| ObjectiveBenzene(BZ,C6H6)is an aromatic hydrocarbon toxic.It is widely used as a solvent for paint,varnish,shoes,drugs,glue,and leather industry.Being highly volatile,it is a common environmental pollutant generated by the processing of indoor contamination,evaporation of vehicle and tabacoo smoke.There is an opportunity for people to develop chronic BZ poisoning following both environmental and occupational exposure.Long-term and chronic exposure to low concentrations of BZ is associated with a decrease in the leukocytes and/or platelets counts.However,severely chronic BZ poisoning can result in aplastic anemia/bone marrow failure(BMF)and acute myeloid leukemia(AML).BZ-induced BMF is an acquired AA and is characterized by a decrease in all three cell types defined as pancytopenia,which in the case of BZ-induced irreversible bone marrow depression.BZ-induced BMF may be caused by metabolites such as hydroquinone(HQ)and phenol(P)mediated hypocellular bone marrow occur as a result of damage to hematopoietic stem/progenitor cells,but its mechanism of toxicity is uncertain.Epidemiological studies in humans have showed that BZ-induced acute myeloid leukemia(BZ-AML)usually subsequent to BMF.So the development of BZ-AML is known as secondary AML after BZ exposure.BZ-AML as secondary leukemia has different character from primary leukemia.So we explore the mechanism of BZ-AML which will contribute to the prevention and treatment for BZ-AML.The inflammasome forms a large multiprotein complex family of intracellular sensor which is able to identify different stimulation signals.The family members of inflammasome are numerous,but the nucleotide-binding oligomerization domain like receptor protein 3(NLRP3)inflammasome is one of the most extensively and the best characterized inflammasome at present.NLRP3 inflammasome is activated by stimulation with endogenous 'danger signals' such as ROS generation,and subsequent leads to an immune response or inflammatory response.Upon activation,NLRP3 is thought to play critical roles in adaptive immune and oxidative stress.Current studies have shown that BZ can cause excessively production of ROS.As all known ROS is an endogenous activator of NLRP3 inflammasome.For these reasons,future work is required to clarify the role of NLRP3 inflammasome in BZ-induced oxidative impairment and T lymphocytes immune function involved BZ-induced hematotoxicity including bone marrow depression and leukemogenesis.Furthermore,an unknown pathway directs NLRP3 inflammasome activation in BZ-induced bone marrow toxicity.In addition,are there any differences of the role of NLRP3 inflammasome between BZ-induced BMF and AML?The problem remains unclear.BZ as an aromatic hydrocarbon toxic induces hematotoxicity by binding to aryl hydrocarbon receptor(AhR)in cellular.As a ligand-dependent transcription factor in the cytoplasm,AhR can combine with exogenous ligands such as BZ and subsequent is activated.An activating AhR translocates into nucleus and further promotes the target gene expression.It has been reported that BZ-induced hematotoxicity of bone marrow was not exhibited in AhR-knockout mice,which indicates that AhR might likely play an important role in BZ-induced hematotoxicity as a cytoplasmic transcription factor.Currently,there are two mechanisms for BZ-induced hematotoxicity:1.Binding of BZ and AhR in bone marrow cytoplasm directly cause hematopoietic stem cells(HSC)functional abnormalities in cell cycle,gene regulation,apoptosis and oxidative stress,which modify HSC proliferative and stationary phases;2.Metabolites of BZ,like benzoquinone(BQ)and HQ,exert their cytotoxicity effects by binding directly to AhR of bone marrow cells.Therefore,AhR plays an important role in the pathogenesis of BZ-induced hematotoxicity.However,AhR-mediated detailed molecular regulation mechanism is still unclear.A great number of animal model of BZ-induced hematotoxicity have been performed in different species,varying the dose,route,period and time of exposure to BZ.However,these BZ exposure schemes were not enough practical as reproducible models for BMF,AML induction.On the basis of concluding previous experimental alternatives,the aim of our present study was to optimize the route,dose,period and time of BZ exposure,and to establish reproducible and the