Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better under-stand the function...Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better under-stand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNProW-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNProE(3+5+7)M-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3―1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep my-ostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin promoter.展开更多
目的:探讨二氯化钴(cobaltous chloride,Co Cl2)诱导缺氧对肌细胞萎缩的调控机制。方法:本研究采用C2C12小鼠成肌细胞系作为细胞模型,分为正常组、Co Cl2组、正常+3-Methyladenine(3MA)组、Co Cl2+3MA组。正常组不作处理,Co Cl2组加入20...目的:探讨二氯化钴(cobaltous chloride,Co Cl2)诱导缺氧对肌细胞萎缩的调控机制。方法:本研究采用C2C12小鼠成肌细胞系作为细胞模型,分为正常组、Co Cl2组、正常+3-Methyladenine(3MA)组、Co Cl2+3MA组。正常组不作处理,Co Cl2组加入200μM Co Cl2诱导缺氧,正常+3MA组加入5 m M 3MA,Co Cl2+3MA组加入200μM Co Cl2及5 m M 3MA。使用吉姆萨染色观察肌管形态,多功能酶标仪检测活性氧(reactive oxygen species,ROS)表达,电镜观察自噬体形成情况,实时定量聚合酶链反应(quantita-tive real time polymerase chain reaction,QRT-PCR)和免疫印迹技术(western blotting,WB)检测缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)、Bcl2/腺病毒E1B 19k Da相关蛋白3(Bcl2/adenovirusE1B 19k Da interacting protein 3,BNIP3)、微管相关蛋白1轻链-3(microtubule associated protein1 lightchain 3,LC3)m RNA及蛋白表达;另外,通过抑制自噬观察肌肉萎缩盒F基因(muscle atrophy F-box,MAFbx)蛋白表达的变化。结果:正常组可见长条状肌管形成,Co Cl2处理后肌管萎缩、断裂。Co Cl2组ROS含量较正常组升高(t=-4.965,P=0.008),电镜可观察到Co Cl2诱导自噬体形成,同时HIF-1α、BNIP3、LC3表达增加(P<0.05)。Co Cl2与3MA共处理可减少MAFbx蛋白的表达(F=18.246,P=0.001)。结论:Co Cl2诱导C2C12骨骼肌细胞萎缩,可能与HIF-1α/BNIP3信号通路促进自噬发生有关,抑制缺氧诱导的自噬可部分减少肌萎缩。展开更多
早期生长反应蛋白1(early growth response protein 1,Egr1)作为转录因子在细胞增殖、分化及凋亡等许多生物学过程中都扮演着重要角色,但是其对小鼠成肌细胞C2C12分化的影响尚不明确。该研究采用Western blot和免疫荧光技术检测Egr1在C2...早期生长反应蛋白1(early growth response protein 1,Egr1)作为转录因子在细胞增殖、分化及凋亡等许多生物学过程中都扮演着重要角色,但是其对小鼠成肌细胞C2C12分化的影响尚不明确。该研究采用Western blot和免疫荧光技术检测Egr1在C2C12细胞分化过程中的表达规律及定位。利用CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9技术分别激活和抑制Egr1的表达,进而探讨Egr1对C2C12细胞分化的影响。结果显示,随着C2C12细胞分化的进行,Egr1的表达量在分化的第5 d达到峰值,随后呈下降趋势。Egr1表达定位于C2C12的细胞核与细胞质中,随着分化的进行,其在细胞核和细胞质中的表达量均显著升高。分别激活或抑制Egr1后,C2C12细胞肌管融合率以及肌肉分化标志分子肌细胞生成蛋白(myogenin,MYOG)和肌球蛋白重链2(myosin heavy chain 2,MYH2)水平均显著增加或降低。该研究结果表明,Egr1能够促进体外小鼠成肌细胞C2C12的分化。展开更多
Global demand for farm animals and their meat products i.e.,pork,chicken and other livestock meat,is steadily incresing.With the ongoing life science research and the rapid development of biotechnology,it is a great o...Global demand for farm animals and their meat products i.e.,pork,chicken and other livestock meat,is steadily incresing.With the ongoing life science research and the rapid development of biotechnology,it is a great opportunity to develop advanced molecular breeding markers to efficiently improve animal meat production traits.Hippo is an important study subject because of its crucial role in the regulation of organ size.In recent years,with the increase of research on Hippo signaling pathway,the integrative application of multi-omics technologies such as genomics,transcriptomics,proteomics,and metabolomics can help promote the in-depth involvement of Hippo signaling pathway in skeletal muscle development research.The Hippo signaling pathway plays a key role in many biological events,including cell division,cell migration,cell proliferation,cell differentiation,cell apoptosis,as well as cell adhesion,cell polarity,homeostasis,maintenance of the face of mechanical overload,etc.Its influence on the development of skeletal muscle has important research value for enhancing the efficiency of animal husbandry production.In this study,we traced the origin of the Hippo pathway,comprehensively sorted out all the functional factors found in the pathway,deeply analyzed the molecular mechanism of its function,and classified it from a novel perspective based on its main functional domain and mode of action.Our aim is to systematically explore its regulatory role throughout skeletal muscle development.We specifically focus on the Hippo signaling pathway in embryonic stem cell development,muscle satellite cell fate determination,myogenesis,skeletal muscle meat production and organ size regulation,muscle hypertrophy and atrophy,muscle fiber formation and its transformation between different types,and cardiomyocytes.The roles in proliferation and regeneration are methodically summarized and analyzed comprehensively.The summary and prospect of the Hippo signaling pathway within this article will provide ideas for further improving 展开更多
