Research on Syringin protecting C2C12 myotube viability through regulating NF- κB/PPAR γ1 pathway / 国际中医中药杂志

Objective:To discuss the protective effect of Syringin (SYR) on myotube cell atrophy induced by lipopolysaccharide (LPS) and its molecular mechanism.Methods:After C2C12 myoblasts were differentiated into myotubes, they were divided into normal control group, model group and syringin group according to the random number table method. The cultured medium of model group and syringin group were added with LPS with a concentration of 200 ng/ml; the cultured medium of the syringin group was also added with 10 μmol/L syringin for 24 h. CCK8 was used to detect cell viability. In cell supernatant, NO release was detected with Griess and TNF-α level was detected by ELISA kit. The expression of NF-κB, PPAR γ1, MyHC were detected by Western blot.Results:Compared with the model group, the viability of cells [(101.08±8.92)%, (79.53±5.19)% vs. (69.07±7.16)%] in the 10 μmol/L and 100 μmol/L syringin groups were significantly increased ( P<0.01 or P<0.01), of which 10 μmol/L syringin had better effect. Compared with the model group, the level of NO [(2.92±0.33) μmol/L vs. (3.57±0.41) μmol/L] in the syringin group was significantly decreased after 6 hours of intervention ( P<0.01), and the cells in the syringin group after 24 hours of intervention, the level of TNF-α [(2.73±0.29) pg/ml vs. (4.15±0.29) pg/ml] was significantly decreased ( P<0.01), and the protein expression of cellular NF-κB (0.95±0.24 vs. 1.16±0.28) was significantly decreased ( P<0.05), the protein expression of MyHC (0.79±0.15 vs. 0.70±0.16) was increased ( P<0.05). Conclusion:SYR could inhibit the inflammatory response induced by LPS, promote the activity of myotubes, and antagonize the damage of LPS to myotube cells.

Myostatin coordinating the proliferation and differentiation of adipose and skeletal muscle cells and energy metabolism balance based on gene chip technology / A miostatina na coordenação da proliferação e diferenciação de células musculares adiposas e esqueléticas e no equilíbrio do metabolismo energético com base na tecnologia de chips genéticos / Msrn, basado en la tecnología de chips genéticos, coordina la fertilización y el metabolismo de las células musculares grasas y óseas

ABSTRACT Myoblasts fuse into multinucleated muscle fibers to form and promote the growth of skeletal muscle. In order to analyze the role of myostatin (MSTN) in body fat, skeletal muscle cell proliferation and differentiation and energy metabolism, this study will use the antisense RNA technology of gene chip technology to study it. The results showed that the MSTN gene regulated the growth and proliferation of myoblasts and affected the development of skeletal muscle by affecting the expression of Cdc42, bnip2, p38 and other genes; knockout or overexpression of the MSTN gene would lead to a trend of fat-related genes from fat synthesis to fat decomposition; after the MSTN gene was knocked down, the expression levels of cpti-b, PPARG and other genes in the cells were corresponding after MSTN overexpression, the relative expression of the PPARG gene decreased. It is suggested that the knockout or overexpression of MSTN may affect lipid accumulation, and cpti-b and PPARG may directly regulate lipid level. It is hoped that this experiment can provide a reference for the study of MSTN effect on fat deposition.

RESUMO Os mioblastos se fundem eM fibras musculares multinucleadas para formar e promover o crescimento do músculo esquelético. A fim de analisar o papel da miostatina (MSTN) na gordura corporal, proliferação de células musculares esqueléticas e diferenciação e metabolismo energético, este estudo utilizará a tecnologia anti-RNA de chips genéticos para estudá-la. Os resultados mostraram que o gene MSTN regulava o crescimento e a proliferação de mioblastos e afetava o desenvolvimento do músculo esquelético, afetando a expressão de Cdc42, bnip2, p38 e outros genes; a eliminação ou sobrexpressão do gene MSTN conduziria a uma tendência de os genes adiposos sintetizarem a gordura até sua decomposição; após a eliminação do gene MSTN, os níveis de expressão de cpti-b, PPARG e outros genes nas células mostraram-se correspondentes após a sobrexpressão do gene MSTN, e a expressão relativa do gene PPARG diminuiu. Sugere-se que a eliminação ou sobrexpressão da MSTN possa afetar a acumulação de lipídeos, e o cpti-b e o PPARG podem regular diretamente o nível lipídico. Espera-se que esta experiência possa fornecer uma referência para o estudo do efeito da MSTN sobre a deposição de gordura.

