| ObjectiveThrough the establishment of bone marrow suppression in C57BL/6 animal model after chemotherapy,spleens and blood cells were detected in different groups,blood plasma factors,Scal、C-kit、Lamin and CD48+CD150+in bone marrow cells,as well as peripheral blood monocytes cells and CD proteins in thymus and spleens,and further observated antioxidant factors in thymus and splenic tissue with Jianpibuxue method,which were investigated the expression of hematopoietic and immune factors in peripheral blood cells,these aimed at the improving mechanism of Jianpibuxue method in bone marrow suppression and immune function after chemotherapy.Methods1.Pre-experimental observation of different CTX doses was on the bone marrow suppression in mice.By the weight of mice after chemotherapy,thymus and spleen organ index and HE staining,peripheral white blood cells,red blood cells and platelets were counted,expression of Scal and CD34 in bone marrow cells,these made clear that the appropriate dose of CTX in mice led to bone marrow suppression.2.The use of alcohol extraction method,and analyzed the main ingredients of traditional Chinese medicine prescription by HPLC.3.The changes of the normal circumstances,thymus and spleens histopathology in mice were observed after chemotherapy.4.Using blood analysis to detect types and quantity of peripheral blood cells in mice after chemotherapy.5.Using the enzyme-linked immunosorbent assay(ELISA)to detect the plasma levels of GM-CSF,TPO,EPO,IL-2 in mice.6.Using flow expression to detect fluorescence of Seal,Lamin,C-kit and CD48+CD150+ in hematopoietic stem cells and CD3,CD4 and CD8a in peripheral blood mononuclear cells(PBMCs).7.Using Western blot method to detect the protein expression of CD3,CD4 and CD8a and Nrf2,Nqol and Ho1 in thymus and spleens.8.Using RT-PCR method to detect the expression of Nrf2,Nqo1 and Ho1 mRNA in spleens.9.Using HE staining method to detect the local structural changes in femur,thymus and spleen tissues,expression of Nqo1 and Ho1 simultaneous detection in splenic tissues.Result1.Effect of CTX on body weight in miceEach group was the same weight before 5 days,at the beginning of the 5th day,body weight of CTX groups decreased gradually,high dose of CTX group decreased significantly.2.Effect of CTX on thymus organ index and HE staining in miceCompared with thymus index of normal mice,two doses of CTX decreased significantly,the organ index of thymus was significantly at 2th day(P<0.01).CTX 50mg/kg decreased significantly at 7th and 14th day(P<0 01).HE staining showed normal thymic cortex,medulla,cysts and diaphragm are clearly visible,but thymus structure in CTX groups were damaged,50mg/kg group was more obvious.3.Effect of CTX on spleen organ index and HE staining in miceCompared with the normal group,the spleen indices of CTX 50mg/kg group declined significantly at 2th day(P<0.01).Compared with CTX 25mg/kg group,CTX 50mg/kg group was significantly decreased(P<0.05).At 7th day,CTX 50mg/kg group was significantly higher than the other two groups(P<0.05).At the 14th day,changes of the three groups were no significant.HE staining showed that white pulp atrophy,hemorrhage and necrosis in spleen of CTX 50mg/kg group were destoried at 2th day.Spleens were recovery at 7th day.4.Effect of CTX on peripheral blood cells in miceCompared with the normal group,the white blood cells and red blood cells in CTX 25mg/kg and 50mg/kg dose group were decreased significantly at 2th,7th,14th day(P<0.01).Platelets in three groups was increased significantly(P<0.05 or P<0.01).5.Effect of CTX on Scal and CD34 of bone marrow cells in miceCompared with the normal group,the expression of Scal in bone marrow cells were significantly decreased in CTX groups at 2th day(P<0.01),the expression of CD34 were significantly increased(P<0.01).Compared with 25mg/kg group,the expression of Scal in 50mg/kg group was decreased more significantly(P<0.01).The expression of CD34 in 50mg/kg group was increased.There were no significant difference of Scal in the three groups at 7th day,the expression of CD34 was decreased.The expression of Scal in bone marrow cells were significantly increased in CTX two dose groups at 14th day(P<0.05 or P<0.01),the expression of CD34 were on the contrary.Compared with 25mg/kg group,the expression of Scal in 50mg/kg group was more significantly increased(P<0.01),the expression of CD34 were on the contrary.Confocal laser scanning microscope can observe nuclear staining(DAPI)was no significant change in the three groups.After incubated Scal-FITC(green)and CD34-PE(red),compared with the normal group,CTX groups had a significant reduction in green and red fluorescence on bone marrow cells.6.HPLC analysis Chinese medicine of Jianpibuxue methodThat herbal compound astragalus,orange peel,ginger,licorice compounds based.7.Effect