Study On The Protective Mechanism Of Codonopsis Pilosula Aqueous Extract On Intestinal Mucosa Of Ulcerative Colitis Based On PI3K/AKT Pathway Mediated Oxidative Stress Injury

In this paper,we investigated the potential mechanism of Codonopsis pilosula in the intervention of ulcerative colitis（UC）using network pharmacology and molecular docking;By establishing a rat model of UC induced by 2,4,6-trinitrobenzene sulfonic acid（TNBS）/ethanol,the effects of PI3K/AKT on the regulation of colon tissue differential metabolites by Codonopsis pilosula aqueous extract（CPAE）in the intervention of UC were investigated using network pharmacology combined with metabonomics,with the focus on oxidative stress induced intestinal mucosal injury;Using transcriptomics to explore the role of PI3K/AKT and its significantly enriched differential genes in the intervention of CPAE in UC;Using animal experiments,we explored the oxidative stress damage mechanism of CPAE based on PI3K/AKT regulatory intervention in downstream signaling pathways to improve hypoxia,mucosal cell iron death,and abnormal energy metabolism in UC tissue.Finally,a comprehensive analysis was made of the characteristics of Codonopsis pilosula’s multi-dimensional intervention in oxidative stress injury of intestinal mucosa in UC model rats with"multi point significant effect and synergistic effect";To explore the mechanism of Codonopsis pilosula in the treatment of UC and provide reference for further exploring the potential application value of Codonopsis pilosula.1 Pharmacodynamic evaluation of CPAE in the treatment of UCRat UC models were established and randomly divided into model group,sulfasalazine（SASP,0.3 g·kg-1）group,CPAE low dose（DSL,4.5 g·kg-1）group,CPAE medium dose（DSM,9 g·kg-1）group,CPAE high dose（DSH,18 g·kg-1）group,and iron inhibitor（Fe,0.8mg·kg-1）group.Rats in each group were administered orally once a day for 7 consecutive days,and daily physical signs such as animal diet,drinking water,defecation,hair color,weight,and mental status were recorded.HE staining was used to observe the pathological condition of colon tissue,and TDI and DAI scores were performed.Detection of serum inflammatory factor IL-1β,IL-6,IL-8,IL-17,TNF-α,NF-κB,CRP,INOS,PCT,D-LA by ELISA;The content of MPO in colon tissue,serum GSH,MDA and SOD activity were measured by biochemical methods.The results showed that CPAE could restore the body weight of UC model rats to varying degrees,and improve the condition of bloody and watery stools;It can improve mucosal congestion and edema,inflammatory cell infiltration at the base,goblet cell reduction,accompanied by mucosal erosion,crypt structure changes,and reduce DAI and TDI scores in UC model rats to varying degrees;It can improve serum GSH level and SOD activity;Reduce serum IL-1β,IL-6,IL-8,IL-17,TNF-α,NF-κB,CRP,PCT,INOS,D-LAand MDA levels reduce MPO content in colon tissue.The results suggest that the therapeutic effect of CPAE on UC may be related to inhibition of inflammatory cytokines and antioxidant stress.2 Study on the potential mechanism of Codonopsis pilosula in treating UC through network pharmacologyDatabases such as TCMSP,Gene Card,DisGeNET,and String mine and screen potential active ingredients,targets,and disease targets of Codonopsis pilosula,and construct a PPI network;Network topology analysis screened drug disease core targets and conducted GO and KEGG enrichment analysis.Molecular docking predicts the stability of target binding of traditional Chinese medicine Codonopsis pilosula in interfering with UC components.The results showed that there were 134 chemical components,21 active components,73therapeutic targets,and 37 key targets in Codonopsis pilosula;The results of GO analysis and KEGG signal pathway enrichment analysis showed that the key targets mainly involved infection,apoptosis,immune,inflammatory pathways,AKT,IL-6,VEGF,EGFRand other closely related targets;Pathways such as IL-17 and PI3K/AKT are potential mechanisms for the treatment of UC with Codonopsis pilosula.The results showed that PI3K/AKT was a significantly enriched signal pathway in the treatment of UC with Codonopsis pilosula;Molecular docking showed that the active components of Codonopsis pilosula bind stably to potential targets.3 Study on the mechanism of CPAE in treating UC by metabonomicsUsing metabonomics to detect poor metabolic foreign bodies in colon tissue,combining PCA,OPLS-DA to screen for poor metabolic foreign bodies,GO analysis,and KEGG enrichment to analyze possible metabolic pathways.The differential metabolites are mapped through a database to find key targets,and interconnected with the core targets predicted by network pharmacology to enrich and analyze the associated bioinformatics processes and signal pathways.The results showed that 662 metabolites were identified in UPLC-MS/MS positive ion mode and 287 metabolites were identified in