Aim This study sought to investigate the effect of chronic nicotine exposure on vascular function and to identify the underlying mechanisms. Methods Isolated organ bath studies were performed to examine the effects of...Aim This study sought to investigate the effect of chronic nicotine exposure on vascular function and to identify the underlying mechanisms. Methods Isolated organ bath studies were performed to examine the effects of chronic nicotine exposure on vascular reactivity of the aorta in Sprague-Dawley rats. We used various analogues and blockers of the cGMP-dependent protein kinase （PKG） pathway as well as molecular techniques to identify the un- derlying mechanisms. Results Chronic nicotine exposure reduced periaortic fat and specifically enhanced smooth muscle relaxation, although aortic adventitial fat and endothelium function were not affected. The soluble guanylyl cyclase inhibitor ODQ or PKG inhibitor Rp-8-Br-PET-cGMP abolished the difference in relaxation between the sa- line and nicotine group, and the cGMP analogue 8-Br-cGMP mimicked the difference in relaxation. PKG protein expression and activity were not altered after nicotine treatment. Conclusion Chronic nicotine exposure enhances vascular smooth muscle relaxation through a cGMP-dependent PKG pathway. Our findings provide novel insights in- to nicotine pharmacology.展开更多
Background and Aim Vascular smooth muscle cell （SMC） phenotype change is a hallmark of vascu-lar remodeling, which can be regulated via MicroRNAs （miRNAs）-dependent mechanism. We recently identified Asymmetric dim...Background and Aim Vascular smooth muscle cell （SMC） phenotype change is a hallmark of vascu-lar remodeling, which can be regulated via MicroRNAs （miRNAs）-dependent mechanism. We recently identified Asymmetric dimethylarginine （ADMA） positively correlates to vascular remodeling-based diseases. Here, we hy-pothesized that ADMA induces SMC phenotypic change via a miRNA-dependent mechanism. Methods and Results Microarray analysis enabled the identification of 7 deregulated microRNAs in ADMA-treated human aortic artery smooth muscle cells （hASMCs）. miR-182 was validated by real-time-PCR. Isobaric tags for relative and absolute quantitation （iTRAQ） based analysis of the hASMC proteome revealed that transfection of an miR-182 inhibitor sig- nificantly increased myeloid-associated differentiation marker （MYADM）, which was verified using Western blot and reporter activity quantization with the MYADM 3＇-UTR dual-luciferase reporter system, miR-182 knockdown further repressed Sprouty2 and enhanced MYADM, leading to ERICZMAP kinase-dependent and MYADM-depend- ent hASMC phenotypic change including proliferation, migration and differentiation marker gene expression change. In vivo, adeno-miR-182 markedly suppressed carotid neointimal formation by using balloon-injured rat carotid artery model, specifically via decreased MYADM expression. Atherosclerotic lesions from patients with high ADMA plas- ma levels exhibited decreased miR-182 expression levels and elevated MYADM expression levels. In patients with coronary heart disease （n- 164）, the miR-182 expression level in plasma was negatively correlated with the plas- ma ADMA levels. Conclusions miR-182 is a novel SMC phenotypic modulator by targeting MYADM and can be a potential therapeutic target combating vascular remodeling-associated diseases. Reduced plasma miR-182 levels might be a new predictor of high vascular remodeling risk especially in patient with coronary heart disease.展开更多
Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C ch...Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C changingin aonormai cyclic stretcn and its roles in VSMC apoptosis.Methods and results Our previous vascular proteomics study revealed that LaminA/C is mechano-sensitive molecule.When VSMCs are subjected to cyclic stretch,the expression of LaminA/C is significantly changed which participates dysfunctions of VSMCs during hypertension.However,the molecular mechanism involved in regulation of LaminA/C expression and the role of LaminA/C in the VSMC apoptosis during cyclic stretch application are still unclear.In the present study,VSMCs were subjected to different amplitudes of cyclic steetch in vitro:5%cyclic stretch(physiological strain)or 15%cyclic stretch(pathological strain).The expression of 2 different selective cleavage isomers of LaminA/C,i.e.LaminA and LaminC,and the apoptosis of VSMCs were detected.The results showed that compared with 5%group,15%cyclic stretch significantly decreased the expression of LaminA and LaminC,and promoted the apoptosis of VSMCs.Using specific small interfering RNA(siRNA)transfection which targets on LMNA the encoding gene of LaminA/C,the expression of LaminA and LaminC in VSMCs was significantly decreased,and the apoptosis was significantly increased.In order to study the molecular mechanism involved in cyclic stretch regulating the expression of LaminA/C,we focused on the microRNA(miR).Bioinformatics analysis showed that the 3’untranslated region(3’UTR)of LMNA has two potential binding sites to miR-124-3p.Double luciferase reported system revealed that both sites have binding abilities to miR-124-3p.Under static condition,miR-124-3p inhibitor significantly up-regulated the expression levels of LaminA and LaminC,while the miR-124-3p mimics significantly down-regulated them.RT-PCR results showed that 15%cyclic stretch significantly up-regulated the expression of miR-124-3p compared with 5%cyclic stretch.Furthermore,in order to study the role of changeed LaminA/C in VSMC apoptosis,LMNA-specific siRNA was transfected to repress the expression of LaminA/C in VSMCs,and Protein/DNA microarray was used to detecte the activity of transcription factors.The transcription factors whose activity were changed significantly(increase or decrease more than 2 times)were analyzed by cluster analysis and ingenurity pathway analysis(IPA).Six transcription factors associated with apoptosis were screened,in which TP53 was activated by the specific siRNA transfection and the other 5 were inavtived,including TP53,CREB1,MYC,STAT1/5/6 and JUN.Using abdominal aorta coarctation hypertensive model,the change of miR-124-3p in VSMCs was explored in vivo.A marked increase of miR-124-3p in thoracic aorta was revealed compared with the sham-operated controls,and in situ FISH revealed that this increase was mainly in the VSMCs.Conclusions The present study suggest that abnormally increased cyclic stretch(15%)up-regulates the expression of miR-124-3p in VSMCs,which subsequently targets on the 3’UTR of LMNA and decreases the expression of nuclear envelope protein LaminA/C;the repressed LaminA/C may play an important role in the apoptosis of VSMCs by regulating the activity of virious transcription factors,such as TP53,CREB1,MYC,STAT1/5/6 and JUN.The present study may provide a new insight into understanding the molecular mechanisms of vascular remodeling.展开更多
Norepinephrine(NE)endogenously released following electrical field stimulation(prazosin and TTX sensitive responses),produced a biphasic contraction of the rat vas deferens(RVD).The initial transient contraction was d...Norepinephrine(NE)endogenously released following electrical field stimulation(prazosin and TTX sensitive responses),produced a biphasic contraction of the rat vas deferens(RVD).The initial transient contraction was decreased by 30μmol/L ryanodine andμmol/L nifedipine while the secondary component was abolished by 2μmol/L nifedipine but increased by 30μmol/L ryanodine.Exogenously added NE produced biphasic contractions of the RVD.These contractions were inhibited by 2μmol/L nifedipine.Ryanodine(30μmol/L)decreased both phases by about 50%.We conclude that ryanodine binding sites reside in RVD endoplasmic reticulum(ER).There was a lack of uniformity in the effect of ryanodine against different phases of alpha-adrenergic stimulation may be indicative of two modes of stimulation-contraction coupling process related to this stimulation.展开更多
