Effects Of Xiao-chai-hu-tang And Its Cold And Hot Herb-subtracted Formulae On Human Colon Carcinoma Cell And Its Mechanism

Background:Colorectal cancer is the third most common malignant tumor in the world,which poses great harm to human health,so it’s urgent to develop effective drugs.The research shows that cold and heat disorder is the main pathogenesis of many kinds of digestive tract malignant tumors.The research shows that cold and heat disorder is the main pathogenesis of many kinds of digestive tract malignant tumors.Xiao-chai-hu-tang is a classic recipe for cold and heat use.A large number of studies have proved that XCHT is used to inhibit cancer,but there are few reports for the treatment of colorectal cancer.The mechanism of anti-cancer effect of a combination use of cold and hot herbs in XCHT was also not reported.Alkaline phosphatase and lipid are the main components of cell membrane and closely related to the fate of cells.Based on the relationship between alkaline phosphatase,lipid and lipid peroxidation and the proliferation and apoptosis of cancer cells,the aim of this study is to explore the effects of XCHT and its cold and hot herb-subtracted formulae on colon cancer cells and its mechanism,and the mechanism of XCHT decoction combined with cold and heat.Method:We measured the cell viability,lipid accumulation,alkaline phosphatas e,lipid peroxidation and cell death mode of XCHT,XCHT-Chai Hu,XCHT-Huang Qin,XCHT-Ban Xia,XCHT-Sheng Jiang treated HCT116 cell at a series final drug concentration of 0.5g/L,1.0g/L,1.5g/L,and colchicine positive cont rol group(colchicine: at a series final drug concentration of 1.0 μmol/L,2.5μmol/L,10.0μmol/L).The cell viability of HCT116 cells was detected by MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)in 24 h,48 h and 72 h,and the lipid accumulation was detected by Sudan red IVstaining and microscope counting method;Alkaline phosphatase kit detecte denzyme activity,heat stable test and intestinal alkaline phosphatase specific inhibitor(L-phenylalanine)inhibition test to distinguish alkali ne phosphatase isoenzyme(Intestinal alkaline phosphatase(IAP)and tissue nonspecific alkaline phosphatase(TNAP)).Detection of lipid peroxidation products(malondialdehyde and conjugated diene)in cells by thiobarbituric acid and ultraviolet spectrophotometer.The effects of XCHT and its cold and hot herb-subtracted formulae on HCT116 cell death mode were observed by double staining of acridine orange /ethidium bromide.Result:1.Effect of XCHT and its cold and hot herb-subtracted formulae on proliferation of HCT116 cells:XCHT and its cold and hot herb-subtracted formulae all had obvious inhibitory effect on the cell proliferation activity of HCT116 human colon carcinoma cells and the inhibitory effect of XCHT was more obvious.2.Effect of XCHT and its cold and hot herb-subtracted formulae on lipid accumulation of HCT116 cells:The cell percentage of lipid accumulation in HCT116 cell group was significantly higher than that in all the five chinese herbal medicine group(P <0.01),the cell percentage of lipid accumulation in XCHT group is the lowest(P <0.01),the cell percentage of lipid accumulation in XCHT-Chai Hu group was significantly lower than that in XCHT-Ban Xia group and XCHT-Sheng Jiang group(P <0.01),The cell percentage of lipid accumulation in XCHT-Huang Qin was significantly lower than that in XCHT-Ban Xia group and XCHT-Sheng Jiang group(P <0.01),there was no lipid accumulation in the colchicine positive control group.3.Effect of XCHT and its cold and hot herb-subtracted formulae on alkaline phosphatase and its isozymes in HCT116 cells:The IAP activity in all the five chinese herbal medicine group was higher than TNAP activity(P <0.01),and the TNAP activity in culture medium was higher than IAP activity(P <0.01).The IAP activity in XCHT-Chai Hu group was higher than that in XCHT-Ban Xia group and XCHT-Sheng Jiang group;and the IAP activity in XCHT-Huang Qin group was higher than that in XCHT-Ban Xia group and XCHT-Sheng Jiang group(P <0.01),and in the culture medium,the TNAP activity in XCHT-Ban Xia group and XCHT-Sheng Jiang group was higher than that in XCHT-Chai Hu group(P <0.05)and XCHT-Huang Qin.4.Effect of XCHT and its cold and hot herb-subtracted formulae on lipid peroxidation of HCT116 cells:The content of malondialdehyde(MDA)in the five chinese herbal medicine groups was less than that in the HCT116 cell group(P <0.01).In the chinese herbal medicine groups:the highest MDA content in the XCHT-Huang Qin group was the highest(P <0.05).The content of MDA was lower in XCHT group,the lowest in XCHT-Chai Hu group,XCHT-Ban Xia group,XCHT-Sheng Jiang group.5.Effect of XCHT and its cold and hot herb-subtracted formulae on cell apoptosis of HCT116 cells:Compared with the HCT116 cell group,the five groups of chinese herbal medicine showed obvious apoptosis(P <0.01).XCHT group showed obvious apoptosis compared with the other four chinese herbal medicine groups.6.The correlation between lipid accumulation,alkaline phosphatase,lipid peroxidation and proliferation,apoptosis : lipid accumulation was positively correlated with HCT116 cell proliferation(P <0.01),and negatively correlated with HCT116 cell apoptosis(P <0.01).In HCT116 cells,IAP was negatively correlation with HCT116 cell proliferation and positively correlated with HCT116 cells apoptosis(P <0.01),the correlation between TNAP and HCT116 cells proliferate,the apoptosis was not obvious;malondialdehyde were positively correlated with HCT116 cell proliferation,and negatively correlated with HCT116 cell apoptosis;the correlation between conjugated diene and HCT116 fine cell proliferation,the apoptosis was not obvious.In the culture medium of HCT116 cells,IAP was negatively correlated with HCT116 cell proliferation(P <0.01),and was positively correlated with apoptosis of HCT116 cells(P <0.01);the correlation between TNAP and proliferation and apoptosis(P <0.05)of HCT116 cells was not obvious;the correlation between malondialdehyde,conjugated diene and HCT116 cell proliferation and apoptosis was not obvious.Conclusion:1.Xiao-Chai-Hu-Tang(XCHT)and its cold and hot herb-subtracted formulae inhibited HCT116 cell proliferation and induced HCT116 cell apoptosis by increasing IAP activity and inhibiting lipid accumulation and lipid peroxidation.2.XCHT’cold herb-subtracted formulae could obviously increased IAP activity and inhibited lipid accumulation,and XCHT’hot herb-subtracted formulae could obviously inhibited lipid peroxidation.3.Xiao-Chai-Hu-Tang(XCHT)and its cold and hot herb-subtracted formulae can effectively inhibited HCT116 cell proliferation and and induced HCT116 cell apoptosis,XCHT(a combination use of cold and hot herbs)exhibited the strongest effect.4.By a combination use of cold and hot herbs in XCHT,act on TNAP and IAP respectively,regulating lipid transport and metabolism,reducing the level of lipid peroxidation,inhibited HCT116 cell proliferation and promoted HCT116 cell apoptosis.