most appropriate animal models for the development of BMF and AML similar to human using two different treatment schemes.Using the preferable animal models,we further demonstrated how NLRP3 activity is regulated in oxidative impairment in bone marrow cells following AhR bind to BZ.Furthermore,to combat the BZ-induced BMF and AML,we aimed to determine the effect of the EPS and Brusatol on BZ-induced hematotoxicity including BMF and AML,and we also evaluated NLRP3 inflammasome abnormal expression effects on BZ-induced BMF and AML mouse models.Therefore,the aim of the study was to analyze the AhR mediated NLRP3 activation in BZ-induced hematotoxicity,and the treatment effects of EPS and Brusatol on BZ-induced BMF and AML mouse models.The present study includes the following two sections:I.The first section:The Role and Mechanisms of NLRP3 Pathway Induced by Aryl Hydrocarbon Receptor in Benzene-induced BMF and Reversal StrategyBZ-induced BMF mouse model has been established successfully by subcutaneous injection of BZ in CD1 mice.At the end of the experiment,both AhR and NLRP3 gene and protein expression levels are increased in bone marrow cells.Our results showed that the AhR and NLRP3 inflammasome abnormally activation was associated with the BMF.EPS treatment can effectively reverse BMF related oxidative stress,exerts anti-apoptosis,enhancing T-cell function,and modulating hematopoietic factors function.?.The second section:The Role and Mechanisms of NLRP3 Pathway Induced by Aryl Hydrocarbon Receptor in Benzene-induced AML and Reversal StrategyBZ-induced AML mouse model has been established successfully by inhalation of BZ in CBA/Ca mice.At the end of the experiment,both NRF2 and NLRP3 gene and protein expression levels are increased in bone marrow mononuclear cells.Our results showed that the NRF2 and NLRP3 inflammasome abnormally activation were associated with the BZ-induced leukemogenesis.Brusatol treatment can effective reverse BZ-AML related the inhibition NRF2 and NLRP3 activation both in mRNA and protein levels and increased apoptosis in bone marrow mononuclear cells.Methods?.The first section:1.Fifty mice were randomly assigned to two different groups,a control group in which 10 mice were injected subcutaneously(in the dorsal region)with equal volume corn oil;and an experimental group in which 40 mice were treated with BZ+corn oil by subcutaneous injection(dorsal region).Four subgroups of 10 mice(BMF,EH,EL,CsA)in experimental group were treated three times a week(Mondays,Wednesdays and Fridays)until 25 doses were completed.The period of experimental,the body weights and the symptom of each group mice were documented.2.Peripheral blood was collected by cutting the tail method.One part of collected blood was used to evaluate total and differential count of WBC(White blood cell),RBC(Red blood cell),PLT(Platelet),and RC(Reticulocyte)percentage,and Hgb(Hemoglobin)concentration by animal hemocytometer.Another part of blood was used for blood smear by using hematoxylin-eosin(HE)stain.Bone marrow smear and femur histological examination were observed by HE staining.By blood counts and histological analysis,the BZ-induced BMF mice model was determined.3.Gene-specific primers for AhR and CYP1A1 were designed.The difference of AhR and CYP1A1 gene/protein expression was determined by qPCR and Western blot in both liver and BMMNCs in BZ-induced BMF mice,after murine bone marrow mononuclear cells(BMMNCs)were separated by erythrocytes lysis and centrifugation.4.The production of ROS in BMMNCs and the percentage of CD4+and CD8+T lymphocytes in peripheral blood were determined by flow cytometry.The gene and protein expression of NLRP3 was detected by qPCR and Western blot.5.The changes of cell cycle and apoptosis rate in BMMNCs were evaluated by flow cytometry.The expression of CyclinDl and CDK4 was detected by Western blot6.After modeling of the BZ-induced BMF model group,EH,EL,CsA subgroups mice were treated orally daily with high-dose EPS(100 mg/kg),low-dose EPS(20 mg/kg)and 10mg/kg dose CsA for 4 weeks,respectively.Both EPS and CsA were dissolved in saline.Equal volume saline was given orally daily to BMF and control subgroups mice.The body weights of each group mice were documented.Blood samples were collected in tubes containing anticoagulant(EDTA)and