Muscle-Derived Estrogen Exerts Antioxidant Protective Effect In The Skeletal Muscle Of Mice

ObjectiveSkeletal muscle is not only a vital motor organ in the body but also has endocrine functions.Aromatase,a rate-limiting enzyme that can synthesize estrogen,has been found in skeletal muscle and catalyzes estradiol（E₂）synthesis from testosterone.Our group’s research has demonstrated that exercise can enhance skeletal muscle quality and promote skeletal muscle aromatase expression and E₂ production in low estrogen conditions.Furthermore,we have found that mechanical stretch stimulation of appropriate intensity can elevate E₂ levels in C₂C₁₂ myocytes in mice.Despite this progress,whether myogenic E₂ maintains redox homeostasis in skeletal muscle under low estrogen conditions remains unclear.In this study,we aimed to explore the impact of myogenic E₂ deficiency on the antioxidant capacity of mouse skeletal muscle under conditions of reduced overall estrogen levels,as seen in postmenopausal women.We created an ovariectomy mouse model and employed the Loxp-Cre system to conditionally knock out the mouse muscle aromatase gene（Cyp19a1）.In addition,C₂C₁₂ cells were used as experimental subjects to induce cellular oxidative damage using hydrogen peroxide（H₂O₂）,mechanical stretching to simulate the contraction-diastole activity of myocytes,and aromatase inhibitor intervention.The above experiments aimed to investigate the protective effect of E₂ produced by mechanical stretch stimulation in skeletal muscle cells against oxidative damage and the potential mechanisms.Our study provides relevant experimental evidence for the antioxidant protective effects of myogenic E₂ on skeletal muscle and the improvement of antioxidant capacity through exercise.Methods1 Animal experimentsTwelve female wild-type mice（genotype:fl/fl;Cre-）at eight weeks of age were randomly allocated into a sham-operated group（Sham group）and an ovariectomy group（OVX group）.At eight weeks of age,six female Cyp19a1 knockout mice（genotype:fl/fl;Cre+）were selected for the knockout+ovariectomy group（KO+OVX group）.We performed the bilateral ovarian removal surgery to construct the ovariectomy mouse model.After that,we measured the mice’s body weight,body composition,and grip strength.The mice were euthanized,and blood and skeletal muscle samples were collected after 18 weeks of the ovariectomy surgery.Serum E₂ levels were measured by the Elisa method,and skeletal muscle superoxide dismutase（SOD）activity was determined by the WST-1 method.The ammonium molybdate method was employed to measure catalase（CAT）activity.Glutathione peroxidase（GSH-Px）activity was measured by the colorimetric method,and Malondialdehyde（MDA）production was measured using the TBA method.We use the western blot to determine the phosphorylated nuclear factor E₂ in skeletal muscle（p-Nrf2）,as well as nuclear factor E₂-related factor 2（Nrf2）and heme oxygenase-1（HO-1）protein expression changes.2 Cellular experimentsC₂C₁₂ cells were cultured in an estrogen-depleted medium to mimic low estrogen conditions.The study comprised five experimental groups:control group（C group）,mechanical stretch group（S group）,H₂O₂ group（H group）,mechanical stretch+H₂O₂ group（S+H group）,and aromatase inhibitor+mechanical stretch+H₂O₂ group（I+S+H group）.For the mechanical stretch group,a 15%,0.5 Hz stretch stimulus was applied for 6 hours,whereas for the aromatase inhibitor group,200 μg/ml anastrozole（aromatase inhibitor）was added to the medium 24 hours before the stretch intervention.The H₂O₂ group was exposed to 400 μM H₂O₂ for 4 hours following the stretch intervention,leading to oxidative cell damage.At the end of the intervention we examined the changes in cell viability by the CCK8 method.Intracellular E₂ levels were measured using the Elisa assay.SOD viability was measured by the WST-1 method,CAT viability by the ammonium molybdate method,GSH-Px viability by the colorimetric method,and MDA production by the TBA method.Changes in intracellular protein kinase B（Akt）/Nrf2/HO-1 protein expression were detected using Western blot.Results1 Animal experimental part（1）Formation of a muscle-specific Cyp19a1 knockout mouse model:Aromatase expression was absent in the skeletal muscle of Cyp19a1 knockout mice.Furthermore,the concentration of E₂ in the gastrocnemius muscle of fl/fl;Cre+mice was significantly lower than that of fl/fl;Cre-mice（P<0.01）.（2）Establishment of the ovariectomy mouse model:Vaginal smears of OVX mice stained with crystalline violet showed leukocytes upon microscopic examination,while uterine morphology was significantly atrophied.Serum E₂ levels were significantly reduced（P<0.01）.