Background and Objective In-stent restenosis(ISR)remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents,whereas the mechanism of ISR has not be...Background and Objective In-stent restenosis(ISR)remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents,whereas the mechanism of ISR has not been fully clarified.In the present study,we sought to investigate the potential association of serum soluble TREM-1(sTREM-1)levels with the incidence of ISR.The role of TREM-1 was evaluated in cultured vascular smooth muscle cells(VSMCs).展开更多
Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches ...Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches had found out that atherosclerotic le-展开更多
Observation of stilbene dropping pill and yiqi drug-containing serum influence mechanism of vascular smooth muscle proliferation， cell cycle and Cyclin D1 and CDK4Choose male SD rats were randomly divided into 2 gr...Observation of stilbene dropping pill and yiqi drug-containing serum influence mechanism of vascular smooth muscle proliferation， cell cycle and Cyclin D1 and CDK4Choose male SD rats were randomly divided into 2 groups， lavage qishen yiqi pill and the gastric saline group，extract the drug-containing serum and normal serum；To set the two groups of serum respectively different concentrations，concentration in different time by CCK8 detection effects on vascular smooth muscle cell proliferation， select best concentration and action time.Flow cytometry instrument and high-throughput screening detect serum medicated effect on vascular smooth muscle cell cycle；Western blot detect the drug-containing serum of cell cycle protein Cyclin D1 and CDK4 expression.Result is 5%， 10% medicated serum inhibits cell proliferation significantly higher than the normal serum concentrations of same within 24 h， 48 h.G1 phase cells 5% medicated serum group was obviously higher than that of 5% in normal group (P<005)， serum and cell proliferation index significantly less than 5% normal serum group (P<005)，At the same time， Cyclin D1 and CDK4 expression significantly less than 5% normal serum group (P<005).Conclusion serum of qishen yiqi pill can inhibit vascular smooth muscle cell proliferation， may be through inhibiting cell cycle protein Cyclin D1 and CDK4 expression， block the cell cycle G1 process is closely related to the role.展开更多
Objective To establish a body composition analysis system based on chest CT,and to observe its value for evaluating content of chest muscle and adipose.Methods T7—T8 layer CT images of 108 pneumonia patients were col...Objective To establish a body composition analysis system based on chest CT,and to observe its value for evaluating content of chest muscle and adipose.Methods T7—T8 layer CT images of 108 pneumonia patients were collected(segmented dataset),and chest CT data of 984 patients were screened from the COVID 19-CT dataset(10 cases were randomly selected as whole test dataset,the remaining 974 cases were selected as layer selection dataset).T7—T8 layer was classified based on convolutional neural network(CNN)derived networks,including ResNet,ResNeXt,MobileNet,ShuffleNet,DenseNet,EfficientNet and ConvNeXt,then the accuracy,precision,recall and specificity were used to evaluate the performance of layer selection dataset.The skeletal muscle(SM),subcutaneous adipose tissue(SAT),intermuscular adipose tissue(IMAT)and visceral adipose tissue(VAT)were segmented using classical fully CNN(FCN)derived network,including FCN,SegNet,UNet,Attention UNet,UNET++,nnUNet,UNeXt and CMUNeXt,then Dice similarity coefficient(DSC),intersection over union(IoU)and 95 Hausdorff distance(HD)were used to evaluate the performance of segmented dataset.The automatic body composition analysis system was constructed based on optimal layer selection network and segmentation network,the mean absolute error(MAE),root mean squared error(RMSE)and standard deviation(SD)of MAE were used to evaluate the performance of automatic system for testing the whole test dataset.Results The accuracy,precision,recall and specificity of DenseNet network for automatically classifying T7—T8 layer from chest CT images was 95.06%,84.83%,92.27%and 95.78%,respectively,which were all higher than those of the other layer selection networks.In segmentation of SM,SAT,IMAT and overall,DSC and IoU of UNet++network were all higher,while 95HD of UNet++network were all lower than those of the other segmentation networks.Using DenseNet as the layer selection network and UNet++as the segmentation network,MAE of the automatic body composition analysis system for predicting SM,SAT,IMAT,VAT and MAE was 27.09,6.95,6.65 and 3.35 cm 2,respectively.Conclusion The body composition analysis system based on chest CT could be used to assess content of chest muscle and adipose.Among them,the UNet++network had better segmentation performance in adipose tissue than SM.展开更多
Aim Angiotensin II （AngII） induces vascular smooth muscle cell （VSMC） migration and growth, which is responsible for vascular remodeling during some cardiovascular diseases. It has been demonstrated to activate a ...Aim Angiotensin II （AngII） induces vascular smooth muscle cell （VSMC） migration and growth, which is responsible for vascular remodeling during some cardiovascular diseases. It has been demonstrated to activate a C1 current, but the underlying mechanism is not clear. Methods Whole-cell patch clamp, co-immunoprecipitation （co-IP）, site-specific mutagenesis, angiotensinII-infusion hypertensive mice model were used. Results In VSMCs, AngII could induce a C1C-3-dependent C1- current that was abolished in C1C-3 null mice. The activation mechanism of this AngII-induced C1- current was ascribed to the interaction between C1C-3 and Rho-kinase 2 （ROCIL2）, as re- vealed by N-terminal or C-terminal truncation of C1C-3, ROCIC2 siRNA and Co-IP experiments. Then we searched for and identified the phosphorylation site of C1C-3 at threonine 532 is critical for AngII-induced C1- current and VSMC migration through ROCK. The C1C-3 T532D mutant （mutation of threonine 532 to aspartate）, mimicking the phos- phorylation state of C1C-3, significantly potentiated AngII-induced C1- current and VSMC migration; while C1C-3 T532A （mutation of threonine 532 to alanine） had the opposite effects. Furthermore, we found a remarkably de- creased AngII-induced VSMC migration in C1C-3 null mice that is insensitive to Y27632, an inhibitor of ROCIL2. In addition, AngII-induced cerebrovascular remodeling was ameliorated in C1C-3 null mice, possibly by ROCIL2 path- way. Conclusions C1C-3 protein phosphorylation at threonine 532 by ROCIL2 is required for AngII-induced C1- cur- rent and VSMC migration that are involved in AngII-induced hypertensive vascular remodeling.展开更多
Muscle satellite cells,as muscle stem cells,play a critical role in the process of muscle regeneration,and effective muscle regeneration helps to restore muscle function and maintain the homeostasis of muscle tissues....Muscle satellite cells,as muscle stem cells,play a critical role in the process of muscle regeneration,and effective muscle regeneration helps to restore muscle function and maintain the homeostasis of muscle tissues.In damaged muscle,muscle satellite cells are activated to form new myofibers through the process of cell proliferation,migration,differentiation and fusion to complete muscle tissue regeneration.Meanwhile,this process is mainly affected by endogenous gene expression and many exogenous factors.Researches in recent years have shown that vitamins,as important nutrients,play an extremely important role in the process of muscle regeneration.Therefore,this article reviewed the roles of vitamins in the regeneration of muscle satellite cells,according to the latest research progress.It would provide more theoretical and data support for the regeneration and repair of muscle damage,muscle atrophy and other muscle diseases,so that it could be better applied in the field of muscle regeneration researches and serve human health.展开更多
