| With the intensification of the "greenhouse effect" and the development of intensive breeding,oxidative stress in broilers caused by heat stress has become an important issue for broiler breeding industry,restricting the healthy and rapid development of this industry.Nrf2 is an upstream regulatory gene of many antioxidant enzymes.Previous research by this research group shows that dietary supplementation with Resveratrol can significantly increase the antioxidant enzyme activity in heat-stressed broiler muscles and improve muscle quality,but the relationship with Nrf2 signaling pathway needs further research.The purpose of this study was to explore the protective effect of resveratrol on heat stress injury of broiler skeletal muscle cells and clarified its mechanism.First,primary broiler myoblasts were isolated and purified from broiler chicken embryos,and the culture conditions were optimized.Cells were identified by real-time fluorescence quantification and immunofluorescence,and induced to differentiate into skeletal muscle cells by a medium containing 2% horse serum.Cell vitality,cell antioxidant function,and gene expression abundance of Nrf2 signaling pathway were used to establish a heat stress model of broiler skeletal muscle cells and determine the optimal concentration of resveratrol.Subsequently,resveratrol,tert-butyl hydroquinone(t-BHQ,a specific activator of Nrf2)or ML385(a specific inhibitor of Nrf2)and resveratrol were used to pretreat broiler skeletal muscle cells.To compare the effects of resveratrol,the activation of the Nrf2 and resveratrol after inhibiting Nrf2 on cell viability,cellular antioxidant function,and the abundance of Nrf2 signaling pathway-related genes and proteins under heat stress.The results showed that the broiler myoblasts were successfully isolated in this experiment,and it was found that they had better proliferation ability under 39.5℃,and skeletal muscle cells could be formed after myogenic differentiation.Compared with the 39.5℃ group,the cell viability and antioxidant function of broiler skeletal muscle cells were significantly reduced after heat treatment at 44.5℃ for 1 hour(P<0.05).The difference level was greater than that of other temperature-treated groups,so the cells were treated with 44.5℃ for 1 hour to establish a heat stress model of broiler skeletal muscle cells.25 μmol/L resveratrol pretreated broiler skeletal muscle cells without significantly affecting their cell viability(P>0.05),significantly increased the cellular antioxidant function and the expression of some genes m RNA related to Nrf2 signaling pathway(P<0.05)Therefore,the pretreatment concentration of resveratrol in this experiment was set to 25 μmol/L.Compared with the appropriate temperature culture groups,the cell viability,the enzyme activity of T-AOC,TSOD and CAT,the expression abundance of m RNA of Nrf2,HO-1 and CAT genes,and the expression of Nrf2 nuclear protein and total protein,HO-1 total protein and NQO1 total protein of broiler skeletal muscle cells after heat stress treatment were significantly reduced(P<0.05),PC,MDA and ROS content were significantly increased(P<0.05);Compared with the heat stress group,Resveratrol pretreatmented significantly increased cell viability,TAOC,CAT enzyme activity(P<0.05),significantly increased the expression abundance of m RNA of HO-1、NQO1、SOD1、SOD2 and CAT genes(P<0.05),significantly increased the expression of Nrf2 nuclear protein and total protein,HO-1 total protein and NQO1 total protein(P<0.05),and significantly reduced PC,MDA and ROS content(P<0.05)of broiler skeletal muscle cells after heat stress.Activation of Nrf2 significantly increased the cell activity,the enzyme activity of T-AOC,T-SOD and CAT,the expression abundance of m RNA of HO-1,NQO1,SOD1,SOD2,CAT and GSH-PX genes,the expression of Nrf2 nuclear protein and total protein,HO-1 total protein and NQO1 total protein expression(P<0.05),significantly reduced the content of MDA,ROS and PC(P<0.05)of broiler skeletal muscle cells under heat stress,the protective effect on heat-stressed broiler skeletal muscle cells is consistent with the resveratrol pretreatment effect trend.After inhibiting Nrf2,resveratrol pretreatment only caused a significant decrease in ROS content(P<0.05),and a significant increase in HO-1 total protein expression(P<0.05)in skeletal muscle cells of broilers after heat stress,the difference of other indicators were not significant(P>0.05).In summary,this experiment successfully isolated myoblasts from broiler chicken embryos,which have better proliferation capacity at 39.5℃,providing an cell model in vitro for further research on broiler muscle growth and development and nutritional regulation mechanisms;Heat treatment at 44.5 ℃ for 1h is a suitable condition for constructing a heat stress model of broiler skeletal muscle cells,providing a cell model for further research on broiler skeletal muscle heat stress;resveratrol mediates the Nrf2 signaling pathway to relieve the oxidative damage caused by heat stress in broiler skeletal muscle cells,which provides a theoretical basis for its rational use in relieving heat stress in broilers. |
