Inhibitory Effect Of Li Ginseng Powder On Colitis-Associated Colorectal Cancer

Colorectal cancer(CRC)is the third most common cancer in the world,and its incidence remains high worldwide,with more than 1 million people developing colon cancer each year.In the early stages of colorectal cancer,surgery can be performed,however,most patients with colorectal cancer do not have early symptoms and about 25% of patients have metastases by the time of diagnosis.Colitis-associated cancer(CAC),a subtype of colon cancer,is one of the most common malignancies of the intestine.Studies on colitis-associated cancer have shown that dietary habits,genetic factors,inflammatory bowel disease,intestinal microecology and other diseases may be the main oncogenic factors of CAC.Inflammatory bowel disease(IBD)is a chronic,nonspecific inflammatory lesion that occurs in the colorectum.Compared to the normal population,patients with inflammatory bowel disease have an approximately 10 to 40 times higher risk of colorectal cancer.The incidence of IBD has been increasing in recent years,and the incidence of CAC is expected to increase as well.Most of the drugs clinically applied to colitis-associated cancers are singletargeted,which can produce various adverse effects with long-term use and are highly susceptible to drug resistance.Therefore,there is an urgent need to develop new therapeutic agents for CAC.Chinese medicines generally contain several components that can act on multiple targets.These components produce relatively warm effects on each target and thus are characterized by effective efficacy and low toxicity.Ginsenoside is the main active component of ginseng,which has various effects including anti-inflammatory,inhibiting the growth of cancer cells and enhancing immunity.Recent studies have shown that disorders of the gut microbiota play an important role in the development of colorectal adenomas and adenocarcinomas,leading to the suggestion that colon tumors are “bacteria-related diseases”.A number of studies have found that the components in ginseng can also modulate the effect of intestinal microflora by inhibiting or promoting the growth of some genera and changing the structure of the intestinal flora.Li-Ginseng Powder(LGP)used in this study is a ginseng deep-processed product obtained by special processing of Jilin ginseng,which is characterized as rich in rare ginsenosides Rh4,Rg3,Rg5,Rk1 and Rk3,accounting for 60% of the total saponins.The complex interaction mechanisms between the regulatory effects of ginseng and its secondary metabolites on gut microflora and its anti-inflammatory and anti-tumor effects are still unclear.This study aims to reveal the target genes and action pathways of Li-Ginseng Powder(LGP)on colitis-associated cancer through network pharmacology analysis,verify the antitumor effect and action mechanism of LGP using the AOM/DSS animal model,and finally elaborate the mechanism of LGP action in inhibiting colitisassociated cancer by regulating intestinal microflora and inflammatory response.In this study,we used an AOM/DSS-induced mouse CAC model to investigate the inhibitory effect of LGP on colitis-associated cancer.It was observed that LGP significantly inhibited the weight loss and tumor numbers in AOM/DSS-induced group mice.Pathological analysis of colon tissues using the H&E staining method revealed that the LGP-fed group showed significantly less structural destruction of tissues and showed relatively well-preserved crypt structures.To elucidate the mechanism of action of LGP on colitis-associated cancer,we predicted the target genes and possible pathways of action of the rare ginsenoside group,the main active ingredient of LGP,on colitis-associated cancer by a network pharmacology approach.We obtained 348 potential targets of rare ginsenoside groups and 709 CAC-related genes,and further screened 49 intersecting genes by intersection analysis.The key genes for LGP intervention in CAC including TNF-α,CASP3,IL-6,STAT3,EGFR,etc were screened by topological analysis of the intersecting genes.The results of Gene Ontology(GO)analysis showed that the anti-CAC targets of LGP were mainly related to nitric oxide biosynthetic process,inflammatory response,cell proliferation,regulation of IL-6 expression,regulation of apoptotic process,and NF-κB binding.In addition,pathway enrichment analysis of target genes using the KEGG(Kyoto Encyclopedia of Genes and Genomes)public database showed that common genes were mainly focused on the TNF signaling pathway,HIF-1 signaling pathway,NF-κB signaling pathway,inflammatory bowel disease,JAK-STAT signaling pathway,and apoptosis regulatory processes.The expression levels of inflammation-related genes in colon tissues were detected based on the results of network pharmacology analysis,and the expression of proinflammatory factors,TNF-α,IL-6,IL-1β,and inflammation-related enzymes,iNOS,and COX-2,were significantly reduced in the LGP-treated group compared with AOM/DSS group.Furthermore,the LGP treatment significantly reduced the phosphorylation levels of NF-κB p50(Ser337),IκBα(Ser32),and STAT3(Tyr705)in colon tissues which was markedly