Aging Fibroblasts Mediated Autophagy Induced Skeletal Muscle Atrophy Through The TGF-β/mTORC1 Pathway

Objective: Aging is one of the causes of sarcopenia,which leads to physical and cognitive deterioration during aging,exacerbating other underlying diseases and ultimately reducing patient life expectancy.Aging may be stimulated by chronic lowgrade inflammation,induce fibroblast senescence in skeletal muscle,and thereby influence the progression of sarcopenia.The transforming growth factor beta(TGF-beta)signaling pathway has pleiotropic roles in muscle development,adaptation,and disease.Elevation of TGF-β1 under aging conditions leads to muscle atrophy by blocking muscle regeneration and inducing fibrosis in human and mouse muscle tissue.Autophagy is a conserved intracellular process that wraps and transports macromolecular substances such as proteins and organelles into lysosomes for subsequent degradation.Both excessive and defective autophagy are highly associated with the loss of skeletal muscle during aging,loss of basal autophagy leads to abnormal aggregation of misfolded proteins,and excessive autophagy can also cause cellular stress leading to increased protein degradation lead to loss of skeletal muscle.The ubiquitin–proteasome system(UPS)is the best-known cellular proteolytic system responsible for the degradation of most misfolded or defective cellular proteins.The muscle-specific E3 ubiquitin ligases muscle RING fnger 1(Mu RF1)and muscle atrophy F-Box(MAFbx)are involved in the regulation of skeletal muscle protein degradation,and their expression is involved in skeletal muscle atrophy A significant increase.Mammalian mechanistic target of rapamycin(m TOR)complex 1(m TOR complex 1,m TORC1)is one of the basic regulators of cell growth and metabolism,its main role is to integrate growth factors,amino acids,oxygen One of its key functions is through p70 ribosomal protein S6 kinase(p70S6K)and ribosome-associated 4E-binding protein 1(Phosphorylation of proteins such as 4E binding protein 1,4EBP1)promotes ribosome biogenesis to drive protein translation.In addition,another major role of m TORC1 is to inhibit autophagy.Genetic or pharmacological inhibition of m TORC1 is by far the most effective intervention to delay age-related pathophysiological changes.m TORC1 inhibition slows aging by reducing protein toxicity and accumulation of oxidative stresscausing a general decrease in m RNA translation.Studies have shown that m TORC1 is overactivated in sarcopenic muscles,and partial inhibition of the m TORC1 pathway can counteract sarcopenia.In this study,we simulated the sarcopenia model in aging mice,induced human embryonic lung fibroblast WI-38 cells to senesce by H2O2,and cultured mouse myoblast C2C12 cells with aging fibroblast conditioned medium to explore whether aging fibroblasts secreted TGF-β affects skeletal muscle atrophy in sarcopenic mice.In addition,this study also explored the effect of sarcopenia on the ubiquitin-proteasome system and autophagy of skeletal muscle in mice,and explored the relationship between TGF-β,ubiquitin-proteasome system and autophagy in sarcopenia and the role of m TORC1 pathway in sarcopenia.Methods: C57BL/6J mice of different months were selected and divided into young group(YC group,6-8 weeks old),middle-aged group(MC group,8 months old)and old group(OC group,20 months old).The gastrocnemius muscle mass index(GMI),gastrocnemius muscle mass(GMM),grasping force and relative grasping force of mice were measured.HE staining was used to observe skeletal muscle atrophy changes,and Sirius red staining was used to observe skeletal muscle fibrosis changes.Western blot and RT-q PCR were used to detect the expression of autophagy-related factors LC3 B,P62,Beclin 1,ubiquitin-proteasome system-related factors Atrogin-1,Mu RF1 and m TORC1 pathway proteins in mouse skeletal muscle.H2O2 induced the senescence of embryonic lung fibroblasts WI-38 cells.Western blot and RT-q PCR were used to detect the expressions of senescence-related factors p16,p21 and p53.Flow cytometry was used to detect the cell cycle,and ELISA to detect the secretion of TGF-β.Mouse myoblast C2C12 cells were induced to differentiate into myotubes,and C2C12 cells were cultured with TGF-β or senescent WI-38 cell conditioned medium.Western blot and RT-q PCR were used to detect the expressions of autophagy-related factors LC3 B,P62,and Beclin 1 in C2C12 cells.Western blot The expression of atrophy-related proteins MHC,Atrogin-1,Mu RF1 and m TORC1 pathway proteins were detected,and TGF-β blocker was administered.Western blot and RT-q PCR were used to detect the expressions of MHC,Atrogin-1,Mu RF1,Myf5 and Myo D in C2C12 cells.Autophagy activator was given,Western blot and RT-q PCR were used to detect the expressions of ubiquitin proteasomesystem-related factors Atrogin-1 and Mu RF1,and Western blot was used to detect the protein expressions of atrophy-related proteins MHC,Atrogin-1 and Mu RF1.The m TORC1 pathway inhibitor rapamycin was administered,Western blot and RT-q PCR were used to detect the expression of autophagy-related factors LC3 B,P62,Beclin 1 and ubiquitin proteasome system-related factors Atrogin-1 and Mu RF1 in C2C12 cells,and Western blot was used to detect atrophy-related proteins MHC,Atrogin-1,Mu RF1 expression.Results: 1.With the increase of age,the grasping ability of forelimbs decreased,skeletal muscle volume decreased,and skeletal muscle atrophy and fibrosis aggravated.H2O2 induced increased protein and m RNA expression levels of P16,P21 and P53 in WI-38 cells,increased the proportion of cells in G1 phase,and increased TGF-β secretion.The expression of myosin heavy chain MHC was decreased,the expression of atrophic genes atrogin-1 and Mu RF1 was increased,and the expression of atrogin-1 and Mu RF1 was decreased in myoduct cells treated with conditioned medium of aging fibroblasts and different concentrations of TGF-β.Increased MHC expression.2.With age,LC3II/LC3 I ratio and Beclin 1 expression decreased,P62 expression increased,atrogin-1 and Mu RF1 expression increased.With the increase of TGF-β dose,LC3 B II/LC3 B I ratio and Beclin 1 expression decreased,P62 expression increased,MHC,Myf5 and Myo D expression decreased.TGF-β stimulation increased the expression of atrogin-1 and Mu RF1 in myoblasts,autophagy activator down-regulated the expression of atro Gin-1 and Mu RF1 in myoblasts,and increased the expression of MHC,Myf5 and Myo D.3.With the increase of age,the expression of p-RPS6 and P-S6K1 proteins downstream of m TORC1 in skeletal muscle of mice significantly increased,and the expression of p-4EBP1 protein significantly decreased.TGF-β stimulation increased the expression of p-RPS6 and pS6K1 in C2C12 cells,and decreased the expression of p-4EBP1.MTORC1 inhibitor rapamycin increased the expression of LC3II/LC3 I and Beclin-1 in C2C12 cells,and decreased the expression of P62.Decreased cell atrogin-1 and Mu RF1 expression increased cell MHC,Myf5 and Myo D expression.Conclusion: TGF-β secreted by senescent fibroblasts by activating mTORC1 signaling pathway to inhibits autophagy of skeletal muscle cells and increases proteasome activity,thereby promoting skeletal muscle atrophy.