IRM0002 Ameliorates Myocyte Atrophy Via Inhibiting Protein Degradation Induced By Inflammation

Background:Skeletal muscle atrophy is a common complication of a variety of injuries and inflammatory diseases(such as rheumatoid arthritis,chronic obstructive pulmonary disease,inflammatory myopathy,etc.),which is characterized by the loss of mass caused by muscle protein catabolism and the disorder of muscle physiology,accompanied by the persistence of systemic inflammation and the elevation of inflammatory mediators.Skeletal muscle atrophy mainly depends on the balance of two physiological activities of muscle protein synthesis and protein degradation.As the main inducing factor of muscle atrophy,inflammation can directly regulate the pathway of protein degradation,and can also indirectly regulate the metabolism of other tissues to affect skeletal muscle atrophy.In an inflammation-activated environment,inflammatory mediators released by immune cells can directly cause metabolic abnormalities of muscle cells and promote the expression of atrogenes.Inflammation is the inducing factor of muscle atrophy,and the excessive activation of inflammation is the pathological process of muscle atrophy.It is very important to eliminate the inflammatory response of muscle tissue and reduce the global level of inflammatory mediators to alleviate muscle atrophy.Effective anti-inflammatory drug therapy can effectively prevent the development of muscle atrophy by inhibiting the over-activation of pro-inflammatory pathways and down-regulating the protein degradation system in vitro and in vivo models,but most drugs have numerous side effects and drug toxicity.Therefore,it is of great therapeutic value to develop an antiinflammatory drug with high safety and efficiency for alleviating muscle atrophy caused by inflammation.Purpose:The anti-inflammatory small molecule inhibitor IRM0002(Immune Regulatory Molecules)was screened from small molecule library.To study the effect and molecular mechanism of IRM0002 on innate immune response,and to explore the protective effect and physiological mechanism of IRM0002 on muscle cell atrophy caused by inflammation.Methods:To determine the anti-inflammatory effects of IRM0002,mouse peritoneal macrophages were stimulated with different innate immune ligands.The protein expression and transcription levels of inflammatory factors and upstream signal related genes were analyzed by ELISA,Western blot and real-time PCR,respectively.The effect of IRM0002 on inflammation-induced skeletal muscle atrophy was investigated in LPS-stimulated C2C12 myotubes,and the expression of inflammatory mediators and protein degradation markers was analyzed by real-time PCR.Results:IRM0002 could effectively inhibit the sensitivity of Tlr1/2,Tlr4,Tlr7 and Tlr9 signaling pathways in peritoneal macrophages,and significantly reduce the release of inflammatory cytokines(IL-6,TNF-α and IL-1β)by stimulation with different innate immune ligands.Results showed that IRM0002 inhibited the activation of Tlr4 downstream proteins,and further downregulated the expression of most family of pattern recognition receptors.IRM0002 reduced Stat3 phosphorylation at Ser727 in cells and mitochondria.IRM0002 exerted anti-inflammatory effect in LPS-treated C2C12 myotubes by down-regulating inflammatory mediators(IL-6,IL-1β,Ifn-β).IRM0002 ameliorated inflammation-induced muscle atrophy by inhibiting ubiquitinproteasome molecules(atrogin-1 and Mu RF1)and autophagolysosome molecules(LC3-II and Bnip3).Conclusion:IRM0002 broadly inhibited the expression of pattern recognition receptors in macrophages and reduced the expression of inflammatory cytokines,which may be mediated by blocking the phosphorylation site of Stat3 Ser727.IRM0002 can alleviate the atrophy of myotubes by inhibiting the expression of IL-6 and other inflammatory mediators in C2C12 myotubes induced by LPS,and reducing the upregulation of key genes of the ubiquitin proteasome system and autophagy protease system.