Influence Of G6PD Deficiency On Bone Marrow Hematopoietic Damage Induced By Benzene In Mice

About 400 million people around the world lack glucose-6-phosphate dehydrogenase(G6PD),making it the most common human enzyme deficiency.When G6PD deficient individuals contact with oxides,hemolysis may occur,such as fresh broad beans,antimalarial drugs,and so on,so it is also called"broad bean disease".Benzene is a common industrial raw material and an important pollutant in the environment.After entering the organism through different ways,benzene passes through a series of metabolism,and finally produces the final carcinogen 1,4-benzoquinone(1,4-benzoquinone)in the bone marrow.Chronic occupational benzene exposure can affect bone marrow hematopoiesis,leading to leukopenia,aplastic anemia,MDS,and even leukemia.Previous studies of our research group found that G6PD deficiency can enhance the oxidative stress,proliferation inhibition,apoptosis and autophagy levels of K562 cells induced by 1,4-BQ.Transcriptome sequencing of mice with G6PD deficiency found that G6PD deficiency could lead to abnormal high expression of CAMK2B gene in mouse bone marrow cells.Given bone marrow hematopoietic cells is an important target of benzene hematopoietic toxicity,make excessive oxidative stress may affect the renewal and the differentiation of hematopoietic stem cells,therefore,this study proposed in previous research,on the basis of further study on G6PD defects in mice bone marrow hematopoietic stem cell update and differentiation can influence,G6PD defects and relationship between benzene toxicity of hematopoietic susceptibility,and further study of G6PD defect CAMK2B abnormal expression of cytotoxic effect of bone marrow cells in mice.1.Influence of low expression of G6PD on hematopoietic function of bone marrow in miceFirstly,G6PD deficiency(G6pdxa-m1Neu/Y)mice were constructed and their genotypes were identified by generation sequencing and fluorescence quantitative PCR.G6PD deficiency mice and normal control mice were exposed to benzene by subcutaneous injection.the mice exposed to benzene were exposed to 80mg/(kg·d)and150mg/(kg·d),to establish the mouse model of benzene poisoning.By analyzing the proportion of bone marrow hematopoietic stem cells,erythroid progenitor cells,granulocyte progenitor cells and lymphoid progenitor cells in mice and the results of HE staining in bone tissue pathological sections,the effects G6PD deficiency on bone marrow hemopoietic stem cell regeneration and differentiation in mice were investigated.The results showed that the proportion of hematopoietic stem cells(lin~-sca1~+ckit~+)and lin~-sca1~+cells in G6PD deficiency mice was significantly higher than that in control mice.The proportion of granulocyte progenitor cells increased significantly,but the proportion of erythroid progenitor cells and lymphoid progenitor cells did not change significantly.After benzene exposure,the proportion of hematopoietic stem cells,lin-sca1 cells,lin~-ckit~+cells,granulocyte progenitor cells,erythroid progenitor cells and lymphoid progenitor cells in deficiency mice and control mice decreased significantly,and with the increase of benzene exposure dose,the proportion of hematopoietic stem cells,lin~-sca1~+cells,lin~-ckit~+cells,granulocyte progenitor cells,erythroid progenitor cells and lymphoid progenitor cells decreased significantly.The ratio of hematopoietic stem cells to three-line progenitor cells decreased more significantly.Compared with the control mice,the proportion of hematopoietic stem cells and lin~-sca1~+cells and granulocyte progenitor cells in mice with deficiency of G6PD decreased more significantly after benzene exposure.There were a large number of bone marrow cells in the bone marrow cavity of normal mice and G6PD deficient mice,and no pathological changes were found.After high dose benzene exposure,the bone marrow hematopoietic cells of mice decreased significantly.These results suggest that G6PD deficiency has a more significant effect on the regeneration and differentiation of bone marrow hematopoietic stem cells induced by benzene in mice,and shows more obvious hematopoietic toxicity.2.Abnormal expression of CAMK2B in G6PD deficient mice and its involvement in the toxicity of 1,4-benzoquinoneIn the above-mentioned animal experiments,the bone marrow cells of G6PD deficiency(G6pdxa-m1Neu/Y)mice