Studies In Intervention Role Of Al And LBP On Abnormal Lymphocyte Apoptosis

Objective:Clinically,the occurrence of many diseases such as stress-induced diseases,radiotherapy and chemotherapy-induced myelosuppression,AIDS, side effects of hormone therapy and so on is closely related to abnormal apoptosis in lymphocytes.Lymphocytes as the main body' s immune cells is the material basis of strong or weak immune function.It relates to the stability of the body internal environment,its functional status of normal or not is closely related to occurrence,development,prognosis and the prognosis of many diseases.Modern immunology pharmacology research and clinical trials prove that many tonified Chinese medicine and its effectivly extracting components has obvious immune-enhancing and immune regulation.It has significant therapeutic effect on wide range of immunocomprom-ised patients.Therefore,the research and development of drugs associated with immunnsl diseases such as immunoenhancer or immunomodulator, especially anti-apoptotic drugs on abnormal immune cells,but also become a hot research field of medicine.Thymus is a central immune organ of the body,also is the place of differentiation and maturation of T lymphocyte,its function is directly related to the quality of the entire immune function.Building model through glucocorticoids inducing thymocytes abnormal apoptosis in mouse, this experiment studys the intervention role of the astragalus,Lycium barbarum,polysaccharides,Cordyceps sinensis polysaccharides, astragalus polysaccharide,Ganoderma lucidum polysaccharides on abnormal apoptosis of thymocytes,,with a view to reveal their new immune pharm- acological effect and its mechanism,but also to provide experimental basis for clinical development of new drugs.Methods:1.Intraperitoneal injection of DEX methodology used to establish model of lymphocyte apoptosisSelect 18-22 grams of NIH mice with half male and female,they are divided into control group and 2h,4h,6h,8h,l0h group with each group 8.Make intraperitoneal injection in mice by clinical Dexamethasone Sodium Phosphate injection(DEX)diluted twenty-fold,each 0.2ml. Intraperitoneal injection of saline normal in control group.Only 2h,4h, 6h,8h,10h after modeling,removing the mice thymus cells from each group with thymus cells from control group doing cell mears,and then Giemsa stained.Through morphological observation,counting apoptotic thymocytes of each mice and apoptotic rate,and then computing average apoptosis rate,rendering time-apoptosis curve.2.The intervention role of Chinese medicine active ingredient on abnormal lymphocyte apoptosisWith the use of MTT and Gimsa staining,determine absorbance values and the number of apoptotic cells in all experimental groups to calculate the cell activity and apoptosis rate,analyze the intervention role of Astragalus injection(AI),Lycium barbarum polysaccharides,Ganoderma lucidum polysaccharides,astragalus polysaccharide and Cordyceps sinensis polysaccharide on glucocorticoid-induced unusual apoptosis of thymocytes from mice,and then from the above-mentioned five drugs initially screen the drugs with the role of suppressing on thymocyte apoptosis in mice.Finally,to conduct an in-depth study of the efficacy.3.Flow cytometry(FCM)analyzes the inhibition of effective drugs on DEX-induced apoptosis in mouse thymocytesBy flow cytometry,with PI single staining and AnnexinV-FITC/PI dual-staining to further determine the influence of these initial screening of the effective drugs on the rate of apoptosis to quantitatively detect t rate of apoptosis of each experimental group.4.Agarose gel electrophoresis analyze the role of inhibition of effective drugs on apoptosisThis experiment adopted by agarose gel electrophoresis from the view of biochemical characteristics analyzes the role of inhibition of above screening effective drugs on lymphocyte apoptosis. 5.Study in apoptosis inhibition mechanismWith the use of immunohistochemistry and western blot techniques, analyze the inluence of effective drugs above mentioned on mouse thymocytes apoptosis-related proteins bcl-2 and caspase-3 expression, to preliminarily investigate the molecular mechanism inhibition of its inhibition to lymphocyte abnormal apoptosis.Result1.Experimental modelFrom the experimental data and time-apoptosis curve observed,6h mice the highest rate of thymocyte apoptosis.Agarose gel electrophoresis results showed that:all strips with the model group show typical apoptosis DNA Ladder bands,while only single-strip with the normal group,this illustrates the success of this experimental model.2.Lyeium barbarum polysaccharides and astragalus can inhibit glucocorticoid -induced thymocytes apoptosis in mouseThrough analysis of the results with MTT and Gimsa staining,it shows that:Astragalus injection significantly enhances