| Background:Idiopathic inflammatory myopathies (idiopathic inflammatory myopathies, IIMs) is a heterogeneous group of skeletal muscle of autoimmune diseases, characted by progressive muscle weakness associated with skeletal muscle inflammation cells infiltration in clinic. According to the clinical and histopathological features, inflammatory myopathy may be divided into polymyositis (polymyositis, PM), dermatomyositis (dermatomyositis, DM) and inclusion body myositis (inclusion-body myositis, IBM). Widespread around the capillaries (DM), the perimysium and endomysium (PM, IBM) of a variety of inflammatory cell infiltration is a characteristic pathological changes of inflammatory myopathy. Macrophages exudated from muscle tissue, monocytes, neutrophils and the release of a variety of chemokines and chemokine, or synergistic inflammatory muscle fiber (MHC-I) playing a role in antigen presentation (APCs), the mobilization of T cell exudated from inflammatory muscle tissue and in situ cloning, through the release of perforin, particulate enzyme and other cytotoxic factor, will kill nearby or distant muscle fibers, causing muscle fiber necrosis continued and repeated and invalid into muscle regeneration.Clinical features of autoimmune myositis patients are progressive muscle weakness, muscle atrophy and fibrosis (proximal muscle is more serious). Patients eventually lose skeletal muscle contraction capacity, because of muscle fiber loss and muscle tissue fibrosis. Confirmed by magnetic resonance spectroscopy, capillary decreased, mitochondria dysfunction, ATP appeared inflammatory muscle cells (ATP) and creatine (PCr) content decreased, resulted in muscle energy metabolism disorder, continued aggravation of muscle weakness, as well as the muscle hypoplasia[5]. At present mainly corticosteroid combined with immunosuppressive is used for the treatment of IIMs, although some patients’muscle performance is improved after drug treatment, but patents can only restore partal muscle function [6]; the majority of patients after a long period of treatment still have limited functions and low quality of life, and long-term high dose of corticosteroid and immunosuppressive agents can lead to chronic bone and muscle metabolism damage.In recent studies, exercises and stretching trains can improve muscle strength reduce muscle injury and improve muscle performance in patients with IIMs. In addition, the mechanical stimulation is important to maintain cell survival and growth of cells,regulating cell metabolism. And it plays an important role in cell activation, proliferation,differentiation and gene expression in muscle cells.There are a lot of researches confirmed that mechanical stimulation can promote myoblast proliferation, and hepatocyte growth factor (HGF) has been proved to be the only one can activate myogenic cells in the stationary phase into cell cycle in vivo or in vitro. With the detection of DNA incorporation of bromodeoxyuridine, research group Tatsumi R found:mechanical stimulation in cultured myoblasts2h, could lead to the activation of HGF. Further study found that mechanical stimulation coud cause nitric oxide synthase (NOS) to activate, induce HGF to release,and lead to muscle cell activation and proliferation of. So Tatsumi R summed up that he mechanical stimulation might cause the mechanical signal channel for myoblast proliferation to activate:calcium-calmodulin complex formation, nitric oxide synthase (NOS) and metalloproteinase (MMPs) activation, HGF and C-met receptors to release, so as to promote myoblast cells proliferating.Under physiological conditions, the expressions of myositis autoantigen in skeletal muscle tissue are few.Repeated regeneration and immature muscle fibres of inflammatory muscle tissue continued to have high expressions of specific myositis autoantigen, such as histidyl tRNA synthetase (HRS/Jo-1), Mi-2(DM patients), U1-70, and Ku/the catalytic subunit of DNA-dependent kinase (protein DNA-PKcs), in vitro culture muscle cells had high level expressions of autoantigen, but with the myoblast differentiation and fusion and myotube formation, autoantigen gradually reduced, suggesting that during the development of autoimmune diseases, autoantigens mainly expressed in the muscle fibers of regeneration, initiation and development of inflammatory process does not involve mature myotubes. During the development process of inflammatory myopathy, regenerating muscle fibers with