Protective Effect Of Artesunate On Hydrocortisone-induced Immunosuppression Mice And Its Mechanism Preliminary Study

Objective:In this study,the protective effect of artesunate(AS)and its molecular mechanism were evaluated by glucocorticoids(GCs)immunosuppressive mice model induced by hydrocortisone(HC).Methods:1.Mice were injected intramuscular(i.m.)with different doses of hydrocortisone(HC5,10,20 mg/kg/day)for 5 days.EC(1×10~8 colony-formation units[CFU]/Kg)was administered intravenously(i.v.)in the 6th day.By observing the mice mortality;serum TNF-?release level;the spleen index and thymus index to select HC dose.In order to observe the bacterial clearance ability of model mice,Mice were i.m.injected HC(optimal dose)for 5 days,and then i.v.injected with different doses of EC(EC1,1.0×10~7 CFU/Kg;EC2,5.0×10~7 CFU/Kg;EC3,1.0×10~8 CFU/Kg),By observing the serum TNF-?release level and the bacterial loads to evaluate the ability of mice to remove bacteria.2.The HC induced immunosuppressive mice model was established by the above methods.Mice were divided into Control group,HC group and AS group.At the same time of the first HC injection,mice were intraperitoneal injection of AS(20 mg/kg)twice a day,and continued for 1~5 days(AS1~AS5)after the cessation of HC administration).EC(5.0×10~7 colony-formation units[CFU]/kg)was administered intravenously(i.v.)at last.The spleen was weighed to calculate the spleen index and the blood were collected for IL-6 and TNF-?detection,the time of AS to significantly increase the IL-6 and TNF-?level was selected for the next study.Mice were divided into Control group,HC group,AS group,Th group and AS+Th group.AS intraperitoneal injection(20 mg/kg),twice a day,Th intramuscular injection(10 mg/kg),once a day,EC(5.0×10~7 colony-formation units[CFU]/kg)was administered intravenously(i.v.)at last.The blood was collected for white blood cells count,CFU count assays and TNF-?and IL-6 detection,the colon tissue is used for sIgA testing,the above indicators were used to judge the protective effect of AS3.Establishment and evaluation of HC-induced immunosuppressive cell model:Peritoneal macrophages(PMs)were cultured with HC(0.02-43.74?g/mL)for 0.5 h,then the culture supernatant was not replaced followed by the addition of LPS(100 ng/mL),the supernatant was collected for TNF-?detection.HC concentration was selected according to the release of TNF-?.Subsequently,adding different concentrations of AS or adding AS at different time in HC-induced immunosuppressive model,confirmating the best effective dose and administration time window of AS based on the level of TNF-?in the cell supernatant.HC cells were treated according to the optimal administration regimen of AS.The expression of TNF-?and IL-6 mRNA and the level of TNF-?and IL-6 in the supernatant were detected to evaluate the effect of AS.RT-PCR analysis was conducted to determine the mRNA expression of GILZ,TLR4,NF-?B p65 and NF-?B p50.By using Elisa kit detect the protein expression of NF-?B p65 in HC-induced immunosuppressive cell model.Results:1.Establish and evaluate of HC-induced immunosuppression mice model.(1)With the increase of HC dose,the spleen index and thymus index of HC mice decreased,the survival rate goes down,the TNF-?level of HC mice decreased.In summary,HC 20 mg/kg intramuscular injection for 5 days,once per day,could induced immunosuppressive mice model.(2)The levels of serum TNF-?in HC mice were significantly lower than those in control mice,and the EC loads in the blood was significantly increased compared with control mice.2.Protective effect of AS in HC-induced immunosuppression mice model.(1)AS could increase the spleen index and increase the level of TNF-?and IL-6.AS 2 increased the level of TNF-?and IL-6 most significantly.(2)AS could significantly enhance the ability of mice to clear bacteria,increase the release of TNF-?and IL-6 inhibited and significantly increased the number of white blood cells and increased the level of sIgA.In combination with Th,the protective effect on immunosuppressive mice was better than AS treatment.3.Preliminary study of protective mechanisms of AS in HC-induced immunosuppressive macrophages.(1)After HC 0.5?g/mL pretreatment for 0.5 h,cells treated with 100 ng/mL LPS to establish HC-induced immunosuppressive cell model.(2)AS increases the release of TNF-?,IL-6 in HC cells.(3)AS inhibits GILZ mRNA and increases TLR4/NF-?B p65 expressions in HC cells.Conclusions:HC 20 mg/kg was used to establish HC-induced immunosuppression mice.AS has a significant protective effect on HC-induced immunosuppressed mice.AS enhances the ability of mice to clear bacteria and increase the level of TNF-?and IL-6.HC 0.5?g/mL was used to establish HC-induced immunosuppressive cell model,AS increases the release of TNF-?,IL-6 in HC cells,may inhibits GILZ mRNA and increases TLR4/NF-?B p65 expressions.