Study On The Anti-tumor Effect Of Active Fraction And Components From Blaps Rynchopetera On Colorectal Cancer In Vivo

Objectives:On the basis of previous research,the tumor inhibitory activity and possible mechanism on the CT26 colorectal tumor model in mice were investigated for methyl-p-benzoquinone(MBQ),the main ingredient of defense solution with significant proliferation inhibitory activity on tumor cell lines,the B.rynchopetera active fraction(BRAF),with significant immune regulatory activity in vivo and in vitro,and inosine(IS),the main component obtained from B.rynchopetera.Methods:(1)The colorectal tumor model of CT26 in BALB/c mice was established by subcutaneous inoculation of tumor cell line,divided into normal group,model group,positive group(5-Fu,25 mg/kg),MBQ low,medium and high dose groups(i.v.,0.75 mg/kg,1.5 mg/kg and 3 mg/kg)and continuously administered for 14 days.The antit-umor effect of MBQ was investigated by tumor weight and HE staining of tumor tissue.The expression of proliferation-related factor Ki-67 and apoptosis-related protein Caspase-3 in tumor tissues was detected by immunofluorescence method,the proportion levels of CD3~+T cells,CD3~+CD4~+T cells,CD3~+CD8~+T cells and Treg cells in tumor tissues were detected by flow cytometry to explore the possible mechanism.(2)Establishing the same model,administered with BRAF(i.g,2 g/kg)and low,medium,and high(0.5 g/kg,1 g/kg,and 2 g/kg)doses of BRAF combined with 5-Fu(12.5 mg/kg)respectively.Then the general state,tumor weight and immune organ index were observed and determinated,the proportion of dendritic cells in spleen and T cells,B cells,CD3~+CD4~+T cells,CD3~+CD8~+T cells and Treg cells in tumor tissues were determined by flow cytometry to investigate the effects of BRAF combined with 5-Fu in CT26 colorectal tumor mice.(3)Established the same model and administered low and high doses of IS treatment(i.v,5 mg/kg and 50 mg/kg).According to the tumor weight and the expressions of Ki-67,macrophage M1 markers F4/80+CD86 and M2 markers F4/80+CD206 in tumor tissues to explore the inhibitory effect of IS on tumor proliferation and its influence on the polarization of M1 and M2 macrophages in tumor microenvironment.Results:(1)The low-dose group of MBQ had general inhibitory activity for CT26 colorectal tumor with the inhibition rate of 32.56%,while there was no effect for the medium and high-dose groups.MBQ could reduce the proportion of CD3~+T cells and Treg cells,increased the proportion of CD3~+CD4~+T cells and CD3~+CD8~+T cells,reduced the expression of Ki-67protein and increased the expression of Caspaese-3 protein in tumor tissue.(2)5-Fu had significant antitumor activity with the tumor inhibition rate 65.69%,but the body weight of mice was significantly decreased(P<0.01).BRAF administered alone had no inhibitory effect on tumor proliferation,when combined administration with 5-Fu,the tumor inhibition rates of the low,medium,and high dose groups were 64.96%,54.74%,and 63.50%,respectively,having no significantly difference with 5-Fu,but it could significantly improved the weight loss caused by 5-Fu in mice(P<0.05),improved the survival status and quality of life of mice.The results of flow cytometry showed that the combination of BRAF and 5-Fu increased the proportion of dendritic cells,T,and B cells,while reducing the proportion of Treg cells.(3)The tumor inhibition rates of the low and high dose IS groups were 34.35%and 47.33%,respectively.Immunohistochemical results showed that IS could significantly reduce the expression of Ki-67 protein.Immunofluorescence results showed that IS could increase the expression of F4/80+CD86,the M1 polarization phenotype marker in a concentration-dependent manner,while had no significant effect on the expression of F4/80+CD206,the M2marker.Conclusions:MBQ,the main component of the defense solution,had a certain inhibitory effect on CT26colorectal tumor,which may play a role by inhibiting tumor cell proliferation,promoting tumor necrosis and apoptosis.BRAF,active extract of B.rynchopetera had no significant inhibitory effect on CT26 colorectal tumor,but it could significantly improve the body weight loss of mice induced by 5-Fu and partially improved the mice survival status and life quality when combined treatment with 5-Fu.IS had a certain inhibitory effect on CT26 colorectal tumor,possibly through inhibiting tumor cell proliferation and promoting macrophages M1-type polarizationin tumor microenvironment.