Study On The Role Of JNK Signaling Pathway In Nano-TiO₂-induced Abnormal Expression Of BTB Junction Proteins In TM4 Cells

Objective:Nano titanium dioxide（Nano-TiO₂）can destroy blood-testis barrier（BTB）,but the specific mechanism has not been fully elucidated.The purpose of this study is to understand the role of JNK signaling pathway in the abnormal expression of BTB junction proteins induced by Nano-TiO₂ and to explore the related mechanism from apoptosis,so as to provide a theoretical basis for the toxic mechanism of BTB damage induced by Nano-TiO₂.Methods:In this study,mouse Sertoli cells（TM4）were treated with different concentrations（0,50,100,150,200μg/m L）of Nano-TiO₂ for 24 h.Then we used CCK-8 assay to measure cell viability,JC-1 probe to measure mitochondrial membrane potential（MMP）,Annexin V-FITC/PI assay to measure apoptosis,western blot to detect the expression of apoptosis-related proteins（Bax,Bcl-2,Cleaved caspase 9 and Cleaved caspase3）and JNK signaling pathway proteins（JNK and p-JNK）,and immunofluorescence（IF）to detect the expression of BTB-related junction proteins（ZO-1,Claudin-11 andβ-catenin）,so as to analyze the effect of Nano-TiO₂ on BTB junctions,TM4 cell apoptosis,and JNK signaling pathway.Furthermore,TM4 cells were treated with 20μM JNK signaling pathway inhibitor（SP600125）and 150μg/m L Nano-TiO₂ for 24 h to analyze the change of cell viability,MMP,apoptosis,apoptosis-related proteins,BTB junction proteins and JNK signaling pathway in order to explore the role of JNK signaling pathway in Nano-TiO₂-induced abnormal expression of BTB junction proteins and apoptosis.Results:1.The results of CCK-8 assay showed that cell viability in 100,150 and 200μg/m L Nano-TiO₂ groups significantly decreased compared with the control group（P<0.01）.The results of IF showed that the protein levels of ZO-1,Claudin-11 andβ-catenin decreased in 150 and 200μg/m L Nano-TiO₂ groups compared with the control group.In the study of apoptosis,we found that compared with the control group,MMP decreased significantly in 150 and 200μg/m L Nano-TiO₂ group（P<0.05）,the apoptosis increased significantly in 150and 200μg/m L Nano-TiO₂ group（P<0.05）,the expression of pro-apoptotic proteins（Bax,Cleaved caspase9 and Cleaved caspase 3）increased significantly in 150 and 200μg/m L Nano-TiO₂ groups（P<0.05）,and anti-apoptotic protein Bcl-2 decreased significantly in 100,150 and 200μg/m L Nano-TiO₂ groups（P<0.01）.2.Compared with the control group,the protein level of p-JNK was significantly increased in the 100,150and 200μg/m L Nano-TiO₂ groups（P<0.05）,and the ratio of p-JNK/JNK was increased significantly in 150and 200μg/m L Nano-TiO₂ groups（P<0.05）.3.Compared with 150μg/m L Nano-TiO₂group,the combined treatment of Nano-TiO₂ and SP600125 could inhibited the increase of protein level of p-JNK and the ratio of p-JNK/JNK（P<0.01）;Meanwhile,SP600125could alleviate the decrease of cell viability（P<0.05）,the reduction of BTB junction proteins（ZO-1,Claudin-11 andβ-catenin）in TM4 cells,the collapse of MMP（P<0.05）,the increase of apoptosis（P<0.05）,and the abnormal expression of apoptosis-related proteins（Bax,Bcl-2,Cleaved caspase 9 and Cleaved caspase 3）（P<0.05）induced by Nano-TiO₂.Conclusion:Nano-TiO₂ could decrease of cell viability,reduce the expression of BTB junction proteins（ZO-1,Claudin-11 andβ-catenin）,induce abnormal apoptosis and activate the JNK signaling pathway;Moreover,activated-JNK signaling pathway could regulate the decrease of BTB junction proteins level induced by Nano-TiO₂,which may be related to Sertoli cells apoptosis mediated by JNK signaling pathway.