Effects Of Nitric Oxide On Muscle Inflammatory Responses

Background:Acute skeletal muscle injury is a common injury in life, including mechanical damage, biochemical toxins, crush injuries, excessive loads, strains, contusions, etc. Whatever the causes of injury after injury showed early part of the inflammatory response to injury:injury, necrosis of muscle fibers release of pro-inflammatory signals induced by peripheral inflammatory cells (neutrophils, monocytes/macrophages mainly) damage to muscle tissue and muscle infiltration moving to engulf necrotic muscle cells and tissue fragments disintegration, while the secretion of various cytokines and chemokines, maintain muscle inflammation and activation of muscle satellite cells, activation of myoblasts continued proliferation, differentiation, tube formation of new muscle to repair damaged muscle fibers, complete muscle regeneration Muscle inflammation contributes injury degenerated muscle fibers cleared inflammatory environment also contribute to the regeneration of muscle fiber repair. However, severe, persistent muscle inflammation easily lead to muscle tissue hematoma or swelling, intramuscular pressure increases, inhibiting myoblast proliferation, even damage the normal muscle fibers, resulting in prolonged healing period, and even scar repair, affecting patients quality of life. Full skeletal muscle injury mechanism, to explore how to reduce muscle inflammation after injury, inflammation, and the relationship between muscle regeneration coordination, thereby improving the healing quality research in the field of skeletal muscle injury is an important issue. Nitric oxide (Nitric oxide, NO) is in the nitric oxide synthase (NOS) the role of signaling molecules by L-arginine synthesis. In skeletal muscle injury and regeneration, NO way to promote the adoption of the following skeletal muscle regeneration:i) promoting myoblast cell activity, proliferation and differentiation; ii) increase the blood supply to the damaged area (for the damage zone provided by vasodilator adequate nutrition and oxygen); iii) increase the level of glucose transporter GLUT4 to improve glucose uptake in muscle tissue; iv) increase the synthesis of mitochondrial sirtuin-1-dependent enhance muscle energy metabolism .By promoting apoptosis of inflammatory cells, inhibit adhesion molecules (e.g. ICAM, E-selectin, P-selectin) expression of myogenic muscle NO can reduce the inflammatory response after injury, reduce neutrophil-mediated myocyte necrosis dissolve, reducing the formation of peroxide concentration and reaction intermediates . As a value-added signal molecule mechanics of muscle cells, NO involved in skeletal muscle injury satellite cell activation, proliferation and differentiation, and promote tissue repair muscle damage.Currently on the role of NO on the inflammatory response after muscle injury is still controversial:Hickey other confirmed iNOS gene knockout mice skeletal muscle damage is reduced exudation of inflammatory cells; McCafferty and Rigamonti considered After iNOS-derived NO muscle injury can be reduced lymphocyte exudation; research Rovere-Querini P Task Force found that iNOS knockout mice damaged muscle tissue inflammatory cells significantly more than the control group. Our previous in vitro studies confirmed, NO antagonist L-NAME promote the expression of C2C12 cell autoantigen and TLR3, whereas SNP (NO donor) inhibited the expression of these molecules. This suggests that NO may be involved in skeletal muscle inflammation down. To further analyze the interference effect of NO on damage-induced skeletal muscle inflammation, and this study was prepared Notexin acute muscle injury model in mice, the level of NO in vivo chemical interference, analysis of skeletal muscle damage autoantigen (Mi-2, HARS, DNA-PKcs Ku-70), and gene and protein expression TLRs (TLR3 and TLR7), monocytes/macrophages and apoptotic intramuscular exudation, CD8+T cells and regeneration of muscle fibers intramuscular exudation MHC-I expression and, is important in the injured muscle tissue cytokines (TNF-α, TGF-β, IL-6 and IL-10) and chemokines (MIP-1α, MCP-1, MCP-3) are