Effects Of MEHP On DNA Methylation And Cell Cycle Progression Of Mouse Leydig Cells

Objective To investigate the effects of mono（2-ethylhexyl）phthalate（MEHP）on the viability of mouse testicular Leydig cells（TM-3）,DNA methylation level and cycle progression under different exposure doses,and to explore the correlation between them in the reproductive damage of TM-3 induced by MEHP.Methods 1.Mouse Leydig cells were cultured in vitro to the logarithmic growth phase,and the concentration gradient of the pre-experimental toxicant was set to 0,50,100,200,400,and 800μmol/L.After 24 h exposure,CCK-8 method was used to detect cell activity,and Graphpad prism6.0 was used to calculate the IC₅₀ of cells and determine the exposure dose in subsequent experiments.After adding methylation inhibitor 5-aza-deoxycytidine（5-Aza-Cdr）,5-Aza-Cdr exposure group(final concentrations were 0,10^-4,10^-3,10^-2,10^-1,10μmol/L,respectively),MEHP exposure group（final concentration was 400μmol/L）,and MEHP+5-Aza-Cdr intervention group(final concentration was 10^-4μmol/L 5-Aza-Cdr+400μmol/L MEHP)were set.CCK-8 method was used to detect cell viability in each group,and the dose of MEHP+5-Aza-Cdr intervention group was determined.2.Using inverted phase contrast microscope to observe the morphological changes of cells.3.Using5-methylcytosine antibody（5-mc）immunofluorescence method to detect the total methylation level of cells in each exposure and intervention group.4.The m RNA expressions of DNMT1,DNMT3B,FOXO1,CDK2 and P27 were detected by RT-q PCR.5.Western blotting was used to detect the expression levels of DNMT1,DNMT3B and FOXO1 proteins in each exposure and intervention group;6.Flow cytometry was used to detect the phase distribution of each cell cycle in each exposure and intervention group.7.Hoechst 33258fluorescence staining was used to detect apoptosis in each exposure and intervention group.Results CCK-8 results showed that the cell viability decreased gradually with the increase of MEHP concentration（P<0.05）.The IC₅₀ of MEHP was 354.4μmol/L calculated by Graphpad prism 6.0,so the final concentrations of MEHP at 0,200,CCK-8results showed that the cell viability decreased gradually with the increase of MEHP concentration（P<0.05）.The IC₅₀ of MEHP was 354.4μmol/L calculated by Graphpad prism 6.0,so the final concentrations of MEHP at 0,200,400 and 800μmol/L were selected as the exposure concentrations in subsequent experiments.The results of light microscope showed that the cells in the control group showed normal spindle-shaped irregular shape,full cytoplasm,and compact distribution.In the 200μmol/L group,the number and morphology of cells had no significant change,the cells were closely distributed,and the growth state was not abnormal.In the 400μmol/L group,the normal morphology of cells was significantly damaged,and most cells were cord-shaped.The intercellular space increased,edema appeared,and cell debris increased.In the 800μmol/L exposure group,the number of floating dead cells was significantly increased,and the cells had no normal morphological structure,and the cells shrank,and a large number of floating dead cells were floating.The results of 5-mc immunofluorescence staining showed that compared with the control group,the fluorescence signal intensity of 200μmol/L group was enhanced,and the methylation level was increased.The fluorescence signals of 400 and 800μmol/L groups were gradually weakened（P<0.05）,and the methylation level was decreased.The results of RT-q PCR showed that the relative expression levels of DNMT1 and DNMT3B m RNA in 200μmol/L group were higher than those in control group（P<0.05）.DNMT1 m RNA in the 400μmol/L group was lower than that in the control group（P<0.01）,and the relative expression levels of DNMT3B m RNA were higher than those in the control group（P<0.05）.DNMT1 m RNA in the 800μmol/L group was lower than that in the control group（P<0.01）,there was no significant difference in the expression level of DNMT3B m RNA between the two groups（P>0.05）.The relative expression level of transcription factor FOXO1 m RNA increased in200μmol/L group（P<0.01）,and decreased gradually in 400 and 800μmol/L groups（P<0.01）.With the increase of exposure dose,the relative expression level of P27 m RNA increased gradually（P<0.01）,and the relative expression level of CDK2 m RNA decreased gradually（P<0.01）.WB results showed that compared with the control group,the relative content of DNMT1 and DNMT3B protein in 