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CHOP通路在邻苯二甲酸单丁酯和邻苯二甲酸单(2-乙基)己酯联合暴露致小鼠睾丸间质细胞凋亡中的作用

Role of CHOP pathway in apoptosis of cultured mouse Leydig cells induced by combined exposure to monobutyl phthalate and mono(2-ethylhexyl) phthalate

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【作者】 员朋娟李玲德小明李丽萍张亚娟张鹏举刘贺荣李娅辉伊梦楠

【Author】 YUAN Peng-juan;LI Ling;DE Xiao-ming;LI Li-ping;ZHANG Ya-juan;ZHANG Peng-ju;LIU He-rong;LI Ya-hui;YI Meng-nan;Department of Occupational and Environmental Health,Public Health and Management School of Ningxia Medical University,Key Laboratory of Fertility Preservation and Maintenance,Ningxia Medical University,Ministry of Education,Key Laboratory of Reproduction and Genetics in Ningxia Hui Autonomous Region;

【通讯作者】 李玲;

【机构】 宁夏医科大学公共卫生与管理学院职业卫生与环境卫生学系生育力保持教育部重点实验室宁夏回族自治区生殖与遗传重点实验室

【摘要】 目的探讨邻苯二甲酸单丁酯(monobutyl phthalate,MBP)和邻苯二甲酸单(2-乙基)己酯[mono(2-ethylhexyl)phthalate,MEHP]染毒致体外培养的小鼠睾丸间质细胞(TM-3)凋亡过程中CHOP通路的作用。方法将小鼠睾丸间质细胞分别暴露于0(对照)、50、100、200、400、800μmol/L的MBP和(或)MEHP溶液24、36、48 h后,采用CCK-8法测定细胞活性。将小鼠睾丸间质细胞分别暴露于完全培养基(对照)、MBP(400μmol/L)、MEHP(400μmol/L)和MEHP(400μmol/L)+MBP(400μmol/L)溶液24 h后,电镜下观察小鼠睾丸间质细胞内质网超微结构的变化。将小鼠睾丸间质细胞分别暴露于完全培养基(对照)及200、400、800μmol/L MBP和(或)MEHP溶液24 h,流式细胞术检测细胞早期凋亡情况,免疫印迹法检测内质网应激标志性蛋白葡萄糖调节蛋白78(GRP78)和CHOP的表达水平。结果与对照组相比,各浓度MBP染毒组小鼠睾丸间质细胞的抑制率均较高,差异有统计学意义(P<0.05);且随着MBP染毒浓度的升高,小鼠睾丸间质细胞的抑制率呈上升趋势。随着MBP染毒时间的延长,小鼠睾丸间质细胞的抑制率除100μmol/L组呈上升趋势外,其余浓度组均呈先上升后下降的趋势。与对照组相比,各浓度MEHP染毒组小鼠睾丸间质细胞的抑制率均较高,差异具有统计学意义(P<0.05);且随着MEHP染毒浓度的升高,小鼠睾丸间质细胞的抑制率呈上升趋势。随着MEHP染毒时间的延长,小鼠睾丸间质细胞的抑制率在50μmol/L组呈上升趋势,在100μmol/L组呈先下降后上升的趋势,在400μmol/L组呈下降趋势,200μmol/L和800μmol/L组抑制率呈先上升后下降的趋势。与对照组相比,各浓度MBP+MEHP染毒组小鼠睾丸间质细胞的抑制率均较高,差异具有统计学意义(P<0.05);且随着MBP+MEHP染毒浓度的升高,小鼠睾丸间质细胞的抑制率呈现上升趋势。随着MBP+MEHP染毒时间的延长,小鼠睾丸间质细胞的抑制率除200、400μmol/L组呈现逐渐上升趋势外,其余浓度组均呈先上升后下降的趋势。与对照组相比,各浓度MBP和(MEHP)染毒组TM-3细胞的早期凋亡细胞比例均较高,差异有统计学意义(P<0.05);且随着染毒浓度的升高,MBP、MEHP和MBP+MEHP染毒小鼠睾丸间质细胞的早期凋亡细胞比例均呈现上升趋势。在同一染毒浓度下,睾丸间质细胞早期凋亡比例依次为MBP+MEHP<MBP<MEHP,差异有统计学意义(P<0.05)。与对照组相比,各浓度MBP和(或)MEHP染毒组小鼠睾丸间质细胞内GRP78和CHOP蛋白的表达水平均增加,差异有统计学意义(P<0.05);且随着染毒浓度的升高,MBP和(或)MEHP染毒小鼠睾丸间质细胞内GRP78和CHOP蛋白的表达水平均呈逐渐上升的趋势。结论 MBP和(或)MEHP染毒可致内质网损伤,上调GRP78蛋白和CHOP蛋白的表达,这可能是MBP和(或)MEHP引起小鼠睾丸间质细胞凋亡的重要途径。

