Focusing On Th17/Treg To Study The Effects Of Astragalus And Curcuma Wenyujin On Tumor Metastasis In Mice With CT26.WT Orthotopic Transplantation Tumor

Objective:Astragalus and curcuma wenyujin as classic medicines for invigorating qi and activating blood have been proven to have good anti-tumor effects.There have been reports explaining that Th17/Treg balance is closely related to tumor immune regulation.In this paper,guided by the theory of traditional Chinese medicine,the literature studies the role of astragalus and curcuma wenyujin in regulating Th17/Treg balance in the process of colon cancer metastasis.The animal experiments were conducted to select BALB/c male mice to create a CT26.WT colon cancer(CC)orthotopic transplantation tumor model.After the administration,the tumor growth and liver metastasis were observed,and the spleen index and thymus index were calculated.Besides,the expression of helper T cell 17(Th17)/regulatory T cell(Treg)cells,and epithelial-mesenchymal transition(EMT)related molecules were detected to explore the relationship among Thl7/Treg cells,TGF-β/Smad4 and EMT,thus providing a feasible basis for the clinical treatment of colon cancer.Methods:1.Established in BALB/c male mice CT26.WT colon orthotopic transplantation tumor model,the model mice were randomly divided into model by weight 5-FU,astragalus,curcuma wenyujin(2:1)high(12 g·kg-1),(6 g·kg-1),low(3 g · kg-1)group and combined group 5-FU high,medium and low,a preliminary calculation between groups a tumor-burdened mice body weight after the treatment,the average tumor weight;the survival state of mice during administration was observed.The growth of orthotopic transplantation tumor,the tumor inhibition rate of metastasis related index and the liver metastasis rate were calculated.Spleen index and thymus index were calculated.2.Change of Th17/Treg cell percentage in CD4+T lymphocytes in peripheral blood and spleen tissue detected by Flow Cytometry;3.Serum IL-17,IL-23,IL-10,IL-6,IL-21 and IL-22 were detected by ELISA;4.The mRNA expressions of TGF-β,TNF-α,IFN-γ,Smad4,N-cadherin and MMP-7 in the tissues and blood of orthotopic transplanted mice were detected by Real-time PCR;5.The protein expressions of TGF-β,TNF-α,IFN-y,Smad4,N-cadherin and MMP-7 in transplanted tumor tissues and blood of mice were detected by Western Blot.Results:1.CT26.WT orthotopic tumor model of colon cancer was successfully established in BALB/c male mice,and all models were successfully established.After administration,the average tumor weight of mice in each group showed that compared with model group,astragalus and curcuma wenyujin in high,medium and low groups combined with 5-FU group couldsignificantly inhibit tumor growth,with statistical significance(P<0.01).From the results of tumor inhibition rate,the tumor inhibition rate of the combination of astragalus and curcuma wenyujin in the high group combined with 5-FU group reached 72.06%,indicating that thecombination of high group had the best effect of inhibiting tumor growth.The liver metastasis rate of the combined high,medium and low groups as well as the 5-FU group was zero,indicating that the combined group had a prominent effect of inhibiting tumor metastasis.2.Compared with the model group,the spleen index and thymus index of colon cancer mice in each administration group were increased after administration of normal saline.Among them,the spleen index of astragalus and curcuma wenyujin high group,astragalus and curcuma wenyujin medium group,astragalus and curcuma wenyujin low group,combined high group and combined low group was significantly increased(P<0.01);The thymus index of astragalus and curcuma wenyujin high group,astragalus and curcuma wenyujin medium group,astragalus and curcuma wenyujin low group,combined high group,combined medium group and combined low group was significantly increased(P<0.01).The results showed that the immune function of CT26.WT colon cancer mice could be improved both in single and combined groups.3.Compared with the model group,CD4+IL-17+in spleen tissue and peripheral blood of colon cancer mice in each treatment group decreased in varying degrees(P<0.001),and the proportion of CD4+Foxp3+cells increased(P<0.001).4.RT-PCR results suggest that,except that there was no difference in mRNA expression in individual groups,compared with the model group,the combination of astragalus and curcuma wenyujin turmeric combined with 5-FU group can reduce orthotopic transplantation tumors andblood TGF-β,TNF-α,IFN-y,Smad4,N-cadherin and MMP-7 mRNA expressions(P<0.001 or P<0.01),and there were statistical differences.5.Western blot results showed that compared with model group,the protein expressions of TGF-β,TNF-α,IFN-γ,Smad4,N-cadherin and MMP-7 in situ tumor tissue and blood of mice in each group were decreased(P<0.001).Compared with 5-FU group,astragalus and curcuma wenyujin combined with high,medium and low groups could inhibit the protein expressions of TGF-β,TNF-α,IFN-y,Smad4,N-cadherin and MMP-7.The combined histone protein was lower than that in astragalus and curcuma wenyujin group and 5-FU group.The results of protein electrophoresis of TGF-β,TNF-α,IFN-γ,Smad4,N-cadherin and MMP-7 in orthotopic transplanted tumor showed that the expression of combined histone decreased significantly.Conclusion:The combination of astragalus and curcuma wenyujin and astragalus and curcuma wenyujin combined with 5-FU can not only effectively inhibit the growth and metastasis of orthotopic transplanted tumor in CT26.WT model mice,but also improve the immune function of mice and maintain immune microenvironment homeostasis.The mechanism may be related to the maintenance of Th17/Treg balance,the regulation of TGF-β/Smad4 expression,and the subsequent inhibition of TGF-β/Smad4-mediated EMT.