of Jianpibuxue method in mice after chemotherapy7.1 By chemotherapy in mice,the model group was obvioused early symptoms of anorexia,food intake and water intake were decreased significantly,compared with the other groups,the hair gradually lose from their luster,and even hair removal,listlessness,decreased activity,and therefore weight than the other group decreased significantly.From the weight change curve plotted for each group of mice,at the 9th day,the weight showed a progressive decline in the normal control group and positive group,the body weight of mice in each dose group of traditional Chinese medicine was relatively curve flat,respectively.7.2 Compared with the control group,the thymus,spleen weight and organ index of model group were significantly decreased(P<0.01),livers weight and organ index were significantly increased(P<0.01).Compared with the model group,thymus weight of positive drug was increased(P<0.05).But the medium and low dose groups decreased(P<0.05).The thymus index of positive group increased(P<0.01),TCM groups decreased(P<0.01).The spleen weight of positive group and TCM groups increased(P<0.01),spleen index in medium dose of TCM group increased(P<0.01).The livers weight and organ index of positive group and TCM groups decreased(P<0.01).8.The number of peripheral blood cells of Jianpibuxue method in mice after chemotherapyCompared with the control group,the number of WBCs in the peripheral blood cells of the model group was significantly reduced(P<0.01),the number of PLTs and RBCs were significantly increased(P<0.01).The number of WBCs in the positive group and TCM groups was significantly increased(P<0.01).Compared with the model group,the number of PLTs in positive group and meidum dose of TCM group was increased significantly(PK0.01),low-dose of TCM groups was significantly decreased(P<0.01).The number of RBCs in high and medium dose of TCM groups was significantly lower(P<0.01).9.Effect of Jianpibuxue method on hematopoietic growth factors concentration of GM-CSF,TPO,EPO and IL-2 after chemotherapy.Compared with the control group,the levels of GM-CSF,TPO,EPO and IL-2 were significantly reduced in model group(P<0.01),while the positive group and TCM groups can significantly improve the hematopoietic factors concentration(P<0.01).Both positive group and high dose of TCM groups could significantly improve the plasma concentrations of TPO after chemotherapy in mice(P<0.01).Compared with the model group,positive group and TCM groups could significantly increase the plasma concentration of EPO in mice(P<0.01,P<0.05).Compared with the control group,the plasma concentration of IL-2 was decreased significantly in the model group(P<0.01),positive group and TCM groups were significantly increased(P<O.01).10.Effect of Jianpibuxue method on bone marrow cells surface markers after chemotherapyCompared with the control group,it was significantly lower in model group(P<0.01),while the TCM groups could significantly increase(P0.01).At the same time,compared with the control group,C-kit-PE and Lamin-PE on bone marrow cells were significantly decreased in model group(P<0.01).Compared with the model group,high dose of TCM groups can significantly enhance the expression of C-kit on bone marrow cells(P<0.01),the positive group and the low dose of TCM groups were significantly decreased(P<0.01).Positive group and TCM groups can significantly enhance the expression of CD48-PE and CD150-FITC(P<.01).11.Effect of Jianpibuxue method on CD3,CD4 and CD8a of peripheral blood mononuclear cells in mice after chemotherapyCompared with the control group,the expression of CD3,CD4 and CD8a were significantly lower in model group(P<0.01),while the positive group and TCM groups can significantly enhance the expression of CD3,CD4 and CD8a on peripheral blood mononuclear cells(P<0.01).12.Effect of Jianpibuxue method on protein expression of CD3 and CD8a in thymus after chemotherapyCompared with the control group,the protein expression of CD3 and CD8a in model group were significantly lower after chemotherapy(P<0.01),while the positive group and TCM groups could significantly increase(P<0.01).13.Effect of Jianpibuxue method on protein expression of Nrf2,Nqol and Ho1 in thymus after chemotherapyCompared with the control group,the protein expression of Nrf2,Nqol and Ho1 in model group were significantly higher after chemotherapy(P<0.01).Compared with the model group,the protein expression of Nrf2 and Nqol in positive group were significantly reduced(P<0.01),the protein expression of Nrf2 in TCM groups was significantly decreased(P<0.01),the protein expression of Nqol and Ho1 were also significantly increased(P<0.01).14.Effect of Jianpibuxue method on protein expression of Nrf2,Nqol and Ho1 in spleens after chemotherapyCompared with the control group,the protein expression