The Regulation Of Bushen Huiyang Formula On The Immune System Of Kidney-yang Deficiency Mice And The Analysis Of Active Monomers And Screening Of Anti-leukemia Models Of Natural Compounds

Background:In the view of traditional Chinese medicine,hypoimmunity mostly belongs to the category of kidney yang deficiency,to warm yang tonifying the kidney to strengthen the basic treatment of patients with clinical immunity low has a better effect,especially for leukemia patients,can alleviate the side effects of leukemia patients due to drugs and immunosuppression.Cellular immunotherapy is one of the new methods of tumor therapy,which can modify the immune active cells in vitro by various biological techniques,and then return to the patients after amplification,so as to improve the immunity of the patients and inhibit or kill the tumor cells at the same time.In addition,in modern medical treatment of leukemia,small molecule inhibitors play an important role,especially tyrosinase inhibitors.Since the birth of imatinib,it has greatly changed the treatment of chronic myeloid leukemia.Many traditional Chinese medicine or plant medicine with medicinal effect have certain killing effect on leukemia cells.Although many small molecules with therapeutic effect have been reported,the effect of small molecules in many drugs on leukemia cells is still unclear.Early clinical practice has proved that the traditional Chinese medicine prescription of Invigorating Yang is helpful to improve the immunity of patients with kidney yang deficiency and the clue of prognosis of leukemia,but we know little about the main active ingredients and the important traditional Chinese medicine in the prescription.At the same time,many medicinal plants are used to fight against tumor in the folk,but the active molecules which play an anti-tumor role are still unclear.Therefore,this research focuses on the regulation of immune cells and small molecule anti leukemia cells in kidney yang deficiency mice by Bushen Huiyang recipe.Objective:(1)To explore the regulatory effect of the Chinese medicine Bushen Huiyang Recipe on the immune system of kidney-yang deficiency mice provides basic research data for clinical medication.(2)Through the analysis of the main active ingredient monomers in Bushen Huiyang formula,the understanding of the formula is deepened.(3)Screen out new natural small molecules with anti-leukemia cells in medicinal plants to lay the foundation for subsequent work.Methods:(1)The regulatory effect of Bushen Huiyang Recipe on immune cells of kidney yang deficiency mice:the model of kidney yang deficiency mice was established by intraperitoneal injection of 25 mg/kg hydrocortisone,and the control group was injected with the same amount of normal saline every day.After 10 days of continuous administration,the general signs,peripheral blood picture and T lymphocyte subsets of the model group and the control group were detected.After that,the kidney yang deficiency model mice were given 0.25ml/day of Bushen Huiyang formula.The model control group and blank control group were given normal saline once a day for 15 days.At the end of the treatment,the general signs of the mice,the related immune cells of the peripheral blood,the proportion of CD3~+、CD3~-NK1.1~+、CD3~+NK1.1~+in the peripheral blood,bone marrow and spleen of the mice in each group were detected by flow cytometry,and the DC cells were induced and cultured by cytokine in the later stage of the peripheral blood mononuclear cells of the mice,and the expansion in vitro was detected,and the data were analyzed statistically.(2)Analysis of the active ingredient method of Bushen Huiyang recipe:the concentrated solution and the standard of Bushen Huiyang recipe were analyzed by HPLC and LC-MS in series,and the active ingredient in Bushen Huiyang recipe was explored by comparing the standard and database.(3)New small molecule anti leukemic effect:CCK-8 drug sensitivity test was carried out on the new small molecule compounds,and the small molecules with inhibitory effect on leukemic cells were screened.Results:(1)The regulatory effect of Bushen Huiyang formula on immune cells of kidney yang deficiency mice:after the treatment of traditional Chinese medicine by gavage,compared with the model control group,the weight of the mice in Bushen Huiyang group increased significantly;the level of leukocytes and lymphocytes in the peripheral blood increased significantly;the proportion of CD3~+、CD3~-NK1.1~+、 CD3~+NK1.1~+in the peripheral blood,bone marrow and spleen increased significantly;after the treatment of Bushen Huiyang formula by gavage The number of DC cells induced by cytokines from monocytes isolated from peripheral blood was significantly higher than that of model control group.(2)Analysis of the active component method of Bushen Huiyang recipe: combined with the standard and database analysis,it is found that there are three substances in Bushen Huiyang recipe:Glycyrrhizin,Icariin and Hypaconitine.In addition,the components of Epimedium were obtained:Isoquercetin,Icariin II,Icariin A and Icariin D2;Components of Glycyrrhiza:Glycyrrhizin,celery glycyrrhizin, Glycyrrhizin C,Glycyrrhetinic acid;Dry ginger ingredients:6-Gingerenol,6-Gingerol;cinnamon ingredients:Cinnamaldehyde.11 substances with the same molecular formula and molecular weight.(3)YWF-10,YHL-14,YWF-08,XY-24,XY-1,DS-7 and DX-15 had inhibitory effects on THP-1,Jurkat and kg-1 cells,among which yhl-14 and ywf-08 had no significant difference on 32D cells(P>0.05).But there was significant difference in the inhibition of KG-1 and THP-1(P<0.01),and ywf-08 has a strong inhibitory effect on Jurkat cells(P<0.001).The inhibition rates of YWF-10,YWF-08,XY-24,XY-1 and DS-7on Jurkat cells are much higher than those on other cells,and the inhibition rates of Jurkat cells are much higher than those on 32D cells.Conclusion:(1)Bushen Huiyang recipe can increase the number of leukocytes and lymphocytes in peripheral blood,the proportion of CD3~+,CD3~-NK1.1~+,CD3~+NK1.1~+in peripheral blood,bone marrow and spleen,and the number of DC cells induced in vitro,which proves that Bushen Huiyang recipe has the effect of strengthening and restoring immunity in kidney yang deficient mice。(2)There are three substances in Bushen Huiyang recipe,glycyrrhizin,icariin and hypaconitine.In addition,it contains isoquercetin,icariin II,icariin a,icariin D2,glycyrrhizin,apigenin glycyrrhizin,glycyrrhizin C,glycyrrhetinic acid There are 11kinds of substances with the same molecular formula and molecular weight,among which epimedium and roasted licorice have the most active components.In view of the previous studies and the results of this study,epimedium and roasted licorice play an important role in the positive regulation of Bushen Huiyang Recipe on immunity.(3)YWF-08,YWF-10,XY-24,XY-1 and DS-7 have a much higher inhibition rate on Jurkat cells than on other cells.YHL-14 and YWF-08 have no inhibition effect on32D cells,but they have a strong inhibition effect on KG-1 cells and THP-1 cells. Therefore,YHL-14 and YWF-08 have a good advantage in anti leukemic cells and the potential to continue experiments.