Study On Safety Evaluation Of Mongolian Medicine Digda-4 Decoction

Objective:In this study,the subacute toxic dosage and toxic target organs of Digda-4 decoction containing gardenia and nutmeg were preliminarily explored by means of subacute toxicity research,and its subacute toxic mechanism was analyzed by 16SrDN A high-throughput sequencing method and transcriptomics method,so as to provide scientific data for the safe and rational use of Digda-4 decoction.Methods:1.Decomposed research methods:Taking Digda-4 decoction and its disassembled herbs,Gardenia,Nutmeg,Gardenia and Nutmeg,as example drugs,SD rats were gavaged with corresponding solution once a day for 21 days,except for the normal group.The changes of liver,renal function and pathomorphology of rats in each group were observed by biochemical detection and HE staining.To evaluate the subacute toxic dose and toxic target organ of Digda-4 decoction.2.The intestinal flora in the feces of each group was sequenced by 16SrDNA highthroughput sequencing.Various bioinformatics analysis and statistical methods were comprehensively used to analyze the species composition of flora in each group and the species function between groups,and the differential flora and differential pathways related to subacute toxicity of Digda-4 Weitang were screened at the level of genus and purpose.3.RNA was extracted from rat kidney tissue in experiment 1,and the library was established and sequenced.Quality control of the information data to be tested,reference genome and gene expression quantification to obtain differential genes;Analysis of KEGG enrichment pathway of differential genes.Finally,the differential genes corresponding to the key components in Digda-4 flavored soup were verified by molecular docking.Results:1.The results of subacute toxicity test showed that the indexes of ALT,AST,UREA and CREP in DG group were significantly lower than those in normal control group;UREA index in DZ group increased significantly,while ALT index in ZG group decreased significantly.UREA index in ZZ group decreased significantly;ALT index in ZD group increased significantly,and UREA index in RG group decreased significantly.UREA index in RZ group increased significantly;UREA index in ZRG and ZRZ groups decreased significantly.Pathological observation showed that the epithelial cells of renal tubules around blood vessels in rats in the treatment group were denatured,swollen and expanded to varying degrees.2.The detection results of intestinal flora showed that DZ could significantly up-regulate Enterococcus and Bacteroides,and significantly down-regulate Lactobacillus,Bifidobacterium and Blaut.ZZ significantly up-regulated Bacteroides and down-regulated Bifidobacterium,Ackermann and Blaut.RZ can significantly reduce the abundance of Bifidobacterium,Blaut,Lactobacillus and Streptococcus.ZRZ can significantly down-regulate the abundance of Lactobacillus and Blaut,and the order of Bifidobacterium is significantly down-regulated in DZ,ZZ and RZ groups.Through plant hormone signal transduction,RNA transport,lysine biosynthesis,degradation of valine,leucine and isoleucine,methane metabolism,arachidonic acid metabolism,biotin metabolism,longevity regulation pathway,FoxO signal and other nine flora functions cause intestinal flora imbalance.3.The results of transcriptomics show that ZC and DZ,ZZ,RZ and ZRZ groups respectively correspond to 291,612,31 and 5 significantly different genes.ZZ,RZ and DZ were co-expressed in Prkaa2 gene,while ZZ and DZ were co-expressed in Ccn1,Fnip2,Rbm47,Pkhd1,C1 galt1 and Prkaa2 gene.The differential genes in DZ,ZZ and RZ groups were enriched in 15,16 and 3 pathways respectively.Among them,ZC and DZ,ZZ and RZ groups are co-enriched in one pathway of circadian rhythm,while ZC and DZ and ZZ groups are coenriched in seven pathways,including insulin resistance,circadian rhythm,adipocytokine signaling pathway,FoxO signaling pathway,hypertrophic cardiomyopathy,AMPK signaling pathway and oxytocin signaling pathway.4.The results of molecular docking test showed that the active components of Digda-4 decoction showed that the up-regulated genes with the highest geniposide score were Birc5 and Ccnb2;Down-regulated genes:C1galt1,Pkhd1,Rbm47.The docking genes with the highest gentiopicroside content are up-regulated genes:Ccn1,Kif18b and Nphs2;Down-regulated genes:Fnip2,Prkaa2.Swertiamarin was up-regulated after docking with Plkl gene.Conclusion:1.The low dose(clinical equivalent dose)of Digda-4 decoction has no obvious subacute toxic effect,but the middle dose(6 times clinical equivalent dose)group has obvious subacute toxic effect on kidney.Therefore,Digda-4 decoction has a certain subacute toxicity when it is used continuously for 21 days at a dose of 6 times the clinical equivalent dose.2.The subacute toxicity of Digda-4 decoction changes the species number and diversity of intestinal flora in rats,which is related to the changes of intestinal flora abundance of Gardenia and Nutmeg.Significantly up-regulated Bacteroides,significantly down-regulated Lactobacillus,Bifidobacterium,Blaut,Bifidobacterium and other flora abundance.3.Transcriptomics and molecular docking analysis of Digda-4 Weitang showed that the target genes of nephrotoxicity were Birc5,Ccnb2,C1galtl,Pkhd1,Rbm47,Ccn1,Kif18b,Nphs2,Fnip2,Prkaa2 and Plk1,among which the up-regulated genes were Birc5,Ccnb2,Ccn1,Kif18b,Nphs2 and Plk1.It mainly involves the regulation of insulin resistance,circadian rhythm,adipocytokine signaling pathway,FoxO signaling pathway,hypertrophic cardiomyopathy,AMPK signaling pathway and oxytocin signaling pathway.It is preliminarily considered that the molecular mechanism of subacute renal toxicity of Digda-4 decoction.