Inhibitory Effects Of Compound Of Epimedium Preperation On NCI-H460 Cell Xenografts In Nude Mice And Its Effects On Immunocompromised Mice

Objective: To investigate the effect of compound of epimedium preparation(CEP)on human lung cancer cells and mice with immunodeficiency.Methods: 1.NCI-H460 xenograft model of lung cancer cells in nude mice was established.These mice were randomly divided into five groups(n=5):model group,positive group,high does group,middle does group and low does group with CEP,another 5 nude mice not inoculated with cells as the control group.the control group and the model group were given the same volume of distilled water;the CTX group was given intraperitoneal injection at a dose of0.025 g/kg;the groups of CEP were given gavage at a dose of 2.4 g/kg,4.8 g/kg and 9.6 g/kg respectively.The positive group was given every other day,and the other groups were given every day for 13 days.During the administration,the tumor volume was measured every other day.The next day after drug withdrawal,the nude mice were weighed and the transplanted tumor volume was measured.2.Serum levels of Interleukin-6(IL-6)and tumor necrosis factor(TNF-?),Determination of superoxide dismutase(SOD)in tumor tissues,malondialdehyde(MDA)in tumor tissues were measured by ELISA,hydroxylamine method,thiobarbituric acid method respectively.3.The histopathological changes of tumor tissues in each group were observed by HE.Immunohistochemical method to observe the expression of Caspase-3,Caspase-9,Bax,Bcl-2,p53 in the tumor tissues of the nude mice.4.The expression of caspase-3,caspase-9,Bax,Bcl-2,p53 and VEGF gene in the transplanted tumor tissues of nude mice was detected by RT-PCR.5.Mice model of hypoimmunity were established,These mice were randomly divided into five groups(n=8): model group,high does group,middle does group and low does group with CEP,another 5 normal mice as the control group.the control group and the model group were given the same volume of distilled water;the groups of CEP were given gavage at a dose of 2.4 g/kg,4.8g/kg and 9.6 g/kg respectively with every day for two weeks.6.Liver index and spleen index in each group were measured respectively.Monocyte-macrophage function of mice,delayed allergy ability of test mice and humoral immune function of test mice was detected by carbon clearance test,toe thickening method,hemolysin test respectively.7.The content of IgG in serum of test mice was determined.Interleukin2(IL-2)and ?-interferon(IFN-?)levels in serum was measured by ELISA.Results: 1.The NCI-H460 subcutaneous xenograft model of lung cancer cells in nude mice was successfully established,and the tumor formation rate was 100%.2.Compared with the model group,the volume and weight of transplanted tumor of the positive group and the groups of CEP were significantly smaller(p < 0.05 or p < 0.01).The tumor inhibition rate of CTX group and the groups of CEP were 59.77%,58.12%,48.21% and 40.56%,respectively.3.Compared with the model group,The levels of TNF-? in serum of tumor-bearing nude mice of the positive group and the groups of CEP were significantly smaller(p < 0.05 or p < 0.01),and the levels of IL-6 were significantly higher than those in model group(p < 0.01).4.MDA content in tumor tissues of each group was significantly lower than that of the model group(p < 0.05 or p < 0.01).SOD activity was significantly higher(p < 0.05 or p < 0.01).5.Immunohistochemical results showed that: compared with the model group,the expression of caspase-3,caspase-9 and Bax protein in tumor tissues were significantly increased in the CTX group and the groups of CEP(p < 0.05 or p < 0.01),the expression of Bcl-2 protein in tumor tissues were significantly reduced(p < 0.05 or p < 0.01),so there was a dose-dependent relationship.The expression level of p53 protein in CTX group was significantly higher than that in model group(p < 0.05 or p < 0.01),there was no significant difference between the groups of CEP and model group(p > 0.05).6.RT-PCR results showed: the expression of caspase-3,caspase-9 and Bax gene in the CTX group and high does group and medium dose group of CEP were significantly higher,compared with the model group(p < 0.05 or p <0.01),expression of Bcl-2 and VEGF was significantly lower(p < 0.05 or p <0.01).The expression of p53 and VEGF gene in the low dose group were significantly down-regulated compared with the model group(p < 0.05 or p <0.01).7.There is no significant effect on immune organ index,delayed allergy,mononuclear macrophage function,serum hemolysin level and cytokines in the low dose group of CEP.There is significant effect on delayed allergy andhemolysin levels in the medium dose group.There is significant effect on delayed allergy,mononuclear macrophage function and hemolysin level in high-dose group(p < 0.05 or p < 0.01),and also significantly increased IgG and il-2 level in immunocompromised mice(p < 0.05 or p < 0.01).Conclusion: CEP can inhibit the growth of NCI-H460 cell xenograft tumor in nude mice,and its mechanism may be related to the removal of free radicals,the increase of TNF-? and IL-6 secretion,the up-regulation of Bax,caspase-3,caspase-9 protein and gene expression,and the down-regulation of Bcl-2 and VEGF expression to induce tumor apoptosis.CEP can also enhance the immunity of mice with low immunity by inducing apoptosis of tumor cells and improving immune function.