Protective Effect Of 17β-estradiol On Oxidative Damage Of Skeletal Muscle In Mice

Purpose of the study:Oxidative damage of skeletal muscle caused by strenuous exercise has always been a hot issue in sports medicine,and this problem has become increasingly prominent with the rapid development of competitive sports and the rapid development of mass fitness.As a sex hormone,in addition to maintaining women’s secondary sexual characteristics,estrogen also has an important antioxidant effect.After women enter menopause or have their ovaries removed,the level of estrogen decreases,the incidence of skeletal muscle injury is greatly increased compared with younger women,and the recovery speed after injury becomes slow,which seriously affects the normal life of the elderly.However,the research on the protective effect of estrogen on oxidative damage to skeletal muscle is still unclear.Therefore,this study used animal experiments and in vitro cell culture techniques to observe the changes of related antioxidant enzymes and signaling pathways after skeletal muscle oxidative injury,to verify the protective effect of estrogen on skeletal muscle injury,and to explore its mechanism.Research methods:Ⅰ.In vivo experimentsAnimal experiments were performed in two parts.The first part of the experiment:mice were divided into control（C）and injury（I）groups,and mice in the injury group were executed on days 3,7,10 and 15 after muscle injury to assess muscle injury repair,serum E₂ levels and redox status.Part II of the experiment:all mice were randomly divided into 4 groups after bilateral ovariectomy:control group（group C:intraperitoneal injection of 0.1%DMSO）,injury group（group I:animals were injected intraperitoneally with 0.1%DMSO after contusion）,injury+E₂supplementation group（group I+E:mice were injected intraperitoneally with E₂ after contusion）and injury+E₂ combined with ER_α/βantagonist group(I+E+A group:mice were injected with E₂ and ER_α/βantagonist intraperitoneally after contusion),mice were executed and blood and skeletal muscle samples were collected on day 7 after injury for investigating whether estrogen signaling is involved in muscle repair and altered redox status.Ⅱ.Ex vivo experimentAfter C₂C₁₂ mouse myogenic cells were revived and passaged,the cells were inoculated in 6-well culture plates and cultured overnight with estrogen-free serum.After the cells adhere to the wall and reach 80%fusion,the six-well plates were grouped and set up in replicate wells:blank control group,H₂O₂ group（0.8 m M H₂O₂intervention for 24h）,H₂O₂+aromatase inhibitor group（aromatase inhibitor Anastrozole was added 36h before H₂O₂ intervention）,H₂O₂+SIRT1 inhibitor group（H₂O₂ intervention was added 24h before SIRT1 inhibitor Selisistat）.After the end of H₂O₂ intervention in each group,total cellular proteins were extracted,and cell morphology and activity,CAT and SOD activities,and MDA levels were detected,and the protein expression of SIRT1,PGC-1α,Nrf2,and HO-1 in C₂C₁₂ myogenic cells were detected by Western blot.Research results:Ⅰ.In vivo experiments1.The results of H&E staining showed that the skeletal muscle cells were regularly arranged in uninjured mice;on day 3 after skeletal muscle injury,the gastrocnemius cells were swollen,the structural integrity was disrupted,and a large number of inflammatory cells infiltrated;on day 7 after skeletal muscle injury,obvious mononuclear cell infiltration could still be observed in the gastrocnemius;regenerating muscle fibers with nuclei in the center could be observed on days 7 and10.2.The results of serum E₂ levels in mice showed that compared with group C,the serum E₂ levels in group I mice increased significantly on days 3,7 and 10 after skeletal muscle injury（p<0.01）,and the serum E₂ levels in mice reached the peak on day 7;by day 15,the serum E₂ levels in group I mice had rapidly decreased to the level of healthy mice.3.The results of antioxidant enzyme activities in mice showed that contusion-induced muscle injury significantly decreased SOD and CAT activities（p<0.05）and significantly increased MDA content（p<0.01）in group I mice compared with group C mice.4.Western blot results showed that the protein expression of SIRT1,PGC-1α,Nrf2 and HO-1 was significantly decreased in group I mice compared with group C mice（p<0.01,p<0.01,p<0.05,p<0.01）.5.For bilaterally ovariectomized mice,there was no significant difference in initial and final body weights in groups I,I+E and I+E+A compared to group C（p>0.05）,and no significant difference in gastrocnemius muscle mass in the uninjured leg in the four groups（p>0.05）,while gastrocnemius muscle weight in the injured leg was reduced by 8.5%（p<0.01）and relative muscle mass by 10.2%（p<0.01）in group I.In addition,the relative muscle mass of the injured leg in groups I+E and I+E+A was reduced by 10.1%and 11.7%,respectively,compared with group C（p<0.01）.6.For mice with bilateral ovarian removal,the results of serum indexes showed that compared with group C,serum E₂ levels were significantly higher（p<0.05）and serum CK and LDH activities were significantly higher（p<0.01）in group I;compared with group I,serum E₂ levels were significantly higher（p<0.01）and serum CK and LDH activities were significantly lower（p<0.01）in group I+E;while compared with group I+E compared to the I+E+A group,although there was no significant difference in serum E₂ levels（p<0.05）,serum CK and LDH activities were significantly higher（p<0.01）.7.For bilateral ovary removal mice,Western blot results showed that the protein expression of SIRT1,PGC-1α,Nrf2 and HO-1 was significantly lower in group I compared with group C（p<0.01,p<0.01,p<0.01,p<0.05）;compared with group I,the protein expression of SIRT1,PGC-1α,Nrf2 and HO-1 were significantly increased（p<0.01,p<0.01,p<0.01,p<0.01）;the protein expression of SIRT1,PGC-1α,Nrf2 and HO-1 were not significantly different in the I+E+A group compared with the I group（p>0.05）.Ⅱ.Ex vivo experiments1.After H₂O₂ intervention,cell morphology showed cytosolic vacuolization and cell survival rate was about 58%of that of the blank control.Meanwhile,both EX527and Anastrozole treatment significantly exacerbated the H₂O₂-induced growth inhibition and morphological changes.2.The results of related antioxidant enzymes showed that H₂O₂ treatment decreased SOD and CAT activities in cells（p<0.01）and significantly increased MDA production（p<0.05）.In addition,after pretreatment with Anastrozole or EX527,SOD and CAT activities were further reduced and MDA content was further increased in the H₂O₂-intervened cells.3.Western blot results showed that the protein levels of SIRT1,PGC-1α,Nrf2and HO-1 were significantly lower in H₂O₂-intervened cells compared with the control group（p<0.01）.A further downregulation of the SIRT1/PGC-1α/Nrf2/HO-1signaling pathway was found in H₂O₂-intervened cells when Anastrozole or EX527was present.Study conclusion:1.In the low estrogen state,contusion significantly reduced the antioxidant capacity of mouse skeletal muscle and down-regulated SIRT1/PGC-1α/Nrf2/HO-1signaling pathway.And exogenous estrogen supplementation can reverse this phenomenon,indicating that estrogen signaling is involved in the repair process after contusion-induced skeletal muscle injury.2.The locally synthesized estrogen in skeletal muscle can also protect C₂C₁₂myoblasts from oxidative damage by up-regulating SIRT1/PGC-1α/Nrf2/HO-1signaling pathway.