were immediately analyzed using animal hemocytometer for hematological parameters.EPS treatment effect on the BZ-induced BMF was verified through the comparison of blood cell counts,blood and bone marrow cells smears,femur biopsy.7.Apoptosis rate of BMMNCs was analyzed using propidium iodide(PI)/Annexin V and was quantified by flow cytometry method.The mRNA expression of apoptosis-related genes Caspase-9,BCL-2 and BAX were analyzed by qPCR.The protein expression of BAX was detected by Western blot8.Cell cycle of BMMNCs was analyzed by ethanol fixation and PI staining and quantified by flow cytometry.The mRNA and protein expression of cell-cycle regulatory gene CDK4 in BMMNCs was detected by qPCR and Western blot.9.The percentage of CD3+,CD4+,and CD8+T lymphocytes in peripheral blood was analyzed by flow cytometry.Plasma interleukin-2(IL-2),interleukin-11(IL-11)and erythropoietin(EPO)levels were analyzed by ELISA according to the manufacturer's instruction.10.The level of ROS and the expression of NLRP3 in BMMNCs were analyzed by flow cytometry and qPCR,respectively?.The second section:1.Thirty CBA/Ca mice were randomly assigned to three different groups(10 mice/per group):a control group and two subgroups(BZ-AML,Brusatol).BZ-AML and Brusatol subgroups of mice were exposed to BZ vapour at a dose 300ppm × 6h/d×5d/w for 8 weeks,and control group of mice was exposed to ambient air for the same time duration.The period of experimental,the body weights and the symptom of each group mice were documented.2.Establishment of BZ-AML mouse model was confirmed by blood routine test,peripheral blood smear,bone marrow smear,femur histology.The expression of CD34+,CD45+,CD13+,CD 19+immunophenotype was analyzed in BMMNCs using flow cytometry compared with control group.3.The ROS production in BMMNCs and the percentage of CD3+,CD4+,CD8+,CD80+,CD86+T cell subsets in peripheral blood were evaluated by flow cytometry in BZ-AML group mice.4.The concentrations of BZ and its metabolites such as phenol and hydroquinone in blood,liver,kidney and bone marrow was examined by SPE-GC-MS method in BZ-AML group mice.The significant of BZ and metabolites diferent organ distribution in BZ-AML pathological mechanisms was evaluated.5.The apoptosis rate and cell cycle in BMMNCs were examined by flow cytometry in BZ-AML mice.6.The expression of NLRP3 inflammasome in BMMNCs and femur tissue in BZ-AML mice was detected by 1HC and Western blot.The expression of AhR and CYP1A1 in BMMNCs was detected by Western blot.7.After modeling of the BZ-AML model group,Brusatol subgroup mice were treated intraperitoneal injection daily with 4mg/kg dose Brusatol for 10 days.Brusatol was dissolved in sterile water.Equal volume of sterile water was intraperitoneal injected daily to BZ-AML and control subgroups mice for 10 days.At the end of the experiment,the protein expression of AhR,CYP1A1,NRF2,CDK4,CyclinDl and NLRP3 in BMMNCs was detected by western blot.Results?.The role of AhR-mediated NLRP3 signaling pathway in BZ-induced BMF and EPS reversal strategyl.Development of BZ-induced BMF mouse modelBMF mouse model was established by subcutaneous injection of 2 ml/kg BZ in CD1 mice.Blood routine results showed that WBC,RBC,PLT counts,RC percentage and Hgb concentration were significantly decreased in the BMF mice group compared with the control group.The decreased nucleated cells were also verified by blood smears and bone marrow smears.Femur tissue histopathological examination showed that a dysplastic marrow and inadequate hematopoiesis.These results indicated that the BZ-induced BMF mouse model has similar hematological alternations to humans.2.Differential expression of AhR and CYP1A1 in liver and BMMNCs in BZ-induced BMFTo explore the role of AhR and CYP1A1 in BZ-induced hematotoxicity,we investigated the expression of AhR and CYP1A1 in the liver and BMMNCS,respectively.The liver and bone marrow are responsible for BZ and BZ metabolism and exerts tissue-specific effect.The results of qPCR and Western blot showed that the mRNA and protein expression levels of AhR and CYP1A1 in BMF group were significantly increased in both BMMNCs and liver compared with control group.In addition,the CYP1A1 protein expression level was increased more obviously in BMMNCs than in liver.3.The aberrant NLRP3 activity