The Influence Of G6PD On Signaling Pathway In Bone Marrow Cell Induced By Benzene In Mice

Benzene is a common environmental pollutant and widely used in industrial production.Long-term chronic benzene exposure can cause damage to the hematopoietic system including aplastic anemia and myelodysplastic syndrome,even leukemia.Glucose-6-phosphate dehydrogenase(G6PD)is a rate-limiting enzyme on the pentose phosphate pathway.G6PD deficiency is the most common human enzymopathy and affected over 400 million individuals worldwide.The people with G6PD deficiency or low activity will occur hematolysis when exposed to certain triggers such as fresh beans.Our previously study showed that G6PD deficiency could increase the oxidative stress and toxicity induced by benzene exposure in K562 cells and mice.However,the mechanism of how G6PD influence the toxicity of benzene was not clear.In this study,we established G6PD low activity C3H/He mice model(G6pdxa-m1Neu),and the RNA-seq detection of bone marrow cells in mice of G6pdxa-m1Neu and normal C3H/He mice exposed to benzene was carried out.The bioinformatics analysis of RNA-seq results was used to investigate the pathways of G6PD on the regulation of benzene toxicity,and the glutathione metabolic pathway was validated by glutathione intervention.1.The influence of G6PD on mRNA Expression Profiles of Bone Marrow Cells in Mice Exposed to benzene by RNA-seqThe G6pdxa-m1Neu and C3H/He mice were exposed to benzene at the concentions of 0 mg/(kg.d)and 160 mg/(kg.d)by subcutaneous injection.The bone marrow cells were isolated after benzene exposure.The RNA-seq was applied to detect the mRNA expression profile.The accuracy of RNA-seq results was verified by qRT-PCR.The result showed there were 486 differentially expressed genes between G6pdxa-m1Neu and C3H/He mice with 209 genes up-regulated and 277 genes down-regulated.Between the benzene expose and unexposed C3H mice,227 differentially expressed genes were screened out with 122 down-regulated and 105 up-regulated.Between the benznen exposed G6pdxa-m1Neu and benzene exposed C3H/He mice,223 differentially expressed genes were screened out with 105 up-regulated and the 118 down-regulated.The results of qRT-PCR of showed that the relative expression of the target genes were consistent with that of RNA-seq.2.The influence of G6PD on signaling pathway in bone marrow cell induced by benzene in miceIn this part,the differential expression genes were analyzed by GO,KEGG Pathway and gene co-expression,and the possible pathways were obtained to reveal the regulatory network and mechanism how G6PD influence the signaling pathway in bone marrow cell induced by benzene in mice.The differential expression genes of G6pdxa-m1Neu and C3H/He mice were mainly enriched in metabolic,redox process,apoptosis,immunization,pentose phosphate pathway,glutathione metabolism,oxidative stress,hematopoietic cell lineage,NADPH regeneration,HIF-1 signaling pathway,NF-KB signaling pathway,AMPK signal pathway,PI3K-Akt signaling pathway,VEGF signaling pathway,diabetes,tuberculosis,asthma and other related functions and pathways.The differential expression genes of benzene exposed and unexposed C3H mice were mainly enriched in immune,apoptosis,metabolism,oxidative stress,cell cycle,cell growth,T cell differentiation,glutathione metabolism,hematopoietic cell lineage,HIF-1 signaling pathway,P53 signaling pathway,Jak-STAT signaling pathway,PI3K-Akt signaling pathway,NF-KB signaling pathway,acute myeloid leukemia and other related functions and pathways.The differential expression genes of between the benznen exposed G6pdxa-m1Neu and benzene exposed C3H/He mice were mainly enriched in metabolism,pentose phosphate pathway,stress response,redox reaction,NADPH regeneration,apoptosis,glutathione metabolism,fatty acid metabolism,inflammation,MAPK signaling pathway,ErbB signaling pathway,GnRH signaling pathway,HIF-1 signaling pathway,TNF signaling pathway,VEGF signaling pathway,NF-KB signaling pathway,acute myeloid leukemia and other related functions and pathways.The results of gene co-expression showed that the genes with high regulatory ability were mainly G6pdx、Ltb、Siglece、Orai 1、Hck、Lmo4、Atp6v0b、Gm 14010、Cmah、Prrl3.Pde3b、Ckap4、Csrpl、P2ry6.It suggested both G6PD Low activity and benzene exposure could influence the signaling pathway in bone marrow cells in mice.The low activity of G6PD mainly influnenced the glutathione metabolism,pentose phosphate pathway,oxidative stress,apoptosis,metabolism,hematopoietic cell lineage pathway which might be the mechneism of how G6PD influenced the toxicity in bone marrows induced by benzene.3.Effect of GSH intervention on G6PD regulation of 1,4-benzoquinone toxicityIn this part,we studied whether the glutathione metabolism involved in the mechanism of how G6PD influenced the toxicity in bone marrows induced by benzene.First,we treated G6PD low activity k562 cells and normarl k562 cells with 0,20μmol/L 1,4-BQ and intervened with 0.16 mmol/L GSH respectively.The effect of GSH intervention on 1,4-BQ toxicity was detected by MTT assay,ROS detection,apoptosis detection and comet assay.At the same time,the genes of G6PD,Pgkl and Pgd related to carbon metabolism and glutathione metabolism were selected to detect the changes of mRNA level after GSH intervention.The results showed that the inhibition of G6PD could increase the ROS production,apoptosis rate and DNA damage,and decrease the cell viability.GSH could significantly decrease the variaition between G6PD low activity cells and control cells on ROS production,apoptosis rate,DNA damage and cell viability.At the same time,GSH intervention could reduce the variaition of Pgkl,Pgd between G6PD low activity cells and normorl cells.The above results indicated that glutathione metabolic pathway is one of the important pathways involved in how G6PD influenced the benzene toxicity on bone marrow.ConclutionIn this study,G6PD inhibition C3H/He mice and G6PD control C3H/He mice were treated with benzene,and then the bone marrow cells were collected to get RNA-seq detection.The bioinformatics analysis of RNA-seq result showed that the main pathways related to the mechinsm of how G6PD influnced the toxicity of benzene exposure were glutathione metabolism,pentose phosphate,oxidative stress,apoptosis,metabolism and hematopoietic lineage pathway.Furthermore,we studied whether the glutathione metabolism involved in the mechanism of how G6PD influenced the toxicity in bone marrows induced by benzene.The results showed that GSH could significantly decrease the variaition between G6PD low activity cells and control cells on oxidative damage,the mRNA level of Pgkl and Pgd.The above results indicated that glutathione metabolic pathway was one of the important pathways involved in how G6PD influenced the benzene toxicity on bone marrow.