| Objective:In this study,we explored the efficacies of Periplaneta Americana extracts PA-B and CII-3 on chemotherapy-induced bone marrow suppression and their protective effects on chemotherapy-induced hematopoietic stem cells(HSCs)injury and the underlying mechanisms.Methods:(1)Kunming mice were treated with cytoxan(CTX)to establish bone marrow suppression model,and PA-B,CⅡ-3 and ginsenoside Rg 1 were used to intervene for 14 days.The body weight and general state of mice were recorded daily.After 14 days of administration,the organ index and the number of nucleated cells in bone marrow were measured.The number of white blood cells(WBC),red blood cells(RBC),platelets(PLT)and the hemoglobin(HGB)concentration in peripheral blood of mice were detected.The femur of mice was taken for HE staining to detect the pathological changes.The cell cycle of bone marrow nucleated cells was detected.The serum levels of interleukin-3(IL-3),IL-6 and erythropoietin(EPO)were detected by ELISA.The activity of superoxide dismutase(SOD)and the level of malondialdehyde(MDA)in serum were detected.The content of CD3~-Sca-1~+c-kit~+HSCs(LSK HSCs)in mouse bone marrow was detected by flow cytometry.(2)CD117~+HSCs were isolated from mouse bone marrow by magnetic cell separation system(MACS)and the content of CD117~+HSCs was detected by flow cytometry.Western blot(WB)was used to detect the expression of Rb1,p53,p21 and p16 proteins in CD117~+HSCs.Results:(1)The general state of mice:PA-B and CⅡ-3 could improve the hair rough and hair loss induced by CTX,and the mice in PA-B and CⅡ-3 groups were in good shape both physically and spiritually.During the 14 days of drug administration,the body weight of mice in each group continued to increase,with the same trend.(2)Peripheral blood cells count:PA-B and CⅡ-3 could increase the number of WBCs in peripheral blood of mice on the 7th day(P<0.001),and the effect of PA-B was better than CⅡ-3(P<0.05 or P<0.01).PA-B and CⅡ-3 at all doses significantly increased the number of WBCs,RBCs,PLTs and the HGB concentration in the peripheral blood of mice on the 14th day after administration(P<0.001).The effect of PA-B on increasing the number of WBCs in the peripheral blood of mice was better than that of CⅡ-3(P<0.01 or P<0.001).(3)Organ index:PA-B and CⅡ-3 could significantly increase the spleen index,thymus index and lung index of mice(P<0.05 or P<0.01 or P<0.001).All doses of PA-B were more effective in improving the decrease of spleen index induced by CTX than all doses of CⅡ-3(P<0.001),and high dose of PA-B was more effective in improving lung index than high dose of CⅡ-3(P<0.001).There were no significant differences in cardiac index,liver index and renal index among the groups.(4)The number of bone marrow nucleated cells:PA-B and CⅡ-3 at different doses could significantly increase the number of bone marrow nucleated cells in a dose-dependent manner(P<0.01 or P<0.001).The effect of PA-B at low and medium doses on increasing the number of bone marrow nucleated cells was better than that of CⅡ-3 at low and medium doses(P<0.01).(5)Femur HE staining:all doses of PA-B and CⅡ-3 could improve the pathological changes caused by CTX,such as the destruction of bone marrow tissue structure,the disordered arrangement of bone marrow cells,the significant reduction of intercellular density,and the significant increase of adipose vacuoles.(6)Cell cycle of bone marrow nucleated cells:compared with the normal group,the proliferation index(PI)of bone marrow nucleated cells in the model group had no significant difference(P>0.05).PA-B and CⅡ-3 decreased the percentage of G0/G1 phase,increased the percentage of S phase and G2/M phase,and increased the PI index(P<0.05 or P<0.01 or P<0.001).There was no significant difference in PI of bone marrow nucleated cells between PA-B and CⅡ-3 groups.(7)The serum levels of IL-3,IL-6 and EPO:PA-B and CⅡ-3 significantly increased the serum level of IL-3(P<0.05 or P<0.01 or P<0.001).CⅡ-3 increased the serum level of IL-6 in mice significantly(P<0.001),while PA-B had no significant effect on the serum level of IL-6.High dose of CⅡ-3 and medium and high dose of PA-B could significantly increase the serum level of EPO in mice(P<0.05 or P<0.01).The serum levels of IL-3 and EPO in PA-B groups were not significantly different from those in CⅡ-3 groups.(8)The levels of SOD and MDA in serum of mice:PA-B and CⅡ-3 significantly increased the activity of SOD and decreased the level of MDA in the serum of mice(P<0.001).There was no significant difference in SOD activity and MDA level between PA-B and CⅡ-3 groups.(9)The content of LSK HSCs in the bone marrow of mice:PA-B and CⅡ-3 at different doses significantly increased the content of LSK HSCs in the bone marrow of mice(P<0.01 or P<0.001).PA-B was more effective than CⅡ-3 in improving the decrease of LSK HSCs content induced by CTX(P<0.01 or P<0.001).(10)CD117~+HSCs isolated by MACS:The purity of CD117~+HSCs was(87.23±1.78)%after magnetic bead sorting.Magnetic bead sorting improved the purity of CD117~+HSCs significantly(P<0.001).(11)WB:PA-B and CⅡ-3 significantly reduced the protein expression of Rb1,p53,p21and p16 in mouse CD117~+HSCs(P<0.001).The protein levels of p53 and p21 in CD117~+HSCs of PA-B+CTX group were significantly lower than those of CⅡ-3+CTX group,while the protein level of p16 was significantly higher than that of CⅡ-3+CTX group(P<0.01 or P<0.001).Conclusions:(1)Both PA-B and CⅡ-3 can improve bone marrow suppression induced by CTX in mice;(2)PA-B is more effective than CⅡ-3 in improving the hematopoietic damage induced by CTX in mice;(3)Both PA-B and CⅡ-3 can improve the damage of mouse HSCs caused by CTX and restore the hematopoietic function of mice,and the mechanism may be relative to reduce the protein expression of Rb1,p53,p21 and p16 in mouse CD117~+HSCs. |
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