The Therapeutic Efficiency And Mechanistic Studies Of Refined Baohe Granules On Intestinal Protection And Against Colorectal Cancer

Objective: This study investigated the effects of RBG on 5-FU-induced intestinal mucositis,and explored its underlying mechanisms by determination the activation of AKT pathway.To investigate the therapeutic effect of RBG on DSS induced intestinal inflammation,and explore its underlying mechanism by determination the changes of intestinal flora.To explore the therapeutic potential of RBG via network pharmacological analysis,and investigate its potential active compounds,targets and pathways.To verify the effect of RBG on tumor growth,proliferation and apoptosis of CRC cells in vivo,and its regulatory effect on potential targets based on network pharmacological analysis.Methods: 1.CT-26 cells were subcutaneously injected into BALB/c mice,followed by treatment of 5-FU and/or RBG.The therapy efficiency of RBG on diarrhea of mice were evaluated by determination of body weight,diarrhea scores,and observation of feces.Hematoxylin and Eosin(HE)staining,immunohistochemistry(IHC)and Terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)analyses were performed to investigate the effects of RBG treatment on the cell proliferation,apoptosis and activation of AKT pathway,as well as expression of its downstream regulatory effectors.2.The model of DSS induced ulcerative colitis(UC)was constructed and administrated with RBG solution.The therapeutic effects of RBG on body weight,disease activity index(DAI),colon length and colon weight,as well as HE staining were performed.Immunohistochemical(IHC)staining were used to detect the effects of RBG treatment on levels of inflammatory cytokines in intestinal tissues.The high-throughput sequencing technique was used to detect the effect of RBG on the intestinal flora of ulcerative colitis mice.3.The potential active compounds and targets of RBG were selected through TCMIP database or documentation retrieval.The enriched signaling pathways were analyzed based on the all potential targets.Based on the result of Pathway analysis,we focused on the effect RBG on anti-CRC,and downloaded the targets of CRC through Gene Cards database.The intersection of CRC targets and potential targets of RBG were analyzed.To further explore the potential core active compounds,targets and pathways,we further constructed the compound-target network,performed GO and Pathway analyses based on the intersected targets.4.HCT116 cells were subcutaneously injected into nude mice,and were administrated with RBG,the tumor growth was assessed by detecting tumor volume and tumor weight.IHC staining and TUNEL assay were used to detect the effect of RBG treatment on tumor proliferation and apoptosis,as well as expression of Bcl-2 and Bax.Results: 1.RBG treatment significantly alleviated the increased diarrhea index,jejunal mucosa injury and fecal morphology of CT-26 bearing mice caused by 5-FU injection.Furthermore,RBG treatment significantly reduced the cell apoptosis,down-regulated the expression of apoptotic protein Bax,and up-regulated the expression of anti-apoptotic protein Bcl-2 in jejunum tissues of CT-26 bearing mice after 5-FU injection.Moreover,RBG treatment obviously promoted the cell proliferation and upregulated the expression of CDK4 and C-myc in jejunum tissues of cells after 5-FU treatment.In addition,RBG treatment significantly up-regulated both AKT and P-AKT expression in jejunum tissues of CT-26 bearing mice after 5-FU treatment.2.RBG treatment significantly attenuated DSS-induced loss of body weight,shorten of colon length,and injury of colon tissues,as well as elevation of both IL-6 and TNF-? levels in colon tissues.Moreover,RBG treatment didn't significantly change the number of OTUs,alpha diversities,while significantly improved flora composition,beta diversities,and decreased Dubosiella abundances(P<0.05),as well as the increased the number of beneficial bacteria Lactobacillus(Lactobacillus).3.Based on the network pharmacological analysis,22 potential active compounds and 217 predicted targets were selected.Pathway analysis revealed a significantly enrichment of multiple signaling pathways,including Pathway in cancer,Colorectal cancer.The 9329 targets of CRC were downloaded from database of Gene Cards,and 184 intersected targets were selected based on the intersection of CRC targets and potential targets of RBG.Moreover,we constructed the compound-target network of RBG for anti-CRC.Network analysis identified 8 potential active compounds and 48 potential core targets.Furthermore,we identified multiple enriched signaling pathways,including Pathways in cancer,Hepatitis B and Prostate cancer,and multiple enriched functions including enzyme binding,response to drug and negative regulation of apoptotic process via Pathway and GO analyses based on the intersected targets.4.RBG treatment significantly attenuated the growth of HCT116 cells in vivo.Moreover,RBG treatment significantly promoted cell apoptosis and inhibited cell proliferation in tumor tissues,as well as down-regulated anti-apopotic protein Bcl-2 expression,which up-regulated pro-apoptotic protein Bax expression.Conclusions: 1.RBG attenuated 5-FU-induced intestinal mucositis by inhibiting apoptosis and promoting cell proliferation by modulating AKT pathway and its downstream effectors in a CT-26 xenograft mouse CRC model.2.RBG significantly attenuated DSS-induced UC and an its possible regulatory mechanism may be by reducing the expression of inflammatory factors IL-6 and TNF-? and modulating intestinal flora.3.RBG exhibits a potential on anti-CRC with the characteristics of multiple compounds,multiple targets and multiple signaling pathways.4.RBG treatment significantly alleviated tumor growth of HCT116 in null mice,by inhibiting cell proliferation,promoting cell apoptosis via up-regulating Bax expression and down-regulating Bcl-2 expression.