| Objective: Cerebral edema caused by 1,2-dichloroethane(1,2-DCE)poisoning is one of the occupational diseases that endanger the health of workers in China.Cytochrome P450 2E1(CYP2E1)can be effectively metabolized into The 1,2-DCE in the body forms the metabolite 2-chloroethanol(2-CE),which has stronger biological activity than 1,2-DCE,and can cause body damage during 1,2-DCE Play an important role.During metabolism of 1,2-DCE,the body can produce a large number of reactive oxygen species(ROS),resulting in oxidative damage to the body.Therefore,it is necessary to investigate the regulatory mechanism of oxidative damage induced by 2-CE.During the development of cerebral edema,astrocytes(AS)edema in the central nervous system is a prominent feature.AS is a functional nerve cell that supports and protects neurons and helps to form a strong blood-brain barrier.When foreign bodies in the blood enter brain tissue,AS should be the main target cell for its initial accumulation and destruction.The purpose of this study was to conduct in vitro cytotoxicity tests with primary cultured AS as the research object,to explore the mechanism of 2-CE on oxidative damage and apoptosis in AS,and the regulation of CYP2E1 expression during 2-CE-induced AS injury and inflammation The regulatory role of Nrf2 and the role of Nrf2 signaling pathway in regulating AS inflammation provide new research directions and experimental reference data for revealing the mechanism of 1,2-DCE toxic cerebral edema.Methods: 1.Take out Wistar pups that were born for 1-3 days,the brain was cut,the dispersed cells were digested with trypsin,inoculated in a petri dish,and purified after staining with glial fibrillary acidic protein(GFAP)to identify purity.2.Before use,10 M 2-CE stock solution was diluted with the culture solution with DMEM culture solution containing 5% fetal bovine serum to prepare culture solutions with different 2-CE concentrations.Using 2-CE as the poison,the first part of the study on the damage of 2-CE to AS was divided into 4 groups.The concentration of 2-CE in the culture solution was 0,7.5,15 and 30 mM,and the culture was performed for 24 h.The fluorescence expression of ROS in the cells was observed by an inverted microscope,and the value was scanned with a microplate reader.The kit was used to detect intracellular malondialdehyde(MDA),MitoSOX,8-hydroxy-2 deoxyguanosine(8-OHdG)content and mitochondrial membrane potential.Apoptosis rate was measured by flow cytometry.The protein expression of CYP2E1 and NLRP3 in AS was detected by immunofluorescence chemical staining.Western Blot and Real time RT-PCR were used to detect the contents of CYP2E1,Nrf2,HO-1,and ?-GCSc proteins and mRNA,respectively,and phosphorylated ERK1/2(pERK1/2),ERK1/2,Bax,Bcl-2,NLRP3,ASC,Caspase-1,GSDMD-N,GSDMD and IL-18 protein content.The content of IL-18 in the medium was determined by Elisa.3.The second part discusses the role of CYP2E1 in the process of 2-CE induced astrocyte damage.Cells are randomly divided into 4 groups: blank control group,inhibitor control group,exposure group and inhibitor intervention group,including Diallyl sulfide(DAS)intervention group,Vitamin C(Vit C),SCH772984,Mithramycin A(MTM A)and ML385.AS in exposure group was exposed to 30 mM 2-CE for 24 h.After the inhibitor intervention group AS received each inhibitor for 1 h,it was exposed for 24 h according to the AS treatment method of the exposure group.The contents of MDA,MitoSOX,8-OHdG and mitochondrial membrane potential were detected by the kit.Apoptosis rate was measured by flow cytometry.The cells were collected to detect the protein and mRNA contents of CYP2E1,SP1,Nrf2,HO-1,and ?-GCSc,and the protein contents of Bax,Bcl-2,p-ERK1/2,ERK1/2,p-SP1,NLRP3,ASC,Caspase-1,GSDMD-N,GSDMD and IL-18.Elisa method was used to detect the contents of IL-18 and IL-1? in the culture medium.4.A one-way analysis of variance and Student-Newman-Keuls(SNK)test were used to compare at a test level of P <0.05.Results: The research results of the first part of the experiment are as follows:1.The effect of 2-CE exposure on the expression of CYP2E1 in AS.The expression level of CYP2E1 protein in AS increased significantly after 30 mM 2-CE exposure for 12 h,peaked at 24 h,and continued until 48 h.In addition,the expression levels of CYP2E1 protein and mRNA in AS of 15 and 30 mM 2-CE exposed groups were significantly higher than those of the control group,the difference was statistically significant(P <0.05).2.The effect of 2-CE exposure on AS oxidative damage.The content of ROS in AS of 15 and 30 mM 2-CE exposure group was significantly higher than that of the control group,the difference was statistically significant(P <0.05).In addition,the MDA content of AS in the 2-CE exposure group increased significantly with the increase of the 2-CE exposure dose(P <0.05).The mitochondrial membrane potential in AS of 15 and 30 mM 2-CE exposed group was significantly lower than that of control group and 7.5 mM 2-CE exposed group,the difference was statistically significant(P <0.05).The levels of mitochondrial ROS(MitoSOX)and 8-OHdG in AS of 15 and 30 mM 2-CE exposed groups were significantly higher than those of the control group,and the difference was statistically significant(P <0.05).3.The effect of 2-CE exposure on the level of apoptosis of AS.The expression ratio of Bax / Bcl-2 protein in AS of 2-CE exposure group increased significantly with the increase of 2-CE exposure dose(P <0.05).In addition,the apoptosis rate of AS in the 30 mM 2-CE exposed group was significantly higher than that in the control group,and the