OBJECTIVE Skeletal muscle undergoes rapid and profound atrophy in response to decreased mechanical loading,e.g.,limb immobilization and bed rest.Phosphatidylinositol 3 kinase(PI3K)/Akt signaling pathway is critical fo...OBJECTIVE Skeletal muscle undergoes rapid and profound atrophy in response to decreased mechanical loading,e.g.,limb immobilization and bed rest.Phosphatidylinositol 3 kinase(PI3K)/Akt signaling pathway is critical for regulating the balance between protein synthesis and degradation during disuse/inactivity-induced muscle atrophy.The present study aimed to investigate whether natural product Icaritin(ICT)required PI3K/Akt signaling to exert counteractive effect on skeletal muscle atrophy following mechanical unloading.METHODS Two oral dosages of ICT(80and 120mg·kg-1·d-1)were administrated daily to adult male rats with or without daily injection of PI3K/Akt signaling inhibitor wortmannin(15μg·kg-1·d-1)during 28-d hindlimb suspension(HS).Ex vivo muscle functional testing,histological and immunohistochemical analysis were performed to determine the changes of soleus muscle function,mean muscle fiber cross-sectional area(CSA)and fiber type distribution.Western blot and real-time PCR analysis were also performed to evaluate the protein or mRNA expression of the markers involved in PI3K/Akt signaling pathway.RESULTS After 28-d HS,soleus muscle underwent profound muscle atrophy(-52.7% muscle mass vs.pre-HS baseline).The high dose ICT treatment significantly attenuated the decreases in soleus muscle mass(+22.6% vs.HS),muscle fiber CSA(+52.8% vs.HS),as well as the muscle functional testing parameters during the unloading.Molecularly,the high dose ICT treatment significantly attenuated the decreases in protein synthesis markers at protein levels(phosphorylation of Akt and its downstream proteins)during the unloading,whereas the increases in protein degradation markers at mRNA(atrogin-1and MuRF-1)and protein(nuclear FOXO1 and FOXO3a)levels during the unloading were significantly attenuated by the high dose ICT treatment.The low dose ICT treatment moderately attenuated the above changes induced by the unloading.Mechanistically,Wortmannin could abolish the above effects of ICT.CONCLUSION ICT requires participation of PI3K/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading in a dose-dependent manner.展开更多
To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, th...To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.展开更多
The classic metallic Split Hopkinson Pressure Bar(SHPB)cannot capture the transmitted signal accurately when measuring soft biological tissue,because of the very low wave impedance and strength of this material.So the...The classic metallic Split Hopkinson Pressure Bar(SHPB)cannot capture the transmitted signal accurately when measuring soft biological tissue,because of the very low wave impedance and strength of this material.So the dynamic compressive response of porcine muscle has been investigated by using a modified SHPB.The forces on both ends of the sample measured using Polyvinylidene fluor(PVDF)transducers were applied to calculate the stress in the specimen instead of the strain gauge signal on the transmitted bar.Moreover,a circular cardboard disk pulse shaper was applied for generating a suitable incident pulse to achieve stress equilibrium and constant strain rates in the specimens.Then,the dynamic mechanical properties of porcine muscle parallel and perpendicular to the fiber directions were measured,and the stress equilibrium process during loading was analyzed,as well as the inertia-induced extra stress being corrected.Furthermore,quasi-static tests were conducted at two different strain rates to investigate the strain rate dependence using a universal material testing machine.The results show that the stress-strain curves are sensitive to strain rate in the two different loading directions.The compressive stress perpendicular to the fiber direction is stiffer than that parallel to the fiber direction.In addition,a strain rate-dependent constitutive model was developed based on the mechanical response of the muscle at different strain rates and fitted to the experimental data.The results show that the overall fit is good,and the constitutive model could describe the muscle's dynamic mechanical properties.展开更多
OBJECTIVE To assess the effects of polyprenols(isolated from Picea abies L.spruce needles)on muscle strength/tone and coordination,and to investigate whether polyprenols may protect atorvastatin-mediated muscle streng...OBJECTIVE To assess the effects of polyprenols(isolated from Picea abies L.spruce needles)on muscle strength/tone and coordination,and to investigate whether polyprenols may protect atorvastatin-mediated muscle strength/tone weakness in female Wistar rats.METHODS Polyprenols at doses of 1,10 and 20mg·kg-1,atorvastatin 80mg·kg-1 or their combination were administered once daily per os for 16 consecutive days in Wistar female rats(n=9-10 per group).Assessment of muscle strength was performed by grip strength test(on day 15)and wire hang test(on day 16).Rotarod test was used to measured locomotor coordination and muscle tone(on day 16).General locomotor activity was evaluated in open field test(on day 15).Assessment of plasma cholesterol level and creatine kinase activity was done on day 17.RESULTS Atorvastatin-treated rats exhibited a marked decrease in grasping strength and hanging time.PP(20mg·kg-1)significantly protected against atorvastatin-induced muscle weakness in grip strength test,and restored it to control values.At all doses,polyprenols prolonged hanging time which was decreased by atorvastatin in wire hang test.Polyprenols per se at 1 and 10mg·kg-1 did not show difference compared to control group animals,while only at 20mg·kg-1 h anging time was prolonged vs.control in wire hang test.No changes between control and tested groups were observed in rotarod and open field tests.Blood cholesterol level was not changed in any of tested groups in female Wistar rats.Polyprenols(20mg·kg-1)significantly(by 25%)increased plasma creatine kinase activity but it was not affected by the combined treatment.CONCLUSION Since polyprenols acted as protectors of atorvastatin-induced muscle weakness,the combination of polyprenols with atorvastatin may be helpful for reducing muscle-related side effects in patients receiving a long-term atorvastatin therapy.展开更多
Skeletal muscle accounts for 40%~45%of the body weight and is composed of a large number of parallel arrangement of muscle fiber bundles through attaching to the skeleton by the tendon.And robust skeletal muscles prov...Skeletal muscle accounts for 40%~45%of the body weight and is composed of a large number of parallel arrangement of muscle fiber bundles through attaching to the skeleton by the tendon.And robust skeletal muscles provide people the ability of daily activities such as walk,lift and so on.Muscle fibers are generated by multinucleated myotubes which form from the fusion of myoblasts with polarity development,and the synergistic effect between muscle fibers and spontaneous contraction provides support for the movement of the bodies and limbs.However,because of common clinical genetic defect,trauma,tumor,primary myopathy there are so many people cannot exercise like normal people.So it is an urgent task to treat these diseases.Traditional treatment methods are mainly through acupuncture,rehabilitation,drug treatment,etc.But these methods have long treatment times,poor efficacy,and high costs.Recently,the clinical researchers find that these diseases have similar pathological characteristics including structural damage,loss of skeletal muscle function,apoptosis and degradation of muscle fibers Therefore,there is academic and clinical value to study the repair and regeneration of skeletal muscle.Nowadays,tissue engineering provides a new idea for repairing damaged muscle tissue.Tissue engineering uses biological materials as a carrier to regulate the structural properties