Peptides Alleviate The Damage Of C2C12 Cells Induced By H₂O₂ And Delay The Decrease Of Muscle In Aging Animals

Objective:Ageing is a progressive process in which the physiological functions of the body gradually decline,of which muscle loss is an important manifestation of ageing.The causes of muscle loss are complex and are currently thought to include mitochondrial dysfunction,oxidative damage and apoptosis.Mitochondrial dysfunction is considered to be an important factor in muscle loss,and is one of the most important targets of current research to alleviate muscle loss.The peptides used in this paper are newly screened unreported sequences that provide a preliminary experimental basis for the application of peptides in the prevention and treatment of muscle loss in the aging process by assessing mitochondrial function in myogenic cells and animal muscles.Approach:In this research,a cellular aging model was established by H₂O₂-induced damage to C2C12 cells,and C57BL/6 naturally aged mouse were chosen as the animal model for intervention with the addition of active peptides to observe and analyze the effects of peptides on the model.Cell experiment:The peptide was screened by MTT method and the suitable dose of H₂O₂ was explored.Effects of screened peptides on senescence phenotype,cell cycle distribution,mitochondrial membrane potential,cellular reactive oxygen species levels and senescence and mitochondrial autophagy,function-related proteins in C2C12 cells byβ-galactosidase staining,flow cytometry and Western Blot,respectively;MDA,GR and GPx were detected by kit;The effects of the peptides on nuclear morphological changes of C2C12 cells were detected by Hoechst 33342fluorescent dyeing.Animal experiment:8-month-old mice were randomly divided into an aging control group,a drug administration group（peptide 1 unit（100 mg/kg）&GSH unit（50 mg/kg））.After 12 weeks of natural drinking,a group of 8-week-old C57BL/6 mice were selected as young control group.Suspension experiment was used for behavioral test;The mass of gastrocnemius was weighed and the gastrocnemius coefficient was counted;Kits to detect serological parameters in mice:liver function AST and ALT,lipid metabolism HDL-C,TC and TG,renal function BUN and CRE;The levels of MDA,GR and GPx in mouse gastrocnemius were detected by the kit;Hematoxylin-eosin staining was used to detect morphological changes in cross-sectional cells of the gastrocnemius muscle;Western Blot was used to detect changes in mitochondrial dynamics,mitochondrial autophagy and apoptosis and the expression levels of myasthenia-related proteins.Results:Cell experiment:MTT results showed that cells could be pre-protected by polypeptide 1 to resist H₂O₂ injury and thus improve survival rate,and polypeptide 2 and glutathione were added for comparison.Compared with the H₂O₂ model group,polypeptide 1 and polypeptide 2reduced SA-β-gal positive staining in C2C12 cells,reduced cell block ratio in G1/S phase,and up-regulated Cyclin D1 protein expression.The results of flow cytometry showed that polypeptide 1 and polypeptide 2 could reduce the intracellular ROS level,and the related oxidative stress indexes showed that the intracellular MDA content increased and GPx activity decreased after H₂O₂ treatment,and polypeptide 1 and polypeptide 2 pretreatment could improve this phenomenon.The results of flow cytometry and JC-1fluorescence staining showed that polypeptide 1 and polypeptide 2 could partially restore mitochondrial membrane potential.Hoechst 33342fluorescence staining showed that polypeptide 1 and polypeptide 2 could reduce nuclear staining.Western Blot results showed that polypeptide 1 and polypeptide 2 could up-regulate PINK1 and Parkin protein expression and promote mitophagy.Western Blot results showed that polypeptide 1 up-regulated Drp1 protein expression and polypeptide 2 up-regulated Drp1 and MFN-2 protein expression to maintain mitochondrial dynamic balance.Animal experiments:In male mice fed the peptide for 12 weeks,male mice in the senescent control group continued to gain weight,whereas the weight of mice in the drug-administered group remained essentially unchanged;Male mice in the peptide group had higher gastrocnemius muscle mass and coefficients than males in the aged group;In terms of physical recovery,most of the males in the peptide group were stronger than those in the aged group;The results of serological indexes showed that the levels of AST,HDL-C,TC,BUN and CRE in male mice in polypeptide group had a tendency to recover to young age.The levels of AST,BUN and CRE of female mice in polypeptide group had a tendency to recover to young age;The MDA content of male mice in polypeptide group decreased significantly.The activities of GR and GPx in male and female mice in polypeptide group were higher than those in aging group;The results of gastrocnemius HE staining showed that the size of gastrocnemius muscle fibers in the aging control group was different,and there were hypertrophic muscle fibers.Some muscle fibers moved inward,and a small amount of muscle fibers were broken and necrotic,showing a loose arrangement.The muscle fibers of mice in polypeptide group and glutathione group arranged more neatly and closely,and the size was relatively consistent;Western Blot of muscle tissue proteins showed increased expression of mitochondrial kinetics-related proteins Drp1,Opa1 and MFN-2;Decreased expression of mitochondrial autophagy-related proteins AMPKαand LC3 and increased expression of p62 protein;Decreased expression of myasthenia-related proteins Fbx32 and Caspase 3;Decreased expression of apoptosis-related protein Bax,increased expression of Bcl-2 and decreased Bax/Bcl-2 ratio in both male and female mice.Conclusion:Peptides reduce mitochondrial damage by reducing the production of excessive reactive oxygen species,and promote the expression of autophagy-related proteins in mitochondria to maintain the dynamic balance of mitochondrial fission and fusion.The upper limb strength of the mice was partially restored and the muscle fibres showed a rejuvenation trend after consuming the peptide.The above results preliminarily show that polypeptides can protect the mitochondrial function of muscles in aging individuals,thereby improving the muscle quality of the body,and providing candidate prevention schemes for aging individuals or people with muscle degradation in the future.