The Effect Of Fluoride Exposure On Rat Sertoli Cell Tight Junction And Gap Junction In Vitro

[objective]The increase of fluoride in testis as an indicator of the effect of fluoride on the reproductive system has been widely accepted,so we speculate that the fluorine can pass through the blood testis barrier(BTB),however,that how the breakout of the BTB lead to elevated levels of fluoride in testis is unknown.In this study,the rat testicular sertoli cells were selected as object of study,and the three kinds of cell connections(TJ,GJ,Basal ES)was detect to figure out the effect of fluoride on the homeostasis of BTB and the changes of related molecules in sertoli cells.We aim to further reveal the mechanism of reproductive toxicity of fluoride,and provide the basis for the implementation of scientific diagnosis and target drugdesign.[Method]The BTB model of primary sertoli cells was established in vitro,simulating BTB system in vivo.The cell activity was detected by XTT method after exposure to different concentrations of NaF.The TER was used to evaluate the tight junction barrier,the gap junction communication function detected by light bleach fluorescence recovery technique(FRAP).The expression of three cell connection-related genes was quantitatively analyzed by qRT-PCR,Western Blotting and IF techniques,and the quantitative analysis and positioning analysis of TJ,GJ and basal ES-related proteins were also performed,then to evaluate the effect of fluoride on the structure and function of BTB cells.[Results]1.The sertoli cells were successfully cultured in this experiment.The cultured cells were identified by HE staining,GATA4 and WT1 immunofluorescence,and the purity was very high..2.The results of XTT showed that NaF had a significant effect on the sertoli cells in the 0.00 1mg/LNaF group compared with the control group(P<0.01).Compared with the control group,the 20mg/LNaF treatment group had significant effect on the sertoli cells significant inhibitory effect(P<0.001).Indicating that low concentration of NaF on cell activity can promote the role of high concentrations of NaF on cell activity3.Expression of tight junctions and related gene proteins:The results of TER showed that we successfully constructed a blood testosterone model in vitro.After 48 hour of NaF,the resistance value of 10 mg/L fluoride group significantly decrease compared with the control group,indicating that the sertoli cell tight junction barrier function was destroyed.The expression of CAR mRNA in each treatment group was not statistically significant compared with the control group.Compared with the control group,the expression of occludin and F11 mRNA was significantly decreased in the treatment group(P<0.05).However,the expression of mRNA in the lmg/L treatment group was significantly higher than that in the control group(P<0.001).Western blot results showed that the expression of ZO-1 protein was significantly increased in 0.1 mg/L treatment group compared with the control group(P<0.01).Compared with the control group,the expression of Occludin protein was significantly higher in the 1 mg/L treatment group(P<0.05),while the expression of Occludin protein was significantly decreased in 10mg/LNaF treatment group(P<0.01).The expression of occludin protein in the control group and 0.1 mg/L treatment group was mainly expressed on the cell membrane,while the 1 mg/L treatment group occludin protein was expressed in the vicinity of the nucleus,and the expression level The expression of Occludin was significantly decreased in 10 mg/L treatment group.4.The expression of basal ES related genes and proteins:Compared with the control group,the mRNA expression of N-cadherin and a-catenin was significantly decreased only in the 10mg/L treatment group(P<0.05),but compared with the control group(P<0.05).The expression of β-catenin mRNA was significantly increased at 1 mg/L(P<0.05).Western-blot showed that the expression of a-catenin andβ-catenin protein in each treatment group was not statistically significant compared with the control group.The expression of N-cadherin protein in lmg/L treatment group was significantly higher than that in the control group(P<0.05).5.The expression of gap junctions and related gene proteins were significantly lower in the 1 mg/L and 10 mg/L treated groups than those in the control group,indicating that the gap junction function was destroyed.The expression of connexin-43 in the 10 mg/L treatment group was significantly lower than that in the control group(P<0.05).Western-blot results showed that the protein expression of connexin-43 in each treatment group was significantly lower than that in the control group(P<0.01,P<0.001).IF results showed that the expression of connexin-43 protein in each treatment group disappeared from the cell membrane.[Conclusion]Under the experimental conditions,the effect of fluoride on the basal ES was almost no obvious effect;10mg/L NaF lead to break of tight junction barrier function,and the expression of occludin gene and protein was also decreased under the action of fluoride;the expression of connexin-43 gene and protein in the sertoli cells was decreased,and the communication function of gap junction was also destroyed.it was shown that the most important target for the destruction of BTB in the sertoli cells was the gap junction.