Ameliorative Effect Of V_E,IGF-I And HCG On Fluoride-induced The Decreases Of Leydig Cells Function

[Objective]The main function of Leydig cells(LCs)is synthesis and secreting testosterone,fluoride is a kind of environmental toxins,the studies demonstrate that long-term fluoride exposure can lead to changes in testosterone secretion.The purpose of this study was to explore the ameliorative of VE,hCG and IGF-I on that fluoride exposure cause the change of LCs proliferation,secretion of testosterone and expression of the related gene in mice,and provide some theoretical basis and guild for the study and effective prevention of the mechanism of effect of fluoride on reproductive system.[Method]3 to 4weeks old male Kunming mice were chosen for culture LCs in vitro.3?-HSD staining and 3?-HSD immunofluorescence were used to identify its purity,Giemsa staining identify morphological.Fluoride exposure was divided into the three treatment groups,followed by:Omg/L NaF group,5mg/L NaF group and 20mg/L NaF group.After 24h and 48h,determined the cell proliferation by MTT assay,determined testosterone secretion by ELISA assay.Fluoride exposure model was established.The model of fluoride exposure in vitro culture LCs,adds the VE,IGF-I and hCG together with different concentrations of sodium fluoride after 24,48h.To detect the cell proliferation by MTT assay,detect testosterone secretion by ELISA assay,using the QRT-PCR detect mRNA expression changes of secretion of testosterone related genes.[Result](1)Identification of LCs in mice:LCs can be observed from spreading cytoplasm by Giemsa staining,soma is the round,elliptical shape or irregular.3?-HSD staining LCs was specificity dyed dark blue,cells concentrate become round.3?-HSD immunofluorescence analysis of cells by specific dyed green.Prove that the extracted cells were LCs,and the purity can be used to the next step test.(2)VE,IGF-I and hCG dose selection:By adding different concentrations of VE,IGF-I and hCG by MTT method and/or ELISA method were used to detect cell proliferation and secretion of testosterone,selection of VE,IGF-I and hCG dose respectively:10?g/mL,100 ng/mL,100 IU/mL.(3)The model of LCs fluoride exposure in mice:LCs in vitro in mice was added Omg/L,5mg/L,20mg/L of NaF 24 and 48h respectively.MTT and ELISA method was respectively used to detect cell proliferation and secretion of testosterone,results showed that cell proliferation and testosterone secretion are decreased significantly with the increase of fluoride dose,the cytotoxicity of 20mg/L NaF treatment group was observed comparing with the control group.(4)The results of VE,IGF-I and hCG on fluoride exposure LCs cell proliferation show that:24h,comparing with the fluoride group,the level of cell proliferation the markedly increase in VE,IGF-I and hCG treatment groups,comparing with the control group,the level of cell proliferation the markedly increase in VE,hCG treatment groups IGF-I treatment group had no significant change.48h:comparing with the fluoride group,the level of cell proliferation was promoted markedly in all treatment groups,except hCG in 20mg/LNaF treatment group.Comparing with the control,the level of cell proliferation was promoted significantly in VE,IGF-I and hCG all treatment groups.(5)VE,IGF-I and hCG on fluoride exposure LCs cell secretion of testosterone show that:24h:comparing with the fluoride group,the secretion of testosterone were increase markedly in VE,IGF-I,hCG all treatment groups,except IGF-I in 20mg/L NaF treatment group.Comparing with the control,not any significant change was observed.48h:comparing with the fluoride group,the secretion of testosterone were increase markedly in VE,IGF-I and hCG all treatment groups,comparing with the control,testosterone levels were increased markedly in IGF-I treatment group,then have no any significant change was observed in VE,hCG treatment groups(6)VE,IGF-I and hCG on fluoride exposure mRNA expression of LCs testosterone secretion relative genes Star,Cyp11a,17?-Hsd,3?-Hsd by using QRT-PCR technique to detect,the results showed that adding VE compared with fluoride group,the expression levels of the mRNA of Star,Cyplla,17?-Hsd significantly increased,the mRNA of 3?-Hsd have no obvious increased change;Adding IGF-I compared with fluoride group,the expression levels of the mRNA of Star,Cypl la significantly increased,17?-Hsd,3?-Hsd without change;Adding hCG compared with fluoride group,the expression levels of the mRNA of Star,Cypl la and 3?-Hsd significantly increased,17?-Hsd without change.[Conclusion]The test results show that the fluoride exposure can induce lower mice leydig cells proliferation and secretion of testosterone decline trend,add the VE,IGF-I and hCG have the effect of the remission and protection,and the LCs secrete key genes and testosterone Star,Cyp11a raised.