Role Of Autophagy In Lysophosphatidylcholine-induced Apoptosis In Mouse Leydig TM3 Cells

Objectives:Lysophosphatidylcholine(LPC)results from hydrolysis of phosphatidylcholines by phospholipase A2,which is a bioactive lipid generated under pathological condition.LPC can affect the integrity of endothelial cells and induce apoptosis.However,it is still unknown whether LPC can induce apoptosis of mouse Leydig cells.This study was to investigate the effects of LPC on apoptosis of mouse TM3Leydig cells and the role of autophagy in LPC-induced apoptosis.Methods:We used MTT assay to observe the effect of LPC on viability of mouse TM3Leydig cells.The reactive oxygen species test kits were used to detect the impact of LPC on oxidative stress in mouse TM3 Leydig cells.Western blot and annexin V-FITC/PI double staining were utilized to investigate whether LPC could induce apoptosis of mouse TM3 Leydig cells.Western blot was used to detect the effect of LPC on autophagy of mouse TM3 Leydig cells.Results:Mouse TM3 Leydig cells were treated with 0,2.5,5,10,20,40,80 and 160μM LPC for 48 h;then MTT assay was utilized to investigate cell viability.Compared with the control group,cell viability was inhibited by LPC at concentration of 2.5,5and 10μM;while cell viability was significantly inhibited by LPC at higher concentration(20,40,80 and 160μM)(P<0.05).Then we treated mouse TM3 Leydig cells with 0,2.5,5 and 10μM LPC for 48 h,and detected the levels of apoptosis related proteins by Western blot.We found that LPC significantly up-regulated the protein levels of cleaved Caspase-3,cleaved Caspase-8 and Bax as well as decreased Bcl-2 level,which indicated that LPC could induce apoptosis of mouse TM3 Leydig cells.Annexin V-FITC/PI double staining showed that LPC could significantly increase the total numbers of AnnexinV~+/PI~-(early apoptosis)and AnnexinV~+/PI~+(late apoptosis)cells.These results suggested that LPC could induce apoptosis of mouse TM3 Leydig cells.Compared with the control group,the MDA content significantly increased in the LPC-treated cells in a dose dependent manner,whereas the enzyme activities of GSH-PX and SOD and the level of GSH significantly decreased,implying that LPC could induce oxidative stress in the mouse TM3 Leydig cells.Inhibition of oxidative stress by N-acetyl-L-cysteine(NAC)could rescue the inhibition of viability and induction of apoptosis by LPC.These results indicated that oxidative stress was involved in LPC-induced apoptosis of mouse TM3 Leydig cells.Autophagy related proteins were detected by Western blot after mouse TM3 Leydig cells were treated with 0,2.5,5 and 10μM LPC for 48 h,LPC was showed to significantly inhibit the protein level of LC3-II and the ratio of LC3-II/LC3-I as well as the protein levels of autophagy protein Atg5 and Beclin1,indicating that LPC might inhibit autophagy of mouse TM3 Leydig cells.Furthermore induction of autophagy by Rapamycin could rescue the inhibition of viability by LPC.Compared with the LPC-treated group,there was a significant decrease in the protein levels of cleaved Caspase-3 and Bax and a dramatic increase in the protein level of Bcl-2 in the Rapamycin plus LPC-treated group.These results indicated that LPC induced apoptosis of mouse TM3 Leydig cells via inhibiting autophagy.Conclusion:LPC could inhibite viability and induced apoptosis of mouse TM3 Leydig cells.LPC could induce oxidative stress of mouse TM3 Leydig cells;and oxidative stress was involved in LPC-induced apoptosis of mouse TM3 Leydig cells.LPC could inhibite autophagy of mouse TM3 Leydig cells and induction of autophagy could rescue the induction of apoptosis by LPC.