Objective To investigate the apoptosis induced by monobutyl phthalate (MBP) and monoethyl hexyl phthalate (MEHP) in mouse leydig cells and the possible mechanism of GRP78, ATF4 and CHOP proteins.
Methods Mouse leydig cells (TM-3 cells) were cultured and the cell growth curve was drawn. Cell viability was measured by CCK-8 assay at 400 μmol/L. Cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI staining. The changes of GRP78, ATF4, and CHOP were detected by Western blot. One-way ANOVA was used to analyze the relationship between the exposed group and the control group, and the 2×2 factorial design method was used to judge the interaction of the combined exposure. When|(MBP+MEHP group-control group value)| < |(MBP group value-control group value)|+|(MEHP group value-control group value)|, it can be determined that the type of interaction is antagonism.
Results The cells were in logarithmic growth phase from 24 to 60 h after designed treatment and the number of apoptotic cells in normal state cells was stable. The results of cell viability assay showed that the inhibition rate induced by MEHP was higher than that by MBP, and the MBP+MEHP combined exposure showed an antagonism effect on cell viability24 h:|(53.21±7.68)%| < |(42.99±8.23)%|+|(65.46±8.29)%|; 36 h:|(79.87±3.76)%| < |(53.12±4.33)%|+|(74.80±4.96)%|; 48 h:|(85.97±6.07)%| < |(38.98±9.59)%|+|(80.73±4.99)%|, which was statistically significant (P < 0.05). The apoptosis rate was higher in the MEHP groupearly apoptosis:(17.44±0.82)%, total apoptosis:(44.24±0.89)% than in the MBP groupearly apoptosis:(14.58±0.97)%, total apoptosis:(29.46±0.58)%, and the apoptosis rate in the MEHP+MBP combined exposure groupearly apoptosis:(8.58±0.35)%, total apoptosis:(32.28±0.41)% was lower than the sum of individual exposure groups, also indicating an antagonism effect. The expression levels of ATF4 and GRP78 in the MEHP group were higher than those in the MBP group, and the expression level in the MEHP+MBP combined exposure group was lower than that in the MEHP group and higher than that in the MBP group, showing an antagonistic effect. The expression level of CHOP also changed after the designed exposure as compared with the control group. The expression level of CHOP in the MEHP group was higher than that in the MBP group, and the expression level in the MEHP+MBP combined exposure group was lower than that in the individual exposure group, showing an antagonistic effect, but the MBP group had no main effect.
Conclusion MBP and MEHP has the main effect on the apoptosis of TM-3 cells, the combination of MBP and MEHP has an antagonistic effect. Moreover, the GRP78-ATF4-CHOP pathway may be involved in the apoptosis of TM-3 cells induced by MBP and MEHP.
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