differentially expressed.Under physiological conditions, muscle satellite cells and mature muscle fibers lack MHC molecule expression. However, research has shown that people vitro cultured myoblasts expressing MHC-I (human leukocyte antigen I, HLA class I), pro-inflammatory cytokines (eg, IFN-γ, TNF-α, IL-1α, IL-1β, or MIP-1α) will further increase myoblast HLA-I expression. After receiving IFN-y stimulated differentiation of myoblasts and myotubes were expressed MHC-Ⅱ molecules (HLA class-Ⅱ) Therefore, some scholars have pointed out, skeletal muscle cells are a part-time (non-professional) antigen-presenting cells (APC).Research on immunological properties of skeletal muscle cells are made of human myoblasts and differentiated myotubes. Rarely reported in the literature is widely used today myoblast cell lines (such as C2C12, L6 cells), as well as more convenient sources and amplified mouse primary myoblasts whether the expression of MHC molecules and with APC function. This paper attempts to proliferation or differentiation of cultured C2C12 myoblasts into IFN-y-induced inflammatory environment, Detection of IFN-γ stimulation of cultured myoblast/myotube differentiation whether they have the characteristics of inflammatory cells (whether upregulated MHC-I, and the expression of MHC-Ⅱ?).NLRP3 inflammasome are innate immunity and acquired immunity to contact molecules. APC activation of inflammatory cells within the small body is released IL-1β, IL-18, and IL-33 in an autocrine manner to promote APC antigen-presenting cell to improve capacity, upregulation of costimulatory molecules and MHC-II expression, to activate antigen-specific T, B cell responses. Normal activation of inflammatory small body in maintaining the immune system plays an important role in stabilizing, but continued activation of chronic inflammatory and autoimmune diseases will result in Mishra found that NO can inhibit the activation of inflammatory corpuscle NLRP3, reduce the secretion of IL-1β to inhibit the immune response by tuberculosis; Kairui Mao demonstrated that NO can inhibit excessive activation NLRP3 inflammatory corpuscle. Although inflammation and autoimmune diseases bodies (such as rheumatoid arthritis, multiple sclerosis) correlation has been widely discussed, but has not been reported whether the impact of inflammation bodies and skeletal muscle inflammation development of. In order to investigate whether the inflammatory environment of muscle cells and expression of inflammatory corpuscles cytokine secretion, and further clarify whether the interference NO activation of inflammatory cells within the muscle bodies, we try to stimulate the use of IFN-y and mouse C2C12 myoblasts Analysis of the formation and secretion of inflammatory activation of small bodies, IL-1β in. The use of L-NAME and SNP-induced IFN-y treatment C2C12 cells, whether inflammatory characterization analysis NO muscle cells.Our main contents include:Three parts of our research.Part one:The Change of NO and NOS Levels in Notexin-Damaged TA MuscleObjective:Notexin acute skeletal muscle injury induced by intramuscular injection, analysis degeneration of muscle tissue damage, inflammatory exudate, reproduction characteristics; and comparative analysis to detect NO antagonist before (L-NAME)/ stimulants (SNP) treatment after injury muscle tissue concentration of NO, NOS level change.Methods:Clean female C57BL/6 mice (4-8w) conventional breeding, tibialis anterior muscle (tibialis anterrior, TA) injection notexin (0.1 mg/kg), respectively Od,4d,7d, lOd removal of the mouse TA, frozen sliced, H & E and Dystrophin immunofluorescent staining. Notexin after injection, respectively the first and four days by intraperitoneal injection of L-NAME (100 mg/kg) and SNP (0.5 mg/kg), respectively, in the first 4 days and 7 days after injection removal notexin TA, total RNA was extracted, Oligo (dT) RT obtain cDNA, through specific primer (eNOS, iNOS, nNOS) PCR amplification; NO concentrations measured after tissue. The results were expressed as mean ± standard deviation (x±s) that the groups were compared using ANOVA (One-way ANOVA), if the difference was statistically