| Background As one of the products of synthesis or catabolism of perfluorooctane compounds,perfluorooctane sulfonate（PFOS）is widely used in industry,production and life.It can cause multi system toxicity to mammals and its reproductive toxicity has attracted more and more attention.In addition,how to improve and treat the reproductive toxicity induced by PFOS has become a difficult problem in the world.Objective 1.To investigate the protective effect of icariin（ICA）on Sertoli cell junction dysfunction induced by PFOS in vivo;2.To explore the mechanism of ICA in protecting Sertoli cell junction dysfunction induced by PFOS in vitro.Methods 1.Animal experiment:Fifty two 7-week-old SPF C57BL/6 male mice were randomly divided into normal control group,model group,low-dose ICA（5 mg/kg）group and high-dose ICA（20 mg/kg）group,with 13 mice in each group.The latter three groups were given PFOS by intragastric administration at 10 mg/kg dose every day,while the normal group was given dd H2O containing 2%Tween 80 in the same volume ratio as the model group.The normal control group and model group were fed with common maintenance feed,while the low and high dose groups were fed with 5 mg/kg and 20 mg/kg ICA containing feed respectively.After continuous feeding for 4 weeks under the same environment,the mice body mass was weighed,the rats were killed in cervical vertebra and the testis tissue was taken out quickly.The following indexes were detected:（1）The weight of testis and epididymis was weighed and the index was calculated;（2）The density and viability of sperm were detected;（3）The histomorphological changes of testis were observed by HE staining,and the thickness of spermatogenic epithelium and the diameter of seminiferous tubules were statistically analyzed;（4）The Sertoli cells in testis were labeled by immunofluorescence method,and the number of Sertoli cells in testis was observed;（5）The integrity of blood testis barrier was detected by biological tracer;（6）The expression of Sertoli cell junction related protein CX43,p-CX43,ZO-1,Occludin and Claudin-11were detected by Western blot;（7）Sertoli cells were observed by double labeled immunofluorescence The expression and localization of ZO-1,CX43 and p-p38MAPK;（8）The expression and colocalization of matrix metalloproteinase 9（MMP9）and CX43 were detected by double labeled immunofluorescence.2.Cell experiment:2.1 after TM4 cells were treated with different concentrations of PFOS for24 hours,MTT assay was used to detect cell viability in order to select the appropriate concentration of PFOS.The following indexes were detected:（1）morphology of TM4 cells was observed under light microscope;（2）Western blot was used to detect the protein expression of connexin related proteins in TM4 cells,including CX43,p-CX43,ZO-1,Occludin and Claudin-11;（3）The protein expression of p38MAPK,p-p38MAPK and MMP9in TM4 cells treated with different concentrations of PFOS were detected by Western blot.2.2after TM4 cells were treated with different concentrations of ICA and PFOS for 24 hours,MTT assay was used to detect the cell viability and select the appropriate concentration of ICA,then detect the related indicators of 2.1（1）-2.1（3）;2.3 After adding p38 inhibitor SB203580,TM4 cells were divided into control group,model group,SB203580 group,PFOS+SB203580 group and PFOS+ICA group.After 24 hours of culture,the following indexes were detected:（1）MTT assay was used to detect cell viability;（2）Western blot was used to detect the expression of junction related proteins,such as CX43,p-CX43,ZO-1,Occludin,and Claudin-11;（3）Double labeled immunofluorescence was used to detect the expression and localization of CX43,p-p38MAPK and MMP9 protein,respectively.Results 1.Animal experiments:（1）ICA could significantly increase the weight of testis,epididymis weight,epididymis index,sperm density and viability of epididymis in mice induced by PFOS;（2）ICA could significantly improve the morphological changes of testis in mice induced by PFOS,significantly increase the thickness of spermatogenic epithelium,but had no significant effect on the diameter of seminiferous tubules and the number of Sertoli cells in mice;（3）ICA could significantly improve the damage of testicular blood testis barrier caused by PFOS in mice;（4）Western blot results showed that ICA could significantly up regulate the expression levels of CX43,p-CX43 and tight junction proteins ZO-1,Occcludin and Claudin-11 in Sertoli cells of mice induced by PFOS;（5）Immunofluorescence results showed that ICA could significantly up regulate the expression levels of CX43 and ZO-1,and down regulate the expression levels of p-p38MAPK and MMP9 in testis of mice induced by PFOS.2.Cell experiment:2.1 The results showed that when PFOS concentration was below150μM,with the increase of PFOS concentration,the viability and the number of TM4 cells was not significantly changed,but the number of abnormal cells increased;meanwhile,the expression of connexin CX43,p-CX43 and tight junction proteins ZO-1,Occludin and Claudin-11 in TM4 cells decreased gradually,while the expression of p-p38MAPK and MMP9 increased gradually;2.2 When ICA concentration was below 16μM,the viability of TM4 cells did not change significantly after treated with ICA and PFOS for 24 h,but ICA significantly reduced the morphological damage of TM4 cells induced by PFOS,and the number of abnormal round or oval cells decreased;Western blot results showed that compared with the normal control group,the protein expression levels of junction related proteins,such as CX43,p-CX43,ZO-1,Occludin and Claudin-11,were significantly decreased in the model group while the protein expression levels of p-p38MAPK and MMP9 were significantly increased;Compared with the model group,ICA significantly increased the expression of junction related proteins in TM4 cells induced by PFOS while The protein expression of p-p38MAPK and MMP9 was down regulated;2.3 After adding p38MAPK inhibitor SB203580,the viability of TM4 cells did not change significantly;Western blot results showed that p38MAPK inhibitor SB203580 or ICA could up-regulated gap junction proteins CX43,p-CX43 and tight junction proteins ZO-1,Occludin and Claudin-11 in TM4 cells induced by PFOS.The results of double labeled immunofluorescence assay showed that compared with the normal control group,the expression of CX43 protein decreased and the expression of p-p38MAPK and MMP9 protein increased in the model group,while the expression of CX43 protein increased and the expression of p-p38MAPK and MMP9 protein increased after the addition of p38MAPK inhibitor SB203580 or ICA.Conclusion ICA could improve the junction damage of testis Sertoli cell induced by PFOS,and its mechanism may be related to the regulation of p38MAPK/MMP9 signaling pathway. |
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