RESUMEN Los mioblastos se funden en fibras musculares multinucleadas para formar y promover el crecimiento del músculo esquelético. A fin de analizar el papel de la miostatina (MSTN) en la grasa corporal, proliferación de células musculares esqueléticas y diferenciación y metabolismo energético, este estudio utilizará la tecnología anti-RNA de chips genéticos para estudiarla. Los resultados mostraron que el gen MSTN regulaba el crecimiento y la proliferación de mioblastos y afectaba el desarrollo del músculo esquelético, afectando la expresión de Cdc42, bnip2, p38 y otros genes; la eliminación o sobreexpresión del gen MSTN conduciría a una tendencia de que los genes adiposos sinteticen la grasa hasta su descomposición; después de la eliminación del gen MSTN, los niveles de expresión de cpti-b, PPARG y otros genes en las células se mostraron correspondientes después de la sobreexpresión del gen MSTN, y la expresión relativa del gen PPARG disminuyó. Se sugiere que la eliminación o sobreexpresión de la MSTN pueda afectar la acumulación de lipídos, y el cpti-b y el PPARG pueden regular directamente el nivel lipídico. Se espera que esta experiencia pueda proveer una referencia para el estudio del efecto de la MSTN sobre el depósito de grasa.

Platelet-derived growth factor-bb promotes proliferation, differentiation and migration of skeletal muscle myoblast / 中国组织工程研究

BACKGROUND: Skeletal muscle myoblasts differentiate and fuse to form polynuclear muscle tubes and muscle fibers to complete the repair of muscle injury when skeletal muscles were injured. However, the repair process is slow and incomplete. Platelet-derived growth factor-BB can stimulate the proliferation, differentiation and migration of a variety of tissue cells, and plays an important role in the process of tissue repair after various injuries. OBJECTIVE: To explore the effects of different concentrations of platelet-derived growth factor-BB on proliferation, differentiation and migration of skeletal muscle myoblast C2C12 cells and the action mechanism. METHODS: The mouse skeletal muscle myoblast C2C12 cells were cultured with platelet-derived growth factor-BB 0, 5, 10, 20 and 40 μg/L and platelet-derived growth factor receptor inhibitor imatinib. The expression of platelet-derived growth factor receptor in C2C12 cells was detected by immunocytochemistry and western blot assay. After 1, 2, 3, 4, and 5 days of culture, CCK8 method was used to detect cell proliferation. After 4 days of induction and differentiation culture, the formation of myotubes in each group was observed by light microscope. The expression of myosin heavy chain and MyoG Gene was observed by immunofluorescence and western blot assay. After 48 hours of culture, Transwell method was used to detect cell migration. RESULTS AND CONCLUSION: (1) Immunofluorescence chemistry and western blot assay indicated that platelet-derived growth factor receptor expression could be detected in C2C12 cells, and semi-quantitative statistical analysis showed no significant difference in platelet-derived growth factor receptor expression between groups with different platelet-derived growth factor-BB concentrations (P > 0.05). (2) CCK8 assay demonstrated that the proliferation of C2C12 cells showed no significant change in platelet-derived growth factor-BB groups compared with the control group (platelet-derived growth factor-BB 0 μg/L group). (3) Immunofluorescence chemistry showed that compared with control group, number of myosin heavy chain positive cells increased in platelet-derived growth factor-BB groups; 40 μg/L platelet-derived growth factor-BB concentration got the highest; the mature muscle tubes up to (27.00±0.76) per field of vision, and MyoG expression populations was most obviously compared with the control group (P < 0.05). (4) Transwell results showed that compared with the control group, the migration number of C2C12 cells in the platelet-derived growth factor-BB group increased, and the migration number in the 40 μg/L group was up to 144.00±13.03 (P < 0.05). (5) It is concluded that platelet-derived growth factor-BB promoted the migration, differentiation and myotube formation of C2C12 cells, and the pro-differentiation mechanism is related to its enhanced binding with platelet-derived growth factor receptor, so as to improve the expression of differentiation related gene MyoG.

