The Role Of GRP78-ATF4-CHOP Pathway In The Apoptosis Of Mouse Leydig Cells Induced By MBP Or (and) MEHP

Objective: To investigate the monobutyl phthalate(MBP)and mono(2-ethylhexyl)phthalate(MEHP)in different testosterone concentrations in mice.Toxic effects of stromal cells(TM-3),as well as activation of endoplasmic reticulum stress and through the glucose-regulated protein 78(GRP78)-activated transcription factor 4(ATF4)-C/EBP homologous protein(CHOP)pathway The process of apoptosis,and then explore the effect of combined exposure of MBP and MEHP on apoptosis in the process of apoptosis.Methods: 1.TM-3 cells were cultured in vitro with 10% fetal bovine serum and double anti-(penicillin and streptomycin)and DMEM high glucose complete medium and the cell density was measured every 12 hours and the cell growth curve was drawn;2,the mouse Leydig cells were treated with MBP,MEHP and MBP+MEHP at different initial concentrations of 0(control),50,100,200,400,800 ?mol/L on mouse Leydig cells.36,48 hours;3,using Cell Counting Kit-8(CCK-8)method to detect the effect of the exposure group on cell viability,according to the inhibition rate calculation formula to calculate the effect of exposure on cell inhibition rate,refer to the improved The half-lethal dose calculated by the method is used to determine the subsequent dose and time of exposure,and then observe the morphological changes of the cell state after exposure;4,observe the ultrastructural changes of the endoplasmic reticulum under the transmission electron microscope;5,with Annexin V-FITC/PI double staining flow cytometry was used to detect early apoptosis and total apoptosis;6.Western blot was used to detect glucose-regulated protein 78(GRP78)and activated transcription factor 4 in cell lysate.(ATF4)and DDIT3/GADD153 Change White(of CHOP)levels.Results: 1.The TM-3 cells were cultured under normal conditions,and the cell density was measured every 12 hours.The cell growth curve showed that the number of cells did not increase significantly in 0-12 hours,and the cells were in the adherent stage at 12-24 h.During the time period,the cells adhered and the state became better.The boundary was clear and the cytoplasm was transparent,and the number began to increase.The cells were in the logarithmic growth phase in the 24-60 h period and the number of apoptosis in the normal state of the cells was relatively stable,60-108 h.The total number of cells is increasing,but the number of apoptotic cells is also increasing;2,compared with the control group,MBP,MEHP and MBP + MEHP exposure groups can cause TM-3 cell activity to decrease,the difference is statistically significant(P<0.05),and the cell inhibition rate was stable at the doses of 24 h and 200,400 and 800?mol/L.The variance analysis of 2×2 factorial design was carried out under the conditions of exposure to 24 h and 400?mol/L.The MBP+MEHP combined exposure group had an interaction with the inhibition rate of TM-3 cells,and the type of action was antagonistic.3.Under transmission electron microscopy,MBP,MEHP and MBP+MEHP were combined at 400 ?mol/in comparison with the control group.L-induced 24 h can cause endoplasmic reticulum structure Injury,mainly manifested as swelling of the endoplasmic reticulum,decreased in number,ribosome shedding,and later became vacuolar-like structure;4,compared with the control group(0),at 24 h and 200,400,800 ?mol/L MBP,MEHP and MBP+MEHP could increase the early apoptosis and total apoptosis of TM-3 cells(P<0.05).Under the conditions of 24 h and 400?mol/L,MBP?MEHP and MBP+ MEHP with the 2×2 factorial design analysis of variance had an interaction,and the interaction mode was antagonistic(P<0.05);5,Compared with the control group,the expression of GRP78 protein,ATF4 protein and CHOP protein in MBP,MEHP and MBP+MEHP groups increased at 24 h and 200,400 and 800?mol/L,and the change was statistically significant(P <0.05),under the condition of 24 h,400?mol/L exposure,the expression of GRP78 protein,ATF4 protein and CHOP protein in MBP,MEHP and MBP+MEHP group were higher than those in the control group and the change was statistically significant(P <0.05),the analysis of variance by 2×2 factorial design had an interaction and was antagonistic,which was statistically significant(P<0.05).Conclusion: 1.The TM-3 cells cultured in vitro were in good condition for 24 h culture,the cells were in logarithmic growth period within 24-60 h,and the experiment could be carried out.2.The cell viability was observed in MBP,MEHP and MBP+MEHP groups.There is inhibition,and there is interaction,the type is antagonistic;3,MBP,MEHP and MBP + MEHP can damage the endoplasmic reticulum structure;4,MBP,MEHP and MBP + MEHP can cause Apoptosis,and the number of cell apoptosis increases with increasing dose,the change is statistically significant,there is also interaction,the type of action is antagonistic;5,MBP,MEHP and MBP+MEHP can increase the expression of GRP78 protein,ATF4 protein and CHOP protein in TM-3 cell lysate.Moreover,the expression levels of GRP78 protein,ATF4 protein and CHOP protein were interactive at 400?mol/L for 24 h,and the type was also antagonistic..6.The infection of MBP,MEHP and MBP+MEHP may activate the GRP78-ATF4-CHOP pathway through endoplasmic reticulum stress response,which may cause TM-3 cells apoptosis.