expressions of autoantigen become cytotoxic targets, recurring muscle fiber necrosis; and muscle cells’ regeneration and differentiation would lead to consistently producing autoantigens, persistently causing pathological process and immune responses of myositis. As a type I transmembrane protein, Toll like receptor family (Toll-like receptors, TLRs) members reacts in the innate immunity, especially in the regulation of phagocytic cells (such as macrophages) specifically in recognizing microbial pathogens antigen, the secretion of proinflammatory cytokines, upregulation of costimulatory molecules and playing an important role to induce acquired immunity reaction process, known as the innate and acquired immune regulation of the auxiliary receptor. Recent research found that (Tournadre A,2010)[18] TLR3and TLR7showed the positive expressions in skeletal muscle tissue of autoimmune myositis patients,especially highly expressed in muscle cells, and through the TLR signaling pathway, the Thl and Thl7cytokine released, accelerated loss of muscle fiber necrosis and systolic function.In view of the above research background, this article precisely launches the research that C2C12cells were mechanical periodic stimulation in vitro, detected the expressions of C2C12cell autoantigen and TLR3, Through the interference of mechanical signal molecules,it will have an effect on expressions of C2C12cell autoantigen and TLR3, and could provide a new way for the treatment of idiopathic inflammatory myopathy. Three parts of our research.Part one:Screening stress loading conditions to promote proliferations of C2C12cells in vitroObjective:Various mechanical conditions of cyclic stretch were practiced by Flexercell5000loading system, and mechanical stimulus conditions promoting the proliferation of C2C12cells were screened by flow cytometry.Methods:After thawed by general procedure, C2C12cells were implanted in the culture bottle and cultivated in DMEM/F12complete medium supplemented with100mg/L penicillin,100mg/L streptomycinand10%fetal bovine serum. They were routine cultured in the incubator at37℃and with5%CO2. C2C12cells according to the density of1×105/mL were seeded on the coated collagen type I Bioflex6Hole culture plate and cultured for24h.Then, cells were stretched at a frequency of0.25Hz,10%tensile range for2days,4days and6days. Cell culture groups:2days in the control group:routine culture in5%CO2cell culture box.2days in the stretching group:cells were stretched in Flexercell5000flexible substrate loading system for2hours a day, then cells were put into5%CO2cell culture box.4days in the control group:cells were cultured in5%CO2cell culture box for2days, then mediums were replaced.4days in the stretching group:cells were stretched in Flexercell5000flexible substrate loading system for2hours a day,after2days, mediums were replaced.6days in the control group:cells were cultured in5%CO2cell culture box, and mediums were replaced after2and4days.6days in the stretching group:cells were stretched in Flexercell5000flexible substrate loading system for2hours a day,after2and4days, mediums were replaced. In addition, cells were stretched for1h at a frequency of1Hz,15%tensile range, and were collected after23h. cell morphology, growth and cell loss of C2C12were observated under a phase contrast microscope every day. Then in the number of days the cells were collected for flow cytometry cycle. Results showed as mean±standard deviation (x±s), and comparisons between groups used the independent sample t test (Independent-Samples T Test),P<0.05considered statistically significant differences.Results:C2C12cells were adherent after4h inoculation, oval or irregular in shape, after12h full extension to spindle and polygonal, cell numbers gradually increased; Under the tensile loading conditions of0.25Hz frequency,10%tensile range:2days, C2C12cells in the stretching group proliferated significantly compared with control group;4days, the control group and the stretching group showed differentiated C2C12cells, and6days, two groups are visible cell fusion. Under the tensile loading conditions of1Hz frequency,15%tensile range,the stretching group showed exfoliated cells. By flow cytometry (flow cytometry, FCM) proliferation cycle3samples of cells in each group were detected, and the proliferations of C2C12cells were expressed with DNA proliferation index [DPI%=(S%+G2/M%)/(S%+G2/M%+G0/G1%)], the sencond day, DPI%of the stretching