upregulated in colon tissues of AOM/DSS treated model groups.Impairment of apoptotic pathways is a common event in colon carcinogenesis.To elucidate the role of apoptosis in colon tissue tumorigenesis,the occurrence of apoptosis and the expression of apoptosis regulatory proteins were examined in colon tissues.TUNEL analysis showed that a large number of apoptotic cells occurred in the colon tissue of LGP-treated mice,while no TUNEL-positive cells were observed in the normal group and the AOM/DSS model group.Western blotting(WB)and IHC analyses observed an upregulation of anti-apoptotic proteins Bcl-2 and Bcl-x L in the colon tissue of the AOM/DSS group,while the treatment of LGP significantly decreased the expression of these two proteins.Meanwhile,significant activation of Caspase-9 and Caspase-3 was observed in the LGP-treated group,suggesting that LGP triggers apoptosis by initiating the endogenous apoptotic pathway.In addition,WB and immunohistochemical analysis showed that LGP treatment significantly inhibited the expression of cell proliferation factor Ki67 in colon tissue.The above findings suggest that AOM/DSS promotes cell proliferation in mouse colon tissues while increasing the anti-apoptotic ability of cells through the expression of anti-apoptotic proteins,which seems to be acquiring two hallmark features of tumors: hyperactivation of cell proliferation cycle and blockage of the apoptotic pathway;and LGP treatment well reverses these two cellular variants.Studies have reported that gut microbiota plays a key role in CRC development.To assess whether the antitumor effect of LGP involves the gut microbiota,we analyzed the changes in tumorigenesis and expression of inflammatory factors by antibiotic treatment in the AOM/DSS mice.Antibiotic pretreatment significantly reduced the antitumor effect of LGP.The inhibitory effect of LGP on iNOS and COX2 expression was also attenuated by the pretreatment with antibiotics.These results suggest that the antitumor effects and the anti-inflammatory effects of LGP in AOM/DSS mice may be mediated through the modulation of gut microbiota.To investigate whether LGP alters the composition of the gut microbiota and its metabolites in AOM/DSS-induced CAC mice,16 S sequencing and metabolomic analysis were performed.The results of correlation analysis between gut microbiota and metabolites showed that LGP treatment significantly down-regulated the level of creatine in serum,and the change in creatine content was highly negatively correlated with Roseburia and highly positively correlated with Turicibacter,Erysipelotrichaceae;LGP treatment up-regulated the level of azelaic acid in serum,and the level of azelaic acid in serum was highly positively correlated with Roseburia,Bifidobacterium,and negatively correlated with Alistipes,Turicibacter;LGP upregulated L-aspartic acid in serum,and L-aspartic acid was positively correlated with Bifidobacterium and highly negatively correlated with Turicibacter.Our correlation analysis revealed that the differential metabolite by LGP treatment,creatine was highly negatively correlated with Roseburia and positively correlated with both Turicibacter,Erysipelotrichaceae,while azelaic acid and L-aspartic acid were highly positively correlated with Roseburia,Bifidobacterium,and negatively correlated with both Alistipes,Turicibacter,these metabolites are strongly associated with CRC occurrence.This study investigated the anti-inflammatory effects of ginsenoside Rh4(G-Rh4),a main active component of LGP.Combining network pharmacology and molecular and cell biology studies,the anti-inflammatory action targets and action pathways of G-Rh4 were elucidated.We screened 58 anti-inflammatory action targets of G-Rh4 using network pharmacology analysis and identified TNF-α、IL-6、IL-1β and STAT3 as key action targets of G-Rh4 anti-inflammation based on core gene analysis.The results of GO analysis showed that the target genes of the anti-inflammatory effect of G-Rh4 were mainly associated with the regulation of nitric oxide biosynthetic process,inflammatory response,IL-6 production,lipopolysaccharide-mediated signaling pathway,and cell proliferation.KEGG pathway enrichment analysis showed that the common targets were mainly focused on the NF-κB signaling pathway,TNF signaling pathway,JAK-STAT signaling pathway,and PI3K-AKT signaling pathway.To validate the potential mechanism of G-Rh4 predicted based on network pharmacology analysis,the anti-inflammatory effect of G-Rh4 was confirmed in LPS-stimulated RAW264.7cells.The results showed that G-Rh4 significantly inhibited the expression of TNF-α,IL-6,and IL-1β,and effectively suppressed the activity of key pro-inflammatory transcription factors NF-κB and STAT3.Taken together,the favorable anti-CAC activity of LGP may result from a combination of mechanisms including modulation of intestinal microflora,regulation of inflammation,and stimulation of apoptosis.This study provides a reliable theoretical and practical basis for the clinical application of ginseng and will provide effective targets and lead compounds for the development of anti-CAC and anti-inflammatory drugs.