Mechanism Of ER Stress In Palmitic Acid-induced Apoptosis In GC-1 Cells

Background Obesity has become a widespread epidemic worldwide,leading to a serious impact on the reproductive function of men of childbearing age.This is primarily manifested as excessive apoptosis of spermatogenic cells;Endoplasmic Reticulum Stress（ERS）induced by high-fat obesity is a key factor in the induction of spermatogenic cell apoptosis,but the mechanism behind its induction of spermatogenic cell apoptosis remains unclear.Objective Explore the mechanism of Palmitic Acid（PA）-induced apoptosis of GC-1cells.Methods GC-1 cells were treated with or without different concentrations of PA(0,100,150,200,250μmol·L^-1).（1）The cell viability was detected by MTT assay;（2）The cell apoptosis rate was detected by flow cytometry;（3）The activity of caspase-3 was detected by Caspase-3 Activity Assay Kit;（4）The levels of apoptosis proteins were detected by Western blot;（5）The difference in gene expression was measured by RNA-seq;（6）Q-PCR was used to verify the differential expression of CHOP and Clu;（7）The protein expression levels of CHOP and Clu were detected by Western blot;（8）The protein expression levels of CDX2were detected by Western blot;（9）The levels of ERS-related proteins were detected by Western blot;（10）Mitochondrial morphology was observed by Confocal Laser Scanning Microscope;（11）The levels of Mitochondrial dynamics-related proteins and Cytochrome c（Cyt c）were detected by Western blot.Results（1）The viability of GC-1 cells was significantly decreased after PA treatment;（2）The apoptosis rate was remarkably increased in PA-treated cells in a concentration-dependent manner;（3）The activity of caspase-3 was remarkably increased after PA treatment;（4）PA significantly increased the expression levels of Cleaved Caspase-3 and decreased the expression levels of BCL2;（5）The results of RNA-seq confirmed that the expression levels of CHOP was significantly higher than normal group,meanwhile,Clu was lower than normal group;（6）The results of Q-PCR were consistent with those of RNA-seq,PA significantly increased the m RNA levels of CHOP and decreased the m RNA levels of Clu;（7）PA significantly increased the expression levels of CHOP and decreased the expression levels of Clu;（8）PA significantly decreased the number of CDX2 nuclei;（9）PA remarkably upregulated the expression levels of ERS-related proteins（GRP78,CHOP,Cleaved Caspase-12/Caspase-12,p-IRE1α/IRE1αand XBP1）;（10）PA remarkably caused the morphological damage of the mitochondria;（11）Cyt c was released into the cytosol after PA treatment,meanwhile,PA significantly increased the expression levels of Drp-1 and MFN1decreased the expression levels of OPA1 and MFN2.Conclusion 1.The expression levels of CHOP increase by activation of IRE1-XBP1pathway after PA treatment,mitochondrial fragmentation,Cytochrome c（Cyt c）release and apoptosis;2.The number of CDX2 nuclei decreased after PA treatment,which decrease the m RNA and protein expression levels of Clu,increase the susceptibility of GC-1 cells to CHOP mediated apoptosis.