significant, the use of LSD pairwise comparison test, P<0.05 considered the difference Statistical significance protein.ResultsHE and Dystrophin staining showed, notexin injection 4 days, showing a lot of muscle fiber necrosis and inflammatory exudate,7 days a central necrotic area was owned nuclear regenerated muscle fibers instead.10 days regenerated muscle fibers almost complete. Notexin four days after injection, the mice muscle tissue NO concentration than the control group significantly increased (P<0.05, n= 3); after SNP treatment of muscle tissue concentration of NO increased further than a single injury group (P<0.05, n= 3); on the contrary, L-NAME treatment will be lowered intramuscular NO levels. qPCR results showed that, L-NAME induces muscle damage TA eNOS, iNOS, nNOS gene downregulated (compared with the simple injury group, P<0.05, n= 3), especially in the most significant iNOS down. SNP treatment does not affect the NOS gene expression.ConclusionNotexin intramuscular injection can induce skeletal muscle necrosis and inflammatory exudate. Because notexin-induced skeletal muscle necrosis in four days after injury most widely used, seven days after the regeneration of muscle fibers completely replace degenerated tissue, so we were selected 4 and 7 days sampling time point as necrosis, regeneration. Acute muscle injury, NOS, and myogenic NO concentration rapidly increase. SNP can further increase damage muscle tissue concentration of NO, L-NAME NO concentration is lowered intramuscular. L-NAME inhibits NOS gene expression intramuscular, especially inhibition of iNOS gene level.Part two:Effects of Nitric Oxide on Notexin-Induced Muscle Inflammatory ResponsesObjective:NO vivo interference level (NO agonist SNP, or intraperitoneal injection antagonist L-NAME). Analysis of autoantigens within TA muscle tissue damage (Mi-2, HARS, DNA-PKcs, Ku-70), TLRs (TLR3 and TLR7) altered expression, oozing characteristic changes in muscle and apoptosis of lymphocytes, intramuscular cytokines (TNF-α, TGF-β, IL-6 and IL-10) and chemokines (MIP-1α, MCP-1, MCP-3) of factor level changes. Through these studies, a clear change would not affect the concentration of NO in vivo acute inflammatory response in skeletal muscle injury.Methods:TA intramuscular injection notexin (0.1mg/kg), the first two days after muscle injury intraperitoneal injection of L-NAME (100 mg/kg) or SNP (0.5 mg/ kg),7 days animals drawn in the first four days after injury again intraperitoneal injection of L-NAME or SNP. Were the first four days, seven days after the removal of mouse TA muscle injury, total RNA was extracted muscle tissue, Oligo (dT) RT obtain cDNA, PCR amplification primers to detect autoantigens, TLRs, cytokines (TNF-α, TGF-β, IL-6 and IL-10) and chemokines (MIP-1α, MCP-1, MCP-3); TA muscle extract total protein, SDS/PAGE electrophoresis, transferred to a membrane separation, ECL chemiluminescence detection autoantigen protein expression of TLRs; TA muscle frozen sections, immunofluorescence intramuscular oozing CD11b, F4/80, CD3/CD8, MHC-I (H-2Kb), caspase-3 positive cells. Groups were compared using ANOVA (One-way ANOVA), if the difference was statistically significant, the use of LSD pairwise comparison test, P<0.05 was considered statistically significant.ResultsNotexin injection of 4 days, TA muscle autoantigens (Mi-2, HRS and Ku-70) and TLR3 mRNA and protein levels were significantly upregulated. L-NAME treatment further increases autoantigens and TLR3 expression levels, but SNP deal will be cut above the molecular level. H & E staining and immunofluorescence observed damage 4 days, extensive necrosis of muscle fibers disintegration, shows a large number of inflammatory cell exudate, exudate cells mostly monocytes/macrophages (CD11b+, F4/80+).7 days, is still leaking a small amount of visible monocytes/macrophages, Dystrophine positive regenerated muscle fibers appeared in large numbers Quantitative fluorescence image analysis showed that four days muscle injury CD11b, or mean fluorescence intensity F4/80 was significantly increased at 7 days decreased, but still higher than normal muscle tissue. L-NAME-treated group 4 