PRDX6 Alleviates DEHP-induced Oxidative Stress And Apoptosis In Testicular Peritubular Cells Of Forest Musk Deer

The insufficient intake of essential trace elements and vitamins in captive breeding of forest musk deer(FMD,M.berezovskii)may cause omnivory in M.berezovskii.Some M.berezovskii with omnivory gnaw on foreign objects in the enclosure,especially some plastic products,which may lead to plasticizer deposition in the body.Di(2-ethylhexyl)phthalate(DEHP)is an endocrine disruptor widely found in plastic products that causes cellular stress damage,while peroxide reductase 6(PRDX6)is a cellular antioxidant factor that resists cellular damage caused by oxidative stress.In this study,we investigated the effects of DEHP exposure on oxidative stress and apoptosis in the testicular peritubular cells(TPCs)of M.berezovskii;we analyzed the role of overexpression of PRDX6 in alleviating DEHP-induced apoptosis in the TPCs of M.berezovskii.The main findings were obtained as follows:1.DEHP induced oxidative stress and apoptosis in TPCs of M.berezovskiiFirstly,TPCs of M.berezovskii were isolated and cultured in vitro.The TPCs of M.berezovskii were dealt with different concentrations of DEHP(0 μM,5 μM,50 μM,500 μM and 1000 μM)for 24 h.The CCK-8 assay showed that 500 μM DEHP significantly inhibited cell viability without significant neurotoxicity.Further analysis TPCs of M.berezovskii treated with 500 μM DEHP for 24 h showed a significant increase in intracellular ROS levels and MDA content,and a significant decrease in GSH-Px activity and SOD activity;RT-q PCR assay showed down-regulation of PRDX1,PRDX4,PRDX5 m RNA expression levels and significantly down-regulated PRDX6 m RNA expression;western blot assay showed that 500 μM DEHP significantly decreased PRDX6 and BCL-2 protein expression,but significantly upregulated BAX,Cleaved-Caspase3 and Cytochrome C protein expression;flow cytometry assay of Annexin V-FITC/PI stained cells showed that 500 μM DEHP significantly induced apoptosis in TPCs of M.berezovskii.In conclusion,500 μM DEHP significantly induced oxidative stress and apoptosis in TPCs of M.berezovskii.2.PRDX6 alleviated DEHP-induced oxidative stress and apoptosis in TPCs of M.berezovskii.Bioconfidence analysis showed the highest affinity between M.berezovskii PRDX6 and sheep,goats and cattle,reaching 100%;PRDX6 was mainly localized in the cytoplasm of TPCs of M.berezovskii.500 μM DEHP treatment of TPCs of M.berezovskii overexpressing PRDX6 for 24 h significantly reduced cellular ROS levels and MDA content and significantly increased SOD activity and GSH-Px activity compared with 500 μM DEHP treatment of TPCs of M.berezovskii without PRDX6 target fragments attached for 24h;significantly inhibited the expression of BAX,Cleaved-Caspase3 and Cytochrome C and promoted the expression of BCL-2,indicating that overexpression of PRDX6 significantly alleviated DEHP-induced apoptosis in TPCs of M.berezovskii.In conclusion,DEHP at 500 μM significantly increased the level of oxidative stress and induced apoptosis in the TPCs of M.berezovskii.Overexpression of PRDX6 significantly alleviated DEHP-induced oxidative stress levels and inhibited apoptosis in TPCs of M.berezovskii.The above results provide a basis for further understanding of testicular damage due to plasticizers in M.berezovskii,and provide a theoretical basis for the feeding management of M.berezovskii in captivity.