Effect Of Compound Fufangteng Mixture On NK Cells In CTX Immunosuppressive Mice

Objective:To investigate the immunomodulatory effect of Compound Fufangteng Mixture(CFM)on CD49b~+NK cells of immunosuppressive mice induced by cyclophosphamide(CTX),and to provide experimental basis for the clinical use and development of CFM by detecting the activation and exhaustion level of CD49b~+NK cells.Methods:(1)the model of CTX immunosuppressive mice was established:12 healthy SPF BALB/c male mice were randomly divided into Blank group and CTX group.Mice in CTX group were intraperitoneally injected with CTX(70 mg/kg)for 3 days to establish immunosuppressive model;The Blank group was intraperitoneally injected with the same dose of normal saline,weighed 24 hours after the last administration,and the eyeballs were taken for blood,and the spleen and thymus were taken;The indexes of spleen and thymus were calculated,and the counts of WBC,LYM,Mono,Gran,RBC and PLT in peripheral blood were detected;The histological structure of spleen was observed by HE staining(2)Effects of CFM on CD49b~+NK cells and their activation and exhaustion in healthy and CTX immunosuppressed mice:120 healthy BALB/c male SPF mice were randomly divided into Blank group,CTX group,CTX+CFM-1 group,CTX+CFM-2 group,CTX+CFM-3 group,CFM-1 group,CFM-2 group and CFM-3 group,with 15 mice in each group.Blank group,CFM-1 group,CFM-2 group and CFM-3group were intraperitoneally injected with normal saline(0.1ml/10g)once a day for three consecutive times;Mice in CTX group,CTX+CFM-1 group,CTX+CFM-2 group and CTX+CFM-3group were intraperitoneally injected with CTX(70 mg/kg)once a day for three consecutive times;From the fourth day,Blank group and CTX group were given normal saline(0.15 ml/10 g);CFM-1 group and CTX+CFM-1group were given CFM 15g/kg;CFM-2 group and CTX+CFM-2 group were given CFM 7.5g/kg;CFM-3 group and CTX+CFM-3 group were given CFM 3.75g/kg.The drug was given by gavage once a day for 10consecutive times.24 hours after the last administration,the weight was weighed,the eyeballs were taken for blood,and the spleen and thymus were taken.The body weight and thymus and spleen index were recorded;At the same time,WBC,LYM,Mono and Gran in peripheral blood were detected;The expression levels of CD49b~+NK cells in spleen and peripheral blood were detected by flow cytometry,and the expression levels of CD2,CD69,CD95 and PD-1 on CD49b~+NK cells were further detected.Results:(1)Three times of intraperitoneal injection of CTX(70mg/kg)mice were given weight loss,decreased thymus index and spleen index,WBC,LYM,Mono,Gran absolute value of peripheral blood,HE staining of spleen tissue showed that the red pulp and white marrow structure of CTX group were disordered,the boundary was unclear,there were slight swelling and inflammatory cell infiltration.(2)CFM has a regulatory effect on the immune function of CTX immunosuppressive mice:compared with Blank group,the thymus index of CTX group is decreased,and the middle and low dose CFM has the tendency to improve the thymus index of immunosuppressive mice;Compared with Blank group,the percentage of CD49b~+NK cells increased(P<0.001),the percentage of CD49b~+CD2~+NK cells decreased(P<0.001),the percentage of CD49b~+CD95~+NK cells increased(P<0.001),and the percentage of CD49b~+PD-1~+NK cells increased(P<0.01);The high and medium dose CFM can reduce the percentage of CD49b~+NK cells in peripheral blood of CTX immunosuppressive mice(P<0.05,P<0.01),and the effect of medium dose CFM is better;The results showed that the low dose CFM could improve the percentage of CD49b~+CD2~+NK cells in peripheral blood of CTX immunosuppressive mice(P<0.01,P<0.001),and the effect of low dose CFM was better;The percentage of CD49b~+CD69~+NK cells in peripheral blood of CTX immunosuppressive mice was increased by medium dose CFM(P<0.05);The percentage of CD49b~+CD95~+NK cells in peripheral blood of CTX immunosuppressive mice was reduced by medium dose CFM(P<0.05);Compared with Blank group,the percentage of CD49b~+NK cells in spleen of CTX group increased(P<0.001),CD49b~+PD-1~+NK cell percentage increased(P<0.001),and the percentage of CD49b~+NK cells in spleen of immunosuppressive mice was decreased by medium dose CFM(P<0.05).High dose CFM could increase the percentage of CD49b~+CD2~+NK cells in spleen of immunosuppressive mice(P<0.05).The percentage of CD49b~+PD-1~+NK cells in peripheral blood of immunosuppressive mice was reduced by high and medium dose CFM(P<0.05,P<0.05).Conclusion:(1)Male BALB/c mice were intraperitoneally injected with CTX(70 mg/kg)for 3 days to establish the immunosuppressive model.(2)CFM can regulate the expression of CD49b~+NK cells in peripheral blood of CTX immunosuppressive mice,and further regulate the expression of CD49b~+CD2~+NK cells,CD49b~+CD95~+NK cells and CD49b~+PD-1~+NK cells.(3)CFM can regulate the expression of CD49b~+NK cells in spleen of CTX immunosuppressive mice and further regulate the expression of CD49b~+PD-1~+NK cells in mice spleen.