Experimental Study On The Function Of Anti Hepatoma And Immunoregulation Of Aconite

Background As the second leading cause of cancer-related death in our country,hepatocellular carcinoma(HCC)poses a serious threat to human health.It is characterized by occult onset,high malignancy,easy recurrence and metastasis.Traditional Chinese medicine has long been proved to exert benefits in treating HCC,with majority of them focusing on heat clearance and detoxification.Whereas,in advanced HCC,the body constitution of patients presents as the co-deficiency of Yin and Yang,that give rise to the application of Yang-warming methods.As the typical herb of Yang-warming methods,Aconitum carmichaeli Debx.has shown efficiency in treating advanced HCC clinically,with limited experimental research on it.Recently,the immunoregulatory potency of TCMs is gathering increased interest.This dissertation focuses on the HCC theatment effeciency of A.carmichaeli experimentally and explore the potential mechanism on the immunoregulatory effect of this herb.Objective A.carmichaeli,a kind of Wenyang Sanhan herb,which is often used in the late stage of liver cancer,is taken as the research object,based on the principle that syndrome differentiation and treatment of traditional Chinese medicine.The treatment effect of A.carmichaeli on liver cancer and the mechanism of its anti liver cancer effect on immune regulation is preliminarily discussed through the experiments in vivo and in vitro,hoping to provide some data support for the treatment of liver cancer by the rule of Wenyang Sanhan.Methods 1.The therapeutic effect of A.carmichaeli on HCC mice model.A total of 50 BALB/c mices were divided into 5 groups randomly as blank control group,model group,low dose group,middle dose group and high dose group.Except for blank control group,other group were inoculated with H22 tumor in groin of left lower extremity subcutaneous.Normal saline and A.carmichaeli were administratered by gavage thereafter for 3 weeks.The weight of tumor and mice,tumor inhibition rate,pathological sections of tumor tissues were recorded.A Kaplan-Meier curve were drawed by another group of 50 BALB/c mice.2.The effect of A.carmichaeli on general immune condition of subcutaneous tumor mice model.A total of 30 BALB/c mices were divided into groups and treated as aforementioned.The spleen index and thymus index of mice in each group were calculated.The expression levels of tumor necrosis factor(TNF-?),interleukin(IL)-1?,interferon(IFN)-? in serum were detected by enzyme-linked immuno sorbent assay(ELISA),and the number of B and T lymphocytes in peripheral blood was detected by flow cytometry.3.The effect of A.carmichaeli on NK cells of of subcutaneous tumor mice model.A total of 30 BALB/c mices were divided into groups and treated as aforementioned.The number of NK cells in peripheral blood and spleen of each group mices was detected by flow cytometry;the expression level of CD107 a in spleen was detected to evaluate the killing activity of NK cells.The NK cell receptors(NKp46,NKG2 D,TIGIT,TACTILE)in peripheral blood and spleen of mice were detected.4.The effect of A.carmichaeli on human HCC cell line.MTT colorimetry was used to detect the effect of A.carmichaeli on the proliferation of human HCC cell line.Annexin V-fifc/PI double staining was used to detect the effect of A.carmichaeli on apoptosis of human HCC cell line.Transwell chamber test was used to detect the effect of A.carmichaeli on the migration ability of human HCC cell line.We also use Western blot to detect the effects of A.carmichaeli on the expression of ERK,p-ERK,JNK,p-JNK,p38 and p-p38 in human HCC cell line.Results A.carmichaeli can improve the general living conditions of mice,inhibit the growth of tumor and prolong the survival time of mice.In general immune condition,A.carmichaeli can improve the thymus index and reduce the spleen index of mice,promote the expression level of TNF-?,IL-1?,IFN–? in serum,and increase the number of T lymphocytes in the peripheral blood of mice.In NK cell immunity,A.carmichaeli can increase the number of NK cells in the peripheral blood and spleen of mice,A.carmichaeli can also enhance the killing activity of NK cells in the spleen.It reduced the expression of NKp46,NKG2 D and TIGIT receptors in the peripheral blood,and increase the expression of inhibitory receptors TIGIT and TACTILE in the spleen of of H22 mice model.A.carmichaeli can inhibit the proliferation and migration of human HCC cell line.It promote the apoptosis,downregulate the expression of p-ERK,and upregulate the expression of p-JNK and p-p38 in human HCC cell line.Conclusion 1.A.carmichaeli has a certain therapeutic effect on HCC in tumor bearing mice;It can be attributed to immunolatory effect of this herb,the NK immunity in especial.2.A.carmichaeli can inhibit the proliferation and migration,and promote the apoptosis of human HCC cell lines,which may be relevant to ERK/JNK/P38 MARKs signal transduction pathway.