Experimental Study Of The Modulatory Effect Of Hemarisin On Mouse Immune Function

Lipopolysaccharide (LPS) or endotoxin is a lipid-containing polysaccharide. It shows wide range of biological activities, such as immune enhancement. However, LPS also shows strong toxic reaction (Shwartzman reaction, pyrogenic reaction, etc) that hinders its clinical application. Many researchers have been committed to the toxicity attenuation of LPS. Bacterium Prodigiosum LPS (BP-LPS) is an immunoreactive substance obtained for this purpose. BP-LPS not only has wide range of biological activities, such as immunopotentiating effect, anti-cancer action, etc, but also shows low toxicity for it is extracted from non-pathogenic Gram-negative bacteria, a pigment producing Bacillus strain NO.BP-S003. This research was carried out to study the modulatory effect of BP-LPS on mouse immune function in order to provide a reliable basis for the rational clinical application.Objective:To study the modulatory effect of hemarisin (Bacterium Prodigiosum lipopolysaccharides, BP-LPS) on the immune function of normal, immunosuppressed and S180 tumor-bearing mice.Methods:1. Mouse models of humoral immune response were established by using rabbit red blood cells as antigen.2. Immunosuppressed mouse models were established by subcutaneous administration of cyclophosphamide. 3. Tumor-bearing models were established by subcutaneously implanting S180 tumor cells to mice.4. Micro-hemolysis spectrophotometry with microplate reader was used to measure the level of serum hemolysin in normal, immunosuppressed and S180 tumor-bearing mice.5. Delay ed-type hypersensitivity (DTH) models induced by dinitro-fluorobenzene (DNFB) were applied for evaluation of the cellular immune function of normal, immunosuppressed and S180 tumor-bearing mice.6. Carbon clearance test was employed to measure the phagocytic activity of mononuclear phagocytes and macrophages in normal, immunosuppressed and S180 tumor-bearing mice.7. Peripheral white blood cells were counted under microscope to estimate the total number of peripheral white blood cells in normal and immunosuppressed mice.8. The mouse splenocyte metabolic activity was assayed by MTT colorimetry, and the amount of TNF-αand IFN-γwas determined by ELISA.Results:1. The peak time needed for the formation of hemolysin against RRBC was six days (The day when the mice were primed was designated as d0) and the optimal amount of antigen was 20%RRBC with each animal intraperitoneally injected with 0.2ml.2. Hydrocortisone (25mg/kg) and cyclophosphamide (20mg/kg) inhibited the production of hemolysin against RRBC. Mannatide (4mg/kg) had no significant effect on serum hemolysin level in normal mice, but it potentiated the hemolysin production significantly in immunosuppressed mice induced by cyclophosphamide (20mg/kg).3. BP-LPS slightly increased the level of serum hemolysin, intensity of reaction of DTH, macrophage phagocytosis and peripheral white blood cell count in normal mice, but there was no significant difference vs control group.4. When the doses were 40U/kg and 20U/kg, BP-LPS markedly increased the level of serum hemolysin, the intensity of reaction of DTH, macrophage phagocytosis and peripheral white blood cell count in immunosuppressed mice. There was significant difference vs control group.5. When the doses were 40U/kg and 20U/kg, BP-LPS markedly increased the level of serum hemolysin, the intensity of reaction of DTH and macrophage phagocytosis in S180 tumor-bearing mice, exhibited considerable anti-tumor effect and raised the spleen index. There was significant difference vs control group.6. When the final concentrations were 20 U/ml and 10 U/ml, BP-LPS improved the splenocyte metabolic activity and obviously increased the amount of TNF-αand IFN-γin the supernatants of splenocyte cultures of S180 tumor-bearing mice. There was significant difference vs control group.Conclusion:1. Rabbit red blood cells as antigen are feasible for establishment of mouse models of humoral immune response and the model is fit for the study of drugs with humoral immune activity.2. Measurement of hemolysin by micro-hemolysis spectrophotometry with microplate reader has advantages such as simplicity in procedure, less experimental errors and convenience.3. BP-LPS has little effect on the immune function of normal mice.4. BP-LPS improves the specific and non-specific immune function of immunosuppressed mice induced by CTX.5. BP-LPS improves the immune function of S180 tumor-bearing mice, promotes release of TNF-αand IFN-γ, and inhibits tumor growth.