Nickel Chloride Impairs Blood-Testis Barrier In Mouse Via The ROS-p38 MAPK Pathway

Nickel（Ni）is an essential trace element for animals as well as a common environmental pollutant.Nickel is harmful to the reproduction of male animals,and our previous study found that nickel chloride（NiCl₂）significantly reduced sperm quality in mice,while the exact mechanism of toxicity remains unclear.The Blood-Testis Barrier（BTB）is a barrier structure between blood vessels and spermatogenic tubules in the testis,and plays an important role in testicular spermatogenesis.Sertoli Cells are one of the constituent components of the BTB.At the basal membrane of the seminiferous tubule of the testis,tight junctions,gap junctions and adherens junction between adjacent sertoli cells work together to maintain the structural and functional integrity of the BTB.In order to damage spermatogenic cells,toxic substances need to pass through the sertoli cells first,or affecting the function of the sertoli cells,which in turn act on the germ cells.Therefore,the sertoli cells are the target of many environmental toxicants.In this study,7-week-old male ICR mice as well as mouse testicular sertoli cells（TM4）were used to investigate the damage of NiCl₂on mouse testicular sertoli cells junctions and its toxicological mechanism.The results are as follows:1.Effect of NiCl₂on the Blood-Testis Barrier in miceOne hundred and twenty 7-week-old ICR male mice were selected and gavaged with different doses of NiCl₂（0/7.5/15/30 mg/kg）to establish a subacute toxicity test for NiCl₂.Samples were taken on days 14 and 28 of the test.Subsequently,we examined the changes in the structure and function of testicular BTB and the levels of relevant connecting factors between BTB sertoli cells.In vitro,we selected mouse testicular sertoli cells（TM4）,added different concentrations of NiCl₂（0/0.75/1.5/3 mM）to the medium of sertoli cells,and collected sertoli cells at 12 h and 24 h of the test to detect changes in the levels of relevant connecting factors between the sertoli cells.In vivo,transmission electron microscopy results showed that in the NiCl₂-treated group,the continuous BTB structure was broken,tight junctions were disintegrated,actin filament bundles were missing,and the endoplasmic reticulum was significantly swollen;Biotin tracing results showed that green fluorescence was visible in the seminiferous tubule of the NiCl₂-treated group,and the fluorescence intensity and diffusion range were enhanced with increasing dose,indicating that BTB integrity was disrupted;Immunofluorescence results showed that the expression positions of Occludin,N-cadherin and Connexin-43 were not changed,but the protein expression levels decreased with increasing NiCl₂dose;The protein and mRNA expression levels of the tight junction proteins ZO-1,Occludin,F11R and Claudin-11,the adherens junction protein N-cadherin,and the gap junction protein Connexin-43 were significantly decreased after NiCl₂treatment compared to the control（p<0.05）.In vitro,NiCl₂treatment significantly reduced TM4 cell activity.WB and qRT-PCR results showed that both protein and mRNA expression levels of intercellular junctions in sertoli cells were significantly reduced by NiCl₂treatment when compared to the control（p<0.05）.2.Effect of NiCl₂on ROS and p38 MAPK in mouse testis and sertoli cellsIn vivo,we measured oxidative stress indicators and p38 MAPK expression levels in mouse testicular tissues in the NiCl₂subacute toxicity assay established above.In vitro,we measured ROS expression levels and p38 MAPK expression levels in cells in the NiCl₂toxicity assay established above in TM4 cells.In vivo,NiCl₂induced a significant increase in MDA content and a significant decrease in T-AOC activity,SOD activity and GSH content in testis compared to the control group（p<0.05）;Meanwhile,the protein expression level of p-p38 was significantly upregulated compared to the control group（p<0.05）.In vitro,NiCl2 treatment significantly elevated ROS（p<0.05）.Meanwhile,the protein expression level of p-p38 was significantly up-regulated in the NiCl₂-treated group compared with the control group（p<0.05）.3.Role of ROS-p38 MAPK signaling pathway in NiCl2-induced Blood-TestisBarrier damageTo investigate the role of ROS,p38 MAPK in NiCl₂-induced disruption of sertoli cell junctions and the relationship between ROS and p38 MAPK,we used the antioxidant N-acetylcysteine（NAC）co-treated with NiCl₂to establish an oxidative stress inhibition intervention assay in mice and TM4 cells.To further investigate the role of p38 MAPK,the p38 MAPK inhibitor SB203580 was added to the above in vitro cytotoxicity assay and co-treated with NiCl₂to establish a p38 MAPK signaling pathway inhibition intervention assay.In vivo,the results of NAC inhibition assay showed that NAC could alleviate NiCl₂-induced oxidative testicular damage;transmission electron microscopy and biotin demonstration tracing showed that the degree of damage to the structure and integrity of BTB was significantly improved in the NAC co-treatment group compared to the NiCl₂group;compared to the NiCl₂-treated group,NAC co-treatment enhanced Occludin,N-cadherin,and Connexin-43 fluorescence intensity,and the protein and mRNA expression of the BTB were significantly elevated（p<0.05）.In addition,the protein expression level of p-p38 was significantly decreased after NAC co-treatment（p<0.05）.In vitro,the results of the NAC inhibition assay showed that:compared with the NiCl₂-treated group,the level of ROS in sertoli cells was significantly decreased in the NAC co-treatment group（p<0.05）,and the expression of BTB junction-related proteins and mRNA was significantly increased（p<0.05）.p-p38 protein expression level was significantly decreased in the NAC co-treatment group compared with the NiCl₂group（p<0.05）.P38 MAPK signaling pathway inhibition assay showed that SB203580co-treatment could alleviate the effects of NiCl₂on cell activity and cell morphology;p-p38 expression was significantly decreased and BTB junction related protein and mRNA expression were significantly upregulated in SB203580 co-treatment group（p<0.05）.In conclusion,NiCl₂could damage the BTB in mice by disrupting the sertoli cells intercellular junctions.And the effect of NiCl₂to impair the sertoli intercellular junctions could depend on the ROS-p38 MAPK pathway.