and 7 days or mean fluorescence intensity CD11b F4/80 were significantly higher than simple injury; SNP treatment is to reduce the value of the fluorescence intensity of inflammatory cells. For CDllb+caspase-3+cells were observed and counted the support of SNP promote apoptosis of monocytes. qPCR results showed that, Notexin intramuscular injection induced damage TGF-β (4d and 7d) and IL-10 (4d) mRNA levels increased, but the L-NAME and SNP does not affect the expression of these molecules.4d and 7d, are visible, SNP significantly reduced TNF-α and IL-6, MIP-1α, MCP-1, and MCP-3 mRNA levels, contrary L-NAME effects, increased TNF-α, IL-6, MIP-1α, MCP-1, and MCP-3 mRNA levels.4 days muscle injury, we have not observed CD3ε+ CD8α+ cells, and the H-2Kb+ muscle fibers. When 7d, muscle gap visible CD8α+ T cells and a small amount of H-2K b+muscle fibers. We found that, after L-NAME treatment, the number of muscle fibers significantly increased CD8a+ T cells and H-2Kb+ConclusionsAfter skeletal muscle injury increases myogenic NO interference intramuscular inflammation, inhibit skeletal muscle autoantigens (Mi-2, HRS, Ku-70) and the expression of TLR3, reduces monocyte/macrophage exudate, and promote inflammatory apoptosis. At the same time, NO participation intramuscular down proinflammatory cytokines and chemokines, and may inhibit the expression of CD8a T cells and muscle fibers oozing MHC-I molecules+ regeneration in the injured muscle tissue. These results indicate that, NO down muscle damage involved in inflammation.Part three:Effects of Nitric Oxide on Immune Features of Myoblast/Myotubes Received Inflammatory Stimuli In VitroObjective:Analysis of the inflammatory environment (IFN-y stimulation) can induce C2C12 myoblast/myotube differentiation MHC molecule expression and activation of NLRP3 inflammasome body clear whether NO interference C2C12 cells activate inflammation inside the small body.Methods Recovery of cryopreserved C2C12 cells,37 ℃ 5% CO2 incubator to 1 × 105/mL were seeded in 6-well plates adherent proliferating culture, or a 2% horse serum differentiation culture. Were added IFN-y (0.6ug/ml) to the proliferation, or differentiation of cultured C2C12 cells. Using L-NAME (2.7ug/ml) and the SNP (8.94ug/ml) IFN-y treatment of differentiated cells 48h of interference. Respectively after IFN-y stimulation 24h,48h, or L-NAME and differentiated cells 6h after SNP treatment cells were collected, extracted total RNA and total protein, qPCR and Western blot MHC-Ⅰ (H-2Kb), MHC-Ⅱ, ASC, NLRP3, caspase-1. ELISA technique for detecting the C2C12 cell culture supernatant of IL-1β protein concentration. The results were expressed as mean ± standard deviation (x±s) that the groups were compared using ANOVA (One-way ANOVA), if the difference was statistically significant, the use of LSD pairwise comparison test, P<0.05 considered the difference statistical significanceResultsIFN-γ after stimulation of cultured myoblast proliferation and differentiation of myotubes were significantly upregulated MHC-I mRNA and protein levels, and express MHC-Ⅱ. qPCR, immunofluorescence, Western blotting confirmed, IFN-γ-induced myoblast/myotube differentiation increases NLRP3, ASC and mature-caspase 1-expression levels. ELISA analysis further confirmed that, compared with unstimulated cells, IFN-y induced significantly upregulated C2C12 cell culture medium concentration of IL-1β. SNP treatment after 6h, differentiated myotubes (IFN-y stimulation 48h) internal inflammation bodies (ASC, NLRP3, caspase-1) mRNA and protein levels than simply stimulate cells were significantly reduced, L-NAME is raised ASC, NLRP3, caspase-1 expression levels. Consistent with these results, SNP and L-NAME treatment also cut or increase in IL-1β concentration in the culture medium.ConclusionsInflammatory stimuli can induce myoblast/regeneration of muscle fibers (myotubes) became part of the APC cells, and expression of MHC molecules. Expression of MHC molecules myoblast/myotube differentiation and activation capabilities has synthesized NLRP3 inflammasome body. NO may be one of the factors that interfere with the immune characteristics of muscle cells.