Galectin-1 is a novel factor that regulates myotube growth in regenerating skeletal muscles

Adult skeletal muscles have a vigorous regenerative capacity in response to chemical, mechanical or physical injuries. Muscle satellite cells play a critical role in skeletal muscle regeneration. Activated satellite cells (myoblasts) proliferate and then differentiate. Differentiated myoblasts fuse with each other to form multinucleated myotubes, and the growth of myotubes is induced by both fusion with additional myoblasts and reinnervation of motor neurons. Cellular and molecular events underlying the regenerative processes are regulated by critical factors, which are produced by satellite cells, myoblasts, myotubes, extracellular matrix and inflammatory cells. Galectin-1 is abundantly synthesized in adult skeletal muscles, but its roles in muscle regeneration have not been fully elucidated. We reviewed previous studies on the function of galectin-1 regarding myogenesis in vivo and in vitro, and discussed the roles of this lectin in regenerating skeletal muscles based on our observations. In intact adult muscles, galectin-1 was associated with basement membranes of myofibers. After muscle injury, galectin-1 immunoreactivity was increased within the cytoplasm of activated satellite cells. Thereafter, differentiated myoblasts lost galectin-1 immunoreactivity, but galectin-1 expression associated with basement membranes was detected in myotubes. Administration of anti-galectin-1 antibody, which perturbs the function of galectin-1, decreased the size of myotubes. Furthermore, muscle injury induced abundant expression of galectin-1 in damaged intramuscular nerve axons. We conclude that galectin-1 is a novel factor that promotes both myoblast fusion and axonal growth following muscle injury, and consequently, regulates myotube growth in regenerating skeletal muscles.     

Ginsenoside Rb₁ inhibits the carotid neointimal hyperplasia induced by balloon injury in rats via suppressing the phenotype modulation of vascular smooth muscle cells

This study aims to investigate the effects of ginsenoside Rb(1) on vascular intimal hyperplasia in rats and explore the mechanisms. The rat vascular neointimal hyperplasia model was made by rubbing the endothelia of carotid artery with a balloon and Rb(1) (10 and 30 mg/kg/day) was given the day after surgery for 14 consecutive days. The neointimal hyperplasia level and the degree of vascular smooth muscle cells (VSMCs) proliferation were evaluated by histopathology and by calculating the proliferating cell nuclear antigen (PCNA) positive expression percentage; protein expressions of PCNA, phosphorylation extracellular signal-regulated kinase 1/2 (pERK1/2), smooth muscle α-actin (SM α-actin), and the mRNA expressions of proto-oncogene c-myc, SM α-actin, SM-emb (embryonic smooth muscle myosin heavy chain) and p38 MAPK were detected by immunohistochemistry and Real Time RT-PCR, respectively. Compared with the endothelia rubbing model group, Rb(1) 10 and 30 mg/kg/day medication significantly ameliorated the neointimal hyperplasia (P<0.05), and decreased the positive expression percentage of PCNA(P<0.05). Rb(1) medication also significantly decreased the elevated protein expression of pERK1/2 and the mRNA expression of c-myc(P<0.05), and tended to reduce the expression of p38 MAPK mRNA. Endothelial rubbing increased the SM-emb mRNA expression, but decreased the expression of SM α-actin mRNA which was reversed by Rb(1) (P<0.05). The results indicate that Rb(1) inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats via suppressing the VSMC proliferation, which may be involved in part the inhibition of pERK1/2 protein and related to its inhibition on VSMC phenotype modulation.     

女贞子及其活性成分的骨骼肌药理作用研究进展

女贞子及其活性成分熊果酸、红景天苷等具有胰岛素样作用,对骨骼肌产生正性作用,即促进骨骼肌利用糖和脂质,增强肌糖原和肌蛋白合成,增加骨骼肌质量、快肌纤维和慢肌纤维体积,并促进快肌纤维向慢肌纤维转化。熊果酸还能刺激肌卫星细胞增殖、成肌细胞肌性分化,促进骨骼肌中新的肌细胞生长,促使骨骼肌强壮。其次,女贞子及其活性成分下调骨骼肌特异性的肌肉萎缩素-1和肌肉环状指蛋白表达,及其抗氧化应激和抗炎作用,最终可以防治肌萎缩和疲劳引起的骨骼肌损伤。     