negative ion mode.Metabolite pathway enrichment analysis found that CPAE interferes with UC disease by regulating purine metabolism,glycerol phospholipid metabolism,arachidonic acid metabolism,arginine and proline metabolism,and tyrosine metabolism pathways.Metabolic foreign body related targets and potential network pharmacological targets are ultimately enriched in signal pathways such as PI3K/AKT,MAPK and VEGF.The results indicate that PI3K/AKT is a significantly enriched signaling pathway for differential metabolite regulation in the treatment of UC.4 Study on the Mechanism of Treatment of UC with CPAE by TranscriptomicsTranscriptomics Experimental Methods:Data processing and screening were performed on eight duplicate colon tissue samples from each group of Control,Model and DSH groups,including total RNA extraction and quality inspection,construction and evaluation of sequence analysis libraries,sequencing and screening of RNA-seq samples,reference sequence alignment analysis,gene expression level analysis,and gene expression difference analysis.Differently expressed genes were selected.GO analysis and KEGG enrichment analysis significantly enriched signal pathways.Further quantitative analysis of differential genes enriched in significantly expressed signal pathways;Significant differences in gene expression were detected by qRT PCR.The results showed that a total of 4727 differentially expressed genes were selected from the Control group compared to the Model group,including 2157 up-regulated genes and 2570down-regulated genes;A total of 1058 differentially expressed genes were selected from the DSH group compared to the Model group,including 669 up-regulated genes and 389 down-regulated genes.PCA analysis showed significant separation between the groups.The results of KEGG enrichment analysis showed that the differentially expressed genes in the Model group and the Control group were enriched in 331 KEGG pathways,significantly enriched in145 pathways such as PI3K/AKT and NF-Kappa B（P<0.05）;The differentially expressed genes in the DSH group and the Model group were enriched in 279 KEGG pathways,significantly enriched in 59 signal pathways such as PI3K/AKT,PPAR and so on（P<0.05）.The results showed that PI3K/AKT was a signal pathway that was significantly expressed in both the model group and the DSH intervention group;Col4a6,Lama2,Ngfr,Lamc3,Lamb2,Itga7,Lpar3,EfnαPrlr,Pdpk1 and Ccnd2 are differentially expressed genes that are significantly enriched in the PI3K/AKT signaling pathway;The results of qRT-PCR showed that after the intervention of CPAE,the reverse regulation trend of each index of the above significant difference genes was significant.5 Study on the Mechanism of CPAE Based on PI3K/AKT in Treating Oxidative Stress Damage of Intestinal Mucosa in UCThe content of serum Fe ²⁺in UC model rats was detected by micro method;Detection of serum GPX4,EPO and HIF-1αContent expression by ELISA;The ATP content in colon tissue of UC model rats was measured by phosphomolybdic acid colorimetry;Biochemical methods were used to detect the contents of ATPase,Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase in colon tissue and calculate the activity of related enzymes;WB detection of colon tissue PI3K,AKT,p-PI3K,p-AKT,SIRT1,PGC-1α,FTH1,GPX4,Keap1,Nrf2,HIF-1α,VEGF,DRP1and MIRO Expression of proteins;Detection of PI3K,AKT,SIRT1,PGC-1α,FTH1,GPX4,Keap1,Nrf2,HIF-1αAnd VEGF mRNA gene expression in colon tissue by qRT-PCR.The results showed that serum Fe²⁺,HIF-1αin the CPAE intervention group,EPO content significantly decreased,while GPX4 content increased;The ATP content in colon tissue increased,and the activities of ATPase,Ca²⁺-Mg²⁺-ATPase,Na⁺-K⁺-ATPase increased to varying degrees;WB results showed that in the CPAE intervention group,PI3K,P-PI3K,AKT,P-AKT,Keap1,DRP1,MIRO,HIF-1α,VEGF protein expression decreased to varying degrees,Nrf2,FTH1,SIRT1,PGC-1α,GPX4 protein expression increased to varying degrees;The results of qRT-PCR showed that PI3K,AKT,Keap1,HIF-1αin the CPAE interventiongroup,VEGFproteinexpressiondecreasedtovarying degrees,Nrf2,FTH1,SIRT1,PGC-1αMRNA gene expression increased to varying degrees.The results showed that PI3K/AKT regulated Keap1/Nrf2,HIF-1α/VEGF,SIRT1/PGC-1αEffectively improves the oxidative stress injury state of UC caused by hypoxia,abnormal energy metabolism,and iron death in mucosal cells.The above experimental results indicate that the PI3K/AKT signaling pathway is the core mechanism of UC occurrence.CPAE may be based on regulating differential metabolites to inhibit the expression of PI3K/AKT,thereby inhibiting downstream oxidative stress hypoxia,impaired energy metabolism,and iron dependent death of mucosal cells,thereby improving the antioxidant capacity of colon tissue and inhibiting the excessive secretion of inflammatory cytokines,playing a protective role in protecting UC mucosa.