Effects of nutritional status on mitochondrial Ca2+ handling / Efeitos do estado nutricional no manejo de Ca2+ mitocondrial

Mitochondria are central players in cell metabolism, responsible for the vast majority of ATP production in most cells. Although originally thought to be passive organelles focused only in keeping cellular ATP at adequate levels, complex interplay between mitochondrial function and cell signaling has been largely recognized over the last decades. Not surprisingly, given their role, changes in nutritional status promoted by chronic interventions like caloric restriction or short-term situations like fasting in animals or nutrient deprivation in cultured cells are one of the main factors that can activate those signaling mechanisms. One particular way in which this mitochondria-cell crosstalk can occur is through mitochondrial Ca2+ handling, a process in which Ca2+ signals generated by the cell are able to translate into elevations in mitochondrial matrix [Ca2+] due to the presence of the mitochondrial Ca2+ uniporter in the organelle. While the impact of mitochondrial Ca2+ handling on cellular function has been widely studied, the conditions which can modulate the process of mitochondrial Ca2+ handling itself are still not well characterized. In this work, we sought to test the effects of different interventions linked to nutritional status on mitochondrial Ca2+ handling. We found that caloric restriction, physiological fasting and modulations of mitochondrial dynamics resulted in modulation of mitochondrial Ca2+ handling through changes in their maximal Ca2+ retention capacity or Ca2+ uptake rates. These changes were, measured by following mitochondrial Ca2+ uptake using different strategies, employing the fluorescent Ca2+ probe Ca2+ Green 5N for experiments in isolated mitochondria and permeabilized cells and the cytosolic probe Fura2-AM in intact cells. Caloric restriction resulted in higher calcium uptake and retention in liver mitochondria, protecting against pathological conditions of Ca2+ overload during ischemia/reperfusion. On the other hand, overnight and short term fasting resulted in lower mitochondrial Ca2+ retention and oxidative phosphorylation capacity in the liver. Modulating mitochondrial morpholoy in C2C12 myoblasts showed that more fragmented mitochondria were less capable of taking up Ca2+, while more fusioned mitochondria showed the opposite phenotype. This modulation in Ca2+ handling through changes in mitochondrial morphology interfered with the process of Store-Operated Ca2+ entry in the cells, showing that these modulations can have impacts in physiological contexts as well. Overall, this work both establishes novel mechanisms of modulation of mitochondrial Ca2+ handling and demonstrates their relevance both in pathology and normal cellular physiology

Mitocôndrias possuem um papel central no metabolismo das células, sendo responsáveis pela maioria da produção de ATP na maioria dos tipos celulares. Embora originalmente se pensasse nas mitocôndrias como organelas estáticas, focadas somente em manter os níveis adequados de ATP na célula, a interação entre a função mitocondrial e a sinalização celular tem sido fortemente reconhecida nas ultimas décadas. Dado este papel, não é surpreendente que mudanças no estado nutricional, tanto crônicas como na restrição calórica quanto em situações como o jejum em animais e a privação de nutrientes em cultura de células foram demonstradas como sendo um dos principais fatores que podem ativar estes mecanismos de sinalização. Uma das formas em que esta interação entre a mitocôndria e a célula ocorre é através do manejo de Ca2+ mitocondrial, um processo em que sinais de Ca2+ gerados pela célula podem resultar em aumentos na [Ca2+] na matriz mitocondrial devido à presença do uniportador de Ca2+ mitocondrial na organelaEmbora o impacto do manejo de Ca2+ mitocondrial na função da célula tenha sido amplamente estudado, a regulação do processo de manejo de Ca2+ mitocondrial em si não é bem conhecida. Neste trabalho, nós nos propusemos a testar os efeitos de diferentes intervenções ligadas ao estado nutricional no manejo de Ca2+ mitocondrial e o possível impacto destas modulações nacapacidade de retenção e na taxa de captação de Ca2+ mitochondrial. As intervenções estudadas foram a restrição calórica, jejum e mudanças na dinâmica mitocondrial, e todas elas resultando em mudanças no manejo de Ca2+ mitocondrial, que foram medidos acompanhando a captação de Ca2+ em mitocôndrias isoladas ou células permeabilizadas utilizando a sonda Ca2+ Green 5N e em células intactas utilizando a sonda de Ca2+ citosólica Fura2-AM. Enquanto a restrição calórica resultou em uma maior capacidade de retenção de Ca2+ e em maiores taxas de captação, protegendo contra as condições patológicas de desregulação de Ca2+ observadas durante a isquemia/reperfusão, o jejum curto ou pela duração da noite resultou em uma diminuição na capacidade de retenção de Ca2+ e na oxidação fosforilativa mitocondriais. As mudanças observadas modulando a dinâmica mitocôndria (feitas utilizando-se mioblastos da linhagem C2C12) revelaram que mitocôndrias mais fragmentadas são menos capazes de captar Ca2+, enquanto mitocôndrias mais fusionadas possuem o fenótipo oposto. Essas mudanças no manejo de Ca2+ mitocondrial interferem com o processo de Store-Operated Ca2+ entry nestas células, demonstrando que essas modulações da captação de Ca2+ mitocondrial também podem ser relevantes em contextos fisiológicos. Em resumo, este trabalho ajudou a estabelecer novos mecanismos de modulação do manejo de Ca2+ mitocondrial que podem ser relevantes tanto em condições patológicas quanto na fisiologia normal das células