is related to increased production of ROS in BZ-induced BMF mouse modelTo investigate the role of NLRP3 activation in BZ-induced myelotoxicity and whether BZ induces bone marrow oxidative impairment,the present study first examined the production of ROS by flow cytometry.The ROS level of BMMNCs was elevated in the BMF model group compared with the control group.As the ROS can trigger NLRP3 inflammasome,the expression of NLRP3 gene and protein in BMMNCs was examined by qPCR and western blot.The results showed that NLRP3 gene and protein were markedly increased in BZ-induced BMF model group compared with the control group.Therefore,our data demonstrate that ROS-mediated aberrant NLRP3 inflammasome activation might play an important role in BZ-induced myelotoxicity.4.BZ-induced S-phase cell cycle arrest via downregulating CDK4 and CyclinDl expressionTo investigate whether the BZ exposure has effect on the cell cycle of BMMNCs,the present study examined the cell cycle by flow cytometry.We observed that the S-phase cell cycle arrest of BMMNCs in BZ-induced BMF model group compared with the control group.The protein expression of CDK4 and CyclinDl in BMMNCs was detected by western blot.The results showed that the protein expression of CDK4 and CyclinDl in BMF group were significantly decreased in BMMNCs compared with control group.5.BZ induces dysregulation of IL-11,IL-2,EPO hematopoietic factorsTo explore whether the dysregulation of hematopoietic factors is associated with BZ-induced BMF,we detected the expression of hematopoietic factors using ELISA method according to the manufactures' instructions.Our results identified increased plasma IL-2 and EPO in BMF mouse model compared with the control group.However,IL-11 concentration was significantly decreased in BMF mouse model compared with the control group.Our results suggested that the dysregulation of hematopoietic factors might play an important role in the pathogenesis of BZ-induced BMF.6.EPS improves BZ-induced weight loss in BMF miceBased on the establishment of BZ-induced BMF mouse model,we observed the effect of EPS on BZ-induced BMF.After 4 weeks of EPS orally treatment in different dose groups,we found that compared with untreated BMF mice,EH group and EL group showed a significant increase in body weight.The results indicated that EPS had the effect of alleviating BMF-induced weight loss in mice.7.EPS improves hematotoxicity in BZ-induced BMF miceAfter 4 weeks of EPS treatment,the results of blood parameters showed an increase WBC,PLT,Hgb and RC in the EH and EL treatment groups compared with the untreated BMF mice group,which associated with an increase nucleated cells by peripheral blood/bone marrow smears and femur biopsy.These results indicate that EPS can improve BZ-induced BMF.8.EPS inhibits BZ-induced apoptosis via down-regulating BAX proteinAs the apoptotic rate in BMMNCs was increased in the BZ-induced BMF group.we further detected the apoptosis rate in BMMNCs after EPS treatment.After 4 weeks of EPS orally treatment,the apoptosis rate was decreased compared with the BMF group.The apoptosis-related genes Caspase-9,BCL-2 and BAX were further detected by qPCR.The expression level of apoptosis-related gene BAX was decreased in BMMNCs in EPS treatment groups compared with the control group.But there was no significant difference in Caspase-9 and BCL-2 expression levels.Therefore,we further investigated the effect of EPS on apoptotic-related protein BAX in BMMNCs.By western blot analysis,we found that the protein level of BAX was significantly upregulated in the BMMNCs in BMF group compared with control group.EPS treatment inhibited the activation of BAX compared to BMF group.These data suggest that both high and low-dose EPS treatment have an anti-apoptosis effect by down-regulating BAX protein expression.9.EPS relieves BZ-induced cell cycle arrest in BMF mice via up-regulating CDK4 proteinTo investigate whether EPS improve BMF through regulating cell cycle,we examined cell cycle in BMMNCs.After EPS treatment,the S-phase cell cycle arrest was alleviated significantly.We explored the protection mechanism of EPS against cell cycle arrest.The expression of the cycle-related gene CDK4 was further examined by qPCR.We found CDK4 expression is upregulated in the BMMNCs in the EH group.These