group was (37±1.43)%, compared with the control group (21.09±2.72)%was significantly increased (P=0.001, n=3); the forth day, DPI%of the stretching group (13.89±0.99)%was slingtly increased than that of the control group (11.64±0.80)%(P=0.038, n=3).T Under the tensile loading conditions of1Hz frequency,15%tensile range,the stretching group showed apoptosis cells.Conclusions:Under tensile stress loading conditions of0.25Hz frequency and10%amplitude,periodic mechanical stimulation can promote the proliferationsof C2C12cells significantly. With the increase in cell number and cell differentiation, the effect of proliferation decreased, while the tensile stress loading conditions of the high frequency and the amplitude is greater than15%had obvious damaging effect on cell growth, promoted cell apoptosis.Part two:The effect of Periodic stress loading conditions promoting proliferation on the expression of C2C12cell autoantigen and TLRs and mechanical signal moleculesObjective:By stimulating proliferation of mechanical conditions of0.25Hz frequency,10%elongation, cycle mechanical stimulation were practiced for2days and4days,then qPCR and Western blot were used for the detections of C2C12cells’ autoantigen (Mi-2, HRS, DNA-PKcs and U1-70) and TLRs gene and protein expression, and Western blot technique was used for the detections of mechanical signal molecules’(Calmodulin, NOS, MMP2, HGF, C-met) protein expression.Methods:C2C12cells according to the density of1×105/mL were seeded on the coated collagen type I Bioflex6Hole culture plate and cultured for24h.Then, cells were stretched at a frequency of0.25Hz,10%tensile range for2days and4days. Cell culture groups:2days in the control group:routine culture in5%CO2cell culture box.2days in the stretching group:cells were stretched in Flexercell5000flexible substrate loading system for2hours a day, then cells were put into5%CO2cell culture box.4days in the control group:cells were cultured in5%CO2cell culture box for2days, then mediums were replaced.4days in the stretching group:cells were stretched in Flexercell5000flexible substrate loading system for2hours a day,after2days, mediums were replaced. In the number of days the cells were collected, total cellular RNA was extracted by Trizol method, using Oligo (dT) reverse transcription cDNA, PCR by specific primer, total proteins wer extracted with KGI protein kit, then were transfered after electrophoresis with SDS/PAGE.after incubation of a resistance to two resistance,ECL chemiluminescence was used to detect proteins.The results were shown by mean±standard deviation(x±s), between group compares the single factor analysis of variance (One-way ANOVA), if the difference was statistically significant, used the LSD inspection comparison between two, P<0.05think differences with a statistical significance.Results:At a frequency of0.25Hz,10%tensile range for tensile loading conditions, the expressions of autoantigens and TLR3of C2C12cells were detected by qPCR and Western blot:2days, stretching group autoantigens (Mi-2, HRS, DNA-PKcs and Ul-70) and TLR3relative gene and protein expression were lower than the control group (P<0.001, n=3);4days, the control group was not significantly difference with tensile. Compared with the control group of2days, the control group of4days had decreased expressions of autoantigens and TLR3(P<0.05, n=3). However, in C2C12cells in vitro, stretching group and the control group were not detected the mRNA and protein levels expression of TLR7. The2day, the mechanical signal molecules’(Calmodulin, NOS, MMP2, HGF, C-met) relative protein expression in stretch group significantly higher than those in control group (P<0.001, n=3),4days, only NOS relative protein expression of stretching group was higher than that of the control group (P<0.05, n=3); and compared with the2days control group,4days control group reduced the expressions of MMP2, HGF and C-met (P<0.01, n=3).Conclusions:Short-term mechanical stimulation can suppress autoantigen and TLR3expression, but with the time prolonging, cell differentiation and fusion and adaptation to mechanical stimulation, resulted in diminished inhibitory effect of autoantigen and TLR3expressions. High expression levels of the mechanical signal molecules were in2days, but with the increase in the number of days, cellular adaptation to stretch stimulation, cell differentiation and fusion, the mechanical signal molecules’expressions decreased, oppositing