Study On Preparation And Immunological Adjuvant Activity Of Glycyrrhetinnic Acid Liposome

Glycyrrhetinic acid (GA) is the main ingredient of glycyrrhiza. GA exhibits wide pharmacological activities, such as anti-ulcerative effect, anti-inflammatory activity, direct and indirect antiviral activity, anti-cancer activity, immuno-modulating and so on, but it has a very low solubility in water, which may result in its poor bioavailability. Liposome is an artifical phospholipids small vesicle with a double membranes, which can be used as artificial membranes and excipients of various types of therapeutic agents. Liposomes could target carrying drugs to lesion, tissues and cells, so as to enhance the efficacy of the drugs. In addition, liposome can be slow-release drug, which extent the drug action time, reduce toxicity and side effect of drug and so on. Many researches found that liposome possessed the sigificant effect of immunological adjuvants. Liposomes got into the focus of scientific interest for its immunocompetence. If GA is encapsulated with liposome, it not only can solve the above problems, but also can play the advantages of GA and liposome. This study is to prepare the glycyrrhetinic acid liposome (GAL) by thin-film dispersion method and get the optimal preparation condition of GAL by Response Surface Methodology (RSM); the immunological adjuvant activity of GAL was researched including the activity of animal lymphocyte proliferation, antibody titers, cytokines, dentritic cells (DCs), macrophage and so on by in vivro or vivo experiments. The aim of this study is to develop high-effect and low-toxicity new-type of immunological adjuvant. The details were divided into seven parts as follows:Experiment 1 Optimization on preparation conditions of glycyrrhetinic acid liposome Glycyrrhetinic acid liposome was prepared by thin-film dispersion method. To optimize the preparation conditions of GAL, ultrasonic time, ratio of soyben phosphatide to drug, ratio of soybean phospholipid to cholesterol, water bath temperature and hydratio madia five factors were tested with two indexes of encapsulation efficiency and drug-loading rate. Based on the single-factor experiment, the influence of ratio of soyben phosphatide to drug, ratio of soyben phosphatide to cholesteral and water bath temperature on the entrapment rate (ER) was studied by response surface methodology. The data was analyzed with Design-Expert software, the optimum preparation conditions were as follows:ratio of soyben phosphatide to GA was 9.0:1, ratio of soyben phosphatide to cholesteral was 2.5:1, water temperature was 31℃. The results showed that GAL was prepared as the optimal preparation condition with the property of high encapsulation efficiencyExperiment 2 Effect of GAL on lymphocyte proliferation and the concentration of immunoglobulins in chickens GAL, GA and blank liposome (BL) were diluted to eleven concentrations with RPMI-1640. Then the drugs were added into the monolayer chick embryo fibroblast to observe the maximal safety concentrations with MTT mathod Five concentrations of GAL, GA and BL were singly or synergistically with PHA added into the peripheral lymphocyte, the same, the three drugs were added into splenic lymphocyte After cultured 48 h, the lymphocyte proliferation was determined with MTT method. These results showed that the maximal safety concentration of GAL was higher than that of GA. At 8.750-0.547 μg·mL-1, GAL could stimulate the peripheral blood lymphocyte proliferation singly or synergistically with PHA, and at 4.375-0.547 μg·mL-1, the effect of GAL was better than GA and BL. At 8.750-0.547 μg·mL-1, GAL could stimulate the splenic lymphocyte proliferation singly or synergistically with LPS, and at 8.750-1.094 μg·mL-1, GAL had better effect than GA and BL. At 8.750 μg·mL-1, GAL could significantly increase the concentration of IgG,; at 8.750-4.375 μg·mL-1, GAL could significantly promote the concentration of IgM.Experiment 3 Effects of GAL on mRNA expression of three cytokines in chickens in vitro In order to study the action mechanism of GAL on immunological adjuvant activity, the primers of IL-2, IL-4 and IFN-γ were designed with Primer Primier 5.0. The chicken peripheral lymphocyte were cultivated, adding GAL, GA and BL at three concentrations. After cultured 36 h, the cells were collected and total RNA was extracted. The transcription of IL-2, IL-4 and IFN-γ mRNA was determined by fluorescence quantitative RT-PCR assay. The results showed that GAL could improve the transcription level of IL-2、IL-4 and IFN-y mRNA, and were significantly better than GA and BL at 8.750 μg·mL-1 and 2.188 μg·mL-1. It might be one of the immune enhancement mechanisms that GAL could promote the animal immunity.Experiment 4 Adjuvant effect of GAL on immune response of ND vaccine in chickens The aim of this study is to investigate whether the activity of inducing immune response of GAL could be enhanced after GA was encapsulated with liposome. Two hundred and ten 14-day-old chickens were randomly divided into 7 groups and vaccinated with New castle disease vaccine, repeated vaccination at 28-day-old. Simultaneously, the chickens in experimental groups were injected with GAL at high, medium and low doses, GA and BL, respectively, in vaccine control groups and blank control groups, physiological saline. On days 7,14,21,28,35 and 42 after the first vaccination, the blood samples were collected from cardiopuncture for determination of peripheral T lymphocyte proliferation in vivo by MTT method and the T lymphocyte subpopulation CD4+and CD8+were examined using flow cytometry., brachial vein for determination of serum HI antibody by β-micro-method andthe concentrations of IgG and IgM were determinated with ELISA. The results showed that GAL not only could enhance significantly the antibody titers, the concentrations IgG and IgM in ND vaccine immunized chicken, but also significantly promote T lymphocyte proliferation and the proportions of CD4+ and CD8+. Moreover, the effects appeared a dose-dependent and a time-dependent manner. These results indicated that GA and liposome could synergistically improve the adjuvanticity and GALM possessed the best efficacy.Experiment 5 Adjuvant effect of GAL on immune response of mice vaccinated with OVA Two hundred and ten 5-weeks-old mice were randomly divided into 7 groups and vaccinated with OVA, repeated vaccination at 6-weeks-old and 7-week-old. Simultaneously, the mice in experimental groups were injected with GAL at three doses, GA and BL, oil-adjuvant (OIL) respectively. On days 7,14,21, 28,35 and 42 after the first vaccination, the peripheral blood samples were collected via the retro-orbital venousplexus in order to measure serum IgG. IL-2 and IFN-y with ELISA. Four mice were sacrificed each group to sterily take spleens off and to separate lymphocyte. The splenic T, B lymphocyte lymphocyte proliferation were determinated by MTT method. The results showed that GAL could not only significantly promote T and B lymphocyte proliferation, but also raise the concentration of IgG、IL-2 and IFN-γ, at suitable dose and some time points. These results indicated that GA and liposome could synergistically improve the adjuvanticity and GALH and GALM possessed the best efficacy.Experiment 6 Effect of GAL on functions of mice dendritic cells In order to study the immunological adjuvant mechanism of GAL, in the experiment, seven concentrations of GAL, GA and BL were added into DCs precursor from mouse bone marrow. DCs precursor proliferation was determinated with MTT method. The immature DCs of the murine bone marrow were induced with rmG-CSF and rmIL-4. Then DCs stimulated GAL, GA and BL for 48 h, OVA and the splenic lymphocytes from mouse were cocultured to examine the effect of GAL on antigen presenting ability of DCs with MTT mathod. In addition, the DCs stimulated GAL, GA and BL were mixed into splenic lymphocytes of mice vaccinated OVA in order to examine the GAL on the effect of DCs on stimulating T lymphocyte proliferation.The results showed that at 0.875 μg·mL-1 and 2.188-0.547 μg·mL-1, GAL could significantly promote DCs precursor proliferation and at 8.750-0.547 μg·mL-1, GAL could not only improve the function of DCs on stimulating T lymphocyte proliferation, but also enhance antigen presenting ability of DCs.Experiment 7 Effect of GAL on mouse peritoneal macrophage phagocytic ability To investigate the effect of GAL on mouse peritoneal macrophage phagocytic ability,6% starch-bouillon was injected into the mice peritoneal. After 3 d, the mice were sacrificed to collect the peritoneal fluid and separate macrophage. The cells were exposed to 5 concentrations of GAL, GA and BL for 48 h. Then neutral red was added into macrophage. After 4 h, the condition of macrophage phagocytic neutral red was measued by neutral red method. At 8.750-0.547 μg·mL-1, the values A490 of GAL group were not significant with those in BC group. The results showed that GAL could not significantly enhance the macrophage phagocytic ability.