模拟高原低氧环境对小鼠脾脏铁代谢的影响*

目的:探讨模拟海拔6 000 m高原低氧环境对小鼠脾脏铁代谢的影响。方法:将C57BL/6小鼠按体重随机分为常压常氧组(Nor)和低压低氧组(HH)。HH组小鼠放置于低压低氧动物实验舱内,模拟急性海拔6 000 m高原低氧环境,控制光照时间比大约12 h∶12 h。Nor组置于同等条件的常压常氧环境。HH组又分为低氧12 h组(HH-12 h)和3 d组(HH-3 d),对照组对应分为(Nor-12 h及Nor-3 d),每组9只小鼠。采用血常规检测、HE染色、组织铁染色、蛋白质免疫印迹(WB)、免疫组织化学(IHC)综合评价模拟高原低氧环境下小鼠脾脏铁代谢情况。结果:与相同时间点Nor组相比:①HH-12 h组小鼠红细胞数(RBC)、血红蛋白量(HGB)、红细胞压积(HCT)均无明显变化。HH-3 d组RBC、HGB及HCT均显著增加(P＜0.05),平均血红蛋白量(MCH)在HH-12 h和HH-3 d组均无显著变化。②与Nor-3 d相比,HH-3 d组小鼠脾脏明显增大,HE染色显示脾窦变窄,铁染色结果显示HH-3 d组脾脏红髓中铁含量明显增加。③WB结果显示,HH-3 d组低氧诱导因子1α(HIF-1α),转铁蛋白受体1(TfR1),铁输出蛋白(Fpn)表达均显著增加,而铁蛋白(Ft-L)的表达显著降低(P＜0.05);IHC结果也与WB结果一致,高原低氧暴露3 d后脾脏红髓TfR1、Fpn表达和分布均明显增多,Ft-L表达分布明显减少。结论:模拟海拔6 000 m高原低氧暴露3 d后小鼠脾脏截留处理RBC增多,脾索铁沉积,脾脏组织细胞内铁动员加速。高原低氧下脾脏铁代谢异常可能是引起高原低氧暴露下红细胞病理性增多甚至造成高原红细胞增多症的主要原因。     

低氧诱导因子-1α在低氧促成肌细胞增殖中的作用

目的:探讨低氧对大鼠骨骼肌成肌细胞(SkMs)增殖的影响及低氧诱导因子(HIF-1α)在低氧促成肌细胞增殖中的相关机制。方法:采用流式细胞仪观察了3、10%O2对SkMs细胞数量和增殖指数的影响;用RT-PCR方法检测了HIF-1αmRNA的表达,用Western blot方法检测了SkMs胞浆、胞核及总HIF-1α蛋白的水平。结果:低氧组较常氧组细胞数量和增殖指数增加(P0.05);HIF-1αmRNA、总蛋白水平在常氧组和低氧组中没有明显差异,常氧下胞浆中HIF-1α蛋白水平高于胞核内,低氧下HIF-1α蛋白水平在胞核内高于胞浆。结论:低氧能够促进SkMs增殖,HIF-1α可能是通过氧浓度调控的核转位的方式参与了低氧促SkMs的增殖。     

低氧经mTOR/4E-BP1和FoxO1/Atrogin-1通路影响骨骼肌蛋白质积累

低氧暴露对骨骼肌蛋白质合成/分解的影响受到广泛关注,但该过程中相关调控通路的研究仍十分有限。本研究拟通过蛋白质相对积累量来研究合成和分解通路的变化。将骨骼肌细胞置于低氧环境中培养,分别在0 h、6 h、12 h和24 h收集细胞,并进行检测。免疫荧光观察肌球蛋白(myosin),翻译表面感应检测蛋白质合成,Western印迹法测试蛋白质合成相关基因(ERK1/2、p-ERK1/2、mTOR、p-mTOR、4E-BP1、p-4E-BP1)、蛋白质分解相关基因(泛素、FoxO1、p-FoxO1、MuRF1和Atrogin-1)表达量。结果发现,随着低氧干预时间延长,肌纤维直径和骨骼肌细胞中蛋白质相对积累量随时间逐渐减小(P<0.01)。与0 h相比,6 h p-4E-BP1/4E-BP1和Atrogin-1的表达显著上调(P<0.05),p-mTOR表达显著高于0 h(P<0.01);6 h和24 h p-mTOR/mTOR的比值显著大于0 h(P<0.05),而p-FoxO1/FoxO1的比值随时间逐渐减小(P<0.01)。上述结果表明,低氧干预能够使骨骼肌细胞直径减少、骨骼肌细胞蛋白质积累减少,并且低氧打破骨骼肌细胞蛋白质合成和分解的平衡,可能是通过调节mTOR/4E-BP1通路活性和FoxO1/Atrogin-1通路的活性实现的。     

