| Objective:To observe the anti-tumor effect Xiao'ai Jiedu Decoction(XAJD)on CT26 cells tumor-bearing mice and the effect of pharmacological serum of Xiao'ai Jiedu Decoction on human colon cancer HCT-116 and HT-29 cells,and to identify the effect and molecular mechanism of Xiao'ai Jiedu Decoction on colon cancer.It provides a scientific basis for the prevention and treatment of cancer in the clinical application of Xiao'ai Jiedu Decoction.Methods:1.Research on the intervention effect of Xiao'ai Jiedu Decoction on colon cancer in vivo.To establish the CT26 cells tumor-bearing model,and to record the changes of tumor and weight in each group during the administration.After the intervention of Xiao'ai Jiedu Decoction,the mice organs were weighed,the tumor tissues were stripped,the tumor volume was measured and the tumor suppressor rate was calculated.Detection of inflammatory factors IL-1?and IL-6 in mice serum by ELISA,and the effects of Xiao'ai Jiedu Decoction were observed.The tumor tissue was stained with HE to observe the effect of Xiao'ai Jiedu Decoction on the tumor histopathology.The effect of Xiao'ai Jiedu Decoction on the apoptosis of tumor tissue was observed by TUNNEL staining.Immunohistochemical staining was used to observe the effect of Xiao'ai Jiedu Decoction on the expression of protein Ki67,apoptotic protein Bax,Bcl-2 and cleaved-caspse-3 protein in tumor tissues.2.The infection of Xiao'ai Jiedu Decoction on the growth of colon cancerPreparation of aseptic pharmacological serum of Xiao'ai Jiedu Decoction.Determination of inhibitory effect of pharmacological serum of Xiao'ai Jiedu Decoction on human colon cancer cell growth by MTT.Inverted microscope was used to observe the influence of pharmacological serum of Xiao'ai Jiedu Decoction on the morphology of human colon cancer cells.Effect of pharmacological serum of Xiao'ai Jiedu Decoction on the apoptosis of human colon cancer HCT-116cells detected by FITC-AnnexinV/PI Double Staining.Effect of pharmacological serum of Xiao,ai Jiedu Decoction on lactic acid and glucose level of human colon cancer cells detected by lactic acid and glucose kits.3.Study on the mechanism of the intervention of Xiao'ai Jiedu Decoction to colon cancer3.1 Study on Xiao'ai Jiedu Decoction in inducing apoptosis of tumor cellsHuman colon cancer HCT-116 and HT-29 cells were cultured in vitro and treated for 48 hours with pharmacological serum of Xiao'ai Jiedu Decoction,Western blot was used to detect the effect of pharmacological serum of Xiao'ai Jiedu Decoction on the expression of KBa,p-IKBa,p65 and p-p65 proteins in human colon cancer HCT-116 cells.3.2 Study on Xiao'ai Jiedu Decoction in regulating energy metabolism of tumor cellsHuman colon cancer HCT-116 and HT-29 cells were cultured in vitro and treated for 48 hours with pharmacological serum of Xiao'ai Jiedu Decoction,Western blot was used to detect the impact of pharmacological serum of Xiao'ai Jiedu Decoction's regulation on the expression of HIF-1?,HK2,PDK,LDHA,mTOR and p-mTOR proteins in human colon cancer cells.Results:1.Research on the intervention effect of Xiao'ai Jiedu Decoction on colon cancer in vivo.On the 14th day after the administration of Xiao'ai Jiedu Decoction,the tumor tissue of the model group was found to be larger in volume and irregular in shape.However,the tumor tissue of Xiao'ai Jiedu Decoction group was oval or irregular in shape,with more limited growth.Increase,the volume of tumor tissue is obviously small.The average tumor weight of Xiaojian Jiedu decoction group was significantly smaller than that of the control group,the anti-tumor rate of Xiao'ai Jiedu Decoction low,medium and high dose group was 19.24%,40.20%and 50.56%respectively,which was significantly lower than that of the blank control group p<0.05).The liver index,spleen index,thymus index,pancreas index of mice in the administration group had no significant difference compared with the normal control group.ELISA results showed that Xiao'ai Jiedu Decoction can reduce the serum levels of inflammatory cytokines