白藜芦醇通过诱导自噬减少肥胖小鼠骨骼肌组织GDF8表达的研究

目的研究白藜芦醇通过诱导自噬减少肥胖小鼠骨骼肌组织中GDF8的表达。方法将8周龄C57BL/6雄性(♂)小鼠随机分为标准饮食组(SCD)、标准饮食+白藜芦醇组(SCD+RES)、高脂组(HFD)及高脂+白藜芦醇组(HFD+RES),白藜芦醇(400 mg·kg~(-1)·d~(-1))胃饲共持续20周。分析血脂指标和骨骼肌组织的形态变化,免疫组化、RT-PCR和Western印迹检测SIRT1,LC3,p62和GDF8的定位和表达。结果 HFD组小鼠体质量、血脂指标TC、TG、LDL-C水平显著升高,HDL-C水平明显降低;骨骼肌纤维间出现脂肪沉积;SIRT1和LC3表达明显减少,GDF8和p62的表达增加,且均定位于胞浆;SIRT1,LC3的表达明显降低。与HFD小鼠相比,HFD+RES组小鼠体质量和血脂指标明显下降,HDL-C水平显著升高,骨骼肌中的脂质沉积明显减少,SIRT1和LC3的表达水平增高,GDF8和p62的表达水平明显降低。结论白藜芦醇有降低骨骼肌GDF8表达的效应,其机制可能与其激活SIRT1进而增加自噬、促进GDF8的降解有关。     

调节自噬,白藜芦醇生物学活性的新机制

自噬是细胞在内部或外部因素的刺激下发生的复杂的"自我吞食"过程。失控的自噬与许多疾病的发病相关,如肿瘤、神经退行性疾病、心血管疾病等。白藜芦醇是一个非黄酮类的多酚化合物,具有抗肿瘤、抗炎、抗衰老、心血管保护、神经保护等多种药理学活性。然其作用机制与靶点尚未完全阐明。最新研究显示,白藜芦醇可显著调节自噬。本文综述了其调节自噬的相关研究,以期为其进一步研发提供新思路。     

铁蓄积对小鼠骨骼肌自噬和凋亡相关蛋白的影响

目的 探讨铁蓄积对小鼠骨骼肌自噬和凋亡相关蛋白的影响.方法 24只雄性ICR小鼠随机分为对照组、枸橼酸铁铵(FAC)组以及FAC+去铁胺(DFO)组.对照组腹腔注射生理盐水12周;FAC组腹腔注射FAC 0.04 g/kg,每周3次,干预8周,随后腹腔注射生理盐水4周;FAC+DFO组按FAC组方案干预8周后,腹腔注射DFO 0.45 g/kg,每周3次,干预4周.测定小鼠腓肠肌肌重/体重比,ELISA法测定血清铁蛋白水平,Western blot测定铁蛋白重链(FHC)、铁蛋白轻链(FLC)、自噬相关蛋白beclin-1、LC3和p62以及凋亡相关蛋白Bcl-2和Bax的表达.结果 与对照组比较,FAC组肌重/体重比降低(P＜0.05);与FAC组比较,FAC+ DFO组肌重/体重比升高(P＜0.05).与对照组相比,FAC组FHC、FLC、 beclin-1、LC3和Bax蛋白表达增加,p62、Bcl-2蛋白表达降低,Bcl-2/Bax比值降低,血清铁蛋白水平升高(P＜0.05);与FAC组相比,FAC+ DFO组FHC、FLC、 beclin-1、LC3和Bax蛋白表达下降,p62、Bcl-2蛋白表达增加,Bcl-2/Bax比值升高,血清铁蛋白水平降低(P＜0.05).结论 铁蓄积导致小鼠骨骼肌自噬和凋亡蛋白表达异常,引起骨骼肌肌量下降,降低铁蓄积可逆转上述现象.     