骨髓间充质干细胞对与平滑肌细胞共培养体系中细胞增殖和自身分化的影响

目的探讨骨髓间充质干细胞（marrow mesenchymal stem cells,MSCs）对联合培养细胞增殖及自身表达平滑肌肌动蛋白（smooth muscle alpha—aetin,α-actin SM）的影响。方法建立MSCs与平滑肌细胞（smooth muscle cells,SMCs）直接共培养体系,对MSCs与SMCs直接共培养组和单纯SMCs组及MSCs组在不同时间点细胞计数。对共培养组和单纯MSCs组行4’6’－二乙酰基－2－苯基吲哚（DAPI）（5ng／ml）标记,利用抗α—actin SM荧光抗体,检测2组MSCs胞浆内-aetin SM表达。结果共培养组细胞计数较单独SMCs培养组明显减少,以第4天为著（P〈0．01）,与SMCs共培养5d,MSCs在共培养组较单纯MSCs组表达α-actin SM阳性率明显降低（P〈0．05）。结论在无干预因素情况下,MSCs与SMCs直接共培养明显抑制共培养体系中细胞增殖,且抑制MSCs表达仪-α ctin SM。     

家兔主动脉平滑肌细胞原代培养的研究

目的探索高效快速的家兔血管平滑肌细胞体外原代培养技术,为分子生物学实验提供足量可靠的细胞来源。方法取家兔主动脉中层,用刀片切成约1mm3,采用不同浓度的Ⅰ型胶原酶以及不同消化时间对组织块进行预消化,然后将组织块贴在培养瓶上进行培养。结果1.5g.L-1胶原酶消化6h后组织块细胞迁出速度及组织迁出率明显提高(P<0.05),组织块贴壁后24h即可见血管平滑肌细胞从组织块周围游离出来,呈梭形或星形,d4～5即可进行传代培养,传代后d3～4即可融合成致密单层细胞,呈典型的峰谷交错状,经免疫组化SP法鉴定有α-actin的表达,进一步确认其为血管平滑肌细胞。结论经1.5g.L-1胶原酶消化6h后组织块细胞迁出速度且迁出率明显提高,从而大大缩短原代培养的时间。     

原代培养的大鼠远端肺动脉平滑肌细胞的功能研究

目的探索原代培养的大鼠远端肺动脉平滑肌细胞(PASMC)是否具有血管平滑肌的功能,为缺氧性肺动脉高压等肺血管疾病发病机制及药物干预研究打下基础。方法采用显微操作和胶原酶消化法分离、培养大鼠远端PASMC,对培养细胞进行形态学观察、平滑肌α-actin免疫荧光染色鉴定,并用荧光显微镜法观测高钾(60 mmol·L-1KCl)溶液对大鼠远端PASMC的细胞内Ca2+浓度([Ca2+]i)的影响。结果培养的大鼠远端PASMC呈血管平滑肌细胞典型的"峰、谷"状生长,平滑肌α-actin免疫荧光染色呈阳性反应,60 mmol·L-1KCl溶液使PASMC的[Ca2+]i明显增高,5μmol·L-1硝苯地平能完全阻断PASMC的[Ca2+]i对KCl溶液的反应。结论原代培养的大鼠远端PASMC具有典型血管平滑肌细胞形态学、免疫学特征,具有功能正常的电压依赖性钙通道(VDCC),可广泛用于缺氧性肺动脉高压等肺血管疾病发病机制及药物干预的研究。     

AvidinOX™ for tissue targeted delivery of biotinylated cells

AvidinOX?, a product containing aldehyde groups, generated by ligand-assisted sugar oxidation of avidin by sodium periodate, maintains the capacity to bind biotin with very high affinity and exhibits the property to chemically link cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks. In recent studies, we have shown that AvidinOX exhibits much higher persistency in the skeletal muscle than native avidin. The aim of the present study is to evaluate whether AvidinOX-biotin interaction might be exploited to target biotinylated cells to an AvidinOX pre-treated muscle. To accomplish this we performed the following experiments: 1) The proliferation and differentiation properties of biotinylated C2C12 myoblasts were tested in vitro upon linkage to AvidinOX; 2) Bone marrow-derived cells (BMDC) were isolated from GFP positive transgenic mice [strain C57 BL/6-tg (UBC-GFP)] and after biotinylation (bBMDC) were intravenously administered to naive and MAVA+ (Mouse anti Avidin Antibody) C57/B6 mice previously injected with AvidinOX in a tibial muscle (TM). Localization efficiency of GFP+ bBMDC was evaluated on serial sections of the AvidinOX- and vehicle-treated (contra lateral limb) TM, 5 days after transplantation. Results show that biotinylated C2C12 cells, once linked to AvidinOX, maintain their proliferation and differentiation capacity, in vitro. Intravenous injection of biotinylated GFP+ bone marrow-derived cells leads to their specific and efficient localization in the AvidinOX-pre-treated, but not contra lateral muscle of both naive and MAVA+ mice. The present data suggest a potential use of AvidinOX to improve tissue targeted delivery of biotinylated cells.     

