The Role And Mechanism Of BDNF/Akt/GSK-3? Signaling Pathway In SK-N-SH Cells Induced By PFOS

Objective:The aim is to investigate the toxicity effect and potential toxicity mechanisms for SK-N-SH cells about PFOS,and try to offer a new sight to study the neurotoxicity of PFOS.Methods: According to precious study,the experiment was divided into control(DMSO)and experiment group.MTT method was used to analyze the toxicity of different concertrations PFOS at24 h,48h,72 h,and then calculated the LD50 of PFOS according to the cell death rate.Optical microscope was used to observe the morphological change of SK-N-SH cells after PFOS exposure.Flow Cytometrymethods was used to detect the apoptosis and cell cycle of SK-N-SH PFOS exposure.DNA damage was observed by comet assay in SK-N-SH cells after PFOS exposure.Sulfite sequencing detected the methylation changes of BDNF promoter in SK-N-SH cells after PFOS exposure.ELISA and Q-PCR were used to detecte the expression of BDNF.Q-PCR was used to analyze the related miRNA-16,miRNA-22,miRNA-30a-5p and m RNA levels of cell cycle dependent protein,CDK2 and Cyclin E,DNMTs,Akt,GSK-3.Western Blot was used to analyze the protein level of DNMTs,Akt,GSK-3,CDK2,Cyclin E after PFOS treatment.Results: The results of MTT showed that PFOS decreased the activity of SK-N-SH cells at high concentration after treatment of 24 h and 72 h.And PFOS decreased the cell viability with a concentration-dependent manner at 48 h exposure.PFOS also caused morphological changes,which shortened synapses and rounded the entire cell in SK-N-SH cells.Flow cytometry showed that PFOS lead to apoptosis and cell cycle arrest in SK-N-SH cells.Alkaline comets suggest that PFOS can cause DNA damage in SK-N-SH cells.Sulfite sequencing revealed that PFOS changed the manner of hypermethylationin BDNF promoter?and IV.ELISA results showed that PFOS decreased the protein level of BDNF,and Q-PCR results also showed that BDNF m RNA expression was decreased.However,BDNF expression increased significantly after pretreatment with inhibitor IGF,Li Cl,NAC.Q-PCR results showed that PFOS significantly increased the m RNA expression of miRNA-16,miRNA-22 and miRNA-30a-5p,decreased the expression of DNMT1 and increased the expression of DNMT3 b.Results of the binding proteins,Western Blot results show that PFOS decreased the protein levels of DNMT1,and increased the expression of DNMT3 B.PFOS decreased the phosphorylation of Akt and GSK-3?,increased theprotein level of Bax,and decreased the expression of Bcl-2 according to Western Blot results.Conclusions:1.PFOS decreased the activity of SK-N-SH cells in a dose-dependent manner,and resulting in the morphological changes.2.PFOS decreased the expression of BDNF,and the probably reason is that PFOS causes hypermethylation of BDNF promoter region(? and ?)and changed the expression of miRNAs (miRNA-16,miRNA-22,miRNA-30a-5p)and DNMTs,related to BDNF.3.PFOS lead to DNA damage and cell cycle arrest in SK-N-SH cells.4.PFOS can injury SK-N-SH cells through regulating the BDNF / Akt / GSK-3?signal pathway.