The Role Of Reactive Oxygen Species-mediated AKAP1 Degradation In 1-nitropyrene-induced Mitochondrial Excessive Fission And Steroidogenesis Disruption In MLTC-1 Cells

Objective 1-Nitropyrene（1-NP）,a key toxic component of fine particulate matter,is a typical representative of NPAHs in diesel vehicle exhaust.Increasing studies suggested that 1-NP has a number of toxicities,such as carcinogenicity,mutagenicity,Neurotoxicity and reproductive developmental toxicity.Epidemiological investigation showed that the T level of men was decreased in recent years,and the reason why the decrease of testosterone level in male population is exposed to adverse environmental factors.Previous study demonstrated that 1-NP inhibited serum and testicular steroidogenesis disruption,however,the underlying mechanisms are not entirely elucidated.This study aims to explore that the role of reactive oxygen speciesmediated AKAP1 degradation in 1-nitropyrene-induced mitochondrial excessive fission and steroidogenesis disruption in MLTC-1 cells.Methods MLTC-1 cell line,stimulated by h CG,was aimed to establish steroidogenesis model in vitro.To observe the effect of 1-NP on steroidogenesis in MLTC-1 cells,supernatant and intracellular T were detected after 1-NP treatment.To explore the role of 1-NP regulated mitochondrial dynamics in MLTC-1 cells,the mitochondrial morphology by transmission electron microscope and the expression levels of mitochondrial fission related proteins（DRP1 and p DRP1（S637））were analyzed.To observe the damage of 1-NP on mitochondrial function of MLTC-1 cells,the expression levels of each subunit of mitochondrial oxidative phosphorylation and mitochondrial membrane potential were detected.To elucidate the regulation of 1-NP on AKAP1 and the role of AKAP1 degradation in 1-nitropyrene-induced mitochondrial excessive fission and steroidogenesis disruption in MLTC-1 cells,the m RNA and protein of AKAP1 were detected,and the antagonistic effect of MG132,a proteasome inhibitor,was observed in 1-nitropyrene-induced mitochondrial excessive fission and steroidogenesis disruption in MLTC-1 cells.Finally,the level of ROS was detected and the protective effect of NAC intervention,an antioxidant,was observed in 1-nitropyrene-induced mitochondrial excessive fission and steroidogenesis disruption in MLTC-1 cells.The role of reactive oxygen species-mediated AKAP1 degradation was clarified in 1-nitropyrene-induced mitochondrial excessive fission and steroidogenesis disruption in MLTC-1 cells.Results In this study,we successfully established steroidogenesis model in vitro,and found that 1-NP treatment reduced supernatant and intracellular T in MLTC-1 cells.It was found that 1-NP treatment increased mitochondrial fission in MLTC-1 cells and significantly reduced the level of negative mitochondrial division protein p DRP1（S637）.What’s more,1-NP decreased the mitochondrial membrane potential and the expression level of each subunit of oxidative phosphorylation in MLTC-1 cells.The level of AKAP1 protein,the upstream regulatory protein of p DRP1（S637）,was significantly decreased by 1-NP treatment,but the m RNA level of AKAP1 was significantly increased.Subsequently,MG132 intervention restored the protein of AKAP1 and p DRP1（S637）by 1-NP treatment,as well as reduced supernatant and intracellular T.In addition,the results showed that 1-NP induced excessive ROS production in MLTC-1 cells,and the intervention of antioxidant NAC can alleviate the down-regulation of AKAP1 protein level and p DRP1（S637）level induced by 1-NP treatment,and protected steroidogenesis disruption induced by 1-NP in MLTC-1cells.Conclusion In conclusion,this study found that the role of reactive oxygen speciesmediated AKAP1 degradation in 1-nitropyrene-induced mitochondrial excessive fission and steroidogenesis disruption in MLTC-1 cells.Further more,it elucidated the mechanism of 1-NP-triggered steroidogenesis disruption in Leydig cells,providing a new theoretical basis for the treatment of T decrease.