MBP、MEHP诱导小鼠睾丸间质细胞自噬过程中PTEN的相关研究

Study on PTEN in autophagy induced by MBP and MEHP in mouse testicular mesenchymal cells

[目的]目前对于邻苯二甲酸酯的研究多集中于其原型,但有学者认为,邻苯二甲酸二丁酯和邻苯二甲酸二乙基己基酯的毒性作用可能是其在体内的代谢产物邻苯二甲酸单丁酯(MBP)和邻苯二甲酸单乙基己酯(MEHP)所致。本研究拟探讨MBP和MEHP对小鼠睾丸间质细胞(TM-3)自噬的影响以及与第10号染色体缺失的同源性磷酸酶-张力蛋白(PTEN)的相关研究。[方法]体外培养TM-3并取其对数生长期,预实验设置毒物浓度梯度为0(对照组)、50、100、200、400、800μmol/L,染毒24 h后,利用改良寇氏法计算MBP、MEHP的半数致死浓度(IC50)后,确定实验染毒浓度为0(对照组)、200、400、800μmol/L。分别采取CCK-8法检测不同毒物剂量和不同染毒时间对细胞存活率的影响;透射电镜观察400μmol/L的MBP、MEHP诱导24 h后具有双层膜状结构的自噬小体;单丹黄酰尸胺(MDC)免疫荧光法检测细胞自噬囊泡的荧光信号强度;采用Western blot技术检测自噬标志蛋白LC3、p62和自噬调节相关蛋白PTEN的表达水平。[结果] CCK-8结果显示:与对照组相比,随着MBP、MEHP染毒剂量增高,细胞存活率降低(P<0.05)。透射电镜结果显示:400μmol/L MBP、MEHP染毒24 h后细胞内可见具有双层膜状结构的自噬小体。MDC法检测结果显示:与对照组相比,200μmol/L MBP、MEHP染毒组荧光信号强度差异无统计学意义(P>0.05);400、800μmol/L MBP染毒组荧光信号强度分别增加了9.0、16.8倍;400、800μmol/L MEHP染毒组荧光信号强度增加了16.3、26.4倍,差异均有统计学意义(P<0.01)。Western blot结果显示:200μmol MBP、MEHP染毒组LC3II/LC3I、PTEN表达水平与对照组相比差异无统计学意义(P>0.05);400、800μmol/L MBP染毒组LC3II/LC3I蛋白表达水平分别增加了7.5、13.6倍,PTEN表达水平增加了4.8、15.4倍;400、800μmol/L MEHP染毒组LC3II/LC3I蛋白表达水平增加了4.5、9.8倍,PTEN表达水平增加了5.7、14.4倍(P<0.05);200μmol/L MBP染毒组p62表达水平与对照组相比差异无统计学意义(P>0.05),400、800μmol/LMBP染毒组p62蛋白表达水平分别降低了43%、72%;200、400、800μmol/L MEHP染毒组p62蛋白表达水平也分别降低了55%、80%、88%(P<0.01)。[结论]一定剂量的MBP、MEHP可提高TM-3细胞自噬水平,并且PTEN表达水平与细胞自噬水平呈正相关趋势。

[Objective]Much research related to phthalates has focused on parent chemicals;however,dibutyl phthalate and diethylhexyl phthalate may exert their toxicities via metabolites monobutyl phthalate(MBP)and mono-ethylhexyl phthalate(MEHP).This study aims to investigate the effects of MBP and MEHP on the autophagy of mouse testicular mesenchymal cells(TM-3)and phosphatase and tensin homology deleted on chromosome ten(PTEN).[Methods]In vitro cultured TM-3 cells in logarithmic growth phase were treated with either MBP or MEHP at concentration gradients of 0(control group),50,100,200,400,and 800μmol/L;after 24 h of treatment,respective median lethal concentrations(IC50)were calculated by improved Karber’s method,and a set of concentrations of 0(control group),200,400,and 800μmol/L were determined appropriate for subsequent experiment.The effects of MBP or MEHP exposure at designed concentrations for different time on cell survival rate were assessed by CCK-8 method;autophagosomes with double membranes induced by 400μmol/L MBP or MEHP for 24 h were observed under transmission electron microscope;the fluorescence signal intensity of autophagic vesicles was detected by monodansyl cadaverine(MDC)immunofluorescence staining;the expression levels of autophagy marker proteins(LC3 and p62)and autophagy regulation protein(PTEN)were assessed by Western blot.[Results]The CCK-8 results showed that compared with the control group,the cell survival rate decreased as the dose of MBP or MEHP increased(P<0.05).Under transmission electron microscopy,the autophagosomes with double membranes were observed in the cells infected with 400μmol/L MBP or MEHP for 24 h.The MDC staining results showed that compared with the control group,the fluorescence intensities of the 200μmol/L MBP group and the 200μmol/L MEHP group had no statistical difference(P>0.05),but the fluorescence signal intensities of the 400 and 800μmol/L MBP groups increased by 9.0 times and 16.8 times respectively,and those of the 400 and 800μmol/L MEHP groups increased by 16.3 times and 26.4 times,respectively(P<0.01).The Western blot results showed that the 200μmol/L MBP and MEHP treatments did not change the LC3 II/LC3 I and PTEN protein expression levels compared with the control group(P>0.05);the protein expression levels of LC3 II/LC3 I in the 400 and 800μmol/L MBP groups increased by 7.5 times and 13.6 times respectively,and the PTEN expression levels increased by 4.8 times and 15.4 times respectively;the protein expression levels of LC3 II/LC3 I in the 400 and 800μmol/L MEHP groups increased by 4.5 times and 9.8 times respectively,and the PTEN expression levels increased by5.7 times and 14.4 times respectively(P<0.05);except the 200μmol/L MBP group(P>0.05),the expression levels of p62 protein in the400 and 800μmol/L MBP groups decreased by 43%and 72%respectively;the expression levels of p62 protein in the 200,400,and 800μmol/L MEHP groups reduced by 55%,80%,and 88%,respectively(P<0.01).[Conclusion]A certain dose of MBP or MEHP can increase the autophagy of TM-3 cells,and the expression level of PTEN is positively correlated with the autophagy level.