不同低氧暴露对大鼠有氧代谢潜能的影响

目的:同时从血液运氧能力和骨骼肌氧化能力的角度,观察不同低氧暴露对机体有氧代谢潜能的影响。方法:雄性SD大鼠30只,随机分成3组(n=10):常氧对照组、12h低氧暴露组(间歇低氧暴露组)和24h低氧暴露组(持续低氧暴露组)。氧浓度为13.6%。6周后测试红细胞(RBC)、血红蛋白(Hb)、红细胞压积(Hct)、2,3-二磷酸甘油酸(2,3-DPG)和腓肠肌柠檬酸合成酶(CS)、琥珀酸脱氢酶(SDH)、苹果酸脱氢酶(MDH)活性。结果:持续低氧能显著提高RBC、Hb、Hct、2,3-DPG和腓肠肌CS、SDH、MDH活性;间歇低氧除了显著提高Hb外,其它指标无明显变化。结论:持续低氧暴露同时提高机体运氧能力和骨骼肌氧化能力,可以充分提高机体有氧代谢潜能;间歇低氧暴露只能提高血氧容量,对血氧亲和力和骨骼肌氧化能力没有影响,提高机体有氧代谢潜能的效果不如持续低氧暴露。     

低氧及低氧预处理时心肌细胞HIF-1α与凋亡相关蛋白表达的变化

目的:观察低氧时心肌细胞HIF-1α表达变化与凋亡相关蛋白表达关系.方法:采用体外心肌细胞培养的方法,将原代培养4～6 d的大鼠乳鼠心肌细胞随机分为对照组、低氧组与低氧预处理组.低氧预处理组在低氧培养箱中通入1%O2、5%CO2、94%N2的低氧混合气体,每天低氧12 h,低氧5 d,第6 d与急性低氧组一同放入0%O2、5%CO2、95%N2的低氧培养箱中进行低氧暴露.低氧48 h后,通过Western blot方法分别检测心肌细胞中HIF-1α、Bcl-2、P53及Bax的表达变化.结果:常氧时细胞不表达HIF-1α,低氧可增加HIF-1的表达,低氧预处理后,能降低HIF-1α的表达.低氧时,Bax的表达变化大致与此相同.p53在低氧时的变化也与其相同,但低氧预处理后似乎没有明显的改变.Bcl-2在低氧时表达下降,低氧预处理后可增加其表达.结论:HIF-1α的表达可协同Bcl-2家族凋亡相关蛋白的表达,在低氧导致的心肌细胞凋亡中发挥重要作用.     

低氧条件下大鼠肺动脉平滑肌细胞中活性氧与低氧诱导因子-1α和细胞增殖的关系

在低氧条件下,观察大鼠肺动脉平滑肌细胞（pulmonary arterial smooth muscle cells,PASMCs）中活性氧（reactive oxygen species,ROS）的变化,探讨ROS的变化是否通过调控低氧诱导因子-4α（hypoxia-inducible factor 1α, HIF-1α）的表达影响PASMCs的增殖。采用组织块法原代培养大鼠PASMCs,分成3组：常氧组（21％O2,24h）,低氧组（5％O2,24h）,低氧＋Mn-TBAP组（5％O2,24h,Mn-TBAP是一种ROS清除剂）。用激光共聚焦显微镜荧光染色法检测细胞内ROS的变化;用RT-PCR和免疫组织化学方法分别测定HIF-1α mRNA和蛋白的表达;用MTT法检测细胞增殖程度。结果显示：（1）低氧组PASMCs内ROS水平明显高于常氧组（P〈0．05）,低氧＋Mn-TBAP组ROS水平明显低于低氧组（P〈0．05）,但仍高于常氧组（P〈0．05）;（2）低氧组及低氧＋Mn-TBAP组的HIF-1α mRNA和蛋白表达均高于常氧组（P〈0．05）,且低氧组表达高于低氧＋Mn-TBAP组（P〈0．05）;（3）低氧组细胞增殖明显高于常氧组和低氧＋Mn-TBAP组（P〈0．05）,低氧＋Mn-TBAP组细胞增殖高于常氧组（P〈0．05）。结果表明：在低氧条件下大鼠PASMCs中ROS水平明显升高,RROS的变化能够调节HIF-1α的表达,进而影响平滑肌细胞的增殖,提示ROS可能在肺动脉高压的发病机制和低氧信号转导中具有重要作用。     

