The Regulatory Mechanism Of Oxidative Stress In Horse Skeletal Muscle Satellite Cells Mediated By Nrf2-ARE Signaling Pathway

In this experiment,by isolating and culturing satellite skeletal muscle satellite cells in vitro,identification and oxidative stress regulation mechanism research,a skeletal muscle satellite cell culture system and oxidative stress model were established to elucidate the regulation mechanism of oxidative stress,which is used for sports horses.The regulation of skeletal muscle oxidative stress provides scientific basis and research basis.This test is mainly divided into the following three parts.Test one isolates and cultures Mongolian horse skeletal muscle satellite cells in vitro and identifies the obtained cells.In the second experiment,skeletal muscle satellite cells with normal value-added and good growth conditions were selected as materials.H2O2 was used to induce oxidative damage to skeletal muscle satellite cells.The damage of different concentrations of H2O2 on skeletal muscle satellite cells was studied to establish skeletal muscle satellite cells.Oxidative damage model,and screen out the optimal induction dose and duration of H2O2.Experiment 3 On the basis of establishing a normal control model of skeletal muscle satellite cells and an oxidative damage model,design gene primers related to the Nrf2-ARE signaling pathway,through the relative expression of genes and the expression of downstream regulated antioxidant genes,and then To determine the mechanism of Nrf2-ARE signaling pathway regulating oxidative damage of Mongolian skeletal muscle satellite cells.This study produced the following results:1.Experiment 1 Culture and identification of horse skeletal muscle satellite cellsA healthy 2-year-old Mongolian horse was selected,and skeletal muscle satellite cells were obtained by a two-step enzyme digestion method using collagenase and pancreatin containing 0.25%EDTA,and the separated cells were filtered through a cell sieve to remove large diameter miscellaneous cells and cells The fragments were purified by using the differential adhesion method,and the muscle satellite cells were identified and analyzed by immunofluorescence,Q-PCR and other methods.In this experiment,Mongolian horse skeletal muscle satellite cells were isolated and cultured from muscle tissue.After differentiation culture medium induction,the cells could differentiate into myotubes.The results of differential adhesion method showed that the adhesion time of skeletal muscle satellite cells was longer than that of horses.Fibroblasts and skeletal muscle satellite cells express significantly different genes compared with fibroblasts.Immunofluorescence results showed that the specific antibodies of muscle satellite cells were positively expressed,of which Pax7 and Myod were expressed in the nucleus,and desmin was distributed in the cytoplasm.2.Experiment 2 Establishment of oxidative stress model of horse skeletal muscle satellite cellsChoose muscle satellite cells that can normally increase in value and grow well as materials,use H2O2 to induce muscle satellite cells to produce oxidative damage,study the effect of different concentrations of H2O2 on muscle satellite cells,and screen out the optimal induction dose and action time of H2O2.An oxidative damage model of muscle satellite cells was established.The results showed that H2O2 had a time and dose-dependent effect on cell viability damage.Combined with the effect of H2O2 on muscle satellite cell apoptosis rate,ROS content and antioxidant indicators,it can be concluded that H2O2 When the concentration of action is 600 umol/L and the treatment time is 6h,the skeletal muscle satellite cells have obvious oxidative damage,which can be used as the best condition for the construction of muscle satellite cell oxidative damage model.3.Experiment 3 Nrf2-ARE regulates the mechanism of oxidative stress by mediating skeletal muscle satellite cells of horsesBased on the establishment of a normal control model of skeletal muscle satellite cells and an oxidative stress model,design gene primers related to the Nrf2-ARE pathway,and determine the Nrf2-ARE signal through the relative expression of genes and downstream regulation of antioxidant enzyme activities The pathway regulates the mechanism of oxidative damage of Mongolian skeletal muscle satellite cells.The results showed that the expression of Nrf2 in the skeletal muscle satellite cells of the H2O2 injured group was significantly lower than that of the control group.The expression results of the antioxidant enzyme genes SOD1,SOD2,GPX1,GPX3,and TrxR1 regulated by the Nrf2-ARE signaling pathway were also lower in the H2O2 treatment group than in the control group.These results indicate that H2O2 inhibits the Nrf2-ARE signaling pathway and causes oxidative stress in cells.