Resveratrol protects muscle cells against palmitate-induced cellular senescence and insulin resistance through ameliorating autophagic flux

Skeletal muscle, a highly metabolic tissue, is particularly vulnerable to increased levels of saturated free fatty acids (FFAs). The role of autophagy in saturated FFAs-induced cellular senescence and insulin resistance in skeletal muscle remains unclear. Therefore, the present study was aimed to explore autophagic flux in cellular senescence and insulin resistance induced by palmitate in muscle cells, and whether resveratrol limited these responses. Our results showed that palmitate induced cellular senescence in both myoblasts and myotubes. In addition, palmitate delayed differentiation in myoblasts and inhibited expression of insulin-stimulated p-AKTSer473 in myotubes. The accumulations of autophagosome assessed by tandem fluorescent-tagged LC3 demonstrated that autophagic flux was impaired in both palmitate-treated myoblasts and myotubes. Resveratrol protected muscle cells from palmitate-induced cellular senescence, apoptosis during differentiation, and insulin resistance via ameliorating autophagic flux. The direct influence of autophagic flux on development of cellular senescence and insulin resistance was confirmed by blockage of autophagic flux with chloroquine. In conclusion, impairment of autophagic flux is crucial for palmitate-induced cellular senescence and insulin resistance in muscle cells. Restoring autophagic flux by resveratrol could be a promising approach to prevent cellular senescence and ameliorate insulin resistance in muscle.     

甲基莲心碱对人脐静脉血管平滑肌细胞增殖及表型调节的影响

目的研究甲基莲心碱对人脐静脉平滑肌细胞增殖及表型调节的影响。方法以0.1,0.5,1.0,5.0μmol·L-1甲基莲心碱处理平滑肌细胞,采用MTT法和流式细胞术观察甲基莲心碱对人脐静脉血管平滑肌细胞增殖的影响。用免疫印迹法检测收缩型平滑肌细胞特异性标志物SM1,calponin 1和α-actin蛋白的表达。结果甲基莲心碱（0.1~5.0μmol·L-1）处理12,24,48 h后,人脐静脉血管平滑肌细胞的增殖具有明显的抑制作用,并具有剂量依赖关系和时间依赖关系。在缺乏甲基莲心碱的干预下,血管平滑肌细胞SM1,calponin 1和α-actin蛋白表达减少,在用0.5,5.0μmol·L-1甲基莲心碱处理48 h后,可显著逆转SM1,calponin 1和α-actin蛋白表达的减少,且具有剂量依赖性。结论甲基莲心碱具有抑制血管平滑肌细胞增殖及表型转化的作用,可用于防治动脉粥样硬化和再狭窄。     

Ethanol decreases agrin-induced acetylcholine receptor clustering in C2C12 myotube culture

We investigated the effect of ethanol on skeletal muscle development using C2C12 cell culture. The ethanol concentrations of 10 mM, 25 mM, and 100 mM, were tested because plasma samples of alcohol-dependent individuals fall within this range. We assessed two specific events in skeletal muscle development, the fusion of myoblasts to form myotubes and the acetylcholine receptor (AChR) clustering associated with neuromuscular synapse formation. We report that ethanol does not effect myotube formation or the viability of myoblasts or myotubes in C2C12 cell culture. However, ethanol does effect AChR clustering on C2C12 myotubes. As motor neurons approach skeletal muscle during development, agrin is released by motor neurons and induces AChR clustering on muscle fibers. In our experiments, agrin was applied to cell cultures during the period when myoblasts fuse to form myotubes. In cell cultures exposed to ethanol during myotube formation, agrin-induced AChR clustering was decreased compared to untreated cultures. In cell cultures exposed to ethanol during myoblast proliferation, with ethanol removed during myotube formation, agrin-induced AChR clustering was unaffected. We conclude that exposure to a physiologically relevant concentration of ethanol during the specific period of myotube formation decreases agrin-induced AChR clustering.     

