Ginsenosides Protects 15p-1 Sertoli Cells Against Cytotoxicity Induced By Bpa

Objective:Ginseng has been used as a general tonic for thousands of years in Asian countries, and has become a popular herbal medicine all over the world. The main active components responsible for the actions of P. ginseng are the ginsenosides(GS).Recent research shows that ginsenosides is helpful in the treatment of Alzheimer's disease, aging, immune disorders, cancer and reproductive system diseases .However, the therapeutic efficacy of ginseng has not been established. The cellular and molecular mechanisms by which ginsenosides induce pleiotropic biological actions remain largely unknown.Now,more and more studies have proved that BPA, one of the endocrine disruptors,could cause reproductive toxicity in humans and rodents. Therefore,we investigated whether or not ginsenosides has the cytoprotective effects in bisphenol A(BPA)-induced 15P-1 sertoli cells, and tried to find out the mechanism involved in.Methods:15P-1 sertoli cells were treated with or without GS for 24h before exposure to BPA for 24h. Cell viability and apoptosis were determined by MTT assay and Flow cytometry,respectively. Cell morphological and ultrastructural changes were assessed by inverted microscopy and electron microscopy,respectively.Vimentin protein expression was tested by immunofluorescence staining. Expression of Clusterin were determined by Q-PCR and western blotting. Bcl-2,bax and phosphorylation of ERK1/2, p38 ,JNK, were examined by western blotting. The antioxidant activity was performed by measuring the total antioxidant capacity (T-AOC), the level of reduced glutathione(GSH), superoxide dismutase (SOD),malondialdehyde(MDA),plutathione peroxidase(GPx) and glutathione reductase(GR). In the last part,we applied miR RNAi technology to construct three vectors targeting Clusterin gene.Then,we used Lipofectamine 2000 to transfect plasmid DNA into 15p-1 sertoli cells, and observed Clusterin expression both in mRNA level and protein level, and treated that cells with BPA or GS,respectively. After that, we used western blotting to determine Clusterin protein expression level,then the cell viability and apoptosis were determined by MTT assay and Flow cytometry.Results:1. In BPA group, MTT assay results showed that 15P-1 sertoli cells viability decrease in a dose dependent manner,100μM BPA can induce cell apoptosis efficiently. We found 15p-1 sertoli cells started to be prolonged and twisted,some became rounded even detached under the inverted microscope.Cell ultrastructures was assessed by electron microscopy,we could find typical changes related with cell apoptosis. Immunofluorescence staining showed a significant loss of Vimentin protein.Q-PCR and western blotting showed a up-regulation followed by a down-regulation of Clusterin production,and BPA could downregulate the level of bcl-2 and upregulate that of bax. After treated with BPA for 4 hours, phosphorylation of ERK1/2 started to emerge and expressed continuely to 24h. The activity of T-AOC and the concentration of SOD,GPx, GR,GSH were decreased and that of MDA was increased significantly. 2. Pretreatment with GS(75μg/ml) could prevent the changs in morphocytology and ultrastructure induced by BPA, and had the inhibition effects on the change of Vimentin. GS also inhibited phosphorylation of ERK1/2 and dual changes on Clusterin production induced by BPA in 15P-1 sertoli cells. The changs of the level of bcl-2 and bax in BPA-treated cells were also stopped through pretreatment with GS. The disorder of the activity of T-AOC and the concentration of SOD,GPx, GR, GSH and MDA were nearly normal or improved significantly.3. The pcDNA6.2-GW/EmGFP-miR-clu were successfully constructed.The expression level of Clusterin mRNA and protein in sertoli cells expressing pcDNA6.2-GW/EmGFP-miR-clu1 were declined compared with the controls. When that cells were treated with BPA,the cell viability was lower than that of cells treated with BPA only. Clusterin protein was recovered partly in GS-treated group compared with the pcDNA6.2-GW/EmGFP-miR-clu plasmid transfected group.Conclusions:1.BPA could induce cell apoptosis by activating the ERK1/2 MAPK pathways,and disturbing the balance between oxidation and antioxidation.2. Ginsenosides have the protective effects on BPA-induced cell damage maybe through preventing phosphorylation of ERK1/2, enhancing the antioxidation capacity ,and maintaining the expression of Clusterin in 15P-1 sertoli cells. Ginsenosides may be beneficial in the prevention of reproduction toxicity induced by BPA existed in environment.