of materials.By applying biological/chemical/physical stimuli to mimic the microenvironment of the living body.And then construct a living tissue and transplant it into the body for wound repair.With the development of tissue engineering,we regulate the micro-structure of the cell matrix and applying mechanical stimulation to reconstruct a functional muscle microtissue in vitro.Such fabrication and mechanical loading strategy provide an easily adaptable platform to create functional muscle tissue constructs.And the mature muscle microtissues provide a useful tool for the clinical application to treat muscle injury.In this work,we regulate ECM microstructure and apply mechanical stimulation to construct three-dimensional muscle cell mechanical microenvironment.We first regulate the collagen fiber orientation by utilizing the characteristics of collagen gelation,which is a slow process and often need half an hour even more.Before gelation,we fabricate a PDMS mould with rectangle chamber and apply a strain along the long axis to a certain extent utilizing its elasticity.Then we pour collagen solution into the chamber and release the force a few minutes later and gelatinize.After gelation,we apply stretching on the end of the rectangle collagen hydrogel to promote further alignment.We adjust the strain loading time and the strain level to form different groups.Determining the optimal experimental conditions for myotube formation by counting the length and area ofthe myotube and immunofluorescence about relevant proteins.Later,we use western blot and RT-PCR to prove our result.It was found that the application of pre-strain and tensile stimulation can promote the alignment of collagen fibers.Results show that strain loading time and strain extent can regulate the morphology and function of myotubes.The muscle mechanical microenvironment we constructed promotes the formation and activity of polar multinuclear myotubes and builds mature muscle tissue,and determines the related molecular pathways and their transmission mechanisms during the mechanical conduction process.展开更多
The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-dow...The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-down was performed by RNA interference(RNAi)in cultured muscle cells and verified by Real-time PCR and Western blotting.In addition,cell viability,GSH content and GPx activity were assayed.The results showed that the mRNA level and protein expression of SelW were decreased successfully by 71.9%and 68.8%relative to control values,cell viability decreased by 21.5%,GSH increased by 29.76%,and GPx increased by 47.58%.WST assay showed that compared with blank control,the value of positive group dropped 21.5%;In GSH and GPx assay,compared with blank control the positive group increased29.76%and 47.58%separately.In conclusion,SelW knock-down by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity.These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.展开更多
Purpose： Fibronectin type III domain-containing protein 5 （FNDC5）, also known as irisin, is a myokine secreted from muscle in response to exercise and improves obesity and glucose homeostasis. However, the molecula...Purpose： Fibronectin type III domain-containing protein 5 （FNDC5）, also known as irisin, is a myokine secreted from muscle in response to exercise and improves obesity and glucose homeostasis. However, the molecular mecha- nisms that regulate FNDC5 expression and the functional significance of FNDC5 in skeletal muscle remain un- known. In this study, we explored the possible pathways that induce FNDC5 expression and delineated its metabol- ic effects on skeletal muscle. Methods： C2C12 myotubes were treated with various concentrations of Sp-cAMP, forskolin, and ionomycin respectively for various durations. FNDC5 and related metabolic genes＇ expressions were measured by quantitative real-time polymerase chain reaction （qRT-PCR）. Cyclic AMP responsive element-binding protein （CREB） phosphorylation was measured by Western blot. Oxidative phosphorylation was quantified by oxy- gen consumption rate （OCR） measurement using XF-96 analyzer （Seahorse Bioscience）. The statistical signifi- cance was calculated by one-way analysis of variance （ANOVA）. Data were considered significant when P 〈 0.05. Results： We found that cAMP and forskolin dose and time dependently increased FNDC5 expression in C2C12 myotubes. A synergistic effect of forskolin and ionomycin on FNDC5 expression was also found. CREB phosphoryl- ation was elevated in myotubes simultaneously upon these treatments. C2C12 myotubes over expressing CREB dis- plays increased FNDC5 expression as well, suggesting CREB was a regulator of FNDC5 expression. Functionally, irisin treatment enhanced mitochondrial biogenesis of C2C12 myotubes through increasing peroxisome proliferator- activated receptor gamma coactivator-1α （PGC-1α）, nuclear respiratory factor 1 （NRF1） and mitochondrial tran-scription factor A （TFAM） expressions, leading to increase myotube mitochondrial respirations and ATP produc- tion. Conclusions Our observation indicates that irisin is a metabolic modulator of skeletal muscle, whose expres- sion is controlled by cAMP pathway and intracellular level of calcium.展开更多
Myoblast differentiation is an essential process during skeletal muscle development.C2C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro.Dehydrogenase/reductase(SDR family...Myoblast differentiation is an essential process during skeletal muscle development.C2C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro.Dehydrogenase/reductase(SDR family)member 3(DHRS3)is a highly conserved member in short-chain alcohol dehydrogenase/reductase superfamily and has been shown to be involved in the metabolism of retinol.Previous experimental results showed that the expression of DHRS3 increased significantly during the differentiation of myoblasts differentiation.However,the effect of DHRS3 on mouse muscle cell differentiation was unclear.The objective of current study was to determine if DHRS3 affected muscle cell differentiation,and if DHRS3 was involved in muscle regeneration.Protein expression was determined by western blot and immunofluorescence analysis.The activation and inhibition of DHRS3 increased and decreased C2C12 myoblast differentiation respectively,which indicated that DHRS3 could affect C2C12 myoblast differentiation.DHRS3 expression was significantly changed during muscle regeneration,with the regeneration of muscle injury,the expression of DHRS3 tended to increase first and then decrease.It suggested that DHRS3 might be involved in muscle regeneration.In summary,this study confirmed the involvement of DHRS3 in C2C12 myoblast differentiation and mouse skeletal muscle regeneration and provided a theoretical basis for further elucidating the molecular mechanism of muscle development.展开更多
文摘Aim This study sought to investigate the effect of chronic nicotine exposure on vascular function and to identify the underlying mechanisms. Methods Isolated organ bath studies were performed to examine the effects of chronic nicotine exposure on vascular reactivity of the aorta in Sprague-Dawley rats. We used various analogues and blockers of the cGMP-dependent protein kinase （PKG） pathway as well as molecular techniques to identify the un- derlying mechanisms. Results Chronic nicotine exposure reduced periaortic fat and specifically enhanced smooth muscle relaxation, although aortic adventitial fat and endothelium function were not affected. The soluble guanylyl cyclase inhibitor ODQ or PKG inhibitor Rp-8-Br-PET-cGMP abolished the difference in relaxation between the sa- line and nicotine group, and the cGMP analogue 8-Br-cGMP mimicked the difference in relaxation. PKG protein expression and activity were not altered after nicotine treatment. Conclusion Chronic nicotine exposure enhances vascular smooth muscle relaxation through a cGMP-dependent PKG pathway. Our findings provide novel insights in- to nicotine pharmacology.