Developing skeletal muscle cells express functional muscarinic acetylcholine receptors coupled to different intracellular signaling systems

This study analyzed the expression of muscarinic acetylcholine receptors (mAChRs) in the rat cultured skeletal muscle cells and their coupling to G protein, phospholipase C and adenylyl cyclase (AC). Our results showed the presence of a homogeneous population of [(3)H]methyl-quinuclidinyl benzilate-binding sites in the membrane fraction from the rat cultured muscle (K(D) = 0.4 nM, B(max) = 8.9 fmol mg protein(-1)). Specific muscarinic binding sites were also detected in denervated diaphragm muscles from adult rats and in myoblasts isolated from newborn rats. Activation of mAChRs with carbachol induced specific [(35)S]GTPgammaS binding to cultured muscle membranes and potentiated the forskolin-dependent stimulation of AC. These effects were totally inhibited by 0.1-1 microM atropine. In addition, mAChRs were able to stimulate generation of diacylglycerol (DAG) in response to acetylcholine, carbachol or selective mAChR agonist oxotremorine-M. The carbachol-dependent increase in DAG was inhibited in a concentration-dependent manner by mAChR antagonists atropine, pirenzepine and 4-DAMP mustard. Finally, activation of these receptors was correlated with increased synthesis of acetylcholinesterase, via a PKC-dependent pathway. Taken together, these results indicate that expression of mAChRs, coupled to G protein and distinct intracellular signaling systems, is a characteristic of noninnervated skeletal muscle cells and may be responsible for trophic influences of acetylcholine during formation of the neuromuscular synapse.     

Biomaterial-based delivery for skeletal muscle repair

Skeletal muscle possesses a remarkable capacity for regeneration in response to minor damage, but severe injury resulting in a volumetric muscle loss can lead to extensive and irreversible fibrosis, scarring, and loss of muscle function. In early clinical trials, the intramuscular injection of cultured myoblasts was proven to be a safe but ineffective cell therapy, likely due to rapid death, poor migration, and immune rejection of the injected cells. In recent years, appropriate therapeutic cell types and culturing techniques have improved progenitor cell engraftment upon transplantation. Importantly, the identification of several key biophysical and biochemical cues that synergistically regulate satellite cell fate has paved the way for the development of cell-instructive biomaterials that serve as delivery vehicles for cells to promote in vivo regeneration. Material carriers designed to spatially and temporally mimic the satellite cell niche may be of particular importance for the complete regeneration of severely damaged skeletal muscle.     

大鼠神经干细胞的分离培养和分化鉴定

目的建立神经干细胞分离、培养和分化鉴定技术。观察神经干细胞生长、增殖特点。方法利用无血清培养,从胚胎大鼠海马、纹状体等区分离神经干细胞,进行体外扩增培养、传代观察。采用荧光免疫细胞化学检测技术,观察鉴定神经干细胞及其分化结果。结果从胚胎大鼠海马、纹状体等区分离的细胞具有增殖能力,可进行传代培养,获得的细胞克隆中有巢蛋白(nestin)表达阳性细胞,显微镜下观察见典型的干细胞特征。分化为3种神经细胞类型:神经元、胶质细胞、少突胶质细胞。结论用上述方法分离培养的神经干细胞具有自我更新和增殖能力,通过鉴定确为神经干细胞,并经过分化鉴定确认。     

Arsenic induces apoptosis in myoblasts through a reactive oxygen species-induced endoplasmic reticulum stress and mitochondrial dysfunction pathway

