Effects And Mechanism Of TGF-? Signaling On Skeletal Muscle Inflammation/Immune Response

BackgroundDifferent from the chronic inflammatory muscle injury,the acute muscle injury,cells exuded from the muscle tissue were mainly monocytes/macrophages,and a small number of CD4+/CD8+T cells exuded and disappeared rapidly.The regenerated muscle fibers rarely expressed MHC ?/? molecules.The regeneration and repair of muscle tissue were efficient and fast.TGF-?,as an anti-inflammatory growth factor,can inhibit the expression of MHC-II membrane of mononuclear/macrophage and induce the synthesis of proinflammatory cytokines.Previous studies showed that TGF-? mRNA continuously up regulated,and reached its peak at the late regeneration phase.Whether the TGF-? signal of muscle fiber is directly involved in the regulation of intramuscular inflammation and affects the immune behavior of muscle fibers,there is still a lack of relevant literature.Objective1.To analyse the changes of TGF-? level in the inflammatory environment induced by acute injury.2.To analyze the intervention of TGF-? signal to acute intramuscular inflammation.3.To analyse whether the TGF-? signal of skeletal muscle affects the intramuscular inflammation.4.To analyse whether the TGF-? signal of skeletal muscle affects intramuscular inflammation by interfering with the immune characteristics of the muscle fibers.Methods1.The model of acute muscle injury constructed by injection of Cardiotoxin(CTX)(50ul,50ug/mL)at the tibial anterior muscle(TA)of C57BL/6 mice.Hematoxylin eosin staining(HE)and immunofluorescence(Immunofluorescence,IF)double standard staining used to observe the inflammatory cell exudation in acute muscle injury.Real time quantitative PCR(qPCR),Western Blot(WB)and IF used to detect the expression of TGF-? in muscle tissues of 4 days,7 days and 10 days after injury.2.To establish TA damage model by CTX injection.Anti-TGF-? neutralization antibody(11D11,0.5ug/ul,20ul/leg)injected in situ at 2,4,and 6 days,after TA injection of CTX.Frozen section,RNA and protein were prepared for 4,7 and 10 days after injury.HE staining and IF observation of inflammatory exudation and muscle regeneration.qPCR used to detect the expression of cytokine IL-10,IL-6,chemokine MIP-1a,MCP-1 and so on.To analyse the intervention effects of TGF-?signal to intramuscular inflammation by compared with CTX injection alone.3.Construction,identification and breed of skeletal muscle specific TGF-? R ?knockout B6 mice(TGF?R ?flox/flox/MCK-Cre+).4.To establish TA damage model of TGF-?R? KO mouse by CTX injection.Frozen section,RNA and protein were prepared for 4,7 and 10 days after injury.Detection of inflammatory exudation by H&E staining,IF,flow cytometry and so on.qPCR used to detect the expression of cytokine IL-10,IL-6,chemokine MIP-1a,MCP-1 and so on.WB used to detect the expression of TLR3 and TLR7 protein.To analyse the TGF-? signal of skeletal muscle affects the intramuscular inflammation by compared with CTX injection of wild type mouse alone.5.To establish TA damage model of TGF-?R? KO mouse by CTX injection.IF detecte the MHC-I/II molecular expression in regenerated muscle fibers and the exudation of T cell.In vitro culture of TGF-?R? knockout mouse primary myoblasts(MPCs),induced differentiation,and then IFN-gamma(60ng/ml)stimulates for 48h.Observation of myotube fusion under the microscope.Extraction of cell protein and RNA.WB detected the expression of MHC-?/? and TLRs protein in muscle fibers.qPCR used to detect the expression of cytokine IL-10,IL-6,chemokine MIP-1a,MCP-1 and so on.To analyse whether the TGF-? signal of skeletal muscle affects the muscle fiber immune characteristics by compared with CTX injection of wild type mouse alone.Results1.In the process of acute muscle injury-regeneration,the expression of TGF-?in muscle tissue is continuous up regulated.2.TGF-? signaling directly affects the inflammatory response of damged skeletal muscle tissue.3.The TGF-? signal of skeletal muscle directly affects intramuscular inflammation of acute injury.4.The TGF-? signal of muscle its self could interfere with the muscle fiber immune characteristics.ConclusionTGF-? expression is continuous up regulated in acute injury of skeletal muscle tissue.TGF-? signal of muscle fiber it self can directly interfere with the immune characteristics of muscle fiber,and affect the proinflammatory cytokine/chemokine secretion in muscle fibers.It suggest that the TGF-? signal of muscle cells may be involved in the immune tolerance of skeletal muscle after acute injury.