低氧运动对Nrf2基因敲除大鼠运动能力和氧化应激的影响

Nrf2可调节多种抗氧化酶的表达,Nrf2的缺失可能影响机体的运动能力,而低氧可提高机体的抗氧化能力并改善运动能力。为了考察低氧运动对Nrf2基因敲除大鼠运动能力和氧化应激的影响,本研究分别在常氧和低氧环境(12%氧浓度)中对野生型大鼠和Nrf2敲除大鼠进行4周的跑台运动。研究显示,低氧运动可提高野生型大鼠的跑台运动力竭时间,Nrf2敲除可缩短大鼠的力竭时间;低氧运动可上调大鼠的Nrf2 m RNA表达量;Nrf2敲除明显抑制HIF-1α蛋白表达,而低氧运动可上调野生型和Nrf2敲除大鼠的HIF-1α蛋白表达;Nrf2敲除大鼠的骨骼肌ROS水平明显升高,并且低氧均可降低野生型和Nrf2敲除大鼠骨骼肌ROS水平。低氧运动可上调Nrf2敲除大鼠的CAT和GSH-PX蛋白表达。苏木精和伊红(HE)染色显示,Nrf2敲除大鼠在力竭跑台运动完成后出现更严重的骨骼肌病理改变,而低氧运动可减轻骨骼肌损伤。本研究认为,Nrf2敲除导致了大鼠骨骼肌中抗氧化酶的抑制及ROS的过量累积,从而造成了骨骼肌损伤并降低了运动能力。此外,低氧可通过上调Nrf2的表达,进而激活HIF-1α及抗氧化酶活性,从而提高运动能力,并防止骨骼肌损伤。     

低氧对胚胎干细胞增殖的影响

目的:观察间歇性低氧和持续性低氧对体外培养的胚胎干细胞(ES细胞)增殖的影响.方法:利用细胞记数法和BrdU (5-溴脱氧尿苷)掺入的流式细胞分析检测细胞增殖,并用RT -PCR的方法检测低氧诱导因子(HIF-1a)的表达变化.结果:①将ES细胞分别放在低氧(3%～10% O2)和常氧(20% O2)的环境中培养24 h后,在低氧环境中培养的ES细胞数较常氧组明显减少;②将ES细胞分别给予间歇性低氧刺激(3%～10% O2),每天10 min,连续4 d后,发现3%低氧组较常氧对照组的细胞增殖明显升高.③用RT-PCR方法观察HIF-1a的表达与细胞增殖的关系,发现在常氧环境中培养的ES即有HIF-1a的表达,ES细胞在持续低氧24 h或间歇性低氧(3%～10% O2)刺激4 d后对HIF-1a的表达均无明显影响.结论:间歇性低氧(3% O2)可明显促进体外培养的ES细胞增殖,而持续性低氧抑制ES细胞增殖,间歇性低氧(3% O2)刺激促进ES细胞增殖的机制尚有待于进一步的研究.     

