pills(JUN,IL6,MAPK8,AKT1,MAPK1,VEGFA,PTGS2,MMP9,IL1B,CXCL8,EGF,MYC,CASP3,EGFR,FOS,RELA,STAT1,CCND1 and ESR1)are considered to be potential targets for treatment.Combined with literature research and network pharmacology analysis,we believe that the NF-?B signaling pathway may be the potential mechanism of Wumei Pills on chemotherapy-induced intestinal mucositis,which will be verified later.2.Research on the alleviating effect and mechanism of Wumei Pills on chemotherapy-induced intestinal mucositisWumei Pills can significantly reverse the body weight,food intake reduction,colon shortening and pathological damage caused by 5-Fu,reduce the diarrhea score and inflammatory cytokines(IL-1?,MPO,IL-6,TNF-?)of mice in the 5-Fu group,and further reverse the high expression of protein in TLR4/Myd88/NF-?B signaling pathway,which is more obvious in the 22650mg/kg Wumei Pill group,thereby alleviating the inflammatory damage of the intestinal mucosa.Further research found that Wumei Pills can reverse the high leakage of the intestinal in 5-Fu mice,reduce expresiion of tight junction proteins(ZO-1,Claudin-1,E-cadherin),and increase the thickness of the mucous membrane and the mucin-2 secretion,thereby protecting the intestinal mucosal barrier.The final intestinal flora results show that Wumei pills can increase the composition and abundance of the intestinal flora in the 5-Fu mice,increase the abundance of Lactobacillus,and reduce the Bacteroides abundance.Fecal short chain fatty acids(SCFAs)detection found that Wumei Pills can reverse the decrease of SCFAs caused by 5-Fu and increase the content of butyric acid.Finally,the experimental results of fecal microbiotatransplantation show that fecal bacteria transplantation can significantly alleviate the Inflammatory pathology state of intestinal mucosal in 5-Fu mice,repair the intestinal barrier damage,and promote the synthesis and metabolism of short-chain fatty acids in the intestine of 5-Fu mice.It is revealed that Wumei Pills can affect the synthesis and metabolism of short-chain fatty acids by changing the composition of the intestinal flora,thereby inhibiting the occurrence and development of inteatinal inflammation.Conclusions:1.Based on network pharmacology to explore the mechanism of Wumei pill in the treatment of chemotherapy-induced intestinal mucositisThis study provides a theoretical basis for the clinical application of traditional Chinese medicine Wumei Pills in the treatment of CIM.And this study fully demonstrates that traditional Chinese medicine has the characteristics of multi-compound,multi-target,multi-pathway active prescription,which is of great significance to the study of the efficacy of traditional Chinese medicine prescription.2.Research on the alleviating effect and mechanism of Wumei Pill on chemotherapy-induced intestinal mucositisBased on experimental results,we found that intragastrically administered mice with 11325 mg/kg and 22650 mg/kg Wumei Pills can alleviate the intestinal mucosal inflammation induced by 5-Fu,and 22650 mg/kg Wumei Pills are more of efficacy.Wumei Pills can change the abundance and structure of the intestinal flora in 5-Fu mice,increase the abundance of Lactobacilli,thereby increase the synthesis and metabolism of SCFAs,repair the intestinal mucosal barrier,and play a role in relieving intestinal mucosal inflammation.Fecal microbiota transplantation also confirmed that the intestinal flora may be the mechanism of Wumei pills in the treatment of chemotherapy-induced intestinal mucositis.This study expounds the scientific connotation of Wumei Pills in the treatment of CIM from the perspective of intestinal flora,and provides theoretical basis for the clinical application and secondary development of Wumei Pills.