文摘Background and Aim Vascular smooth muscle cell （SMC） phenotype change is a hallmark of vascu-lar remodeling, which can be regulated via MicroRNAs （miRNAs）-dependent mechanism. We recently identified Asymmetric dimethylarginine （ADMA） positively correlates to vascular remodeling-based diseases. Here, we hy-pothesized that ADMA induces SMC phenotypic change via a miRNA-dependent mechanism. Methods and Results Microarray analysis enabled the identification of 7 deregulated microRNAs in ADMA-treated human aortic artery smooth muscle cells （hASMCs）. miR-182 was validated by real-time-PCR. Isobaric tags for relative and absolute quantitation （iTRAQ） based analysis of the hASMC proteome revealed that transfection of an miR-182 inhibitor sig- nificantly increased myeloid-associated differentiation marker （MYADM）, which was verified using Western blot and reporter activity quantization with the MYADM 3＇-UTR dual-luciferase reporter system, miR-182 knockdown further repressed Sprouty2 and enhanced MYADM, leading to ERICZMAP kinase-dependent and MYADM-depend- ent hASMC phenotypic change including proliferation, migration and differentiation marker gene expression change. In vivo, adeno-miR-182 markedly suppressed carotid neointimal formation by using balloon-injured rat carotid artery model, specifically via decreased MYADM expression. Atherosclerotic lesions from patients with high ADMA plas- ma levels exhibited decreased miR-182 expression levels and elevated MYADM expression levels. In patients with coronary heart disease （n- 164）, the miR-182 expression level in plasma was negatively correlated with the plas- ma ADMA levels. Conclusions miR-182 is a novel SMC phenotypic modulator by targeting MYADM and can be a potential therapeutic target combating vascular remodeling-associated diseases. Reduced plasma miR-182 levels might be a new predictor of high vascular remodeling risk especially in patient with coronary heart disease.
基金supported by grants from the National Natural Science Foundation of China( 11572199 and 11625209)
文摘Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C changingin aonormai cyclic stretcn and its roles in VSMC apoptosis.Methods and results Our previous vascular proteomics study revealed that LaminA/C is mechano-sensitive molecule.When VSMCs are subjected to cyclic stretch,the expression of LaminA/C is significantly changed which participates dysfunctions of VSMCs during hypertension.However,the molecular mechanism involved in regulation of LaminA/C expression and the role of LaminA/C in the VSMC apoptosis during cyclic stretch application are still unclear.In the present study,VSMCs were subjected to different amplitudes of cyclic steetch in vitro:5%cyclic stretch(physiological strain)or 15%cyclic stretch(pathological strain).The expression of 2 different selective cleavage isomers of LaminA/C,i.e.LaminA and LaminC,and the apoptosis of VSMCs were detected.The results showed that compared with 5%group,15%cyclic stretch significantly decreased the expression of LaminA and LaminC,and promoted the apoptosis of VSMCs.Using specific small interfering RNA(siRNA)transfection which targets on LMNA the encoding gene of LaminA/C,the expression of LaminA and LaminC in VSMCs was significantly decreased,and the apoptosis was significantly increased.In order to study the molecular mechanism involved in cyclic stretch regulating the expression of LaminA/C,we focused on the microRNA(miR).Bioinformatics analysis showed that the 3’untranslated region(3’UTR)of LMNA has two potential binding sites to miR-124-3p.Double luciferase reported system revealed that both sites have binding abilities to miR-124-3p.Under static condition,miR-124-3p inhibitor significantly up-regulated the expression levels of LaminA and LaminC,while the miR-124-3p mimics significantly down-regulated them.RT-PCR results showed that 15%cyclic stretch significantly up-regulated the expression of miR-124-3p compared with 5%cyclic stretch.Furthermore,in order to study the role of changeed LaminA/C in VSMC apoptosis,LMNA-specific siRNA was transfected to repress the expression of LaminA/C in VSMCs,and Protein/DNA microarray was used to detecte the activity of transcription factors.The transcription factors whose activity were changed significantly(increase or decrease more than 2 times)were analyzed by cluster analysis and ingenurity pathway analysis(IPA).Six transcription factors associated with apoptosis were screened,in which TP53 was activated by the specific siRNA transfection and the other 5 were inavtived,including TP53,CREB1,MYC,STAT1/5/6 and JUN.Using abdominal aorta coarctation hypertensive model,the change of miR-124-3p in VSMCs was explored in vivo.A marked increase of miR-124-3p in thoracic aorta was revealed compared with the sham-operated controls,and in situ FISH revealed that this increase was mainly in the VSMCs.Conclusions The present study suggest that abnormally increased cyclic stretch(15%)up-regulates the expression of miR-124-3p in VSMCs,which subsequently targets on the 3’UTR of LMNA and decreases the expression of nuclear envelope protein LaminA/C;the repressed LaminA/C may play an important role in the apoptosis of VSMCs by regulating the activity of virious transcription factors,such as TP53,CREB1,MYC,STAT1/5/6 and JUN.The present study may provide a new insight into understanding the molecular mechanisms of vascular remodeling.
