| Background:Colorectal cancer(CRC)is a common malignancy and is a multifactorial and multistage disease.Many reports indicates that a variety of factors affect the occurrence and development of colon cancer,such as genetic background,environmental factors,eating habits,mental stress and population aging.Recent studies have found that with long incurable disease and continuous inflammation,the risk of colorectal cancer has gradually increased.At present,the mechanism of action and pathogenesis of colon cancer is not clear.In the course of human health and disease progression,intestinal flora is considered a forgotten organ that maintains intestinal balance through a variety of complex mechanisms.A large number of recent studies have shown that intestinal microflora can also serve as a major factor in the development and progression of colon cancer.Many scholars have proposed the role of flora in colorectal cancer according to the results of high-throughput sequencing and animal model construction.However,due to the limitations of the study,we can only understand the pathogenesis of intestinal flora,the effect of certain stages or certain pathogens on the tumor,but the dynamic changes and specific mechanisms of intestinal flora in the colon remain to be further explored.The basic experimental results into clinical applications,reflects the intestinal flora in the diagnosis and treatment of colorectal cancer in the important clinical value.In this era of microbes,we must not only explore the underline mechanism,but also should emphasize the importance of potential clinical practice significance.Objective:We have selected several representative probiotics Lactobacillus acidophilus pro15 and pathogenHelicobacter hepaticus 51448,Fusobacterium nuleatum 25586,enterotoxigenic bacteroides fragilis43860,etc.,through the experimental study,observed the role of these representative intestinal bacterias in the transformation of colitis to colon cancer and its mechanism.Method:(1)We injected mice with intraperitoneal injection of 12.5 mg/kg dose of azoxymethane(AOM),and the mice were given a combination of 2%dextran sulfate sodium(DSS)to induce Colon cancer mouse model,this model can be a good simulation of UC patients with enteritis the malignant transformation of colon cancer process.The C57/B6 mice infected with Helicobacter hepaticus 51448 or Lactobacillus acidophilus pro15 were sacrificed and the colon tissue was fixed The pathology grade of the tumor was identified by HE staining and histological observation with.(2)Construction of acute liver injury model in mice by LPS+D-GalN.LPS/CalN was injectedintraperitoneally at a dose of LPS:35?L/kg,GALN:800?g/kg,6?L/g per mouse.(3)The mice were treated with Fusobacterium nuleatum 25586 and enterotoxigenic bacteroidesfragilis 43860,sacrificed and the colon tissue was fixed.The histological observation was performed by HE staining to identify the pathology gradeof the tumor.In order to explore the underline molecular mechanism,the inflammatory cytokines:interleukin-6(IL-6),interleukin-17(IL-17),interferon-?(IFN-?)and interleukin-1?(IL-1?),tumor necrosis factor(TNF-?)were detected by enzyme-linked immunosorbent assay(ELISA).The mouse mesenteric lymph nodes were stained with anti-mouse CD4,IFN-?,IL-17 and FOXP3 antibody,and the levels of pro-inflammatory and anti-inflammatory responses levels in different groups were detected by flow cytometry response.In vitro co-culture experiments with macrophages with Fusobacterium nuleatum 25586 and enterotoxigenic bacteroides fragilis 43860,respectively,62.5×10~6cfu,31.3×10~6cfu,15.6×10~6cfu,7.8×10~6cfu,0 cfu dose of two Bacteria stimulated macrophages 24 hours later,the supernatant was collected and TNF-?and IL-6 ELISA were detected.Results:(1)In the colon cancer mouse model,the survival rates of the mice were in the Helicobacterhepaticus 51448 group and the Lactobacillus acidophilus pro15 group were higher than those in the control group.There was no difference in colon length between the experimental group and the control group.But the tumor numbers in control group were more than in experimental group.HE staining and histological results showed that there was no significant difference in inflammatory cytokine levels between two groups.(2)In mice with acute liver injury model,Helicobacter hepaticus 51448 group had a higherthe survival rate than that of control group.(3)There were no significant differences in the length of colon and the number of tumorscompared with the control group.However,it was found by ELISA that inflammatory cytokines IL-17,IFN-?,IL-1?,IL-6,TNF-?in colonic tissue cultural supernatant of 25586 treated group were significantly higher than the control group,suggesting that 25586 ccould accelerate the inflammatory response of the colon,promoting the development of colon cancer The 43860 bacteria could promote the expression of some inflammatory cytokines and promote the development of colon cancer,as well.According to the results of flow cytometry analysis,we could see that the levels of IFN-?and IL-17 in the colon of 25586and 43860 teated mice were significantly higher than those in the control group in CD4~+T cells,while T-reg positive cells was significantly lower than that of the control group.According to the ELISA results of co-culture in vitro,25586 bacteria and 43860 bacteria could stimulate macrophages to produce TNF-?and IL-6 compared with the control group without bacterial stimulation,but 25586 bacteria produced TNF-?concentration higher than 43860 bacteria.Conclusion:(1)Helicobacter hepaticus 51448 and Lactobacillus acidophilus pro15 have a protective effectto inhibit the development and progression of colon cancer.But it may not be mainly through reducing the intestinal inflammation.(2)The 51448 can reduce the mortality of mice induced by AOM-induced chronic liver injuryand LPS+D-Gal N-induced acute liver injury,and has a significant protective effect on the liver.(3)Fusobacterium nuleatum 25586 and enterotoxigenic bacteroides fragilis 43860 can promotethe development of colon cancer. |
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