Immunomodulatory Effects Of Fufang HD On Cyclophosphamide-Induced Immunosuppression In Mice

Objective:As a classical immunomoduLatory,FuFang HD showed significant immunomoduLatory effects through many years of clinical experience.However,due to the muLtitudinous composition of Chinese medicine compound preparations,complicated mechanism,unclear mechanism of immune reguLation and the existence of toxicity,it is difficult to be convinced by people.Therefore,we carried out the acute toxicity test to investigate the safety of FuFang HD.In our study,the model of immunosuppressivemicewasinducedbyintraperitonealinjectionof cyclophosphamide,and FuFang HD was given by intragastric administration and then we investigated the mechanism of immune reguLation of FuFang HD by determining the relevant immune indexes.Methods:Experiment one:40 ICR mice,6-8 weeks old,were randomly and equally divided into control group and experimental group,male and female in half.For there was no obvious toxic reaction in the clinical application and the pre-experiment of the compound containing traditional Chinese medicine,therefore,the mice were administered with a maximum volume of 0.4 mL/10 g and a maximum concentration of 2.093 g/mL twice a day for 24 hours.The changes of body mass and reaction of the animals were recorded for 14 days.On the 15th day,the peripheral blood samples from the control and the treated mice were collected by removing the eyeball and the ALT and AST were determined by the microplate method,CRE by creatine oxidase,BUN by urease method,to investigate the effect of FuFang HD on the function of liver and kidney.The main organ index was measured,and histopathological examination was performed if the organ had obvious visual changes.Experiment two:48 male ICR mice,6-8 weeks old,were randomly and equally divided into normal control group,model control group,positive control group,FuFang HD low dose group,FuFang HD medium dose group and FuFang HD high dose group.The positive control group received lentinan at 200 mg/kg.The mice in different oral administration groups were treated with FuFang HD at 5,10 and 20g/kg,whereas the mice in normal control group and model control group were given distilled water via oral administration once daily for 10 consecutive days,and each group(except for noamal control group)were given cyclophosphamide(40 mg/kg)intraperitoneally on days 4-6.On the 11th day,the mice were sacrificed and the spleen and thymus were excised from the animal and weighed.Relative thymus/spleen weights were expressed as the ratio of the thymus/spleen to body mass(mg/g).T cell subsets of CD3~+,CD4~+and CD8~+lymphocytes were detected by flow cytometry.The levels of IFN-?and IL-4 cytokines were measured by ELISA kit and real-time fluorescence quantitative PCR was used to detect the expression of T-bet and GATA-3mRNA in spleen.Results:Experiment one:1.The mice in the experimental group and the normal control group were observed for 14 days with normal state and no death,and the time-weight change curve of the two groups were nearly parallel,and there was no significant difference in body weight between the two groups on the 7th and 14th day(P>0.05).2.There was no significant difference in the organ index of the heart,liver,spleen,lung and kidney between the two groups of mice(P>0.05),and there was no obvious visual change in the anatomy of the mice.3.No statistical difference in plasma AST,ALT,CRE and BUN in the two groups of mice(P>0.05).Experiment two:1.Compared with the normal control group,the body weight of the model group reduced markedly.The thymus index,the percentage of CD3~+cells,CD3~+CD4~+cells,the ratio of CD4~+/CD8~+,IFN-?level and the ratio of IFN-?/IL-4,and the expression of T-bet and GATA-3 mRNA of spleen transcription factor all significantly decreased,P<0.05.2.Compared with the model control group,the weight loss of the mice in FuFang HD low,medium and high-dose groups were significantly reduced,and the recovery of body weight in the FuFang HD mice groups showed a dependence on the concentration.In the FuFang HD medium and high dose group,the thymus index,the percentage of CD3~+cells,CD3~+CD4~+cells and CD4~+/CD8~+ratio,the level of IFN-?,IFN-?/IL-4 ratio and the expression of spleen transcription factor T-bet and GATA-3 mRNA increased significantly,P<0.05.Conclusion:1.This study shows that FuFang HD has little effect on body weight and organ index through acute toxicity test and there was no obvious toxicity for hepatic and renal.2.For the first time,it is proved that the FuFang HD can promote the recovery of the immune function of immunosuppressive mice induced by cyclophosphamide,confirming the certain curative effect of FuFang HD in the prevention and treatment of immune deficiency.The results provided a scientific basis for the further scientific exploration and the clinical application.