Alteration of mitochondrial DNA content modulates antioxidant enzyme expressions and oxidative stress in myoblasts

Mitochondrial dysfunction is closely associated with reactive oxygen species (ROS) generation and oxidative stress in cells. On the other hand, modulation of the cellular antioxidant defense system by changes in the mitochondrial DNA (mtDNA) content is largely unknown. To determine the relationship between the cellular mtDNA content and defense system against oxidative stress, this study examined a set of myoblasts containing a depleted or reverted mtDNA content. A change in the cellular mtDNA content modulated the expression of antioxidant enzymes in myoblasts. In particular, the expression and activity of glutathione peroxidase (GPx) and catalase were inversely correlated with the mtDNA content in myoblasts. The depletion of mtDNA decreased both the reduced glutathione (GSH) and oxidized glutathione (GSSG) slightly, whereas the cellular redox status, as assessed by the GSH/GSSG ratio, was similar to that of the control. Interestingly, the steady-state level of the intracellular ROS, which depends on the reciprocal actions between ROS generation and detoxification, was reduced significantly and the lethality induced by H₂O₂ was alleviated by mtDNA depletion in myoblasts. Therefore, these results suggest that the ROS homeostasis and antioxidant enzymes are modulated by the cellular mtDNA content and that the increased expression and activity of GPx and catalase through the depletion of mtDNA are closely associated with an alleviation of the oxidative stress in myoblasts.

Mitochondrial dysfunction reduces the activity of KIR2.1 K⁺ channel in myoblasts via impaired oxidative phosphorylation

Myoblast fusion depends on mitochondrial integrity and intracellular Ca²⁺ signaling regulated by various ion channels. In this study, we investigated the ionic currents associated with [Ca²⁺]i regulation in normal and mitochondrial DNA-depleted (ρ0) L6 myoblasts. The ρ0 myoblasts showed impaired myotube formation. The inwardly rectifying K⁺ current (I(Kir)) was largely decreased with reduced expression of KIR2.1, whereas the voltage-operated Ca²⁺ channel and Ca²⁺-activated K⁺ channel currents were intact. Sustained inhibition of mitochondrial electron transport by antimycin A treatment (24 h) also decreased the I(Kir). The ρ0 myoblasts showed depolarized resting membrane potential and higher basal [Ca²⁺]ᵢ. Our results demonstrated the specific downregulation of I(Kir) by dysfunctional mitochondria. The resultant depolarization and altered Ca²⁺ signaling might be associated with impaired myoblast fusion in ρ0 myoblasts.

Effect of Bcl-2 and PCNA expression in rat myoblast injury induced by hydrogen peroxide / 中国病理生理杂志

AIM:To explore the role of Bcl-2 and PCNA expression in the injury of rat myoblasts induced by hydrogen peroxide (H2O2).METHODS:Rat myoblasts at growth phase were divided into 4 groups based on basic fibroblast growth factor (bFGF) and H2O2 levels:normal control group, bFGF group, model group (H2O2 group) and treatment group (bFGF+H2O2 group).The expression of Bcl-2 and Bax was observed by immunohistochemistry and fluorescence methods.The protein levels of Bax, Bcl-2 and PCNA were determined by Western blot.RESULTS:Compared with model group, both immunofuorescence and fluorescence in treatment group showed enhanced Bcl-2 and low expression of Bax.Furthermore, the results of Western blot showed up-regulated PCNA and Bcl-2 protein and decreased Bax expression in treatment group.CONCLUSION:Oxidative stress results in the pathologic changes of myoblasts, and the up-regulation of Bcl-2 and PCNA may attenuate myoblast injury.