基金This work was supported bv a granl-in-aid awarded by the Medical Re-search Council of Canadaa Career Investigator Awand(CY Kwan)from the Heart and Stroke Foun-dation of Ontario.
文摘Norepinephrine(NE)endogenously released following electrical field stimulation(prazosin and TTX sensitive responses),produced a biphasic contraction of the rat vas deferens(RVD).The initial transient contraction was decreased by 30μmol/L ryanodine andμmol/L nifedipine while the secondary component was abolished by 2μmol/L nifedipine but increased by 30μmol/L ryanodine.Exogenously added NE produced biphasic contractions of the RVD.These contractions were inhibited by 2μmol/L nifedipine.Ryanodine(30μmol/L)decreased both phases by about 50%.We conclude that ryanodine binding sites reside in RVD endoplasmic reticulum(ER).There was a lack of uniformity in the effect of ryanodine against different phases of alpha-adrenergic stimulation may be indicative of two modes of stimulation-contraction coupling process related to this stimulation.
文摘Background and Objective In-stent restenosis(ISR)remains a major limitation of percutaneous coronary intervention despite improvements in stent design and pharmacological agents,whereas the mechanism of ISR has not been fully clarified.In the present study,we sought to investigate the potential association of serum soluble TREM-1(sTREM-1)levels with the incidence of ISR.The role of TREM-1 was evaluated in cultured vascular smooth muscle cells(VSMCs).
基金supported by grants from the National Natural Science Foundation of China,Nos10732070,10702043,30970703,10972140 and 30470432
文摘Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches had found out that atherosclerotic le-
文摘Observation of stilbene dropping pill and yiqi drug-containing serum influence mechanism of vascular smooth muscle proliferation， cell cycle and Cyclin D1 and CDK4Choose male SD rats were randomly divided into 2 groups， lavage qishen yiqi pill and the gastric saline group，extract the drug-containing serum and normal serum；To set the two groups of serum respectively different concentrations，concentration in different time by CCK8 detection effects on vascular smooth muscle cell proliferation， select best concentration and action time.Flow cytometry instrument and high-throughput screening detect serum medicated effect on vascular smooth muscle cell cycle；Western blot detect the drug-containing serum of cell cycle protein Cyclin D1 and CDK4 expression.Result is 5%， 10% medicated serum inhibits cell proliferation significantly higher than the normal serum concentrations of same within 24 h， 48 h.G1 phase cells 5% medicated serum group was obviously higher than that of 5% in normal group (P<005)， serum and cell proliferation index significantly less than 5% normal serum group (P<005)，At the same time， Cyclin D1 and CDK4 expression significantly less than 5% normal serum group (P<005).Conclusion serum of qishen yiqi pill can inhibit vascular smooth muscle cell proliferation， may be through inhibiting cell cycle protein Cyclin D1 and CDK4 expression， block the cell cycle G1 process is closely related to the role.
文摘Objective To establish a body composition analysis system based on chest CT,and to observe its value for evaluating content of chest muscle and adipose.Methods T7—T8 layer CT images of 108 pneumonia patients were collected(segmented dataset),and chest CT data of 984 patients were screened from the COVID 19-CT dataset(10 cases were randomly selected as whole test dataset,the remaining 974 cases were selected as layer selection dataset).T7—T8 layer was classified based on convolutional neural network(CNN)derived networks,including ResNet,ResNeXt,MobileNet,ShuffleNet,DenseNet,EfficientNet and ConvNeXt,then the accuracy,precision,recall and specificity were used to evaluate the performance of layer selection dataset.The skeletal muscle(SM),subcutaneous adipose tissue(SAT),intermuscular adipose tissue(IMAT)and visceral adipose tissue(VAT)were segmented using classical fully CNN(FCN)derived network,including FCN,SegNet,UNet,Attention UNet,UNET++,nnUNet,UNeXt and CMUNeXt,then Dice similarity coefficient(DSC),intersection over union(IoU)and 95 Hausdorff distance(HD)were used to evaluate the performance of segmented dataset.The automatic body composition analysis system was constructed based on optimal layer selection network and segmentation network,the mean absolute error(MAE),root mean squared error(RMSE)and standard deviation(SD)of MAE were used to evaluate the performance of automatic system for testing the whole test dataset.Results The accuracy,precision,recall and specificity of DenseNet network for automatically classifying T7—T8 layer from chest CT images was 95.06%,84.83%,92.27%and 95.78%,respectively,which were all higher than those of the other layer selection networks.In segmentation of SM,SAT,IMAT and overall,DSC and IoU of UNet++network were all higher,while 95HD of UNet++network were all lower than those of the other segmentation networks.Using DenseNet as the layer selection network and UNet++as the segmentation network,MAE of the automatic body composition analysis system for predicting SM,SAT,IMAT,VAT and MAE was 27.09,6.95,6.65 and 3.35 cm 2,respectively.Conclusion The body composition analysis system based on chest CT could be used to assess content of chest muscle and adipose.Among them,the UNet++network had better segmentation performance in adipose tissue than SM.
文摘Aim Angiotensin II （AngII） induces vascular smooth muscle cell （VSMC） migration and growth, which is responsible for vascular remodeling during some cardiovascular diseases. It has been demonstrated to activate a C1 current, but the underlying mechanism is not clear. Methods Whole-cell patch clamp, co-immunoprecipitation （co-IP）, site-specific mutagenesis, angiotensinII-infusion hypertensive mice model were used. Results In VSMCs, AngII could induce a C1C-3-dependent C1- current that was abolished in C1C-3 null mice. The activation mechanism of this AngII-induced C1- current was ascribed to the interaction between C1C-3 and Rho-kinase 2 （ROCIL2）, as re- vealed by N-terminal or C-terminal truncation of C1C-3, ROCIC2 siRNA and Co-IP experiments. Then we searched for and identified the phosphorylation site of C1C-3 at threonine 532 is critical for AngII-induced C1- current and VSMC migration through ROCK. The C1C-3 T532D mutant （mutation of threonine 532 to aspartate）, mimicking the phos- phorylation state of C1C-3, significantly potentiated AngII-induced C1- current and VSMC migration; while C1C-3 T532A （mutation of threonine 532 to alanine） had the opposite effects. Furthermore, we found a remarkably de- creased AngII-induced VSMC migration in C1C-3 null mice that is insensitive to Y27632, an inhibitor of ROCIL2. In addition, AngII-induced cerebrovascular remodeling was ameliorated in C1C-3 null mice, possibly by ROCIL2 path- way. Conclusions C1C-3 protein phosphorylation at threonine 532 by ROCIL2 is required for AngII-induced C1- cur- rent and VSMC migration that are involved in AngII-induced hypertensive vascular remodeling.