Objective:To evaluate the effects of Catalpa bignonioides fruit extract on the promotion of muscle growth and muscular capacity in vitro and in vivo.Methods:Cell viability was measured using the 3-(4,5-dimethylthiazol...Objective:To evaluate the effects of Catalpa bignonioides fruit extract on the promotion of muscle growth and muscular capacity in vitro and in vivo.Methods:Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Cell proliferation was assessed using a 5-bromo-2’-deoxyuridine(BrdU)assay kit.Western blot analysis was performed to determine the protein expressions of related factors.The effects of Catalpa bignonioides extract were investigated in mice using the treadmill exhaustion test and whole-limb grip strength assay.Chemical composition analysis was performed using high-performance liquid chromatography(HPLC).Results:Catalpa bignonioides extract increased the proliferation of C2C12 mouse myoblasts by activating the Akt/mTOR signaling pathway.It also induced metabolic changes,increasing the number of mitochondria and glucose metabolism by phosphorylating adenosine monophosphate-activated protein kinase.In an in vivo study,the extract-treated mice showed improved motor abilities,such as muscular endurance and grip strength.Additionally,HPLC analysis showed that vanillic acid may be the main component of the Catalpa bignonioides extract that enhanced muscle strength.Conclusions:Catalpa bignonioides improves exercise performance through regulation of growth and metabolism in skeletal muscles,suggesting its potential as an effective natural agent for improving muscular strength.展开更多
Objective:To observe whether CAV3 is involved in the physiological process of IMGU and NIMGU.Down-regulation of caveolin-3(CAV3)protein expression by small interference RNAs(iRNA)has been studied.Insulin-mediated gluc...Objective:To observe whether CAV3 is involved in the physiological process of IMGU and NIMGU.Down-regulation of caveolin-3(CAV3)protein expression by small interference RNAs(iRNA)has been studied.Insulin-mediated glucose uptake(IMGU)is critical in skeletal muscle and cardiac myocytes,but non-insulin-mediated glucose uptake(NIMGU)should not be neglected.Methods:CAV3 siRNAs were designed and transfected in C2C12 cells and H9c2 cells in skeletal muscle and cardiac muscle,respectively,and C2C12 and H9c2 cells were cultured in DMEM medium with and without insulin,respectively.Glucose transporter 4(GLUT4)protein expression was detected by Western blot,and the glucose uptake rate of cells was measured by biochemical kit.Results:Transfection with CAV3 siRNA successfully down-regulated CAV3 protein expression in C2C12 and H9c2 cells.In the absence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.05)after 48 h of transfection in C2C12 cells,and GLUT4 expression was decreased(P<0.05)and glucose uptake was reduced(P<0.01)after 48 h of transfection in H9c2 cells.In the presence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.01)after 48 h of transfection in C2C12 cells,and the downregulation of GLUT4 was not statistically significant and glucose uptake was reduced(P<0.01)after 48 hours of transfection in H9c2 cells.Conclusion:Two different states,IMGU and NIMGU,exist in C2C12 cells and H9c2 cells.Both in the quiet state stimulated by insulin as well as in the absence of insulin stimulation,the cellular uptake of glucose is affected by GLUT4 changes regulated by CAV3.展开更多
基金Supported by the National Hi-Tech R&D Program ("863" Program) of China (Grant No. 2002AA206311)
文摘Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better under-stand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNProW-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNProE(3+5+7)M-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3―1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep my-ostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin promoter.
