Protective Effect Of Tea Polyphenols On The Damage In Mouse Sertoli Cells Induced By Glyphosate

Objective: To establish the apoptosis model of mouse sertoli cell induced byglyphosate in vitro and investigate the protective effect and mechanism of teapolyphenols on oxidative damage of sertoli.Methods: Sertoli cells from18~20day male mouse were prepared and cultured.Then the SC was identified by Fas-L immunocytochemical reaction. After culturing for5~7days, the sertoli cells were passaged and were divided randomly into differentgroups:①control group;②glyphosate model group: sertoli cells were exposed toglyphosate with different dose of60μg/ml,90μg/ml,120μg/ml,150μg/ml and180μg/mlfor24h,The IC50were evaluated by cell inhibition rate in different groups;③teapolyphenols protect group: sertoli cells were pretreated with different concentration of5μg/ml,10μg/ml,20μg/ml,40μg/ml,80μg/ml and160μg/ml of tea polyphenols for12h,then the culture medium was get rid of and the fresh solution with glyphosate IC50wasadded to the above mentioned volume and cultured for another24h.The sertoli cellsviability was measured by MTT.The morphology of sertoli cells and their nuclei wereobserved by phase-contrast and Hochest33342staining, respectively.The sertoli cellsapoptosis index was observed by TdT-mediated dUTP nick end labeling (TUNEL). Theactivity of lactate dehydrogenase (LDH) was determined by a colorimetric method,Theactivity of SOD, GSH-Px and the content of MDA were assayed.The mRNA expressionof sertoli cells ABP mRNA and Vimentin mRNA were detected with Semi-quantitativeRT-PCR.Results:1.The proportion of mouse sertoli cells was about90%identified by Fas-Limmunofluorescence reaction.2. Different concentration of glyphosate (60μg/ml,90μg/ml,120μg/ml,150μg/ml,180μg/ml) were added to cultured cells for24hours, The results showed that thedamage of mouse sertoli cells induced by glyphosate with the relationships ofdose-dependence. According to the different dose of glyphosate inhibition ratecalculated half inhibitory concentration to90μg/ml. 3. The results showed that the time and concentration of tea polyphenols pretreatmenthad different effect on sertoli cells. tea polyphenols40μg/ml,80μg/ml groupspretreatment for12h,The sertoli cells viability could elevate to78%and86%(P<0.01),tea polyphenols20μg/ml,40μg/ml,80μg/ml groups pretreatment for24h and teapolyphenols10μg/ml,20μg/ml,40μg/ml groups pretreatment for48h, could significantlyblock the damage of sertoli cells induced by glyphosate.4. After Hoechst33342staining，the nuclei of normal cells appeared regular contours,round and large in size.After glyphosate90μg/ml for24hours, many cells showedbright blue fluorescence and nuclear chromatin appeared compaction、condensation andsegregation. the cell apoptosis was observed by TdT-mediated dUTP nick end labeling(TUNEL), After treating with tea polyphenols, the morphology of apoptotic wasimprovement, apoptosis rate was decreased significantly, compared with glyphosategroup (P<0.05, P<0.01).5. The index of damage was measured, compared with control group, The activity ofLDH,SOD, GSH-Px was decreased and the contents of MDA were increasedsignificantly treated with glyphosate model group. tea polyphenols could elevated theactivity of LDH,SOD, GSH-Px and reduced the contents of MDA respectively(P<0.05，P<0.01).6. The results of analysis RT-PCR showed that glyphosate model group compared withnormal group there were a lower level expression of ABP mRNA and Vimentin mRNA,tea polyphenols pretreatment group there were a higher level expression of ABP mRNAand Vimentin mRNA (P<0.01).Conclusions:1. The cultured sertoli cells treated with60μg/ml～180μg/ml concentrations ofglyphosate for24h can induce the damage change in dose-dependent manner.2. The10μg/ml～80μg/ml concentrations of tea polyphenols blocked significantly thedamage of sertoli cells induced by glyphosate, Protective effect of tea polyphenols wasrelated to its concentration of dose-dependent manner.3. For the cells treated by glyphosate, tea polyphenols can enhance the cell viabilitysignificantly, improve the sertoli cells condition in morphology, reduce the apoptosisindex of sertoli cells, reduce the apoptosis rate, reduce the damage degree of sertolicells,increase the activity of antioxidant enzyme and decrease the contents of lipid peroxide,and enhancing the cells antioxidative ability.4. Tea polyphenols can reduce the sertoli cell damage induced by glyphosate,Theseeffects of tea polyphenols may be concerned with increasing the expression of ABPmRNA and Vimentin mRNA.