Liraglutide increases FNDC5 expression in C2C12 myotubes via activa-tion of CAMKK2/AMPK signaling pathways / 中国病理生理杂志

AIM:To investigate the effect of liraglutide ( LG) on the expression of fibronectin type Ⅲdomain-containing protein 5 (FNDC5) in the C2C12 myotubes.METHODS:The C2C12 mouse myoblast cell line was induced to differentiation.Differentiated cells were stimulated with gradient concentrations (1 ~1000 nmol/L) of LG for different time (0 ~24 h).The effects of LG on the expression of FNDC5 and the activation of adenosine 5'-monophosphate ( AMP)-activated protein kinase ( AMPK) signaling pathway were determined .After pretreated with glucagon-like peptide-1 ( GLP-1 ) receptor antagonist exendin 9-39 , the inhibitor of Ca 2+/calmodulin-dependent protein kinase kinase 2 (CAMKK2), STO609, or the inhibitor of AMPK, Compound C, the LG-induced FNDC5 expression in C2C12 myotubes was examined.The expression of FNDC5 and the activation of AMPK were determined by Western blot .RESULTS: In C2C12 myotubes, LG promoted the expression of FNDC5 in a dose-and time-dependent manner .LG also activated AMPK signaling pathway .These effects of LG were partly abolished by exendin 9-39 , STO609 and Compound C .CONCLUSION:LG promotes the expression of FNDC5 via GLP-1 receptor in the C2C12 myotubes possibly through activation of the CAMKK2/AMPK signaling pathways .

Músculo esquelético y lesión por reperfusión. ultraestructura, alteración y regeneración: revisión sistemática / Skeletal muscle and reperfusion injury: ultrastructure, disease and regeneration: systematic review

Introducción: La función tisular se basa en la asociación celular y la comunicación mediante uniones intercelulares o la matriz extracelular, que compone el tejido conectivo. La isquemia conlleva a cambios de lesión a los cuales las células responden según duración e intensidad del estímulo de lesión. En periodos cortos de isquemia y prolongados de reperfusión, el tejido muscular estriado esquelético presenta cambios en la predominancia de los tipos de fibras musculares y en los componentes de la matriz extracelular intramuscular. Objetivo: Establecer los cambios que se presentan en el músculo esquelético durante la reperfusión prolongada, tanto en las fibras musculares como en su matriz extracelular. Métodos: Se realizó una revisión sistemática mediante la búsqueda de artículos en inglés y español publicados en revistas indexadas en las bases de datos Ovid Medline, PubMed, Wiley y Science Direct. Los descriptores MESH utilizados fueron skeletal muscle, ischemia, reperfusion, fiber type fast twitch, fiber type slow twitch, sarcomere and myoblast. Se acoplaron los términos histology y tissue. Resultados: Se seleccionaron 81 publicaciones y se complementó con imágenes de músculos esqueléticos provenientes de muestras procesadas en el Laboratorio de Histología de la Universidad del Valle, Colombia. Conclusión: La recuperación del músculo durante la reperfusión seguida de isquemia, tiende hacia el patrón histológico y funcional normal. En algunos casos es un proceso lento y que aún después de varios meses no ha finalizado. Así mismo, pueden persistir alteraciones leves o moderadas en la contracción muscular, dados los cambios que se presentan en la matriz extracelular intramuscular.

Introduction: The tissue function is based on the cell association andcommunication through junctions or the extracellular matrix, which comprisesconnective tissue. Ischemia injury leads to changes to which the cells respondand it depends on duration and intensity of stimulus injury. In short periodsof prolonged ischemia and reperfusion, skeletal striated muscle tissue showschanges in the predominance of muscle fiber types and components of theextracellular matrix intramuscular. Objective: To determine the changes whichoccur in skeletal muscle during prolonged reperfusion in both muscle fibersin its extracellular matrix. Methods: A systematic review was performed bysearching for articles in English and Spanish published in journals indexed indatabases Ovid Medline, PubMed, Science Direct and Wiley. MeSH descriptorsused were skeletal muscle, ischemia, reperfusion, fast twitch fiber type, slowtwitch fiber type, sarcomere and myoblast. The terms tissue and histology werecoupled. Results: 81 relevant publications were selected and supplementedwith images of skeletal muscles from samples processed at the Laboratory ofHistology of the Universidad del Valle, Colombia. Conclusion: The recoveryof muscle during ischemia followed by reperfusion, tends toward the normalhistological and functional pattern. In some cases it is a slow process and evenafter several months has not been completed. Likewise, they may persist mildor moderate alterations in muscle contraction, given the changes that occur inthe intramuscular extracellular matrix.