A pool of myoblasts available for myogenesis is important for skeletal muscle size. The decreased number of skeletal muscle fibers could be due to the decreased myoblast proliferation or cytotoxicity. Identification of toxicants that regulate myoblast apoptosis is important in skeletal muscle development or regeneration. Here, we investigate the cytotoxic effect and its possible mechanisms of arsenic trioxide (As(2)O(3)) on myoblasts. C2C12 myoblasts underwent apoptosis in response to As(2)O(3) (1-10 μM), accompanied by increased Bax/Bcl-2 ratio, decreased mitochondria membrane potential, increased cytochrome c release, increased caspase-3/-9 activity, and increased poly (ADP-ribose) polymerase (PARP) cleavage. Moreover, As(2)O(3) triggered the endoplasmic reticulum (ER) stress indentified through several key molecules of the unfolded protein response, including glucose-regulated protein (GRP)-78, GRP-94, PERK, eIF2α, ATF6, and caspase-12. Pretreatment with antioxidant N-acetylcysteine (NAC, 0.5 mM) dramatically suppressed the increases in reactive oxygen species (ROS), lipid peroxidation, ER stress, caspase cascade activity, and apoptosis in As(2)O(3) (10 μM)-treated myoblasts. Furthermore, As(2)O(3) (10 μM) effectively decreased the phosphorylation of Akt, which could be reversed by NAC. Over-expression of constitutive activation of Akt (c.a. Akt) also significantly attenuated As(2)O(3)-induced myoblast apoptosis. Taken together, these results suggest that As(2)O(3) may exert its cytotoxicity on myoblasts by inducing apoptosis through a ROS-induced mitochondrial dysfunction, ER stress, and Akt inactivation signaling pathway.     

富含微量元素矿泉水对人血管平滑肌细胞生长增殖的影响

目的研究富含微量元素矿泉水对体外培养血管平滑肌细胞（HVSMCs）生长增殖的影响。方法无菌取12例冠状动脉旁路移植术患者术中废弃的大隐静脉,应用金山矿泉水配制的DMEM培养液（MW—DMEM）和双蒸水配制的DMEM培养液（DDW—DMEM）分别培养平滑肌细胞,分为金川矿泉水组和双蒸水组。观察记录2种培养液培养的HVSMCs游出组织块的时间及细胞呈“峰”-“谷”样时间;建立血管紧张素Ⅱ诱导HVSMCs增殖模型,采用MTT比色法观察2种培养液对AngⅡ诱导HVSMCs增殖抑制作用;应用电镜和免疫组织化学染色法观察鉴定HVSMCs。结果金川矿泉水组培养的HVSMCs贴壁时间为（3．7±0．2）d,双蒸水组为（8．5±0．6）d;金川矿泉水组培养的HVSMCs自贴壁后到长成“峰”-“谷”样形态的时间为（8．3±0．2）d,双蒸水组为（7．1±0．5）d;MTT比色法测定金川矿泉水组培养的HVSMCs的增殖活度明显降低;电镜观察MW—DMEM培养的HVSMCs细胞形态完整,表面有丰富的微绒毛。HVSMCs胞浆内富含肌丝,纵向平行排列,胞内有密体;抗-actin平滑肌肌动蛋白免疫组织化学染色后可见胞浆内染成褐色呈细丝网状。结论富含微量元素和矿物质的金川矿泉水具有修复血管平滑肌细胞损伤和抑制血管紧张素Ⅱ诱导HVSMCs增殖的作用。     

Cellular and molecular mechanisms responsible for the action of testosterone on human skeletal muscle. A basis for illegal performance enhancement

The popularity of testosterone among drug users is due to its powerful effects on muscle strength and mass. Important mechanisms behind the myotrophic effects of testosterone were uncovered both in athletes using steroids for several years and in short-term controlled studies. Both long-term and short-term steroid usage accentuates the degree of fibre hypertrophy in human skeletal muscle by enhancing protein synthesis. A mechanism by which testosterone facilitates the hypertrophy of muscle fibres is the activation of satellite cells and the promotion of myonuclear accretion when existing myonuclei become unable to sustain further enhancement of protein synthesis. Interestingly, long-term steroid usage also enhances the frequency of fibres with centrally located myonuclei, which implies the occurrence of a high regenerative activity. Under the action of testosterone, some daughter cells generated by satellite cell proliferation may escape differentiation and return to quiescence, which help to replenish the satellite cell reserve pool. However, whether long-term steroid usage induces adverse effects of satellite cells remains unknown. Testosterone might also favour the commitment of pluripotent precursor cells into myotubes and inhibit adipogenic differentiation. The effects of testosterone on skeletal muscle are thought to be mediated via androgen receptors expressed in myonuclei and satellite cells. Some evidence also suggests the existence of an androgen-receptor-independent pathway. Clearly, testosterone abuse is associated with an intense recruitment of multiple myogenic pathways. This provides an unfair advantage over non-drug users. The long-term consequences on the regenerative capacity of skeletal muscle are unknown.