MBP和MEHP联合染毒致小鼠睾丸间质细胞的凋亡作用及其可能机制
Leydig cell apoptosis in mice induced by combined administration of monoethyl hexyl phthalate and monobutyl phthalate and potential mechanism
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摘要:
目的 探讨邻苯二甲酸单丁酯（MBP）和邻苯二甲酸单乙基己酯（MEHP）染毒致小鼠睾丸间质细胞的凋亡作用及葡萄糖调节蛋白78（GRP78）、活化转录因子4（ATF4）与C/EBP同源蛋白（CHOP）参与的可能机制。
方法 培养小鼠睾丸间质细胞（TM-3细胞）并且测出细胞生长曲线；用CCK-8法检测细胞在染毒浓度400 μmol/L状况下的细胞活性变化；用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡情况；应用免疫印迹法检测GRP78、ATF4和CHOP含量的变化。单因素方差分析法分析染毒组与对照组之间的关系，2×2析因设计法判断联合染毒的交互作用，当|（MBP+MEHP组指标-对照组指标）| < |（MBP组指标-对照组指标）|+|（MEHP组指标-对照组指标）|，可判定交互作用类型为拮抗作用。
结果 细胞在24~60 h时间段内处于对数生长期，且细胞正常状态下的凋亡数量比较稳定。细胞活性检测结果显示MEHP染毒对细胞的抑制率比MBP的大，并且MBP+MEHP联合染毒且具有交互作用，其作用方式为拮抗作用24h：|（53.21±7.68）%| < |（42.99±8.23）%|+|（65.46±8.29）%）|；36h：|（79.87±3.76）%| < |（53.12±4.33）%|+|（74.80±4.96）%|；48h：|（85.97±6.07）%| < |（38.98±9.59）%|+|（80.73±4.99）%|，具有统计学意义（P < 0.05）；细胞凋亡比例也呈现出MEHP组早期凋亡：（17.44±0.82）%，总凋亡：（44.24±0.89）%大于MBP染毒组早期凋亡：（14.58±0.97）%，总凋亡：（29.46±0.58）%，同时出现MEHP+MBP联合染毒组早期凋亡：（8.58±0.35）%，总凋亡：（32.28±0.41）%的凋亡比例小于各单独染毒组之和，呈现出拮抗作用；ATF4和GRP78在MEHP染毒组的表达量高于MBP染毒组，MEHP+MBP联合染毒组表达量低于MEHP染毒组但高于MBP染毒组，并且表现出拮抗作用。CHOP的表达量与对照组相比也有变化，MEHP染毒组表达量较MBP染毒组表达量高，在MEHP+MBP联合染毒组表达量低于单独染毒组，并且表现出拮抗作用，但MBP染毒组没有主效应。
结论 MBP、MEHP染毒致TM-3细胞凋亡具有主效应，MBP和MEHP联合染毒致TM-3细胞凋亡具有交互作用，呈现拮抗作用。GRP78-ATF4-CHOP通路可能参与MBP、MEHP染毒致TM-3细胞的凋亡过程。
Abstract: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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