High Fat Diet-induced Gut Microbiota Dysbiosis Promotes Intestinal Carcinogenesis

Aims:Colorectal cancer(CRC)is one of the most common cancers and the leading cause of cancer deaths in the world.About 80%-90%of sporadic CRC occur after adenomatous polyposis coli(Apc)gene mutation.Related studies have found that increased intake of high fat diet(HFD)is closely related to the occurrence and development of CRC.HFD-induced gut microbiota dysbiosis is considered to play an important role during this process,but the specific mechanism remains to be explored.This study first explored the relationship between HFD and the occurrence and development of CRC from a clinical perspective.Then we used Apc^min/+mice that could develop intestinal adenomas spontaneously to explore the specific mechanism of HFD-induced gut microbiota dysbiosis to promote intestinal adenoma-adenocarcinoma sequence progression.MethodsPart 1:The asymptomatic individuals who underwent a colonoscopy at the Digestive Endoscopy Center of Tianjin Medical University General Hospital were involved in this study.Participants'clinical data were recorded in detail,and then were divided into HFD group and normal diet group according to different dietary patterns.Calculate the incidence of advanced colorectal neoplasia(AN,adenoma?10 mm,adenoma with villous component or high-grade dysplasia,or invasive carcinoma)in the two groups,and analyze the relationship between HFD and the development of AN in combination with clinical data.Immunohistochemical staining was performed to evaluate the expression levels of MCP-1,CCR2 and M2 tumor-associated macrophage(TAM)in CRC patients of these two groups.Part 2:The Apc^min/+mouse model was used to explore the mechanism of HFD-induced intestinal carcinogenesis.Four-week-old Apc^min/+mice were randomized divided into HFD group(60%fat content)and control group(16%fat content).After feeding under the specific pathogen free(SPF)environment for 12weeks,fresh stool pellets were collected for gut microbiota sequencing analysis.Mice were then sacrificed and cecal contents were collected for short-chain fatty acids(SCFAs)detection,and the number and size of intestinal tumors were recorded.Pathological analysis was performed to evaluate tumor malignancy,proliferation and apoptosis.Inflammation of intestinal tissues were determined.The expression of MCP-1/CCR2 signaling pathway and the key signal molecules of the monocyte-macrophage system in the tumor microenvironment were evaluated by Real-time PCR,Immunohistochemistry,Immunofluorescence staining and Western blot.Part 3:To further explore the causal relationship between HFD-induced gut microbiota dysbiosis and intestinal carcinogenesis,four-week-old Apc^min/+mice were used as recipients for a fecal microbiota transplantation(FMT)experiment.The fresh stool pellets collected from the HFD group and the control group after 12 weeks of feeding were used as donors.The recipient mice were treated with streptomycin for 3days to eliminate the native gut microbiota before FMT,and then the feces from the donors were transplanted to the recipient mice by gavage.The recipient mice were divided into FHFD group(transplantation of fecal microbiota from HFD group to a new batch of recipient Apc^min/+mice)and FCON group(transplantation of fecal microbiota from control group to a new batch of recipient Apc^min/+mice).The transfer experiment was carried out for 8 weeks and inoculated 16 times,and fecal samples were collected at the end of the eighth week for gut microbiota sequencing analysis.Mice were then sacrificed and the number and size of intestinal tumors were recorded.Pathological analysis was performed to evaluate tumor malignancy.Real-time PCR and Western blot were performed to detect the m RNA and protein expression levels of MCP-1/CCR2 signaling pathway.ResultsPart 1:(1)A total of 2338 individuals who underwent a colonoscopy were enrolled in this study.According to the dietary patterns,they were divided into HFD group(n=560,24%)and normal diet group(n=1778,76%).Statistical analysis showed that HFD could significantly increased the incidence of AN,especially invasive carcinoma.(2)Then we randomly selected 30 CRC patients with no significant differences in pathological characteristics(TNM classification)from the HFD group(n=15)and the normal diet group(n=15)for immunohistochemical staining to evaluate the difference of MCP-1,CCR2 and M2 TAM(CD163)expression.The results showed that the expression of MCP-1,CCR2 and CD163 in CRC patients with HFD was significantly higher than that in CRC patients with normal diet.Part 2:(1)After feeding Apc^min/+mice for 12 weeks,the body weight of the HFD group was significantly higher than that of the control group.The total tumor numbers in HFD-supplemented mice was significantly increased compared with that in the control group.HFD increased tumor numbers in all the three segments of the small intestine(proximal,middle,distal)and in the colon.The number of tumors of different sizes(?1mm,1-2mm,?2mm)in the small intestine in the HFD group increased significantly,and some large adenomas appeared in colon.Hematoxylin and eosin(HE)staining showed that high-grade dysplasia was detected in 70%HFD-fed Apc^min/+mice,while adenomas with low-grade dysplasia was found only in20%of Apc^min/+mice supplemented with control diet,and no dysplastic adenomas were found in the remaining mice.Ki-67 staining and TUNEL staining showed that HFD could significantly promote intestinal tumor cell proliferation and inhibit apoptosis.(2)HFD could significantly reduce the diversity of microbiota and increase the ratio of Firmicutes/Bacteroides.It was found that the abundance of pathogenic bacteria in HFD group increased significantly,such as Desulfovibrio,Streptococcus,Parabacteroides,Bacteroides acidifaciens,while the abundance of beneficial bacteria including the SCFAs-producing bacteria showed a significant decreasing trend,such as Roseburia,Lachnospiraceae bacterium.With the decrease of the abundance of SCFAs-producing bacteria,the concentration of SCFAs(acetate,propionate,butyrate)in the cecal contents of HFD group also decreased.(3)HFD could significantly up-regulate the expression of inflammatory mediators(IL-1?,TNF-?,IFN-?)in colonic mucosa.(4)The relative m RNA expression levels of MCP-1,CCR2 and M2TAMs markers in intestinal tumor tissues of HFD group were significantly higher than those in the control group.The results of Immunohistochemistry and Western blot showed that HFD could increase the protein expression level of MCP-1 and CCR2.Immunofluorescence results showed that the number of M2 TAMs increased and the number of M1 TAMs decreased in the tumor tissues of HFD group.Part 3:(1)There was no significant difference in body weight between the FHFD group and the FCON group.The total number of intestinal adenomas,the number of adenomas in each intestinal segment(proximal,middle,distal and colon)and the different sizes of tumors in small intestine(<1mm,1-2mm,>2mm)in FHFD group were significantly higher than those in FCON group.HE staining showed that the malignancy of intestinal tumors in FHFD group was higher than that in FCON group.(2)Gut microbiota sequencing analysis showed that the ratio of Firmicutes/Bacteroides in the FHFD group was increased.It was found that the abundance of pathogenic bacteria in the FHFD group was higher than that in FCON group at the levels of genus and species,such as Parabacteroides,Helicobacter,Bacteroides acidifaciens.While the beneficial bacteria such as Roseburia,Lactobacillus and Bifidobacterium showed a downward trend in the FHFD group.(3)The expression levels of m RNA and protein of MCP-1 and CCR2 in intestinal tumor tissues in FHFD group were significantly higher than those in FCON group.ConclusionHFD-mediated gut microbiota dysiosis could induce intestinal low-grade inflammation,reduce the production of SCFAs,and activate the MCP-1/CCR2signaling pathway to promote the recruitment and polarization of M2 TAMs,thereby accelerate the progression of intestinal adenoma-adenocarcinoma sequence.