文摘目的:探讨二氯化钴(cobaltous chloride,Co Cl2)诱导缺氧对肌细胞萎缩的调控机制。方法:本研究采用C2C12小鼠成肌细胞系作为细胞模型,分为正常组、Co Cl2组、正常+3-Methyladenine(3MA)组、Co Cl2+3MA组。正常组不作处理,Co Cl2组加入200μM Co Cl2诱导缺氧,正常+3MA组加入5 m M 3MA,Co Cl2+3MA组加入200μM Co Cl2及5 m M 3MA。使用吉姆萨染色观察肌管形态,多功能酶标仪检测活性氧(reactive oxygen species,ROS)表达,电镜观察自噬体形成情况,实时定量聚合酶链反应(quantita-tive real time polymerase chain reaction,QRT-PCR)和免疫印迹技术(western blotting,WB)检测缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)、Bcl2/腺病毒E1B 19k Da相关蛋白3(Bcl2/adenovirusE1B 19k Da interacting protein 3,BNIP3)、微管相关蛋白1轻链-3(microtubule associated protein1 lightchain 3,LC3)m RNA及蛋白表达;另外,通过抑制自噬观察肌肉萎缩盒F基因(muscle atrophy F-box,MAFbx)蛋白表达的变化。结果:正常组可见长条状肌管形成,Co Cl2处理后肌管萎缩、断裂。Co Cl2组ROS含量较正常组升高(t=-4.965,P=0.008),电镜可观察到Co Cl2诱导自噬体形成,同时HIF-1α、BNIP3、LC3表达增加(P<0.05)。Co Cl2与3MA共处理可减少MAFbx蛋白的表达(F=18.246,P=0.001)。结论:Co Cl2诱导C2C12骨骼肌细胞萎缩,可能与HIF-1α/BNIP3信号通路促进自噬发生有关,抑制缺氧诱导的自噬可部分减少肌萎缩。
文摘早期生长反应蛋白1(early growth response protein 1,Egr1)作为转录因子在细胞增殖、分化及凋亡等许多生物学过程中都扮演着重要角色,但是其对小鼠成肌细胞C2C12分化的影响尚不明确。该研究采用Western blot和免疫荧光技术检测Egr1在C2C12细胞分化过程中的表达规律及定位。利用CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9技术分别激活和抑制Egr1的表达,进而探讨Egr1对C2C12细胞分化的影响。结果显示,随着C2C12细胞分化的进行,Egr1的表达量在分化的第5 d达到峰值,随后呈下降趋势。Egr1表达定位于C2C12的细胞核与细胞质中,随着分化的进行,其在细胞核和细胞质中的表达量均显著升高。分别激活或抑制Egr1后,C2C12细胞肌管融合率以及肌肉分化标志分子肌细胞生成蛋白(myogenin,MYOG)和肌球蛋白重链2(myosin heavy chain 2,MYH2)水平均显著增加或降低。该研究结果表明,Egr1能够促进体外小鼠成肌细胞C2C12的分化。
基金supported by the National Natural Science Foundation of China(31830090)the High-level Talent Project of Shihezi University,China(2022ZK022)the Agricultural Science and Technology Innovation Program,Chinese Academy of Agricultural Sciences(CAAS-ZDRW202006).