to expressions of autoantigen and TLR3.Therefore, the mechanical signal molecules could play a key role in the inhibitory effects of short-term mechanical stimulation on the expressions of autoantigen and TLR3.Part three:The effect of mechanical signal molecules on expressions of autoantigen and TLR3of C2C12cells in vitroObjective:The specific culture mediums were added with the promoters or inhibitors of the mechanical signal molecules, and the gene and protein expressions of autoantigen (Mi-2, HRS, DNA-PKcs and U1-70) and Toll like receptor TLR3of C2C12cells were detected by qPCR and Western blot.Methods:When the cell density was about80%, after cleaning2times with PBS, respectively, specific culture mediums were added to cells,containing calcium ionophore A23187(3μ mol/L), calcium chelating agent EGTA (1.8mmol/L), calmodulin Calmodulin (1μg/m), calmodulin inhibitor Calmidazolium Chloride (0.3μmol/L), SNP (NOS accelerator30μmol/L), NOS inhibitor L-NAME (10mol/L), MMP-2(10ng/ml), MMP-2inhibitor1(250ng/ml), recombinant HGF (20 ng/ml), anti-HGF rabbit IgG (2μg/ml), recombinant c-met (1μg/ml), anti-C-Met rabbit IgG(2μg/ml).After placed in the37℃, the volume percentage of5%CO2cell culture box for4hours.cells were collected.Total cellular RNA was extracted by Trizol method, using Oligo (dT) reverse transcription cDNA, PCR by specific primer, total proteins wer extracted with KGI protein kit, then were transfered after electrophoresis with SDS/PAGE.after incubation of a resistance to two resistance,ECL chemiluminescence was used to detect proteins.The results were shown by meani standard deviation (x±s), between group compares the single factor analysis of variance (One-way ANOVA), if the difference was statistically significant, used the LSD inspection comparison between two, P<0.05think differences with a statistical significance.Results:Detection of the C2C12cells with qPCR and Western blot Technology:I n group A231873relative gene and protein expression levels of autoantigens (Mi-2, HRS, DNA-PKcs and U1-70) and TLR were lower than those in the control group (P<0.001, n=3), group EGTA was on the contrary; Calmodulin group’s relative gene and protein expression levels of autoantigens (HRS and DNA-PKcs) and TLR3were lower than the control group’s (P<0.05, n=3), Calmidazolium Chloride group’s autoantigens (Mi-2, HRS, DNA-PKcs and U1-70) were higher than the control group’s (P<0.05, n=3). Group SNP relative gene and protein expression levels of autoantigens (Mi-2, HRS, DNA-PKcs and U1-70) and TLR3were lower than the control group (P<0.001, n=3),group L-NAME on the contrary. Group MMP-2gene and protein expression levels of autoantigen (Mi-2, HRS, DNA-PKcs and Ul-70) and TLR3had no significant difference compared with the control group, MMP-2inhibitorsl group was lower than control group (P<0.05, n=3). Recombinant HGF group relative gene and protein expression levels of autoantigen (HRS, DNA-PKcs and U1-70) and TLR3were lower than the control group (P<0.05, n=3), anti-HGF rabbit IgG group autoantigen (Mi-2, HRS and DNA-PKcs) and TLR3was higher than the control group (P<0.05, n=3). Recombinant c-met group relative gene and protein expression levels of autoantigen (Mi-2, HRS and U1-70) were lower than the control group (P<0.05, n=3), anti-c-Met rabbit IgG group autoantigen (Mi-2, HRS and U1-70) and TLR3was higher than the control group (P<0.05, n=3).Conclusions:Compared with promoters and inhibitorsof the mechanical specific signal molecules, promoters of mechanical signal molecules (Calcium-Calmodulin complex and NOS) could obviously inhibit gene and protein expression levels of autoantigens,while inhibitors were opposite.Promoters of mechanical signal molecules(MMP-2) had no effect of gene and protein expressions of autoantigens and TLR3,but MMP-2inhibitor1could inhibited.mechanical signal molecules’(HGF and C-Met) promoters can inhibit individual autoantigens(except for Mi-2of Recombinant HGF group and DNA-PKcs of Recombinant C-met group) and TLR3(recombinant C-met had no inhibitory effect on TLR3), while inhibitors can promote expressions of individual autoantigens(except for U1-70of anti-HGF rabbit IgG group and DNA-PKcs of anti-C-Met rabbit IgG group) and TLR3. Therefore, mechanical signal molecules may play an important role in the inhibitory effects of mechanical stimulation on the gene and protein expressions of autoantigens and TLR3. |
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