低氧处理不同时间对人肺腺癌A549细胞增殖的影响 及相应缺氧模型探讨*

目的:观察低氧处理不同时间对人肺腺癌A549细胞增殖的影响,探讨合理的人肺腺癌细胞株A549体外模拟缺氧时间。方法:将人肺腺癌细胞A549细胞株在低氧环境下分别培养12 h、24 h、48 h、72 h,设置常氧对照组,通过CCK8法测定A549细胞存活率,RT-PCR和免疫印迹分别检测细胞缺氧诱导因子-1α(hypoxia-inducible factor-1α, HIF-1α)和血管内皮生长因子(vascularendothelial growth factor, VEGF)mRNA及蛋白的表达。结果:低氧24 h组A549细胞存活率最高,低氧48 h、72 h组A549细胞存活率呈时间依赖性明显下降(P0.001)。自低氧12 h起,A549细胞HIF-1αmRNA和VEGFmRNA的表达开始随低氧时间延长而显著增加(P均0.001);HIF-1α和VEGF蛋白表达自24 h开始随低氧时间延长而显著增加(P均0.001)。结论:低氧诱导的A549细胞存活率呈时间依赖性降低,而HIF-1α、VEGF表达呈时间依赖性增高,人肺癌细胞株A549缺氧模型最适时间为24 h。     

低氧联合LPS刺激对星形胶质细胞中炎性因子和BNIP3表达的影响*

目的:探讨在低氧联合脂多糖(LPS)作用下,星形胶质细胞中B淋巴细胞瘤-2/腺病毒E1B 19-kD相互作用蛋白3(BNIP3)的表达和炎症反应变化。方法:将体外培养的原代星形胶质细胞和神经元进行下列分组:常氧组、LPS组、低氧组和LPS+低氧组(每组设置3个复孔)。LPS处理后,低氧组和LPS+低氧组放入低氧细胞孵箱,LPS组和常氧组放入正常的细胞孵箱。LPS浓度:100 ng/ml,氧气浓度为0.3%。处理时间为24 h。原代的星形胶质细胞进行上述的分组,时间点设为6 h、12 h和24 h。Western blot检测BNIP3的表达变化,RT-PCR和ELISA分别检测星形胶质细胞的肿瘤坏死因子-ɑ(TNF-ɑ)、白细胞介素-1β(IL-1β)和白细胞介素6(IL-6)mRNA水平变化和分泌情况。结果:与常氧组比较,低氧组炎症因子的表达没有变化,LPS组和LPS＋低氧组的炎症因子TNF-ɑ、IL-1β和IL-6 mRNA水平升高(P＜0.01);与LPS组比较,LPS＋低氧组炎症因子IL-1β和IL-6 mRNA水平进一步升高(P＜0.05,P＜0.01)。与常氧组比较,低氧组炎症因子的分泌水平没有变化,LPS组和LPS＋低氧组的炎症因子TNF-ɑ和IL-6 分泌水平升高(P＜0.01),IL-1β的水平没有变化;与LPS组比较,LPS＋低氧组炎症因子TNF-ɑ和IL-6分泌水平没有进一步升高。BNIP3在体外培养的神经元和星型胶质细胞中都有表达;在星形胶质细胞中,与常氧组比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达明显增加(P＜0.01);在神经元中,与常氧组比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达增加(P＜0.05,P＜0.01);与神经元的低氧组比较,星形胶质细胞的低氧组BNIP3的表达增加更明显(P＜0.01)。在星形胶质细胞中LPS联合低氧刺激6、12、24 h后BNIP3蛋白的表达,与常氧组相同时间点比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达增加(P＜0.05,P＜0.01);与低氧组相同时间点比较,6 h和12 h的LPS＋低氧组BNIP3的表达增加的更高(P＜0.01)。结论:低氧联合LPS刺激可以增强星形胶质细胞的炎症反应,LPS能增加低氧下星形胶质细胞中BNIP3的表达,提示BNIP3在星形胶质细胞的炎性反应中可能具有一定的调节作用。     

