Compound 511 Regulate Th1/Th2/Th17/Treg Balance Via Pi3K/AKT/mTOR Pathway In Morphine-induced Immunosuppression

Objective:Based on the PI3K/AKT/mTOR signal pathway,to observe the therapeutic effect of compound 511 on Thl/Th2/Th17/Treg balance influenced by morphine.The purpose of this study is to explore the mechanism of compound 511 in the treatment of morphine induced immunosuppression,to lay a foundation for further elucidating the molecular biological mechanism of compound 511 in the treatment of morphine induced immunosuppression,and to provide a new idea and methods for the treatment of immunosuppression with traditional Chinese medicine.Methods:40 healthy male C57BL/6 mice(bodyweight 20-25g)of 8 weeks old were randomly divided into 5 groups,each group include 8 mice.The name of each group are as follows:(1)control group;(2)model group(MOR group);(3)low dose group(3g/kg)(MOR+511-L group);(4)medium dose group(6g/kg)(MOR+511-M group);(5)high dose group(12g/kg)(MOR+511-H group).The mice in MOR group,MOR+511-L group,MOR+511-M group and MOR+511-H group were all given intraperitoneal injection of morphine.The dosage of morphine injection increased according to the number of days,as follows:the first day:10mg/kg;the second day:20mg/kg;the third day:30mg/kg;the fourth day:40mg/kg;the fifth day:50mg/kg;the sixth day:50mg/kg.From the first day to the fifth day,morphine was injected into abdominal cavity twice a day,and only once on the sixth day.The mice in the control group were injected with the same amount of normal saline at the same site.All mice were given normal saline or compound 511 solution by oral administration for 6 days.The weight of all mice was measured and recorded from the first day to the sixth day,and the peripheral blood,spleen,thymus and lymph nodes of mice were collected on the sixth day for subsequent molecular experiments.The effects of compound 511 on the immunosuppression induced by chronic morphine were observed by measuring the body weight of mice and calculating the spleen index and thymus index.The proportion of macrophages,neutrophils,T cells and lymphocyte apoptosis in spleen were detected by flow cytometry,and the ratio of CD4/CD8 was calculated.The expression levels of IL-1?,IL-10,TGF-?,T-bet,GATA3,STAT3 and Foxp3 in spleen were detected by qPCR.The percentage of Th1,Th2,Treg and Th17 cells in spleen,lymph nodes and peripheral blood were detected by flow cytometry.Western blot was used to detect the expression of p-PI3K,PI3K,p-AKT,AKT,p-mTOR and mTOR in spleen.Results:Experiment 1:1.Compared with the control group,the weight of mice in the model group decreased significantly.2.Compared with the model group,the body weight of mice in the middle dose group increased significantly on the 5th day of administration,and there was no significant change in the low dose and high dose groups.2.The results of spleen index and thymus index showed,compared with the control group,the spleen index and thymus index of the model group decreased significantly.Compared with the model group,the immune organ index in the low dose group increased significantly.There was no significant difference in the middle dose group and the high dose group.Experiment 2:1.Compared with the model group,compound 511 could effectively increase the proportion of macrophages and neutrophils in the spleen and the proportion of CD4/CD8,and reduce the proportion of T cells in the spleen.2.The results of qPCR showed that compound 511 reduced the expression level of IL-1?and TGF-?,and increased the expression level of IL-10.3.The results of flow cytometry showed,compared with the model group,compound 511 could significantly reduce the apoptosis of lymphocytes in spleen.Experiment 3:1.Compared with the model group,compound 511 could balance Thl/Th2/Treg/Th17 in spleen,lymph nodes and peripheral blood.2.The results of qPCR showed that compound 511 increased the expression of T-bet and STAT3,and decreased the expression of GATA3 and Foxp3.3.Western blot showed that the contents of p-PI3K,p-AKT and p-mTOR in the spleen of the compound 511-treated mice were significantly lower than those of the model group.Conclusion:Compound 511 could effectively alleviate the balance of Thl/Th2/Treg/Th17 induced by morphine,and its mechanism may be related to the inhibition of PI3K/AKT/mTOR signaling pathway.