白藜芦醇通过促进线粒体自噬减轻小鼠脑缺血/再灌注损伤的实验研究

目的研究白藜芦醇(resveratrol,Res)对脑缺血/再灌注(ischemia/reperfusion,I/R)损伤的保护作用及其可能作用机制。方法采用线栓法建立小鼠右侧大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型。各组小鼠术前连续腹腔给药5 d,每日1次。术后24 h对梗死体积和神经功能进行评价; Western blot检测沉默信息调节因子1(silent information regulator 1,SIRT1)和线粒体自噬相关蛋白p62、LC3B、PHB2、TOM20表达情况,以及细胞色素C (cytochrome C,CytC)分布情况;流式细胞术检测线粒体膜电位(mitochondrial membrane potential,MMP)变化;酶标仪检测胞内ATP水平变化;透射电镜观察线粒体形态变化。结果Res预处理减少了脑梗死体积和神经功能缺损,提高了SIRT1和线粒体自噬相关蛋白表达,减少了CytC的释放,恢复了MMP,提高了ATP水平。透射电镜结果显示,Res减轻了缺血导致的线粒体损伤。自噬抑制剂3-MA抑制了线粒体自噬,同时也逆转了Res的神经、线粒体保护作用。结论Res减轻小鼠脑组织I/R损伤可能与其对线粒体自噬的调控,最终确保线粒体质量有关。     

白藜芦醇通过调控自噬减轻乳大鼠心肌细胞低氧复氧损伤

目的探讨白藜芦醇(Res)对低氧复氧(H/R)损伤心肌细胞的保护作用。方法采用三气培养箱法制备乳大鼠心肌细胞H/R损伤模型(H12 h/R12 h)。乳大鼠心肌细胞分为5组:细胞对照组、H/R模型组、模型+Res 5和20μmol·L-1组(Res预处理细胞12 h,然后H12 h/R12 h)和模型+Res 20μmol·L-1+氯喹(CQ)10μmol·L-1组(Res预处理细胞12 h,然后含CQ的培养基H12 h/R12 h);CCK-8法检测心肌细胞存活率,比色法测定心肌细胞乳酸脱氢酶(LDH)漏出率,AnnexinⅤ/PI双染法检测细胞凋亡,免疫荧光法检测心肌细胞内微管相关蛋白1轻链3(LC3)阳性细胞率,Western印迹法检测心肌细胞Beclin 1,LC3Ⅱ和P62蛋白表达水平。结果与细胞对照组比较,H/R组心肌细胞存活率下降(P<0.01),细胞凋亡率和LDH漏出率上升(P<0.01),LC3阳性细胞率和Beclin 1表达水平无明显变化,LC3Ⅱ和P62表达水平下降(P<0.05)。与H/R组比较,Res 5和20μmol·L-1可升高心肌细胞存...     

白藜芦醇通过抑制Akt/mTOR信号通路活性促进肺癌细胞自噬发挥抗肿瘤效应的研究

目的 探讨白藜芦醇对肺癌细胞生长活性的抑制效应及可能的机制。方法 稳定培养肺癌细胞系H292,分别加入含10、20、40 μmol·L^-1白藜芦醇的培养基进行干预,采用CCK-8、流式细胞术、细胞集落形成实验检测白芦藜醇对H292细胞生长活性的影响,采用免疫荧光、Western blotting检测白藜芦醇对细胞Akt/mTOR信号通路相关蛋白的影响。结果 （1） CCK-8检测结果显示,白藜芦醇对H292细胞增殖有明显抑制作用（P<0.05）,且呈时间和剂量依赖性,同时对H292细胞周期有明显影响,能阻滞细胞周期于G₂/M期。（2）白藜芦醇可明显抑制H292细胞集落形成（P<0.05）,并呈剂量依赖性。（3） Western blotting检测结果显示,白藜芦醇以剂量依赖的方式诱导H292细胞中LC3-II的累积。免疫荧光检测发现,白藜芦醇可诱导细胞核和线粒体中GFP-LC3显著增加。（4）自噬抑制剂3-MA能有效减弱白藜芦醇对H292细胞活力的抑制作用,溶酶体酸化抑制剂Bafilomycin A1则可促进白藜芦醇导致的LC3-II累积。（5）白藜芦醇可下调H292细胞中p-Akt、p-P70S6K、p-mTOR蛋白的表达（P<0.05）,而对Akt、P70S6K、mTOR蛋白的表达无明显影响（P>0.05）。结论 白藜芦醇可通过抑制Akt/mTOR信号通路抑制肺癌细胞的增殖。     

糖尿病大鼠海马组织自噬减少与SIRT2下调有关

目的探讨自噬与SIRT2在糖尿病大鼠海马组织中的表达及意义。方法采用单次腹腔注射链脲佐菌素(STZ,60 mg/kg)建立1型糖尿病大鼠(T1DM)模型,采用激光共聚焦检测LC3蛋白表达,采用免疫组化检测海马SIRT2蛋白表达水平。结果与正常组相比,T1DM大鼠海马组织自噬减少,SIRT2表达降低,而胰岛素[4 U/(kg·d)]治疗后促进细胞自噬并上调SIRT2表达。结论高血糖下调海马神经元SIRT2表达并抑制自噬发生,这可能与糖尿病脑病的发生与发展相关。     