difference was statistically significant(P <0.05).4.The effect of 2-CE exposure on ERK expression and phosphorylation level in AS,the protein expression and phosphorylation level of ERK1 / 2 in AS of 2-CE exposure group increased significantly with the increase of 2-CE exposure dose(P <0.05).5.The effect of 2-CE exposure on the expression of Nrf2 signaling pathway-related proteins in AS,the expression level of Nrf2 protein in AS of 2-CE exposed group increased significantly with the increase of 2-CE exposure dose(P <0.05),2-CE exposure group The mRNA expression level of Nrf2 in AS was significantly higher than that in the control group,and the difference was statistically significant(P <0.05).The expression levels of HO-1 protein and mRNA in AS of 15 and 30 mM 2-CE exposed groups were significantly higher than those of the control group(P <0.05).The expression levels of ?-GCSc mRNA in AS of 15 and 30 mM 2-CE exposed group and the expression of ?-GCSc protein in AS of 30 mM 2-CE exposed group were significantly higher than those of control group,the difference was statistically significant(P <0.05).6.The effect of 2-CE exposure on the expression of NLRP3 protein in AS.The expression level of NLRP3 protein in AS of 2-CE exposure group increased significantly with the increase of 2-CE exposure dose(P <0.05).In addition,the content of ASC and IL-18 protein in AS of 30 mM 2-CE exposed group was significantly increased compared with the control group,the difference was statistically significant(P <0.05).The Caspase-1 protein content in AS of 15 and 30 mM 2-CE exposure groups increased significantly with the increase of 2-CE exposure dose(P <0.05).Compared with the control group and 7.5 mM 2-CE exposure group,the GSDMD-N protein content in AS of 15 and 30 mM 2-CE exposure group was significantly higher than that of control group and 7.5 mM 2-CE exposure group(P <0.05).The IL-18 content in AS culture medium of 15 and 30 mM 2-CE exposure groups increased significantly with the increase of 2-CE exposure dose(P <0.05).The research results of the second part of the experiment are as follows:1.The effect of DAS intervention on the expression and oxidative damage of CYP2E1 in 2-CE exposed AS.The expression level of CYP2E1 protein and mRNA in AS of DAS intervention group was significantly lower than that in 2-CE exposed group(P <0.05)).In addition,the ROS and MDA content in the AS,the ratio of Bax and Bcl-2,the mitochondrial membrane potential,the protein and phosphorylation levels of ERK1 / 2,and the protein and mRNA expression levels of Nrf2,HO-1 and ?-GCSc in the DAS intervention group Compared with the 2-CE exposed group,the difference was statistically significant(P <0.05).2.The effect of Vit C intervention on CYP2E1 expression and oxidative damage in AS exposed to 2-CE.The expression levels of CYP2E1 protein and mRNA in AS in Vit C intervention group were significantly lower than those in 2-CE exposure group,the difference was statistically significant(P <0.05).In addition,the content of ROS and MDA in the AS of the Vit C intervention group,the ratio of Bax to Bcl-2,mitochondrial membrane potential,protein and phosphorylation levels of ERK1 / 2,and protein and mRNA expression of Nrf2,HO-1 and ?-GCSc The level was significantly improved compared with the 2-CE exposed group,and the difference was statistically significant(P <0.05).3.The effect of SCH772984 intervention on the expression of CYP2E1 in 2-CE exposed AS.The expression level of CYP2E1 protein and mRNA in AS of SCH772984 intervention group was significantly lower than that in 2-CE exposed group,the difference was statistically significant(P <0.05).In addition,the SP1 protein and mRNA expression levels and phosphorylation levels in AS of the SCH772984 intervention group were significantly lower than those of the 2-CE exposure group,and the difference was statistically significant(P <0.05).4.The effect of MTMA intervention on the expression of CYP2E1 in AS exposed to 2-CE.The protein and m RNA expression levels of CYP2E1 in AS of MTMA intervention group were significantly reduced compared with the group exposed to 2-CE(P <0.05).5.The effect of ML385 intervention on the expression of CYP2E1 in 2-CE exposed AS.The expression level of CYP2E1 protein in AS of ML385 intervention group was significantly higher than that in 2-CE exposure group,the difference was statistically significant(P <0.05).In addition,the expression levels of HO-1 and ?-GCSc protein in AS of ML385 intervention group were significantly lower than those of 2-CE exposure group,and the difference was statistically significant(P <0.05).The content of ROS,MitoSOX,8-OHdG and MDA in the AS of the ML385 intervention group and the protein levels of NLRP3,ASC,Caspase-1,GSDMD-N and IL-18 were significantly increased compared with the 2-CE exposure group,and the difference was statistically significant Academic significance(P <0.05).The content of IL-18 and IL-1? in AS culture fluid of ML385 intervention group was also significantly higher than that of 2-CE exposure group,the difference was statistically significant(P <0.05)Conclusions: 1.2-CE exposure leads to the increase of CYP2E1 protein and mRNA content in AS and produces oxidative damage effect and inflammatory response to AS,and finally causes apoptosis.2.The activation of ERK1 / 2 signaling pathway plays an important role in 2-CE-induced upregulation of CYP2E1 expression in AS.3.2-CE exposure activates the inflammatory bodies in AS,and Nrf2 plays a certain role in balance and protection during this process. |
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