IL-1?and IL-6.Immunohistochemical analysis of tumor tissues in mice showed that Xiao'ai Jiedu Decoction can reduce the expression of Ki67 protein and decrease the proliferation activity of tumor cells.At the same time,the anti-apoptotic protein Bcl-2 can be down-regulated,the pro-apoptotic protein Bax can be up-regulated,and the expression of Cleaved-caspase-3 protein can be enhanced,induce apoptosis in tumor tissue and inhibit the growth of tumor.2.Effect of Xiao'ai Jiedu Decoction on the growth of colon cancer cellsMTT method was used to detect the inhibitory rate of pharmacological serum of Xiao'ai Jiedu Prescription with different concentrations in stimulating HT-29 and HCT-116 cell after 24h,48 h.It shows that pharmacological serum of Xiao'ai Jiedu Prescription has a strong inhibitory effect on human colon cancer cells.The inhibitory rates of 15%drug-containing serum on human colon cancer HT-29 and HCT-116 were 42.7 ± 2.9%and 50.2 ± 2.2%respectively.The results of optical inverted microscope showed that the cells in the untreated group grew well and had a clear outline.With the increase of the dosage,the cell growth was inhibited,the number of cells was significantly reduced,and the cells showed irregular morphology.The apoptosis rate of FITC-AnnexinV/PI double staining assay showed that the total apoptosis rates of the cells treated with 5%,10%,15%pharmacological serum of Xiao'ai Jiedu Prescription were 10.86 ± 1.28%26.2 ± 3.45%.43.7 ± 3.23%respectively.The apoptosis rates of 10%and 15%pharmacological serum of Xiao'ai Jiedu Prescription were statistically significant(p<0.05)compared with the control group.Lactic acid and glucose test results showed that compared with the control group,the treated group cells showed a significant decrease in the level of lactic acid,and with the increase in the concentration of lactate decreased,the more obviousand the level of lactic acid decreased with the increase of drug concentration.(P<0.05 or P<0.01).The cells in the administration group showed a significant decrease in glucose level,and the more obvious the decrease of glucose level was with the increase of the drug concentration(P<0.05 or P<0.01).Compared with the control group,the results of the Western blot showed that the expression of HK2,PDK,LDHA and HIF-1?were decreased significantly after treatment with 10%,15%of the pharmacological serum of Xiao'ai Jiedu Prescription(P<0.05).3.Study on the mechanism of the intervention of Xiao'ai Jiedu Prescription to colon cancer3.1 Study on Xiao'ai Jiedu Decoction in inducing apoptosis of tumor cellsPharmacological serum of Xiao'ai Jiedu Prescription induces colon cancer cell apoptosis through the NF-?B signaling pathway and reduces the expression of p-IKBa and p-p65 protein.3.2 Study on Xiao'ai Jiedu Decoction in regulating energy metabolism of tumor cells Pharmacological serum of Xiao' ai Jiedu Decoction can reduce glucose uptake and lactic acid production in human colon cancer cells,and down-regulate the expression of hexokinaseHK2,PDK and LDHA,indicating that pharmacological serum of Xiao'ai Jiedu Decoction inhibit the level of glycolysis in colon cancer cells and the activity of enzymes in key steps of the glycolysis process.In addition,pharmacological serum of Xiao'ai Jiedu Decoction can downregulate the expression HIF-1?,indicating that pharmacological serum of Xiao'ai Jiedu Decoction inhibited the glycolytic activity of colon cancer cells and decreased the expression of HIF-1?.Further study found that pharmacological serum of Xiao'ai Jiedu Decoction can inhibit the expression of p-mTOR,indicating that pharmacological serum of Xiao'ai Jiedu Decoction can affect the process of glycolysis of colon cancer cells through the mTOR signaling pathway and play an anti-colon cancer role.Conclusion:Xiao'ai Jiedu Decoction has better anti-colon cancer effect,anti-colon cancer mechanism may be related to promoting tumor cell apoptosis,affecting the level of glucose metabolism in tumor cells. |
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