Induction Of HO-1 Influences The Microenvironment And Promotes The Proliferation And Differentiation Of Satellite Cells In The Skeletal Muscle Of Aged Mouse

Back ground and Objective:The function and quantity of satellite cell decreases gradually in the process of aging, this is the main reason for decline of muscle regeneration capability in aged animal. And it is reported that the microenvironment of satellite cell has important influence on its proliferation and differentiation. In the present study, to explore new ways to promote the skeletal muscle regeneration capacity and further provide countermeasures for the prevention and treatment of age-related muscular atrophy such as sarcopenia,the oxidative stress and inflammatory microenvironmental changes of skeletal muscle of aged mice was ameliorated through induction of heme oxygenase-1(HO-1), then the proliferation and differentiation potential of muscle satellite cell, and especially the regeneration of skeletal muscle after injury was investigated.Methods:1. Normal adult(2 month) and aged(18 month) male C57/BL6 mice were randomly divided into control group and cobalt protoporphyrin(CoPP) injection group. Co PP group was intraperitoneally injected with CoPP solution(5 mg/kg/d) for 3 consecutive days, the control group injected with saline.2.The expression of HO-1 in normal skeletal muscle of adult, aged mouse and after CoPP induction was examined by Western Blot.3.The level of oxidative stress markers including malondialdehyde(MDA) and hydrogen peroxide(H2O2), and the activity of antioxidant enzyme CuZn-super oxide dismutase(SOD) and so on in skeletal muscle of adult, aged mouse and after CoPP induction was detected through ELISA.4.The gastrocnemius injury model was established in aged mice, the neutrophil and M2 macrophage infiltration were observed by immunohistochemical staining and Western blot with use of myeloperoxidase(MPO) and CD163 antibody respectively in both CoPP treatment and control group.5.The expression of MyoD and Myogenin in Co PP treatment and control group of the injury skeletal muscle in aged mice were detected respectively from the level of protein and RNA by Western Blot and Real-Time PCR.6.The number of regenerating skeletal muscle fibers of Co PP treatment and control group in the injury skeletal muscle in aged mice was examined with HE and embryonic Myosin Heavy Chain(eMHC) immunofluorescent staining.7.The expression of CollagenI(ColI) of Co PP treatment and control group in the injury skeletal muscle in aged mice was examined by Western Blot, and fibrosis was observed through Masson staining.Results:1.In aged group, the expression of HO-1 is higher than that of the adult group, but the increase of HO-1 induced by CoPP in aged animals is much less than that in adult group.2.After induction of HO-1, the activity of SOD,CuZn-SOD, GSH-Px and CAT enzyme increases, while MDA and H2O2 content declines in both adult CoPP and aged CoPP groups; the activity of SOD,CuZn-SOD, GSH-Px and CAT enzyme in aged CoPP group is lower than that in adult Co PP group, whereas the MDA and H2O2 content is higher.3.The neutrophil infiltration is much serious in the control group than that in CoPP group, both at the 3, 7 days after muscle injury in aged mice. The M2 macrophage infiltration appears later than that of neutrophil, and expression of CD163 is much higher in CoPP group than in the control.4.The expression of Myo D and Myogenin increase in 3 d, 7 d, 14 d, in Co PP group than that in the control group after muscle injury.The highest expression of MyoD appears in 7d,while the peak of Myogenin appears in 14 d.5.The number of regenerating skeletal muscle fibers increased in CoPP group compared with that of control group at 3d,7d after muscle injury in aged mice, and e MHC positive neonatal myofibers rised markedly in the 5d of Co PP treatment group.6.The expression of collagen I(Col I)and collagen fiber number decrease in CoPP treatment group than in control group at 7 d, 14 d,21 d,28d after muscle injury in aged mice, and the highest expression and number appear in 21 d.Conclusion:1.The HO-1 responsivity of aging skeletal muscle to extrinsic stimuli falls markedly, and the ability to resist oxidative stress drops.2.Induction of HO-1 reduces peroxidation while enhances the activity of antioxidant enzymes significantly,so ameliorate oxidative stress in the aged skeletal muscle.3.Induction of HO-1 reduces early neutrophils infiltration and promotes M2 macrophages appearence after injury, which may be helpful in accelarating skeletal muscle inflammation progress.4. Induction of HO-1 promotes activation and proliferation of muscle satellite cell after injury of skeletal muscle in aged mice.5.Induction of HO-1 ameliorates oxidative stress and inflammation,which further promote the muscle regeneration, reduce the level of fibrosis after injury of skeletal muscle in aged mice.