results indicated that EPS treatment improved S-phase arrest in BZ-induced BMF mice through upregulation CDK4 expression10.EPS reduces BZ-induced oxidative stress by inhibiting NLRP3 inflammasome activationIn vitro studies showed that EPS as herba epimedii anti-oxidative properties can exert anti-inflammatory effect.We further to identify the anti-oxidative effect of EPS on BZ-induced BMF in vivo.After 4 weeks of EPS treatment,the production of ROS was decreased in BMMNCs in EH and EL groups compared with BMF group.We further explore the anti-oxidative mechanism of EPS in the BMF mouse model.The expression of NLRP3 inflammatory gene was detected via qPCR.Our result showed that the mRNA expression of NLRP3 in BMMNCs of EH and EL treatment groups was significantly decreased compared with BMF group.These results suggest that EPS may exert anti-oxidative effect by inhibiting the expression of NLRP3 inflammasome.11.EPS improves the imbalance of CD4+/CD8+T cell ratio and related hematopoietic factorsPrevious studies prove EPS has an immunomodulatory activity and has the potential to enhance the body's immunity.We further explored EPS treatment effect on BZ-induced BMF immunotoxicity.The present study examined the percentage of T cells and the level of hematopoietic factors using flow cytometry and ELISA methods.Our results showed that the percentage of CD4+T-cell and the CD4+/CD8+ratio in the EH and EL groups were significantly increased compared with the BMF group.An increase plasma level of IL-11 and a decrease plasma level of IL-2 and EPO were observed in EH and EL group compared with BMF group.These results indicated that EPS has the potential to enhance T-cell immune function and regulate the abnormal imbalance of hematopoietic factors.?.The role of AhR-mediated NLRP3 signaling pathway in BZ-AML and Brusatol reversal strategyl.Establishment of BZ-AML mouse model by inhalation routeMale CBA/Ca mice were exposed to benzene in the inhalation chamber to establish BZ-AML mouse model.The blood parameters showed a decrease in WBC,RBC and PLT counts,while an increase in the percentage of neutrophils.The incidence of AML after high-level BZ exposure was observed by peripheral blood and bone marrow smears.Liver histopathological examination showed that the hepatic cell cord filled with atypical mononuclear myeloblastic cell.Femoral bone marrow histopathological examination showed that the increased cellular density for atypical mononuclear cells with heterogeneous size distribution.In addition,BZ and BZ metabolites such as phenol and hydroquinone are analyzed by SPE-GC-MS.The concentration of BZ and BZ metabolites was significantly increased in blood,bone marrow,kidney,spleen and liver in the BZ-AML group compared with the control group.The BZ-AML group showed human similar AML immunophenotype as known myeloid antigens of CD34+,CD13+,CD19-by flow cytometry in BMMNCs.2.Immunosuppressive effect of benzene in BZ-AML miceCompleting the BZ-AML model,BZ-AML group mice exhibited decreased numbers of CD3+,CD4+,CD8+,CD80+and CD86+T lymphocytes in peripheral blood compared with control group using flow cytometry.These results suggest that BZ-induced immunosuppression might be participate in the mechanism associated with BZ-AML.3.BZ induces the activation of NLRP3 and NRF2 of BMMNCs in BZ-AML miceCompared with the control group,the gene and protein expression of AhR,CYP1A1 and NLRP3 in BZ-AML group was increased in BMMNCs.Immunohistochemistry also confirmed the expression of NRF2 and NLRP3 was increased in femur tissue.These results showed that the BZ binding AhR and further activated the NRF2 and NLRP3,which might be associate with BZ-AML pathological mechanisms.4.BZ induces delayed apoptosis and G0/G1 phase arrest in BZ-AML miceCompared with the control group,the delayed apoptosis and G0/G1 phase arrest of BMMNCs were observed in the BZ-AML group by flow cytometry5.Brusatol inhibited the activation of NLRP3 and NRF2 in BZ-AML miceAfter 10 days of Brusatol treatment,the Brusatol group significantly decreased the gene and protein expression of NLRP3,KEAP1 and NRF2 compared with the BZ-AML group.These results demonstrated that Brusatol can inhibit the NLRP3 and KEAP1-NRF2 activation to exert anti-leukemia effect.6.Brusatol induces apoptosis of BMMNCs in BZ-AML