The Effect Of ROS-induced Autophagy On Glucose Metabolism In TM4 Cells

Reactive oxygen species（ROS）are closely related to the occurrence of male reproductive diseases.High levels of ROS,can induce cellular oxidative stress and activate autophagy,and are often associated with male infertility such as cryptorchidism and teratozoospermia.Sertoli cells that provide protection for germ cells are more sensitive to changes of ROS levels in testis.Sertoli cells also provide lactate as an energy metabolic substrate for germ cells through glucose metabolism,and its normal function directly affects the process of sperm production.Although many factors involve in synthesis and secretion of lactate through influencing different signaling pathways,the effect and role of ROS on synthesis and secretion of lactate has not been clarified.Therefore,it is important to study the influence of ROS on sertoli cell function.In present study,the mouse sertoli cell line TM4 cells were used as a model to investigate the effects of ROS-induced oxidative stress on autophagy and glucose metabolism in sertoli cells,and to screen for candidate autophagy genes that play key regulatory roles in this process.It mainly includes the following three aspects:1.Effect of ROS on autophagy of TM4 cellsThe concentration of H₂O₂ on TM4 cells was determined to be 40⁶0μmoL/L by MTT and Hoechst staining,and the treatment time was 8 hours.After detecting a series of intracellular oxidative stress indicators,results showed that compared with the control group,intracellular ROS levels and MDA content significantly were increased,SOD activity and GSH content decreased markly.Western blotting results showed that oxidative stress-related protein Nrf2 was up-regulated whereas Keap1was reduced.The above results indicated that the oxidative stress model of TM4 cells was successfully constructed.The expression characteristics of 15 autophagy-related genes in the cryptorchidism of GEO databases and ROS-induced TM4 cells were compared.It was found that there were 8 genes with consistent expression trends.Then,by observing the distribution of fluorescent spots in TM4 cells transfected with GFP-LC3,and detecting changes in autophagy marker molecule LC3 by WB,it was confirmed that autophagy was activated during oxidative stress induced by ROS in TM4 cells.2.Effect of ROS on Glucose Metabolism in TM4 CellsUnder ROS-induced oxidative stress,metabolic index（such as ATP,LDHA activity,etc.）and key molecules of glucose metabolism（such as HK1,PGK1,etc.）have been changed significantly,confirming that ROS can promote glucose metabolism and production of lactate in TM4 cells.3.Inhibition of ROS-induced autophagy on the glucose metabolism in TM4 cellsThrough detecting the autophagy marker protein by western blotting,the concentration of autophagy inhibitor 3-MA and its inhibition efficiency were determined.After 3-MA inhibition on autophagy,compared with H₂O₂ treatment group,the ATP level of cells in H₂O₂+3-MA group was increased,while the level of glucose uptake and lactate production decreased.The results of western blotting showed that after treatment with 3-MA,the expression of key glycolysis molecules such as HK1,PGK1 and LDHA was significantly down-regulated,and the protein level of the glycolytic inhibitor TIGAR increased.Subsequently,the changes of 15 autophagy-related genes were detected by qRT-PCR after 3-MA treatment.It was found that ATG4B,ATG4D and ATG10 were up-regulated during autophagy activation,and down-regulated after addition of autophagy inhibitor 3-MA.The expression profile of three autophagy genes was similar with that of cryptorchidism GEO database,suggesting that they may play a major role during this process.In summary,ROS-induced sertoli cells autophagy can promote cell glycolytic process,thereby ingesting more glucose to synthesize and produce lactic acid.In this process,ATG4B,ATG4D,and ATG10 may play a key role in meeting the material energy requirements of germ cells under oxidative stress.