| Objective:Titanium dioxide nanoparticles（TiO2-NPs）have applied in food and cosmetics because of its special physicochemical properties.Studies have found that TiO2-NPs has adverse effects on the male reproductive system.The purpose of the study was based on p38 MAPK signaling pathway to explore the toxic mechanism of TiO2-NPs in Sertoli cells,which provides a theoretical basis for the specific mechanism of reproductive damage induced by TiO2-NPs.Methods:To investigate the cytotoxicity of TiO2-NPs and the effect of TiO2-NPs on p38 MAPK signaling pathway,we treated Sertoli cell line（TM4）with 0,50,100,150,and 200μg/mL TiO2-NPs for 24h.We examine the cells viability of TiO2-NPs by CCK-8 assay.Intracelluar reactive oxygen species（ROS）content were detected by DCFH-DA kit.The SOD activity and MDA content were determined by enzyme activity kit.The expression of BTB related protein（ZO-1,Claudin-11,andβ-catenin）were observed by Immunofluorescence.The protein expression of p38 MAPK signaling pathway（p38 MAPK and p-p38 MAPK）was detected by Western Blot.To analyze the role of p38 MAPK and ROS in the cytotoxicity of TM4 cells induced by TiO2-NPs,we respectively treated TM4 cells with NAC and SB203580 for 24h.We detected related index including ROS,SOD activity,MDA content,and the protein expression of p38,p-p38,ZO-1,Claudin-11,andβ-catenin.One-way ANOVA method was used to compare the differences between multiple groups,and Bonferroni method was used for multiple comparisons between groups.P<0.05 was considered as statistically significant.Results:1.Effect of TiO2-NPs on cytotoxicity of TM4 cells.Compared with the control group,when the concentration of TiO2-NPs was higher than 100μg/mL,cell viability obviously lessened with the increase of concentration and the difference was statistically significant（P<0.01）;when the concentration of TiO2-NPs was higher than 50μg/mL,ROS content had a significant increase（P<0.05）.Compared with the control group,the activity of SOD in TM4 cells decreased significantly in the TiO2-NPs groups（P<0.001）,the content of MDA increased significantly in the TiO2-NPs groups（P<0.05）.Moreover,TiO2-NPs exposure could decrease the fluorescence intensity of ZO-1,Claudin-11 andβ-catenin in TM4 cells.2.Effect of TiO2-NPs on p38 MAPK signaling pathway.Compared with the control group,when the concentration of TiO2-NPs was higher than 150μg/mL,the expression of p-p38 MAPK protein in TM4 cells increased significantly（P<0.05）.Compared with the control group,100,150,and 200μg/mL TiO2-NPs increased the ratio of p-p38 MAPK/p38 MAPK in TM4 cells（P<0.05）.p38 MAPK signaling pathway was activated by oxidative stress pathway.Compared with TiO2-NPs group,p-p38 protein expression and the ratio of p-p38/p38 were significantly decreased in NAC combined with TiO2-NPs treatment group（P<0.01）.3.P38 MAPK signaling pathway is activated by oxidative stress.Compared with TiO2-NPs group,the expression of p-p38 protein and p-p38/p38 were significantly decreased in NAC combined with TiO2-NPs treatment（P<0.01）.4.Effect of p38 MAPK signaling pathway on cytotoxicity of TM4 cells induced by TiO2-NPs.Compared with TiO2-NPs group,cell viability increased in SB203580 combined with TiO2-NPs group and the difference was statistically significant（P<0.05）.Compared with TiO2-NPs group,the oxidative stress of TM4 cells induced by TiO2-NPs has alleviated in SB203580 combined with TiO2-NPs group,such as alleviated ROS accumulation（P<0.01）and increased the activity of SOD（P<0.05）,and decreased the content of MDA（P<0.01）.Compared with the TiO2-NPs group,the fluorescence intensity of ZO-1,Claudin-11,andβ-catenin in TM4 cells increased significantly in the co-treatment of SB203580 and TiO2-NPs group.Conclusion:TiO2-NPs could induced the decrease of TM4 cell viability,oxidative damage,and the expression of BTB related proteins.TiO2-NPs also activated of p38 MAPK signaling pathway.Moreover,the activation of p38 MAPK signaling pathway could mediated oxidative damage and BTB related proteins expression of TM4 cells induced by TiO2-NPs. |
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