白藜芦醇诱导人乳腺癌MDA-MB231细胞凋亡及自噬

目的探讨白藜芦醇(resveratrol,Res)诱导人乳腺癌MDA-MB231细胞凋亡及自噬蛋白表达的作用与机制。方法以人乳腺癌MDA-MB231细胞为研究对象,MTT法检测Res(0、1、5、10、20、50、100、200 mg·L~(-1))抑制MDA-MB231细胞增殖情况; Res(0、5、20、60 mg·L~(-1))处理MDA-MB231细胞,划痕实验检测细胞迁移能力,Annexin V/PI双染法检测细胞早期凋亡率,免疫荧光法检测细胞LC3Ⅰ/Ⅱ的表达;Res(0、20、60 mg·L~(-1))处理MDA-MB231细胞,蛋白免疫印迹法检测细胞中自噬相关蛋白p62及Beclin-1的表达。结果Res在体外可明显抑制MDA-MB231细胞增殖,且呈浓度依赖关系。随着Res浓度的增高,乳腺癌细胞迁移能力较对照组分别下降了(8. 7±1. 1)%、(16. 5±2. 9)%和(32. 2±3. 6)%。流式细胞仪检测结果表明,早期凋亡细胞数增加;激光共聚焦检查显示,细胞质中LC3Ⅰ/Ⅱ绿色荧光增加;蛋白免疫印迹法检测出MDA-MB231细胞p62蛋白表达下降,Beclin-1蛋白表达升高。结论 Res可抑制人乳腺癌MDA-MB231细胞增殖,其机制可能与Res诱导细胞凋亡和自噬有关。     

GDF11在棕榈酸诱导骨骼肌细胞胰岛素抵抗中的作用研究

目的探讨GDF11对棕榈酸诱导骨骼肌细胞胰岛素抵抗的影响。方法用棕榈酸构建骨骼肌细胞胰岛素抵抗模型,分为对照组、GDF11干预组、棕榈酸干预组和GDF11联合棕榈酸干预组。CCK-8检测细胞活力,2NBDG检测细胞葡萄糖摄取。实时荧光定量PCR检测肌管标志基因(desmin、myogenin),胰岛素介导葡萄糖摄取相关基因(GLUT-4、IRS-1)及PGC-1α的表达。Western blot检测PGC-1α蛋白水平的表达。结果不同浓度GDF11对骨骼肌细胞活力无明显影响。与对照组相比,棕榈酸干预组葡萄糖摄取及GLUT-4、IRS-1、PGC-1α的表达明显降低(P<0.05)。与棕榈酸干预组相比,GDF11联合棕榈酸干预组葡萄糖摄取及GLUT-4、IRS-1、PGC-1α的表达无明显变化。结论棕榈酸可成功诱导骨骼肌细胞胰岛素抵抗,而GDF11对骨骼肌细胞胰岛素抵抗没有明显改善作用。     

白藜芦醇保护脑缺血/再灌注损伤与自噬关系的研究

目的研究白藜芦醇(resveratrol,Res)预处理对大鼠局灶性脑缺血/再灌注损伤后自噬的影响,探讨Res保护局灶性脑缺血/再灌注损伤的作用机制。方法将64只SD♂大鼠随机分成4组,假手术组(S组)、模型组(M组)、Res低剂量(15 mg.kg-1)干预组(R1组)和Res高剂量(40 mg.kg-1)干预组(R2组),用线栓法制备大鼠大脑中动脉栓塞(MCAO)模型,缺血2 h,再灌注24 h后,观察大鼠神经功能评分;透射电镜法观察神经元的超微结构及其内自噬空泡数量的变化;免疫组化法检测LC3的蛋白表达;RT-PCR法分别检测LC3和Beclin 1的mRNA表达。结果与M组相比,Res可明显减轻大鼠脑缺血/再灌注损伤的神经功能评分(P<0.05);使自噬泡数量明显增加;上调缺血皮质区脑组织LC3和Beclin 1蛋白及mRNA的表达(P<0.05)。结论 Res对大鼠局灶性脑缺血/再灌注损伤后神经元有保护作用,其机制可能与其诱导自噬的生成,上调LC3和Beclin 1的表达有关。     