（3）Impact of ovariectomy and muscle-specific Cyp19a1 knockout on mouse body weight and composition:The body weight of mice significantly increased with age after de-ovulation surgery（starting from the third week after surgery,OVX group vs.Sham group,P<0.05）.However,there was no significant difference in body weight between the KO+OVX and OVX groups（P>0.05）.At week 18 after de-ovulation,body fat percentage increased significantly（P<0.01）,and lean body mass ratio decreased significantly（P<0.01）in the OVX group compared to the Sham group.There were no significant changes in body fat percentage and lean body mass ratio in the KO+OVX group compared to the OVX group（P>0.05）.（4）Impact of ovarian removal and muscle-specific Cyp19a1 knockout on mouse strength:The relative grip strength of mice in the OVX group was significantly lower compared to the Sham group（P<0.01）.However,the relative grip strength of mice in the KO+OVX group did not significantly differ from the OVX group（P>0.05）.（5）Impact of ovarian removal and muscle-specific Cyp19a1 knockout on serum E₂ in mice:Serum E₂ levels in OVX mice were significantly lower compared to the Sham group（P<0.01）.However,there were no significant changes in serum E₂ levels in the KO+OVX group compared to the OVX group（P>0.05）.（6）Impact of ovariectomy and muscle-specific Cyp19a1 knockout on antioxidant capacity of mouse skeletal muscle:Skeletal muscle SOD（P<0.01）,CAT（P<0.05）,and GSH-Px（P<0.05）activities significantly decreased,while MDA production significantly increased in the OVX group compared to the Sham group（P<0.01）.Nrf2（P<0.01）,p-Nrf2（P<0.05）,and HO-1（P<0.01）protein expressions significantly decreased as well.However,there were no significant changes in SOD,GSH-Px,and CAT activities,MDA content,and Nrf2,p-Nrf2,and HO-1 protein expression in the skeletal muscle of mice in the KO+OVX group compared to the OVX group（P>0.05）.2 Cellular experimental sections（1）Effect of mechanical stretching on E₂ levels in C₂C₁₂ cells:Compared with group C,the activity（P<0.01）and protein expression（P<0.01）of aromatase were significantly increased in group S,and intracellular E₂ concentration was significantly increased（P<0.01）.（2）Establishment of H₂O₂-induced oxidative damage model in C₂C₁₂ cells:Compared with group C,cell proliferation was inhibited,cell viability was significantly decreased（P<0.01）,and HO-1 protein expression was significantly decreased（P<0.05）in group H after 4 hours of H₂O₂ intervention.（3）Effects of different intervention methods on cell viability:Compared with group C,cell viability in group H decreased significantly（P<0.01）,while cell viability in group S increased significantly（P<0.01）;compared with group H,cell viability in group S+H increased significantly（P<0.01）;compared with group S+H,cell viability in group I+S+H decreased significantly（P<0.01）.（4）Effects of different interventions on cellular antioxidant capacity:Compared with group C,group H showed a significant decrease in intracellular SOD,CAT,and GSH-Px activities（P<0.01）,a significant increase in MDA production（P<0.01）,and a significant decrease in Akt phosphorylation level（P<0.05）,Nrf2（P<0.05）and HO-1（P<0.05）protein expression.The S group relative to the C group showed that mechanical stretch increased intracellular SOD activity（P<0.05）,decreased MDA content（P<0.05）,and upregulated Akt phosphorylation level（P<0.01）,Nrf2（P<0.01）and HO-1（P<0.05）protein expression.SOD,CAT,and GSH-Px activities were significantly higher（P<0.01）,MDA production was significantly lower（P<0.01）,and Akt phosphorylation level（P<0.01）and HO-1 protein expression（P<0.05）were upregulated in the S+H group relative to the H group.The I+S+H group compared with the S+H group showed that aromatase inhibitors blocked the effects of mechanical stretch on intracellular SOD（P<0.01）,GSH-Px（P<0.01）,CAT（P<0.01）activity,MDA content（P<0.01）,Akt phosphorylation level（P<0.01）,Nrf2（P<0.05）and HO-1（P<0.05）protein expression were affected.Conclusions1.After ovarian removal,the overall estrogen level was reduced leading to a weakened antioxidant capacity in mouse skeletal muscle,while myogenic E₂ deficiency did not further affect the antioxidant level in mouse skeletal muscle;2.Mechanical stretch stimulation under low estrogen conditions increased aromatase activity and expression,and it also promoted endogenous E₂ production in mouse myogenic cells;3.Myogenic E₂ may exert antioxidant protective effects on skeletal muscle by activating the Akt/Nrf2/HO-1 signaling pathway.