or inhibits cell activity or apoptotic rate of thymus cells under the effect of glucocorticoids and there was a dose-dependent relationship trends(P＜0.01 or P＜0.05,compared with model group),the best high-dose;Lycium barbarum polysaccharides also enhances or inhibits cell activity or apoptotic rate of thymus cells under the effect of glucocorticoids(P＜0.01 or P＜0.05, compared with model group),but no obvious dose-dependent relationship,among three dose-group,mid-group is better.3.Flow cytometry(FCM)analysisWhether PI single-stained or double staining method,the results of flow instruments shows:Astragalus injection and Lycium barbarum polysaccharides all significantly inhibit abnormal apoptosis of thymus cells in mouse.Among them,apoptotic rate of the AI high,medium and low dose group:PI single-stained apoptosis rate 2.50%±0.10%,5.67%±1.11%,9.40%±1.31%(P＜0.01,compared with model group);double staining method 3.13%±0.25%4.60%±0.44%,5.13%±0.45%(P＜0.01 or P＜0.05,compared with model group),high-dose group of Ai is the lowest rate of cell apoptosis,and there is a dose-dependent trend; apoptotic rate of the Lycium barbarum polysaccharides(LBP) high,medium and low dose group:PI single-stained apoptosis rate 15.2%±1.66%,11.36%±0.72%,18.5%±1.76%(P＜0.01,compared with model group); double staining method 4.27%±0.15%,3.53%±0.24%,5.60%±0.50% (P＜0.01或P＜0.05,compared with model group).The mid-dose group is the lowest rate of apoptosis,and with the dose increase or decrease,its rate of apoptosis has a rising trend.4.Agarose gel electrophoresis experimentSuch as Figure seven or eight of the body plan shown.LBP and AI experiment have no typical apoptotic DNA Ladder bands.AI high,medium and low dose group all have DNA Ladder stripe,but bands of the high-dose group compared with the model group was not obvious.LBP high,medium and low dose group also appear typical Ladder apoptosis band,but bands of mid-dose group compared with the model group is not obvious.5.Mechanism study(l) The results of AI immunohistochemical experiment show that compared with the model group,the percentage of apoptosis suppressor gene bcl-2 protein positive area with each dose-group of Astragalus injection is significantly increased(P＜0.01),and the percentage of caspase-3 protein positive area is significantly reduced(P＜0.01);Weatern Blot protein bands of gray-value analysis shows:AI high,medium and low-dose group and model group,Bcl-2 gene protein expression,respectively,is 2.20,1.77,1.69,1.29;AI high,medium and low-dose group and model group, caspase-3 gene protein bands of gray value respectively,is 1.18, 1.25,1.40,2.23.The results suggest that AI can promote apoptosis inhibition protein Bcl-2 expression,and reduced extra expession of Caspase-3 protein,and show some dose-dependent relationship.(2) LBP immunohistochemical experiments show that:Compared with model group,Bcl-2 protein positive area the LBP high,modiem and low dose group are larger,the difference was statistically significant(P＜0.01),and the positive area of medium group is the largest;and each dose group of LBP,compared with the model group,Caspase-3 protein in the positive area is smaller,the difference was statistically significant(P＜0.01 or P＜0.05).Weatern Blot results showed that LBP high,medium and low-dose group and model group,the Bcl-2 protein expression are 2.31,2.34,1.18,2.11;the caspase-3 protein expression of high,medium, low-dose group and model group,are respectively 1.70,1.26,1.59,2.1,and mid-dose group is the lowest.The results suggest that LBP can promote apoptosis inhibition protein Bcl-2 expression,and reduced extra expession of Caspase-3 protein,and show no dose-dependent relationship. ConclusionThis study uses DEX to successfully establish the model of thymocyte apoptosis in mice,using MTT method,Gimsa staining,FCM and agarose gel electrophoresis to analyze the intervention role of AI,LBP,Ganoderma lucidum polysaccharides,astragalus polysaccharide,Cordyceps sinensis polysaccharide on the DEX-induced apoptosis in mouse thymocytes apoptos-is in mice,and reserch through Western Blot and immunohistochemical techniques the molecular mechanism of apoptosis inhibition of the effective drugs above-mentioned.The results showed that Lycium barbarum polysaccharides and astragalus injection can inhibit DEX-induced thymocytes abnormal apoptosis,with AI high-dose group best,and there is the dose-dependent trend.Compared with model group,he mid-dose group of Lycium barbarum polysaccharides is the most significant,high-and low-dose group are effective.Part of the mechanism of their role is likely to be through the promotion of anti-apoptotic gene bcl-2 expression and suppress pro-apoptotic gene caspase-3 expression,but do not rule out other mechanisms,other mechanisms may be combined to inhibit apoptosis, which remains to be further studied.