Effect of different concentrations of calpain inhibitor ALLN on proliferation and apoptosis of C2C12 myoblasts / 中华临床营养杂志

Objective To explore the effect of different concentrations of ALLN on proliferation and apoptosis of C2C12 myoblasts.Methods After intervention with Ca2+ and ALLN,methyl thiazolyl tetrazolium and flow cytometry were used to determine the effect of Ca2+ and ALLN on the proliferation and apoptosis of C2C12 cells,respectively.The morphological changes of C2C12 myoblasts were observed using Giemsa staining.Results The absorbance of Ca2 + group was significantly lower than that of the control group (P ＜0.05).After 6,12,24,36 hours of intervention,the absorbance in ALLN groups 1 to 7 (cultured in serum-free media containing 16 mmol/L Ca2+ and ALLN at final concentrations of 3.125,6.25,12.5,25,50,100,200 μmol/L) were all significantly higher than that in the 16 mmol/L Ca2+ group (after 6 hours:0.449±0.024,0.472±0.022,0.513 ±0.008,0.540±0.014,0.588±0.016,0.607±0.030,0.700±0.020 vs.0.355 ±0.012,all P =0.000; after 12 hours:0.407 ±0.007,0.414 ±0.006,0.434 ±0.004,0.441 ±0.003,0.460 ±0.010,0.484 ± 0.006,0.525 ± 0.006 vs.0.368 ± 0.027,all P =0.000; after 24 hours:0.436±0.005,0.431 ±0.015,0.441 ±0.006,0.459 ±0.013,0.527 ±0.009,0.581 ±0.005,0.599 ±0.011 vs.0.386 ± 0.007,all P =0.000 ; after 36 hours:0.464 ± 0.022,0.460 ± 0.018,0.461 ± 0.007,0.434 ± 0.020,0.454 ± 0.028,0.479 ± 0.006,0.524 ± 0.011 vs.0.379 ± 0.011,all P =0.000),while no significant differences were observed after 48-72 hours of intervention.After treatment for 36 hours,the apoptosis rate in ALLN 10,50,100,and 200 μmol/L groups were (6.00 ± 1.20) ％,(5.02 ± 1.13) ％,(4.89±1.11)％,and (2.71 ± 1.15)％,all significantly lower than that in the Ca2+ group [(13.70 ±2.30)％] (all P =0.000).Giemsa staining showed apoptotic morphological changes in the Ca2+ group,which were obviously alleviated in the ALLN group.Conclusions Ca2+ at a concentration of 16mmol/L can induce apoptosis of C2C12 cells.In contrast,ALLN can inhibit cell apoptosis and promote proliferation in a time-and dose-dependent manner.

Cigarette smoke extract promotes senescence by reducing expression of HDAC2 in C2C12 myoblasts / 中国病理生理杂志

AIM:To investigate whether the cigarette smoke extract (CSE) causes senescence of C2C12 myo-blasts and the relationship between senescence and histone deacetylase 2(HDAC2).METHODS: Murine C2C12 cells were induced to differentiate into myoblasts.The HDAC2 activator and inhibitor were used to investigate the effects of CSE in the myoblasts on cell senescence and the expression of HDAC2.The expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blot, respectively, and the positive cell rate of β-galactosidase staining for cell senescence was also detected.RESULTS:The optimal concentration of CSE was 60 mL/L and the intervention time was 24 h.After the intervention of CSE, the positive cell rate ofβ-galactosidase staining was increased, accompanied with the reduction of HDAC2 expression at mRNA and protein levels.The expression of HDAC2 at mRNA and protein levels was increased by 4, 5, 6, 7-tetrabromobenzotriazole (TBB), accompanied with the reduction of positive cell rate ofβ-galacto-sidase staining.Furthermore, when HDAC2 expression at mRNA and protein levels was reduced by HDAC2 inhibitor valp-roic acid, the positive cell rate of β-galactosidase staining was increased.CONCLUSION: CSE promotes the senescence by reducing the expression of HDAC2 in C2C12 myoblasts.

The microRNAs regulating transplantation skeletal myoblasts may improve heart function after myocar-dial infarction / 心血管康复医学杂志

The microRNAs is a kind of endogenous and non-coding small RNA ,which possesses biological function negatively regulating gene expression.Recent researches also found that artificial controlling some microRNAs ex-pressions can improve heart function via regulating skeletal myoblasts through multiple mechanisms,so may become a new breakthrough point in myocardial infarction treatment area.