Exercise ameliorates the detrimental effect of chloroquine on skeletal muscles in mice via restoring autophagy flux

Aim： To study the roles of autophagy in muscle establishment during long-term exercise in mice.
Methods： Female ICR mice were submitted to exercise training with a wheel running regimen： 6 m/min, 15 min/time, 3 times/d （on 8：00, 14：00, and 20：00）, 5 d/week for 2 months. The mice were treated with the autophagy activator trehalose （1% aqueous solution as a daily drinking water） or the autophagy inhibitor chloroquine （10 mg/kg, ip, 5 times a week） before the training. Western blotting analysis, TUNEL staining, H＆E staining and transmission electron microscope were used to evaluate the activity of autophagy and the structure of the muscle fibers.

Results： The exercise training significantly stimulated the formation of autophagosomes, increased the LC3-II, cathepsin L and Bcl-2 levels, lowered the P62 level and increased the antioxidant capacity in the muscles. Meanwhile, the exercise training significantly improved the morphology of mitochondria, reduced the release of cytochrome c from mitochondria to cytoplasm, and slightly decreased the apoptosis rate in the muscles. Administration of trehalose increased the level of autophagy and protected the muscle fibers from apoptosis. Administration of chloroquine blocked autophagy flux and exerted detrimental effects on the muscles, which were ameliorated by the exercise training.

Conclusion： Long-term regular exercise activates autophagy process associated with muscle establishment, and ameliorates the detrimental effects of chloroquine on skeletal muscles via restoring autophagy flux.     

GDF8 inhibits bone formation and promotes bone resorption in mice

Growth Differentiation Factor 8 (GDF8), also called myostatin, is a member of the transforming growth factor (TGF)‐β super‐family. As a negative regulator of skeletal muscle growth, GDF8 is also associated with bone metabolism. However, the function of GDF8 in bone metabolism is not fully understood. Our study aimed to investigate the role of GDF8 in bone metabolism, both in vitro and in vivo. Our results showed that GDF8 had a negative regulatory effect on primary mouse osteoblasts, and promoted receptor activator of nuclear factor κB ligand (RANKL)‐induced osteoclastogenesis in vitro. Intraperitoneal injection of recombinant GDF8 repressed bone formation and accelerated bone resorption in mice. Furthermore, treatment of aged mice with a GDF8 neutralizing antibody stimulated new bone formation and prevented bone resorption. Thus, our study showed that GDF8 plays a significant regulatory role in bone formation and bone resorption, thus providing a potential therapeutic pathway for osteoporosis.     

Use of GLUT-4 null mice to study skeletal muscle glucose uptake

1. The present review focuses on the effects of varying levels of GLUT-4, the insulin-sensitive glucose transporter, on insulin sensitivity and whole body glucose homeostasis. 2. Three mouse models are discussed including myosin light chain (MLC)-GLUT-4 mice which overexpress GLUT-4 specifically in skeletal muscle, GLUT-4 null mice which express no GLUT-4 and the MLC-GLUT-4 null mice which express GLUT-4 only in skeletal muscle. Overexpressing GLUT-4 specifically in the skeletal muscle results in increased insulin sensitivity in the MLC-GLUT-4 mice. In contrast, the GLUT-4 null mice exhibit insulin intolerance accompanied by abnormalities in glucose and lipid metabolism. Restoring GLUT-4 expression in skeletal muscle in the MLC-GLUT-4 null mice results in normal glucose metabolism but continued abnormal lipid metabolism. 3. The results of experiments using these mouse models demonstrates that modifying the expression of GLUT-4 profoundly affects whole body insulin action and consequently glucose and lipid metabolism.     

骨骼肌萎缩信号通路的研究进展

在恶性肿瘤、脓毒血症、糖尿病甚至长期太空飞行等状态下常伴随着骨骼肌萎缩的现象.骨骼肌萎缩是肌蛋白合成下降和(或)分解代谢加速的结果.骨骼肌萎缩的程度与患者的康复、生活能力和生活质量息息相关,防治并逆转骨骼肌的萎缩对上述疾病的治疗具有重要意义.现代医学对骨骼肌萎缩的分子生物学机制进行了大量研究,对近年骨骼肌萎缩相关信号通路及药物对其防治作用做一简要综述,以期对骨骼肌萎缩相关疾病的研究与治疗提供参考.     