抗阻训练通过Akt-FoxO1通路抑制低氧诱导的骨骼肌萎缩

高原低氧环境会引起肌力下降和运动能力退化,而抗阻训练是刺激骨骼肌生长的重要手段,叉头转录因子1（fork head box protein O 1,FoxO1）在调控骨骼肌蛋白质分解通路中承担重要角色。为探究Akt-FoxO1通路是否参与抗阻训练抑制低氧诱导的骨骼肌萎缩,本研究构建低氧诱导骨骼肌萎缩的大鼠模型,并模拟海拔4 000 m低氧环境下（12.4% O2）进行抗阻训练,对比观察大鼠比目鱼肌和趾长伸肌湿重和横截面积,以及蛋白激酶B（protein kinase B,Akt）、叉头转录因子1、泛素蛋白连接酶1（muscle ring finger 1,MuRF1）的表达差异等。结果表明,低氧暴露导致大鼠趾长伸肌湿重显著下降,苏木精-伊红染色组织切片分析肌纤维横截面积、低氧环境下比目鱼肌横截面积明显下降,而低氧抗阻训练后趾长伸肌横截面积明显高于安静组。实时荧光定量PCR和蛋白质免疫印迹结果显示,低氧暴露后FoxO1和MuRF1基因表达明显上调,低氧下抗阻训练后发现,Akt基因表达明显上调而FoxO1、MuRF则明显下调。免疫荧光观察磷酸化FoxO1在细胞核内外表达情况,发现抗阻训练后FoxO1（S256）于细胞核外表达增强。上述结果表明,抗阻训练可以达到抑制低氧诱导骨骼肌萎缩的效果,Akt促进FoxO1磷酸化从而减缓骨骼肌蛋白质分解过程是抗阻训练能够抑制骨骼肌萎缩的分子机制之一。     

Iron homeostasis is maintained in the brain, but not the liver, following mild hypoxia

Alterations in iron metabolism or oxidative damage in response to hypoxic incidents have been examined following re-oxygenation of the hypoxic tissue. To understand the consequences of decreased tissue oxygen on iron load, metal-catalyzed redox activity and oxidative modifications in isolation from re-oxygenation, the present study exposed mice to either normoxia, or mild hypoxia (380 Torr; approximately 10% normobaric oxygen) where the tissue was not allowed to re-oxygenate prior to examination. Brain, liver and skeletal muscle were examined for Fe3+ load, metal-catalyzed redox activity and oxidative modifications to proteins (N(epsilon)-(carboxymethyl)lysine), lipids (4-hydroxynonenal pyrrole) and nucleic acids (8-hydroxyguanosine). Hypoxia induced a 43% increase in the iron content of the liver (P < 0.001) as determined by ICP-MS and a 3.8-fold increase in Fe3+ load (P < 0.001) as determined by Perl's stain. There was a corresponding 2-fold increase in metal-catalyzed redox activity (P < 0.01) in the liver, but no change in the expression of oxidative markers. In contrast, non-significant increases in Fe3+ and metal-catalyzed redox activity were observed in the cerebral cortex, and molecular and granular layers of the hippocampus and cerebellum. Interestingly, hypoxia significantly decreased oxidative modifications to proteins and lipids, but not nucleic acids in most brain regions examined. In addition, hypoxia did not alter the Fe content of skeletal muscle, or the contents of Zn, Cu, Ni or Mn in liver, skeletal muscle, cerebral cortex or hippocampus. Together, these results indicate that there is a tighter regulation of iron metabolism in the brain than the liver, which limits the redistribution of Fe3+ following hypoxia.     

Proteins modulation in human skeletal muscle in the early phase of adaptation to hypobaric hypoxia

High altitude hypoxia is a paraphysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. In man, skeletal muscle, after prolonged exposure to hypoxia, undergoes mass reduction and alterations at the cellular level featuring a reduction of mitochondrial volume density, accumulation of lipofuscin, a product of lipid peroxidation, and dysregulation of enzymes whose time course is unknown. The effects of 7-9 days exposure to 4559 m (Margherita Hut, Monte Rosa, Italy) on the muscle proteins pattern were investigated, pre- and post-exposure, in ten young subjects, by 2-D DIGE and MS. Ten milligram biopsies were obtained from the mid part of the vastus lateralis muscle at sea level (control) and at altitude, after 7-9 days hypoxia. Differential analysis indicates that proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses were significantly (p<0.05) decreased in hypoxia. Parenthetically, hypoxia markers such as hypoxia inducible factor 1 alpha (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) were still at the pre-hypoxia levels, whereas the mammalian target of rapamycin (mTOR), a marker of protein synthesis, was reduced.     