Construction of Human Hepatocyte Growth Factor Gene Recombinant Lentivirus Vectors and Its Expression in MyobIasts / 听力学及言语疾病杂志

Objective To construct a gene recombinant lentiviral vector pCMV -G -U6 -hHGF and detect its expression in C2C12 myoblast cells .Methods hHGF gene fragments were obtained and purified by RT -PCR method ,and were cloned to pCMV -G&NR -U6 ,then the restructured lentiviral vector was transformed into e . coli DH5 alpha ,the positive colonies were identified by BamHI and Hind Ⅲ enzyme digestion .The selected positive colonies were tested by PCR and sequencing analysis .The expression plasmids and packing plasmids were co -trans_fected into 293 T cells ,and virus titer was observed under the fluorescence microscope .Furthermore ,transfected C2C12 cells with lenti virus ,and the expression of HGF was detected by PCR and WB methods .ResuIts PCR and sequencing analysis showed that the lentiviral vector was constructed correctly and successfully ,the virus titer was above 1 x 109 IU/mL .The results of PCR and WB showed that HGF expression level in the lentiviral vector group was much higher than those of in blank control and negative control groups ,and yet the expression was stable after 72 hours .ConcIusion The lentiviral vector pCMV -G﹠NR -U6 -hHGF has been successfully constructed ,and stable expressed in C2C12 cells .It provides references for experimental study in the fields of the denervated skeletal muscle fibrosis and nerve regeneration treatment .

Influence of lentivirus modification of human PERK gene on endoplasmic reticulum stress-mediated apoptosis / 第二军医大学学报

Objective: To construct the lentivirus vector targeting human protein kinase R-like endoplasmic reticulum kinase (PERK) gene, and to study the effect of lentivirus modification of human PERK gene on endoplasmic reticulum (ER) stress-mediated apoptosis in C2C12 cells.

Damage to skeletal muscle cells leads to delayed muscle soreness:how to effectively improve muscle recovery both in recovery speed and quality / 中国组织工程研究

BACKGROUND:There is no simple and effective method to relieve delayed muscle soreness. OBJECTIVE:To conclude the injured mechanism and therapies of delayed muscle soreness by reviewing literature about damage and repair of the skeletal muscle. METHODWanfang and PubMed databases (from January 1991 to January 2014) were retrieved for articles related to morphological structure of the skeletal muscle, mechanism of delayed muscle soreness, and treatment and repair of the skeletal muscle using the keywords of“molecular mechanisms;delayed onset muscle soreness;pain;skeletal muscle;injury”in Chinese and English, respectively. Final y, 24 articles were included in result analysis. RESULTS AND CONCLUSION:Studies have shown that skeletal muscle injury is related to calcium imbalance, energy imbalance and high concentration of active oxygen. Skeletal muscle injury includes metabolic injury, mechanical injury and inflammatory injury. Insulin-like growth factor, peroxisome proliferator-activated receptorγ-coactivator-1αpromoter and tumor necrosis factorαplay important roles in skeletal muscle repair process. Animal experiments have demonstrated that edaravone may reduce secondary damage and inflammatory infiltration by means of directly preventing rapid peroxidation injury of free radicals in the skeletal muscle. Clinical studies have shown that Chinese medicine preparations, massage and acupuncture can delay the occurrence of exercise-induced muscle injury and fatigue, to improve the speed and quality of the recovery of damaged muscles. The treatment of delayed muscle soreness can achieve satisfactory results by combining physiotherapy with traditional Chinese medicine.

Caspase-9 in myoblasts is involved in mechanical signal transdunction under cyclic stretch / 中国组织工程研究

BACKGROUND:The adaptive reconstruction of maxil ofacial muscles would happen when functional orthopedic treatment is done to cure micromaxil ary deformity. The myoblast is the main responder in the process of adaptive reconstruction, and cyclic stretch can induce apoptosis of myoblasts. Caspase-9 is an important factor in the mitochondrial apoptosis pathway. OBJECTIVE:To investigate the expression of Caspase-9 in different cyclic stretch. METHODS:Based on myoblasts cultured in vitro-mechanical stimulation model, the rat L6 myoblasts were loaded stretch for 1, 6, 12 and 24 hours through multi-channel cellstress loading system, while the control group received no stretch. The morphological change and growth of myoblasts were observed under inverted phase contrast microscope;the expression of the mRNA and protein of Caspase-9 were detected by RT-PCR and western blot analysis, respectively. RESULTS AND CONCLUSION:Under inverted phase contrast microscope, the rat L6 myoblasts at cyclic stretch maintained a good growth state and biological characteristics;there was no celldegeneration;and the loss rate was extremely low, which could demonstrate that myoblast in vitro-mechanical stimulation model was established successful y. The results of RT-PCR and western blot analysis showed that, the expression of Caspase-9 mRNA and Cleaved Caspase-9 protein was significantly increased as the loading time prolonged, and the expression of Procaspase-9 protein was significantly decreased as the time. We can conclude that Caspase-9 is involved in the mechanical signal transduction of cyclic stretch.