文摘Muscle satellite cells,as muscle stem cells,play a critical role in the process of muscle regeneration,and effective muscle regeneration helps to restore muscle function and maintain the homeostasis of muscle tissues.In damaged muscle,muscle satellite cells are activated to form new myofibers through the process of cell proliferation,migration,differentiation and fusion to complete muscle tissue regeneration.Meanwhile,this process is mainly affected by endogenous gene expression and many exogenous factors.Researches in recent years have shown that vitamins,as important nutrients,play an extremely important role in the process of muscle regeneration.Therefore,this article reviewed the roles of vitamins in the regeneration of muscle satellite cells,according to the latest research progress.It would provide more theoretical and data support for the regeneration and repair of muscle damage,muscle atrophy and other muscle diseases,so that it could be better applied in the field of muscle regeneration researches and serve human health.
基金The project supported by National Natural Science Foundation of China(81201406)Direct Grant for Research,The Chinese University of Hong Kong(4054138)
文摘OBJECTIVE Skeletal muscle undergoes rapid and profound atrophy in response to decreased mechanical loading,e.g.,limb immobilization and bed rest.Phosphatidylinositol 3 kinase(PI3K)/Akt signaling pathway is critical for regulating the balance between protein synthesis and degradation during disuse/inactivity-induced muscle atrophy.The present study aimed to investigate whether natural product Icaritin(ICT)required PI3K/Akt signaling to exert counteractive effect on skeletal muscle atrophy following mechanical unloading.METHODS Two oral dosages of ICT(80and 120mg·kg-1·d-1)were administrated daily to adult male rats with or without daily injection of PI3K/Akt signaling inhibitor wortmannin(15μg·kg-1·d-1)during 28-d hindlimb suspension(HS).Ex vivo muscle functional testing,histological and immunohistochemical analysis were performed to determine the changes of soleus muscle function,mean muscle fiber cross-sectional area(CSA)and fiber type distribution.Western blot and real-time PCR analysis were also performed to evaluate the protein or mRNA expression of the markers involved in PI3K/Akt signaling pathway.RESULTS After 28-d HS,soleus muscle underwent profound muscle atrophy(-52.7% muscle mass vs.pre-HS baseline).The high dose ICT treatment significantly attenuated the decreases in soleus muscle mass(+22.6% vs.HS),muscle fiber CSA(+52.8% vs.HS),as well as the muscle functional testing parameters during the unloading.Molecularly,the high dose ICT treatment significantly attenuated the decreases in protein synthesis markers at protein levels(phosphorylation of Akt and its downstream proteins)during the unloading,whereas the increases in protein degradation markers at mRNA(atrogin-1and MuRF-1)and protein(nuclear FOXO1 and FOXO3a)levels during the unloading were significantly attenuated by the high dose ICT treatment.The low dose ICT treatment moderately attenuated the above changes induced by the unloading.Mechanistically,Wortmannin could abolish the above effects of ICT.CONCLUSION ICT requires participation of PI3K/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading in a dose-dependent manner.
基金Supported by the Ministry of Agricultural Nuarture of New Varieties Genetically Modified Organisms Significant Special Funding (2008ZX08007-002)
文摘To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.
基金supported by the National Natural Science Foundation of China(Grant No.11872215)the National Defense Basic Scientific Research program of China(Grant No.JCKYS2019209C001)the Fundamental Strengthening Program of the Military Science and Technology Commission Technical Field Foundation(2020-JCJQ-JJ-403).
文摘The classic metallic Split Hopkinson Pressure Bar(SHPB)cannot capture the transmitted signal accurately when measuring soft biological tissue,because of the very low wave impedance and strength of this material.So the dynamic compressive response of porcine muscle has been investigated by using a modified SHPB.The forces on both ends of the sample measured using Polyvinylidene fluor(PVDF)transducers were applied to calculate the stress in the specimen instead of the strain gauge signal on the transmitted bar.Moreover,a circular cardboard disk pulse shaper was applied for generating a suitable incident pulse to achieve stress equilibrium and constant strain rates in the specimens.Then,the dynamic mechanical properties of porcine muscle parallel and perpendicular to the fiber directions were measured,and the stress equilibrium process during loading was analyzed,as well as the inertia-induced extra stress being corrected.Furthermore,quasi-static tests were conducted at two different strain rates to investigate the strain rate dependence using a universal material testing machine.The results show that the stress-strain curves are sensitive to strain rate in the two different loading directions.The compressive stress perpendicular to the fiber direction is stiffer than that parallel to the fiber direction.In addition,a strain rate-dependent constitutive model was developed based on the mechanical response of the muscle at different strain rates and fitted to the experimental data.The results show that the overall fit is good,and the constitutive model could describe the muscle's dynamic mechanical properties.
基金The project supported by Pharma and Chemistry Competence Center of Latvia,Ltd.L-KC-11-0001with the co-financing of the European Regional Development Fund
文摘OBJECTIVE To assess the effects of polyprenols(isolated from Picea abies L.spruce needles)on muscle strength/tone and coordination,and to investigate whether polyprenols may protect atorvastatin-mediated muscle strength/tone weakness in female Wistar rats.METHODS Polyprenols at doses of 1,10 and 20mg·kg-1,atorvastatin 80mg·kg-1 or their combination were administered once daily per os for 16 consecutive days in Wistar female rats(n=9-10 per group).Assessment of muscle strength was performed by grip strength test(on day 15)and wire hang test(on day 16).Rotarod test was used to measured locomotor coordination and muscle tone(on day 16).General locomotor activity was evaluated in open field test(on day 15).Assessment of plasma cholesterol level and creatine kinase activity was done on day 17.RESULTS Atorvastatin-treated rats exhibited a marked decrease in grasping strength and hanging time.PP(20mg·kg-1)significantly protected against atorvastatin-induced muscle weakness in grip strength test,and restored it to control values.At all doses,polyprenols prolonged hanging time which was decreased by atorvastatin in wire hang test.Polyprenols per se at 1 and 10mg·kg-1 did not show difference compared to control group animals,while only at 20mg·kg-1 h anging time was prolonged vs.control in wire hang test.No changes between control and tested groups were observed in rotarod and open field tests.Blood cholesterol level was not changed in any of tested groups in female Wistar rats.Polyprenols(20mg·kg-1)significantly(by 25%)increased plasma creatine kinase activity but it was not affected by the combined treatment.CONCLUSION Since polyprenols acted as protectors of atorvastatin-induced muscle weakness,the combination of polyprenols with atorvastatin may be helpful for reducing muscle-related side effects in patients receiving a long-term atorvastatin therapy.