文摘Global demand for farm animals and their meat products i.e.,pork,chicken and other livestock meat,is steadily incresing.With the ongoing life science research and the rapid development of biotechnology,it is a great opportunity to develop advanced molecular breeding markers to efficiently improve animal meat production traits.Hippo is an important study subject because of its crucial role in the regulation of organ size.In recent years,with the increase of research on Hippo signaling pathway,the integrative application of multi-omics technologies such as genomics,transcriptomics,proteomics,and metabolomics can help promote the in-depth involvement of Hippo signaling pathway in skeletal muscle development research.The Hippo signaling pathway plays a key role in many biological events,including cell division,cell migration,cell proliferation,cell differentiation,cell apoptosis,as well as cell adhesion,cell polarity,homeostasis,maintenance of the face of mechanical overload,etc.Its influence on the development of skeletal muscle has important research value for enhancing the efficiency of animal husbandry production.In this study,we traced the origin of the Hippo pathway,comprehensively sorted out all the functional factors found in the pathway,deeply analyzed the molecular mechanism of its function,and classified it from a novel perspective based on its main functional domain and mode of action.Our aim is to systematically explore its regulatory role throughout skeletal muscle development.We specifically focus on the Hippo signaling pathway in embryonic stem cell development,muscle satellite cell fate determination,myogenesis,skeletal muscle meat production and organ size regulation,muscle hypertrophy and atrophy,muscle fiber formation and its transformation between different types,and cardiomyocytes.The roles in proliferation and regeneration are methodically summarized and analyzed comprehensively.The summary and prospect of the Hippo signaling pathway within this article will provide ideas for further improving
基金supported by Korea Environment Industry&Technology Institute through Project to make multi-ministerial national biological research resources more advanced Project,funded by Korea Ministry of Environment(grant number RS-2023-00230403).
文摘Objective:To evaluate the effects of Catalpa bignonioides fruit extract on the promotion of muscle growth and muscular capacity in vitro and in vivo.Methods:Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Cell proliferation was assessed using a 5-bromo-2’-deoxyuridine(BrdU)assay kit.Western blot analysis was performed to determine the protein expressions of related factors.The effects of Catalpa bignonioides extract were investigated in mice using the treadmill exhaustion test and whole-limb grip strength assay.Chemical composition analysis was performed using high-performance liquid chromatography(HPLC).Results:Catalpa bignonioides extract increased the proliferation of C2C12 mouse myoblasts by activating the Akt/mTOR signaling pathway.It also induced metabolic changes,increasing the number of mitochondria and glucose metabolism by phosphorylating adenosine monophosphate-activated protein kinase.In an in vivo study,the extract-treated mice showed improved motor abilities,such as muscular endurance and grip strength.Additionally,HPLC analysis showed that vanillic acid may be the main component of the Catalpa bignonioides extract that enhanced muscle strength.Conclusions:Catalpa bignonioides improves exercise performance through regulation of growth and metabolism in skeletal muscles,suggesting its potential as an effective natural agent for improving muscular strength.
基金This study was supported by the National Natural Science Foundation of China(No.81660360)The Natural Science Foundation of Guangxi(No.2019JJA140605)。
文摘Objective:To observe whether CAV3 is involved in the physiological process of IMGU and NIMGU.Down-regulation of caveolin-3(CAV3)protein expression by small interference RNAs(iRNA)has been studied.Insulin-mediated glucose uptake(IMGU)is critical in skeletal muscle and cardiac myocytes,but non-insulin-mediated glucose uptake(NIMGU)should not be neglected.Methods:CAV3 siRNAs were designed and transfected in C2C12 cells and H9c2 cells in skeletal muscle and cardiac muscle,respectively,and C2C12 and H9c2 cells were cultured in DMEM medium with and without insulin,respectively.Glucose transporter 4(GLUT4)protein expression was detected by Western blot,and the glucose uptake rate of cells was measured by biochemical kit.Results:Transfection with CAV3 siRNA successfully down-regulated CAV3 protein expression in C2C12 and H9c2 cells.In the absence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.05)after 48 h of transfection in C2C12 cells,and GLUT4 expression was decreased(P<0.05)and glucose uptake was reduced(P<0.01)after 48 h of transfection in H9c2 cells.In the presence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.01)after 48 h of transfection in C2C12 cells,and the downregulation of GLUT4 was not statistically significant and glucose uptake was reduced(P<0.01)after 48 hours of transfection in H9c2 cells.Conclusion:Two different states,IMGU and NIMGU,exist in C2C12 cells and H9c2 cells.Both in the quiet state stimulated by insulin as well as in the absence of insulin stimulation,the cellular uptake of glucose is affected by GLUT4 changes regulated by CAV3.