8-氮鸟嘌呤通过Akt/mTORC1/ULK1诱导细胞自噬增强其在肝癌细胞中的耐药性

细胞自噬是真核生物中进化保守的对细胞内容物进行降解的生理过程,其利用溶酶体将细胞内物质降解再利用,在应激条件下可以促进癌细胞的存活.8-氮鸟嘌呤(8-azaguanine,8-AG)是一种嘌呤核苷酸生物合成的抑制剂,对多种肿瘤细胞具有抗肿瘤活性.然而,耐药性限制了8-AG作为抗癌药物的应用,其耐药性机制尚不清楚.本研究...     

Effect of verbascoside on decreasing concentration of oxygen free radicals and lipid peroxidation in skeletal muscle

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Mechanism of action of HI-6 on soman inhibition of acetylcholinesterase in preparations of rat and human skeletal muscle; comparison to SAD-128 and PAM-2

HI-6 is presently considered the most potent oxime antidote against soman poisoning in mice, rats, dogs and monkeys. However, it is still an open question whether efficiency of HI-6, observed in experimental animals, can be extrapolated to soman intoxicated humans. In this paper efficiency of HI-6 and possible mechanisms of action were compared in rat and human fresh muscle preparations. In rat muscle, about 50% of control AChE activity could be recovered by both therapeutic (5 min after soman) and prophylactic (5 min before soman) application of HI-6. On the other hand, in human muscle therapeutic treatment restored only 5%, while prophylactic application of HI-6 again resulted in about 50% recovery of control AChE activity. As revealed by comparison of the prophylactic effects of HI-6 and the non-oxime bispyridinium compound SAD-128, competitive inhibition of AChE plays a minor role as a protective mechanism. Immediate reactivation of rapidly aging human AChE must therefore be instituted for successful protective treatment by HI-6. Retardation of aging, a direct effect of SAD-128, was roughly estimated to improve reactivation by HI-6 for about 10% of control AChE activity of the human muscle. PAM-2 proved completely inefficient as a therapeutic and as a prophylactic agent on both rat and human muscle preparations.     

丹酚酸B激活自噬改善高磷诱导血管平滑肌细胞钙化的机制研究

目的:探讨丹酚酸B(Salvianolic acid B,SAB)改善高磷诱导的血管平滑肌细胞(vascular smooth muscle cells, VSMCs)钙化的作用及机制。方法:采用大鼠血管平滑肌细胞(A7r5)钙化体外模型,用细胞增殖与活性检测试剂盒(cell counting kit-8,CCK-8)检测SAB对细胞活力的影响;2.6μmol·g^-1高磷诱导VSMCs钙化,加入丹参多酚酸类化合物丹酚酸B、丹酚酸C(Salvianolic acid C,SAC)、迷迭香酸(Rosmarinic acid, RA)及细胞自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine, 3-MA,5 nmol·g^-1),采用邻甲酚酞络合铜法及茜素红S染色检测细胞钙化程度;Western blot检测VSMCs钙化指标Runx2、OPN,自噬相关蛋白Beclin-1和LC3II/LC3I以及平滑肌收缩表型蛋白Calponin、SM22α的表达。结果:(1)钙离子浓度定量实验及茜素红染色实验显示SAB显著减轻高磷诱导的VSMCs钙化;...     

小檗碱对链脲菌素和高糖高脂饲料诱导糖尿病大鼠骨骼肌CDK9和cyclin T1表达的影响

目的观察小檗碱对2型糖尿病大鼠骨骼肌病理结构改变及CDK9和cyclin T1蛋白表达的影响。方法腹腔注射链脲菌素(35 mg·kg~(-1))加高糖高脂饲料喂养16 wk建立2型糖尿病大鼠模型,随后16 wk每天分别拌食给予低中高剂量小檗碱(75、150、300 mg·kg~(-1))、非诺贝特(100 mg·kg~(-1))和罗格列酮(4 mg·kg~(-1)),用HE染色检查骨骼肌结构病变和免疫组化检测CDK9和cyclin T1的表达。结果骨骼肌纤维在各组大鼠中仍正常分布,中高剂量小檗碱部分地改善糖尿病肌纤维的萎缩;中高剂量小檗碱和罗格列酮都能明显恢复糖尿病大鼠骨骼肌中表达降低的CDK9和cyclin T1至正常水平(P<0.01)。结论小檗碱调控骨骼肌CDK9和cyclin T1蛋白的表达可能是其改善糖尿病骨骼肌纤维萎缩的机制之一。