Role Of Autophagy In Lysophosphatidylcholine-induced Apoptosis Of Mouse Ovarian Granulosa Cells

Objective:Lysophosphatidylcholine(LPC),also known as lysolecithin,is one of the major components of oxidized low-density lipoproteins(ox-LDL).LPC can promote the production of proinflammatory factors and reactive oxygen species,and induce apoptosis of many cells,thus promoting the occurrence of diseases,such as bronchial asthma,psoriatic skin,and biliary tract cancer and so on.However,the effect of LPC on mouse ovarian granulosa cells and its mechanism have not been studied.In this study,we explored the effects of LPC on autophagy and apoptosis of mouse ovarian granulosa cells to further elucidated the female reproductive toxicological mechanism of LPC.Methods:After treated with a series of concentrations of LPC,CCK-8 assay was utilized to examine the activity of mouse ovarian granulosa cells;Western blot was used to detect the apoptosis and autophagy of mouse ovarian granulosa cells;The apoptosis and autophagy of mouse ovarian granulosa cells were confirmed by annexin V-FITC/PI double staining and transmission electron microscope,and the oxidative stress indexes of mouse ovarian granulosa cells were detected by reactive oxygen species detection kit.In order to clarify whether oxidative stress is involved in LPC induced autophagy and apoptosis,mouse ovarian granulosa cells were pretreated with or without N-acetyl-L-cysteine(NAC)and then treated with LPC.Western blot was used to detect the apoptosis and autophagy of mouse ovarian granulosa cells,The apoptosis and autophagy of mouse ovarian granulosa cells were confirmed by annexin V-FITC/PI double staining and transmission electron microscopy.To investigate the role of autophagy in LPC-induced apoptosis of mouse ovarian granulosa cells,the cells were pretreated with or without 3-MA and then treated with LPC;the apoptosis of cells was detected by Western blot and further confirmed by annexin V-FITC/PI double staining.Results:After treated with different concentrations of LPC(0,40,80,160 μM)for 24 h,with the increase of LPC concentration,the result of CCK-8 showed that the viability of mouse ovarian granulosa cells decreased more and more significantly(P < 0.05);the result of western blot showed that the protein levels of cleaved caspase-8,cleaved caspase-3,Bax increased,and the protein level of Bcl-2 decreased,suggesting that LPC induced apoptosis in mouse ovarian granulosa cells;The results of Annexin V-FITC/PI double staining showed that the proportion of early and late apoptotic cells increased,which further proved that LPC could induce apoptosis of cells.Meanwhile,we also found that LPC could significantly increase the protein levels of Atg-5,Beclin-1,LC3-II and LC3-II/LC3-I in cells,and the number of autophagic vesicles increased significantly of cells that examined by transmission electron microscope,indicating that LPC induced autophagy in mouse ovarian granulosa cells.The results of oxidative stress-related indicators suggested that LPC could induce oxidative stress in mouse ovarian granulosa cells(P < 0.05).After inhibiting oxidative stress with NAC,the inhibition of cell viability was alleviated and and the induction of apoptosis and autophagy by LPC were significantly attenuated,demonstrating that oxidative stress is involved in the LPC-induced apoptosis and autophagy in mouse ovarian granulosa cells.After inhibiting autophagy with 3-MA,both the inhibition of cell viability and the induction of apoptosis by LPC were significantly reduced of mouse ovarian granulosa cells,suggesting that autophagy promoted the generation of LPC-induced apoptosis of mouse ovarian granulosa cell.Conclusion:LPC induced apoptosis and autophagy of mouse ovarian granulosa cells,and the production of oxidative stress;LPC induced apoptosis and autophagy of cells are related to oxidative stress;Autophagy promoted LPC-induced apoptosis of cells.