and normal control mice were collected and the expression levels of CAMK2B gene and protein in G6PD deficiency mice were studied by RT-PCR,Western blot and immunohistochemistry.The results of RT-PCR showed that the mRNA expression of CAMK2B in G6PD low expression mice was higher than that in control mice.The results of Western blot and immunohistochemistry showed that the expression of CAMK2B protein in mice with low expression of G6PD was higher than that in control mice,and most of them were expressed in cell membrane and cytoplasm.KN93,a chemical inhibitor of CAMK2B,was used to establish K562 cell line with low expression of CAMK2B.MTT cell proliferation assay,RT-PCR and Western blot assay were used to detect the inhibitory effect of KN93 on cell proliferation,and the expression of mRNA and protein in CAMK2B cells.The suitable concentration of KN93 was selected to construct the cell line with low expression of CAMK2B.After the construction of CAMK2B low expression cell line,CAMK2B cells and control cells were treated with 1,4-BQ.Cell proliferation rate,content of reactive oxygen species(ROS),ATP production,mitochondrial membrane potential,calcium concentration,cell apoptosis and cell cycle were measured to study the changes of cell function after 1,4-BQ exposure and to explore the possible mechanism of the effect of low expression of CAMK2B on benzene hematopoietic function.The results showed that the level of ROS in CAMK2B low expression cells was higher than that in negative control cells,and the ROS levels in CAMK2B low expression cells and control cells increased with the increase of 1,4-BQ concentration.The ATP production of CAMK2B low expression cells was lower than that of control cells.With the increase of1,4-BQ concentration,the production of mitochondrial ATP in both kinds of cells decreased,and the ATP production of CAMK2B low expression cells was still lower than that of control cells.The mitochondrial membrane potential of the cells with low expression of CAMK2B was lower than that of the control cells.With the increase of the concentration of 1,4-BQ,the membrane potential of the two kinds of cells decreased,and the membrane potential of the cells with low expression of CAMK2B was still lower than that of the control cells.The intracellular calcium concentration in CAMK2B low expression cells was higher than that in control cells.With the increase of 1,4-BQ concentration,intracellular calcium concentration increased,but the intracellular calcium concentration in CAMK2B low expression cells was still higher than that in control cells.The apoptosis rate of CAMK2B low expression cells was higher than that of control cells.With the increase of 1,4-BQ concentration,the apoptosis rate of both kinds of cells increased significantly,and the apoptosis rate of CAMK2B low expression cells was still higher than that of control cells.CAMK2B low expression cells decreased in G1 phase and S phase,and increased in G2 phase.After exposure to 1,4-BQ,CAMK2B low expression cells showed obvious increase in S phase and decrease in G2phase.The above results suggest that CAMK2B plays an important role in the antioxidant damage of cells,and the high expression of CAMK2B in g6pd-deficient mice may be the protective factor for the antioxidant in g6pd-deficient mice.However,benzene exposure can reduce the expression of CAMK2B,which may be one of the more significant mechanisms of G6PD deficiency on benzene-induced bone marrow hematopoietic toxicity in mice.Conclution1.The proportion of hematopoietic stem cells,lin-sca1 cells and granulocyte progenitor cells in G6PD deficient mice was higher than that in control mice.After the successful establishment of benzene poisoning mouse model,the function of bone marrow hematopoietic stem cell renewal and differentiation in G6PD deficient mice was obviously damaged,showing more obvious hematopoietic toxicity.2.The expression of CAMK2B gene in G6PD deficient mice was abnormally high.In the CAMK2B inhibitory cell model,after exposure to 1,4 CAMK2B,the low expression of CAMK2B cells showed more obvious mitochondrial damage.CAMK2B is of great significance to the antioxidant damage of the cells.Exposure to benzene can reduce the expression of CAMK2B,which may be one of the more significant mechanisms of G6PD deficiency on benzene-induced bone marrow hematopoietic toxicity.