200μmol/L group was increased,and the difference was not statistically significant（P>0.05）.The relative content of DNMT1protein in 400 and 800μmol/L groups decreased gradually（P<0.01）,and the relative content of DNMT3 B protein increased gradually（P<0.01）.With the increase of exposure dose,the relative expression level of FOXO1 protein increased gradually（P<0.01）.The flow pattern results showed that 200μmol/L and 400μmol/L groups of cells were blocked in G1 phase,and 800μmol/L groups of cells were blocked in S phase.The results of Hoechst fluorescence staining showed that the cells in the control group showed weak blue fluorescence,and the fluorescence signal in the 200μmol/L exposure group was weak,while the fluorescence signals in the 400 and 800μmol/L groups were significantly enhanced.Obvious chromatin condensation and crescent nuclei were observed.With the increase of exposure dose,the apoptosis signal gradually increased.CCK-8 results showed that after the methylation inhibitor was added,the cell survival rate of the 5-Aza-Cdr exposure group showed a trend of first increase and then decrease,and there was no significant difference in the cell survival rate between the 10^-4μmol/L group and the control group,so 10^-4μmol/L was determined as the intervention dose of the inhibitor group;CCK-8 method was further used for detection:compared with the control group,the cell survival rate of MEHP+5-Aza-Cdr intervention group decreased,and the difference was statistically significant（P<0.01）.The results of light microscopy showed that compared with the control group,400μmol/L MEHP exposure group and MEHP+5-Aza-Cdr intervention group showed serious cell damage,cell loss of normal morphological structure,cell gap was significantly larger,more cord-like distribution,floating death cells increased significantly.The results of 5-mc immunofluorescence staining showed that compared with the MEHP exposure group,the fluorescence signal of the MEHP+5-Aza-Cdr intervention group was weakened and the methylation level was decreased（P<0.01）.The results of RT-q PCR showed that compared with MEHP group,the relative expression level of DNMT1m RNA decreased（P<0.01）,the relative expression level of DNMT3Bm RNA decreased（P<0.05）,the relative expression level of transcription factor FOXO1m RNA increased（P<0.01）,the relative expression level.WB results showed that compared with the 400μmol/L MEHP exposure group,the relative expression levels of DNMT1 and DNMT3B in the MEHP+5-Aza-Cdr intervention group were decreased（P<0.05）,and the relative expression level of FOXO1 was increased（P<0.01）.The results of flow cytometry showed that compared with the control group,the cells in MEHP exposure group and MEHP+5-Aza-Cdr intervention group were blocked in G1 phase（P<0.01）.The results of Hoechst fluorescence staining showed that compared with the cells in the control group,the fluorescence signals of the cells in the 400μmol/L MEHP exposure group and the MEHP+5-Aza-Cdr intervention group were stronger.Compared with the 400μmol/L MEHP exposure group,the fluorescence signals of the cells in the MEHP+5-Aza-Cdr intervention group were enhanced,and significant nuclear condensation and nuclear staining were observed.The level of apoptosis was increased.Pearson correlation analysis showed that the transcription factors FOXO1,P27 and DNMT1 were negatively correlated,and CDK2 and DNMT1 were positively correlated 24 h after MEHP exposure.After treated with methylation inhibitor（5-Aza-Cdr）for 24 h,transcription factors FOXO1,P27 and DNMT1 were still negatively correlated,CDK2 and DNMT1 were positively correlated.Conclusion 1.MEHP exposure can reduce the viability of TM-3 cells,damage the normal morphological structure,and induce apoptosis.2.MEHP exposure can reduce the total methylation level of cells by inducing the change of DNMTs expression.3.MEHP exposure promoted the expression of cyclin inhibitor P27 and inhibited the expression of cyclin-dependent kinase CDK2 by inducing the expression level of transcription factor FOXO1,resulting in cell division arrest at G1/S phase.4.MEHP exposure may cause changes in methylation modification of TM-3 cells,decrease in total methylation level of cells,increase in the expression level of transcription factor FOXO1,further promote the expression of P27,and ultimately inhibit the expression of CDK2 m RNA,failing to enter the DNA synthesis phase,resulting in cell division blocked and blocked in the G1/S phase.