文摘Skeletal muscle accounts for 40%~45%of the body weight and is composed of a large number of parallel arrangement of muscle fiber bundles through attaching to the skeleton by the tendon.And robust skeletal muscles provide people the ability of daily activities such as walk,lift and so on.Muscle fibers are generated by multinucleated myotubes which form from the fusion of myoblasts with polarity development,and the synergistic effect between muscle fibers and spontaneous contraction provides support for the movement of the bodies and limbs.However,because of common clinical genetic defect,trauma,tumor,primary myopathy there are so many people cannot exercise like normal people.So it is an urgent task to treat these diseases.Traditional treatment methods are mainly through acupuncture,rehabilitation,drug treatment,etc.But these methods have long treatment times,poor efficacy,and high costs.Recently,the clinical researchers find that these diseases have similar pathological characteristics including structural damage,loss of skeletal muscle function,apoptosis and degradation of muscle fibers Therefore,there is academic and clinical value to study the repair and regeneration of skeletal muscle.Nowadays,tissue engineering provides a new idea for repairing damaged muscle tissue.Tissue engineering uses biological materials as a carrier to regulate the structural properties of materials.By applying biological/chemical/physical stimuli to mimic the microenvironment of the living body.And then construct a living tissue and transplant it into the body for wound repair.With the development of tissue engineering,we regulate the micro-structure of the cell matrix and applying mechanical stimulation to reconstruct a functional muscle microtissue in vitro.Such fabrication and mechanical loading strategy provide an easily adaptable platform to create functional muscle tissue constructs.And the mature muscle microtissues provide a useful tool for the clinical application to treat muscle injury.In this work,we regulate ECM microstructure and apply mechanical stimulation to construct three-dimensional muscle cell mechanical microenvironment.We first regulate the collagen fiber orientation by utilizing the characteristics of collagen gelation,which is a slow process and often need half an hour even more.Before gelation,we fabricate a PDMS mould with rectangle chamber and apply a strain along the long axis to a certain extent utilizing its elasticity.Then we pour collagen solution into the chamber and release the force a few minutes later and gelatinize.After gelation,we apply stretching on the end of the rectangle collagen hydrogel to promote further alignment.We adjust the strain loading time and the strain level to form different groups.Determining the optimal experimental conditions for myotube formation by counting the length and area ofthe myotube and immunofluorescence about relevant proteins.Later,we use western blot and RT-PCR to prove our result.It was found that the application of pre-strain and tensile stimulation can promote the alignment of collagen fibers.Results show that strain loading time and strain extent can regulate the morphology and function of myotubes.The muscle mechanical microenvironment we constructed promotes the formation and activity of polar multinuclear myotubes and builds mature muscle tissue,and determines the related molecular pathways and their transmission mechanisms during the mechanical conduction process.
基金supported by China Postdoctoral Science Foundation(LRB06-262)Funds of Northeast Forestry University
文摘The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-down was performed by RNA interference(RNAi)in cultured muscle cells and verified by Real-time PCR and Western blotting.In addition,cell viability,GSH content and GPx activity were assayed.The results showed that the mRNA level and protein expression of SelW were decreased successfully by 71.9%and 68.8%relative to control values,cell viability decreased by 21.5%,GSH increased by 29.76%,and GPx increased by 47.58%.WST assay showed that compared with blank control,the value of positive group dropped 21.5%;In GSH and GPx assay,compared with blank control the positive group increased29.76%and 47.58%separately.In conclusion,SelW knock-down by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity.These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.
文摘Purpose： Fibronectin type III domain-containing protein 5 （FNDC5）, also known as irisin, is a myokine secreted from muscle in response to exercise and improves obesity and glucose homeostasis. However, the molecular mecha- nisms that regulate FNDC5 expression and the functional significance of FNDC5 in skeletal muscle remain un- known. In this study, we explored the possible pathways that induce FNDC5 expression and delineated its metabol- ic effects on skeletal muscle. Methods： C2C12 myotubes were treated with various concentrations of Sp-cAMP, forskolin, and ionomycin respectively for various durations. FNDC5 and related metabolic genes＇ expressions were measured by quantitative real-time polymerase chain reaction （qRT-PCR）. Cyclic AMP responsive element-binding protein （CREB） phosphorylation was measured by Western blot. Oxidative phosphorylation was quantified by oxy- gen consumption rate （OCR） measurement using XF-96 analyzer （Seahorse Bioscience）. The statistical signifi- cance was calculated by one-way analysis of variance （ANOVA）. Data were considered significant when P 〈 0.05. Results： We found that cAMP and forskolin dose and time dependently increased FNDC5 expression in C2C12 myotubes. A synergistic effect of forskolin and ionomycin on FNDC5 expression was also found. CREB phosphoryl- ation was elevated in myotubes simultaneously upon these treatments. C2C12 myotubes over expressing CREB dis- plays increased FNDC5 expression as well, suggesting CREB was a regulator of FNDC5 expression. Functionally, irisin treatment enhanced mitochondrial biogenesis of C2C12 myotubes through increasing peroxisome proliferator- activated receptor gamma coactivator-1α （PGC-1α）, nuclear respiratory factor 1 （NRF1） and mitochondrial tran-scription factor A （TFAM） expressions, leading to increase myotube mitochondrial respirations and ATP produc- tion. Conclusions Our observation indicates that irisin is a metabolic modulator of skeletal muscle, whose expres- sion is controlled by cAMP pathway and intracellular level of calcium.
基金Supported by the Natural Science Foundation of Heilongjiang Province(C2017025)。
文摘Myoblast differentiation is an essential process during skeletal muscle development.C2C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro.Dehydrogenase/reductase(SDR family)member 3(DHRS3)is a highly conserved member in short-chain alcohol dehydrogenase/reductase superfamily and has been shown to be involved in the metabolism of retinol.Previous experimental results showed that the expression of DHRS3 increased significantly during the differentiation of myoblasts differentiation.However,the effect of DHRS3 on mouse muscle cell differentiation was unclear.The objective of current study was to determine if DHRS3 affected muscle cell differentiation,and if DHRS3 was involved in muscle regeneration.Protein expression was determined by western blot and immunofluorescence analysis.The activation and inhibition of DHRS3 increased and decreased C2C12 myoblast differentiation respectively,which indicated that DHRS3 could affect C2C12 myoblast differentiation.DHRS3 expression was significantly changed during muscle regeneration,with the regeneration of muscle injury,the expression of DHRS3 tended to increase first and then decrease.It suggested that DHRS3 might be involved in muscle regeneration.In summary,this study confirmed the involvement of DHRS3 in C2C12 myoblast differentiation and mouse skeletal muscle regeneration and provided a theoretical basis for further elucidating the molecular mechanism of muscle development.
