Optimization Of In Vitro Cultivation Of Engineering Bacteria For Bezoar Production And Pharmacological Prediction Of Bezoar’s Therapeutic Potential For Periodontitis

Objective:The objective of this study is to prepare a new engineering strain of bezoar for in vitro cultivation through heavy ion radiation mutagenesis,and to explore the metabolic characteristics of the new engineering strain during fermentation based on metabolomics.This study aims to provide new insights for improving the preparation of bezoar through in vitro cultivation.In addition,in vitro anti-inflammatory experiments of bezoar will be conducted on human periodontal ligament cells induced by LPS,and network pharmacology will be applied to predict the pharmacological effects and related molecular mechanisms of bezoar in the treatment of periodontitis.Method:Bezoar engineering bacteria were cultured and screened through heavy ion radiation mutagenesis.The bacterial growth curve was determined using the WST-8assay,and bacterial properties were examined by dilution and plating on agar.The safety of the engineering bacteria was evaluated through antimicrobial susceptibility testing,and the β-glucuronidase activity of the bacterial strain was measured.Bezoar was produced through the biofermentation of the engineering bacteria,and the content of bilirubin,free bilirubin,and bile acids was determined.Metabolomics analysis was conducted on the above bacterial strains using quality control analysis,principal component analysis,data clustering,OPLS-DA analysis,and differential metabolite screening.The metabolic characteristics of the engineering bacteria during fermentation were studied,and functional annotation and enrichment analysis were performed on significant differential metabolites.An in vitro inflammatory model of human periodontal ligament cells induced by LPS was established,and anti-inflammatory experiments were conducted using bezoar.Network pharmacology and molecular docking technology were employed to predict the molecular mechanism of bezoar in treating periodontitis.Key targets related to bezoar and periodontitis were screened from the database and intersected to obtain core targets.Protein-protein interaction analysis was performed using the STRING database.The core targets were subjected to GO and KEGG enrichment analysis using the DAVID database,and Autodock was used to validate the molecular docking of the effective components of bezoar with key targets.Results:This study obtained an engineered strain of Escherichia coli that can be used for in vitro cultivation of bezoar.Compared with the wild-type strain,this strain showed increased proliferation activity in bile environments,while the activity of β-glucuronidase remained unchanged.We also analyzed the content of bezoar cultivated by the engineered strain,and found that the levels of bilirubin,free bilirubin,and bile acids all met the requirements of the Chinese Pharmacopoeia,with higher levels than those obtained by the wild-type strain.A total of 6972 metabolites were detected,with the most common category being benzene and its derivatives(18.43%).Compared with the bile domestication group(Bo E group),there were 109 different metabolites significantly up-regulated in the heavy ion beam domestication group(HIB group)under positive ion mode,including melatonin,spermine,boldenone,creatine,L-valine,and methylglucamine,which were enriched in the pathways of histidine metabolism,protein digestion and absorption.Under negative ion mode,there were 173 different metabolites significantly up-regulated,including topiramate,gallic acid,glucuronic acid,succinic acid,4-methoxyphenylacrylic acid,metformin,and thymol,which were enriched in the pathways of metabolism.In vitro experiments showed that bezoar significantly inhibited the inflammatory response of human periodontal ligament cells,indicating that bezoar at a concentration of 0.1 mg/ml has good anti-inflammatory effects,and confirming its potential value for treating periodontitis.Network pharmacology was conducted,and24 effective ingredients of bezoar and 665 related genes were identified in the database,along with 1112 potential target genes for periodontitis.The intersection of predicted target genes and potential target genes resulted in a list of 137 genes.Various active components in bezoar acted on core target genes,such as TNF,EGFR,INS,and AKT1,which were associated with periodontitis.Molecular docking experiments showed that the binding energy between the effective ingredients of bezoar and core target genes was less than 0 kcal/mol,indicating stable binding between them.Conclusion:The use of heavy ion radiation-induced mutagenesis technology to prepare engineered bacteria for in vitro cultivation can effectively improve the yield and quality of bezoar.The good anti-inflammatory efficacy of bezoar provides preliminary evidence for its application in inflammatory diseases such as periodontitis.