miceAfter 10 days of Brusatol treatment,an increase of the percentage of late stage apoptotic BMMNCs was observed by flow cytometry,2.14%±0.28 in Brusatol group compared with the Control group 4.27±0.42%and BZ-AML group 1.20±0.50%,respectively.These results showed Brusatol-induced apoptosis might be related to anti-leukemia effect.Conclusions?.AhR-mediated NLRP3 inflammation activation plays an important role in BZ-induced BMF1.BZ induced ROS production and further activated NLRP3 inflammasome in the BMMNCs2.An increase of apoptosis rate and an arrest of S-phase in the BMMNCs were detected in BMF mice after BZ exposure.3.Elevated levels of AhR and CYP1A1 were detected in liver and BMMNCs after BZ exposure.In addition,AhR and CYP1A1 were highly expressed in BMMNCs.The differential expression might be an important mechanism for BZ-induced BMF hematotoxicity.4.A decrease of CD3+,CD4+ T lymphocytes and the disorder of hematopoietic factors 1L-11,IL-2 and EPO in the peripheral blood were closely related to BZ-induced BMF immunotoxicity.?.Treatment effect of EPS against BZ-induced BMF via anti-apoptosis,anti-oxidative stress,alleviating cell cycle arrest and enhancing T cell immune function1.EPS can exert anti-apoptotic effect via downregulating of BAX.2.EPS can exert anti-oxidative stress effect via inhibiting the activation of NLRP3 inflammasome and reducing ROS production.3.EPS can alleviate the S-phase arrest through an upregulation the expression of CDK44.EPS can exert an immune-enhancing effect via increasing the percentage of CD3+,CD4+T lymphocytes and balancing the hematopoietic factors IL-11,IL-2 and EPO in the peripheral blood?.The BZ-AML mouse model was successfully established by inhalation in CBA/Ca mice1.The in vivo humanized modeling of BZ-AML has been modeled by the use of CBA/Ca mice exposed to BZ vapor.The model was verified by peripheral blood and bone marrow smear,liver and femur histology examination and combined with leukemia immunophenotype analysis of CD34+,CD13+,CD 19-.2.The number of CD3+,CD4+,CD8+,CD80+,and CD86+T lymphocytes was obviously lower in the peripheral blood in BZ-AML mice after BZ inhalation.3.A decrease of the apoptosis rate and an arrest of G0/G1 phase were observed in BZ-AML mice after BZ inhalation.?.BZ binds and activates AhR,regulating the activation of NLRP3 inflammasome in BZ-induced AML1.The expression of AhR,CYP1A1,NRF2 and NLRP3was increased in BMMNCs in BZ-AML mice,which indicated that BZ binds to AhR and resulted in the activation of NRF2 and NLRP32.Anti-leukemia effect of Brusatol on BZ-AML through inducing apoptosis of BMMNCs in mouse model of BZ-AML.Brusatol significantly lowed the expression of NLRP3,KEAP1,and NRF2 genes and proteins,which indicated that anti-leukemia mechanism of Brusatol might be closely related to the inhibition of NLRP3 inflammasome and KEAP1-NRF2 signaling pathway activation.Originality and Significance1.Our results clarify that BZ exposure induced excessive ROS production and further activated NLRP3 inflammasome in BMMNCs in BZ-induced BMF mouse model2.Our study suggests that treatment effect of EPS and Brusatol on BZ-induced BMF and AML in different mouse model.3.Our study suggests that EPS and Brusatol may improve bone marrow depression and anti-leukemia through inhibiting NLRP3 activation in BMF and AML different mouse model.4.Our study suggests that an increase expression of AhR and CYPIA1 in liver and BMMNCs and highly expression in BMMNCs,which may provide a new perspective insight for BZ-induced hematotoxicity.5.Our study suggests that EPS and Brusatol may become a new target therapy to attenuate hematotoxicity induced by BZ.Our findings provide a significant in vivo foundation for further clinical research in terms of EPS and Brusatol treatment effects.Limitations1.This study proposes a possible in vitro cellular model to explore the dose-effect relationship between the ROS accumulation and NLRP3 activation.Further study will explore the mechanisms of NLRP3 inflammasome in BZ-induced hematotoxicity.2.Due to time limitation,the molecular mechanism NLRP3 activation has not been clarified.Further investigations are certainly needed on the substantial cross-talk between the activation NLRP3 and AhR signaling pathway. |
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