【Abstract】 Objective To understand the role of CHOP pathway in apoptosis of testicular stromal cells(TM-3) induced by MEHP and MBP. Methods Based on the test of cell viability,the mouse testicular stromal cells(TM-3) were exposed to complete medium(control),MBP(400 μmol/L),MEHP(400 μmol/L) and MEHP(400 μmol/L) + MBP(400 μmol/L) solution,respectively.After 24-hour exposure,the ultrastructural changes of endoplasmic reticulum in mouse Leydig cells were observed under electron microscope. Mouse Leydig cells were exposed to complete medium(control),200,400 and 800 μmol/L MEHP and/or MBP solution for 24 hours,respectively,flow cytometry was used to detect early apoptosis of cells,and the endoplasmic reticulum stress-signaling protein,glucose-regulated protein 78(GRP78) and CHOP were detected by Western blot. Results Compared with the control group,the inhibition rate of Leydig cells in the MBP-treated group was significantly higher(P<0.05),with a dose dependent manner. With the prolongation of MBP exposure time,the inhibition rate of Leydig cells increased in the100 μmol/L group,increased first and then decreased in other dose groups.Compared with the control group,the inhibition rate of Leydig cells in the MEHP-treated group was significantly higher( P<0.05),with a dose dependent manner. With the prolongation of MEHP exposure time,the inhibition rate of Leydig cells increased in the 50 μmol/L group,decreased in the 400μmol/L group,decreased first and then increased in the 100 μmol/L group,increased first and then decreased in the 200 μmol/L and 800 μmol/L groups. Compared with the control group,the inhibition rate of Leydig cells in MBP +MEHP group was significantly higher(P<0.05),with a dose dependent manner. With the prolongation of MBP+MEHP exposure time,the inhibition rate of mouse Leydig cells was gradually increased in the 200 and 400 μmol/L groups,increased first and then decreased in other dose groups. Compared with the control group,the proportion of early apoptotic cells in MBP and/or MEHP groups was significantly higher(P <0.05),with a dose dependent manner.At the same exposure concentration,the proportion of early apoptosis of Leydig cells was ranked as MBP+MEHP<MBP<MEHP,the difference was statistically significant(P<0.05). Compared with the control group,the expression levels of GRP78 and CHOP protein in the Leydig cells increased significantly in alone and joint exposure groups of MBP and MEHP respectively(P<0.05),with a dose dependent manner. Conclusion MBP and/or MEHP can cause damage to the endoplasmic reticulum and up-regulate the expression of GRP78 protein and CHOP protein,which may be an important pathway for MBP and/or MEHP to induce apoptosis of mouse Leydig cells.

【基金】 宁夏医科大学校级科研项目(XM2018003)
  • 【文献出处】 环境与健康杂志 ,Journal of Environment and Health , 编辑部邮箱 ,2018年10期
  • 【分类号】R114
  • 【下载频次】25
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