Exploring The Mechanism Of Qiwei Baizhu Powder In The Treatment Of T2DM Based On Small RNA Transcriptome Sequencing

Previous experiments have proved that Qiwei Baizhu Powder can effectively improve the intestinal barrier of T2 DM,improve blood sugar level and insulin resistance,but the impact on liver steatosis is still unclear.Therefore,we explore the pathogenesis of liver mi RNA m RNA as a regulator of Metabolic disorder in T2 DM at the molecular level and the therapeutic mechanism of Qiwei Baizhu Powder,providing more scientific evidence for the study of Qiwei Baizhu Powder in treating T2 DM.ObjectiveBased on small RNA transcriptome sequencing,this study explored the mechanism of Qiwei Baizhu Powder in treating T2 DM.Method1.Effect of Qiwei Baizhu Powder on T2 DM model rats.Four-week-old SD rats were fed adaptively for 7 days,and then randomly divided into blank group(10 rats)and model group according to their weight.The blank group rats were fed with common feed,while the model group rats were fed with high-sugar and high-fat feed.Four weeks later,the T2 DM rat model was established by intraperitoneal injection of STZ(streptozotocin)combined with high-sugar and high-fat feed.After 3-5days,the blood sugar level was measured,and the fasting blood sugar value > 11.1mmol/L was selected as the successful model,with a total of 50 rats.The model group was randomly divided into five groups,namely model group,metformin positive drug control group,low dose group of Qiwei Baizhu powder,middle dose group of Qiwei Baizhu powder and high dose group of Qiwei Baizhu powder.Ten rats in each group were given corresponding drugs by gavage for 4 weeks.During the period,the blank group and the model group were given the same amount of normal saline,and the weight changes of rats were monitored every week.FBG was detected in the second and fourth weeks,and rats were killed four weeks later.Liver tissue and serum samples were taken,and FBG and fasting insulin content(FINS)were measured,and HOMA-IR,HOMA-IS and Homa-βwere calculated.Total cholesterol(TC),triglyceride(TG),high density lipoprotein(HDL-C)and low density lipoprotein(LDL-C)were measured.Alanine aminotransferase(ALT)and aspartate aminotransferase(AST);Serum creatinine(CREA)and urea nitrogen(BUN).Morphological observation of liver tissue,HE staining and oil red O staining.2.small RNA transcriptome sequencingOn the basis of the above experiments,the blank group,model group and high-dose group of Qiwei Baizhu Powder were selected,and the liver tissues of 3 rats were randomly selected for transcriptome sequencing.Screening its mi RNA differential genes,predicting the target genes through five databases such as mi RDB,and constructing mi RNA-m RNA relationship pairs;After crossing the m RNA of model group vs.blank group and treatment group vs.model group,the target gene was obtained.After human-mouse homologous gene transformation,PPI,GO and KEGG were analyzed.3.Experimental verificationThe m RNA of Qiwei Baizhu Powder collected by TCMSP database and the target gene obtained by sequencing intersect again,and the active components corresponding to the target gene are obtained by reverse network pharmacology,and the targets corresponding to the core components are screened according to the Degree value,which is verified by molecular docking.Immunohistochemical staining was used to verify the expression of Mapk8 and Tfrc in liver and q RT-PCR experiment was used to verify the expression of mir-204-5p,mir-224-5p,Mapk8 and Tfrc.ResultCompared with the model group,Qiwei Baizhu Powder can significantly reduce FBG,serum FFA and HOMA-IS levels(P < 0.01),significantly increase ISI and HOMA-β levels(P < 0.01),reduce body weight(P < 0.05)and significantly improve ALT,AST and CREA(P < 0.01)in T2 DM rats,with the best curative effect.HE staining showed that the liver tissue structure in the model group was disordered,hepatocytes were swollen,vacuoles appeared in a large number of cells,some necrosis and inflammatory cell infiltration appeared,showing diffuse steatosis;The results of oil red O staining showed that a large number of lipid droplets appeared in the liver tissue of the model group.However,after the drug intervention of Qiwei Baizhu Powder,the liver pathological morphology of rats was improved in different degrees under the action of different doses,and the vacuolar degeneration of liver cells was significantly reduced,especially in the high-dose group of Qiwei Baizhu Powder.The sequencing results of small mi RNA transcriptome showed that the quality control was good,and there were 14 significantly different genes involved in the pathogenesis of T2 DM,of which 9 were up-regulated and 5 were down-regulated.There are two significantly different genes in the treatment of Qiwei Baizhu Powder.According to the prediction of the website,there are target genes regulated by mi RNA in the model group vs.blank group,and the PPI analysis of these target genes shows that the key target genes are FOXO1,IGF1,MAP2K1 and NR3C1 according to the degree value.GO analysis shows that the functions are mainly enriched in positive regulation of early endosome to late endosome transport,positive regulation of cytopathic transport,The regulation of early endosome to late endosome transport,KEGG results show that it is mainly related to Prostate cancer,Colorectal cancer,Fox O signaling pathway and EGFR tyrosine kinase inhibitor resistance pathways.The target genes of the model group were intersected with those of the high-dose group of Qiwei Baizhu Powder,and 194 crossed target genes were obtained.PPI analysis showed that the key target genes were MAPK8,TFRC,SORT1,NTRK2,HNRNPC and AP1S2;according to the degree value.GO analysis showed that the therapeutic effect of Qiwei Baizhu Powder was mainly concentrated in the mesenchymal cell development;of BP.KEGG is mainly concentrated in Pathways in cancer and PI3K-Akt signaling pathway.The active components obtained from reverse network pharmacology are lappadilactone,Cerevisterol and Genkwanin,and the molecular docking results show that the key active components have good binding with the core targets.Immunohistochemical results showed that Qiwei Baizhu Powder significantly decreased the expression of Mapk8 and increased the expression of Tfrc(P < 0.05).The results of q RT-PCR showed that Qiwei Baizhu Powder could significantly down-regulate MAPK8(P < 0.05),while the other genes were not statistically significant,but there was still a trend.Conclusion1.Qiwei Baizhu Powder can significantly improve blood glucose and lipid levels,improve IR,and improve liver and kidney function related indicators in T2 DM model rats.2.mir-204-5p and mir-224-5p are involved in the mechanism of Qi Wei Bai Zhu San in strengthening spleen and promoting dampness at the non coding RNA level.3.Tfrc and Mapk8 are involved in the mechanism of Qi Wei Bai Zhu San in strengthening spleen and promoting dampness at the coding RNA level.