Protective Effect And Mechanism Of Melatonin On Oxidative Stress Of Pilose Antler Mesenchymal Stem Cells

Velvet antler has high medicinal and economic value,and its growth depends on the proliferation and differentiation of pilose antler mesenchymal stem cells（MSCs）,but it may be affected by various adverse factors in the artificial rearing environment,resulting in oxidative stress and cell damage.Melatonin（MLT）has a wide range of physiological functions,and many studies have shown that it has significant antioxidant function.In this study,the oxidative stress indexes of deer antler mesenchymal cells were detected by using reactive oxygen species（ROS）detection kit,and the oxidative stress model of deer antler MSCs induced by H₂O₂ was established to explore the protective effect of MLT on H₂O₂-induced oxidative stress of deer antler MSCs.The mechanism of MLT was studied by q PCR,flow cytometry,Western Blot and fluorescence intensity analysis.The experimental results are as follows:（1）Establishment of MSCs oxidative stress model:400μmol/L H₂O₂ treatment for 2h induced oxidative stress of MSCs,including decreased cell viability（P<0.01）,increased intracellular ROS level（2.524±0.056 times,P<0.05）,increased intracellular malondialdehyde（MDA）level（1.176±0.046 times,P<0.05）,the intracellular ATP level,mitochondrial copy number and mitochondrial membrane potential were significantly decreased（P<0.05）.（2）Pretreatment with 100 ng/mL MLT for 12 h significantly alleviated the oxidative stress of Pilose antler MSCs,significantly increased cell viability,inhibited the decrease of intracellular ATP level,maintained the number of intracellular mitochondria,protected the mitochondrial function,and inhibited apoptosis（P<0.05）.（3）The physiological function of melatonin receptor was blocked by luzindole（Luz）.The results showed that the levels of ROS,MDA and other oxidative stress phenotypes in cells were significantly increased compared with those before the blockade.The number of mitochondria and mitochondrial membrane potential were significantly decreased compared with those before receptor blockade（P<0.05）.（4）The results showed that MLT could activate Nrf2 pathway and up-regulate the expressions of Nrf2,Keap1 and HO-1 genes,which were inhibited after receptor blockade（P<0.05）.At the same time,the expression of apoptotic genes was significantly increased compared with that before receptor blockade（P<0.05）.In conclusion,MLT can maintain the mitochondrial number and function of pilose Antler MSCs,activate Nrf2 pathway through the melatonin receptor pathway,and inhibit the oxidative stress and apoptosis of pilose Antler MSCs.