of Nrf2,Nqol and Hol in model group was significantly higher after chemotherapy(P<0.01).Compared with model group,the protein expression of Nrf2 in positive group and the high and low dose of TCM groups was no significant changed,the medium dose of TCM group was significantly increased(P<0.01).Compared with model group,the protein expression of Nqol in positive group and high and low dose of TCM groups was increased significantly(P<0.01).The protein expression of Ho1 in positive group and TCM groups was reduced significantly(P<0.01).15.Effect of Jianpibuxue method on mRNA expression of Nrf2,Nqol and Ho1 after chemotherapyThe mRNA expression of Nrf2,Nqol and Ho1 had significant changes after chemotherapy,compared with the control group,the mRNA expression of Nrf2 and Ho1 were significantly increased in the model group(P<0.01),mRNA expression of Nqol decreased significantly(P<0.01).Compared with model group,the mRNA expression of Nrf2 was significantly higher in positive group and high dose and medium dose of TCM groups(P<0.01),it was significantly decreased in low dose of TCM group(P<0.01).Compared with the model group,the mRNA expression of Nqol was significantly higher in positive group and high and low dose of TCM groups(P<0.01).Compared with the model group,the mRNA expression of Ho1 was significantly increased in positive group(P<0.01),medium dose of TCM groups was significantly decreased(P<0.01).16.Effect of Jianpibuxue method on the femur,spleen,liver tissues stained with HE after chemotherapyFemoral organization nuclear staining of the control group was more visible,femoral nuclear staining suggested less tissue in model mice,nuclear of cells were visible reduced.Femoral nuclear staining tissue of mice treated with TCM group was better than the model group,significantly increased intracellular mitotic count,the low dose of TCM groups was more visible.In spleen tissue sections,the control group was more nuclear staining,nuclear staining deepened,less nuclear staining,reducing the number of nuclear chromatin.Spleen nuclear staining tissues of mice treated with TCM were better than the model group,significantly increased the number of nuclear staining.In liver tissue sections,the control group had more nuclear staining,nuclear staining deepened,deformed nuclear,irregular,small vacuoles,with around cell infiltration,the model group was on the contrary.Nuclear staining of mice treated with TCM were better than the model group,significantly increased the number of vacuoles.17.Effect of Jianpibuxue method on Nqol and Ho1 with immunohistochemistry after chemotherapyImmunohistochemistry showed that Nqol and Ho1 in the control group were positive,showing the cytoplasm and cell gap with pale brown uniform staining.In the model group,the expression of Nqol and Ho1 was weakly positive,showing a small amount of brown staining in the cytoplasm and cell gap.The expression of Nqol and Ho1 were significantly increased in TCM groups.Positive group also significantly increased the expression of Nqol and Ho1 in spleens.Conclusions1.Long-term with low-dose CTX produce more stable bone marrow inhibitory effect on mice.2.Jianpibuxue method could maintain the body weight and food intake and improve the state in mice after chemotherapy.Jianpibuxue method has a protective effect on the structure of femur,spleens,livers in mice after chemotherapy.Jianpibuxue method may have a protective effect on the vital organs of the body.3.Jianpibuxue method has a regulatory role of WBCs,PLTs and RBCs after chemotherapy.4.Jianpibuxue method can improve plasma concentration of GM-CSF,TPO and EPO after chemotherapy,in order to reduce the incidence of leukopenia.At the same time significantly increased plasma concentration of IL-2,suggesting the Jianpibuxue method may have anti-inflammatory effects.5.Jianpibuxue method can significantly enhance the expression of Sca1,C-kit,lamin and CD48+CD150+ on bone marrow cells,suggesting Jianpibuxue method have enhanced the number of hematopoietic stem cells in bone marrow cells after chemotherapy,it might improve the role of bone marrow suppression.6.Jianpibuxue method have enhanced expression of CD3,CD4 and CD8a in peripheral blood mononuclear cells,and enhanced expression of CD3 and CD8a in thymus after chemotherapy,thereby to reduce the occurrence of suppressing immune function after chemotherapy,may play a role on enhancing immune function.7.Jianpibuxue method can enhance mRNA and protein expression of Nrf2,Nqo1 and Ho1 in thymus and spleen after chemotherapy,while enhancing immunohistochemical expression of Nqo1 and Ho1 in spleens,Jianpibuxue method may participate in Nrf2 signal transduction pathways,thereby reducing the possibility of oxidation after chemotherapy,enhancing the body resilience. |
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