Effects of cyclic stretch on in vitro culture-tensile stimulation models of myoblasts / 中国组织工程研究

BACKGROUND:Endoplasmic reticulum stress participates in the occurrence and development of many diseases, such as atherosclerosis, diabetes, and Alzheimer’s disease. GRP78 is a marker of endoplasmic reticulum stress. The expression of GRP78 reflects the degree of endoplasmic reticulum stress. OBJECTIVE:To investigate the effect of cyclic stretch on GRP78 expression of L6 rat myoblasts, and to identify the relationship between cyclic stretch and endoplasmic reticulum stress. METHODS:In vitro culture-tensile stimulation models of myoblasts of L6 rats were established successful y. The expression of GRP78 of myoblasts exposed to cyclic stretch was determined by reverse transcription-PCR and western blot assay. Stretch groups were subjected to 15%surface elongation at a frequency of 10 cycles per minute, over a period of 1, 6, 12 and 24 hours. cells were simultaneously seeded on a plate in the control and experimental groups with no stimulation. RESULTS AND CONCLUSION:The expression of GRP78 mRNA was continuously elevated over time after stretched treatment, and significant differences were detected as compared with the control group (P<0.05). GRP78 protein expression began to increase at 1 hour after stretched treatment, was significantly increased at 6 hours, peaked at 24 hours, and significant differences were visible as compared with the control group (P<0.05). In conclusion, cyclic stretch induced the occurrence of endoplasmic reticulum stress, which was enhanced with prolonged time. However, prolonged stretch caused severe endoplasmic reticulum stress and leaded to apoptosis of myoblasts.

Exogenous VEGF165 regulates skeletal myoblasts transplantation induced Th17/Treg imbalance / 中华胸心血管外科杂志

Objective To investigate whether Th17/Treg imbalance existed,and whether VEGF165 attenuated the imbalance in allogeneic skeletal myoblasts transplantation(allo-SMT) for acute myocardial infarction (AMI).Methods There were 60 C57BL/6 male mice,3 to 4 weeks old,weighting 0.016-0.020 kg.On the day 1,2,4,and 7 after allo-SMT,the percentages of Th17 and Treg cells,and the ratios of Th17/Treg cells were analyzed by FCM in AMI group,AMI-S group(alloSMT) and AMI-Ⅴ group(with VEGF165 treatment).Subsequently,related proinflammatory and regulatory cytokines and key transcription factors ROR-γt mRNA and Foxp3 mRNA expression were examined by Bio-plex and RT-PCR respectively.Results On the day 1,2,4,and 7 after allo-SMT,the percentage of Treg,related cytokines concentrations and transcript factor Foxp3 mRNA in AMI-S group were lower than those in AMI group,while the AMI-Ⅴ group were higher than those in AMI groups.However,the percentage of Th17 cells,related cytokines concentrations,and ROR-γt mRNA in AMI-S group were higher than those in AMI group; but the AMI-Ⅴ group were lower than those in AMI group.Apparently,compared with AMI group,the ratios of Th17/Treg significantly ascended in AMI-S group and decended in AMI-Ⅴ group.Conclusion Th17/Treg imbalance participated in the formation and development of inflammation and immune response after allo-SMT.However,transfected VEGF165 could relieve the severity of Th17/Treg imbalance.

Bone morphogenetic protein 2-induced C2C12 and MC3T3-E1 osteoblast differentiation and autophagy / 中国组织工程研究

BACKGROUND:A series of studies indicate that autophagy is closely linked with differentiation. Bone morphogenetic protein 2 (BMP-2) is the classical pathway for C2C12 and MC3T3-E1 osteoblast differentiation, and the ideal model to study osteogenic differentiation process. OBJECTIVE:To observe the relationship between autophagy and BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. METHODS:Real-Time PCR was applied to detect osteogenic differentiation and autophagy related index after C2C12 and MC3T3-E1 were induced with BMP-2 (100μg/L) for 72 hours. The osteogenic index alkaline phosphatase in BMP-2-induced MC3T3-E1 and C2C12 cultured with different concentrations of 3-methyladenine (0, 1, 5, 10 mmol/L) was determined with alkaline phosphatase staining. Western blot analysis was applied to detect LC3-I/II expression levels in C2C12 and MC3T3-E1 induced with BMP-2 for different time points (0, 12, 24, 48, 72, 96 hours). RESULTS AND CONCLUSION:The autophagy-related mRNA and protein expression showed an increasing tendency and autophagy-related protein LC3 levels was increased, which was associated with the time, during the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. Meanwhile, alkaline phosphatase expression levels were inhibited by autophagy in the process of osteogenic differentiation. Therefore, there is a close relationship between autophagy and the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation.