Study On The Immunoregulation Mechanism Of An Optimal Chinese Herbal Monomer Compound On Splenic Lymphocytes Of Allergic Rhinitis Mouse Model

Background:Allergic rhinitis (AR).which is mediated by immunoglobulin E. is an chronic inflammation disease in atopic individual’s nasal mucosa. Many immunocytes and cell factors take part in this process of disease. It is called "BiQiu" in traditional Chinese medicine which has special advantages in prevention and treatment of AR. Our research group have been in research of the treatment of AR and designed an effective Chinese compound called "Bimin". This compound demonstrated excellent immunoregulation in clinical trials and animal experiments. In order to study the relationship between the constituent and the dosage of the compound, we selected three herbal monomers——baicalin(Bai), icarrin(Ica) and astragalus saponin Ⅰ (As Ⅰ), from this compound in early research days. And we conducted the three monomers into an optimal proportion experiment by structuring an mathematical model——the uniform design method. Then the effect was tested and verified by some experiments in cellular level. Finally we came to a conclusion that the optimal dose combination could inhibit the proliferation of the splenocyte of AR mice and regulate the imbalance of the Th1/Th2cells. However, the detailed mechanism remained unknown to us. So our research is on the base of former study results and we should explore further to study the immunoregulation mechanism of this optimal herbal monomer compound by doing researches as followes: Detecting the proliferation of splenocytes by cell counting kit-8(CCK-8) method at different time points; Observing the cell cycle distribution by flow cytomentry(FCM) at different time points; Measuring the intracellular calcium concentration by fluorescence microplate reader at different time points.Objective:To study the immunoregulation mechanism of an optimal Chinese herbal monomer compound, which consists of three monomer Baicalin (Bai), Icariin (Ica) and Astragulus Saponin Ⅰ (As Ⅰ), on splenic lymphocytes of allergic rhinitis mouse model. Methods:Firstly, an classic modeling method of AR was established by intraperitoneal interjection of ovalbumin(OVA) and aluminum hydroxide gel suspension. On day1.day7and day14, the mice were sensitized by injecting intraepitoneally with10%OVA, and the sensitized mice were sunjected to5%OVA inhalation challenge on day21to day28. Secondly,the mice were killed and the splenocytes were isolated and cultured in vitro. The cells were divided into three groups:the blank control group, the concanavalin A(Con A) group, the compound group. Then CCK-8method was used to detect the effect of splenocytes proliferation at five different time points such as the12h.18h,24h,48h and72h. The result was measured by microplate reader in the form of optical density value(OD). In the end,the inhibition rate was calculated in order to definite the time-effect relationship and the most marked time point. Cell cycle distribution was observed by FCM also at four different time points as12h,18h,24h,48h in exploring the characteristic of cell cycle distribution with the changing time and definiting the most marked time points of the compound. The changes of intracellular calcium concentration of splenic lymphocyte were measured by fluorescence microplate reader after cells were incubated with fluorescence probe Fluo-3/AM in12h,18h,24h,48h. The characteristic of intracellular calcium concentration and the most marked time point were eventually definite. In addition, photos were taken by fluorescence microscope.Results:The Chinese herbal monomer compound could inhibit cell proliferation of allergic rhinitis mouse model effectively, which were stimulated by Con A previously. The significant difference just emerged in12h(P<0.01). With extending of the action time, the inhibition rate were gradually increasing and reaching peak in48h. And the inhibition presented certain time-effect relationship. In72h the OD value of three groups decreased. Cells in microscope showed a poor situation and this inferred an decline of cellular activity. FCM tests could reveal the fact that ConA could promote cells to enter into the mitosis by reducing the percentage of cells which in GO/Gl phases while rising the percentage of cells in S and G2/M phases. This effect enhanced with time and showed significant difference by comparing with blank control group(P<0.01or P<0.05). Compared with the ConA group, the compound group could rising the percentage of cells in G0/G1and at the same time reducing the percentage of cells in S and G2/M phases at different time points, with the effect being most marked in24h. The results of the tests taken by the fluorescence microplate reader could reveal that the fluorescence value of the ConA group was increasing with the time going on in the previous24hours (vs blank control group P<0.01)while the compound group could reduce this increase obviously, thus reduce the intracellular calcium concentration.Conclusion:The Chinese herbal monomer compound could inhibit the splenocyte proliferation of allergic rhinitis mouse model. The effects of the compound for lowering intracellular calcium concentration and arresting cell cycle at GO/Gl phases from entering into S and G2/M phases might responsible for its anti-proliferation activity.