Physiological and biochemical correlates of increased work in trained iron-deficient rats

We investigated physiological and biochemical factors associated with the improved work capacity of trained iron-deficient rats. Female 21-day-old rats were assigned to one of four groups, two dietary groups (50 and 6 ppm dietary iron) subdivided into two levels of activity (sedentary and treadmill trained). Iron deficiency decreased hemoglobin (61%), maximal O2 uptake. (VO2max) (40%), skeletal muscle mitochondrial oxidase activities (59-90%), and running endurance (94%). In contrast, activities of tricarboxylic acid (TCA) cycle enzymes in skeletal muscle were largely unaffected. Four weeks of mild training in iron-deficient rats resulted in improved blood lactate homeostasis during exercise and increased VO2max (15%), TCA cycle enzymes of skeletal muscle (27-58%) and heart (29%), and liver NADH oxidase (34%) but did not affect any of these parameters in the iron-sufficient animals. In iron-deficient rats training affected neither the blood hemoglobin level nor any measured iron-dependent enzyme pathway of skeletal muscle but substantially increased endurance (230%). We conclude that the training-induced increase in endurance in iron-deficient rats may be related to cardiovascular improvements, elevations in liver oxidative capacity, and increases in the activities of oxidative enzymes that do not contain iron in skeletal and cardiac muscle.     

Chronic hypoxia attenuates resting and exercise-induced VEGF, flt-1, and flk-1 mRNA levels in skeletal muscle.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic mitogen. However, chronic hypoxia is generally not found to increase mammalian skeletal muscle capillarity. We sought to determine the effect of chronic hypoxia (8 wk, inspired O2 fraction = 0.12) on skeletal muscle gene expression of VEGF, its receptors (flt-1 and flk-1), basic fibroblast growth factor, and transforming growth factor-beta1. Wistar rats were exposed to chronic hypoxia (n = 12) or room air (n = 12). After the exposure period, six animals from each group were subjected to a single 1-h treadmill exercise bout (18 m/min on a 10 degrees incline) in room air while the remaining six animals served as rest controls. Morphological analysis revealed that chronic hypoxia did not increase skeletal muscle capillarity. Northern blot analyses showed that chronic hypoxia decreased resting VEGF, flt-1, and flk-1 mRNA by 23, 68, and 42%, respectively (P < 0.05). The VEGF mRNA response to exercise was also decreased (4.1- and 2.7-fold increase in room air and chronic hypoxia, respectively, P < 0.05). In contrast, neither transforming growth factor-beta1 nor basic fibroblast growth factor mRNA was significantly altered by chronic hypoxia. In conclusion, prolonged exposure to hypoxia attenuated gene expression of VEGF and its receptors flt-1 and flk-1 in rat gastrocnemius muscle. These findings may provide an explanation for the lack of mammalian skeletal muscle angiogenesis that is observed after chronic hypoxia.     

藏羚羊骨骼肌肌红蛋白含量及乳酸脱氢酶、苹果酸脱氢酶活力的研究

目的:探讨藏羚羊骨骼肌对低氧环境的适应机制。方法:以生活在同海拔高度(4 300 m)的藏绵羊和低海拔绵羊(1 800 m)为对照,用分光光度法测定三种动物骨骼肌中肌红蛋白(Mb)含量、乳酸(LA)含量,酶活力法测定三种动物骨骼肌中乳酸脱氢酶(LDH)和苹果酸脱氢酶(MDH)活力。结果:藏羚羊骨骼肌中Mb含量明显高于藏绵羊和低海拔绵羊(P<0.05),而藏绵羊和低海拔绵羊间无明显差异。LA含量和LDH活力明显低于藏绵羊和低海拔绵羊(P<0.05),而MDH活力及MDH/LDH比值显著高于藏绵羊和低海拔绵羊(P<0.05),藏绵羊和低海拔绵羊间无明显差异。结论:藏羚羊可能通过增加骨骼肌中Mb的含量,提高其在低氧环境获取氧的能力,且藏羚羊骨骼肌组织中有氧代谢比例高,这可能与肌肉中Mb含量较高有关,推测藏羚羊较高的Mb含量可能是其适应高原缺氧条件的分子基础之一。     

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.     

Skeletal muscle capillarity and angiogenic mRNA levels after exercise training in normoxia and chronic hypoxia.

Gene expression of vascular endothelial growth factor (VEGF), and to a lesser extent of transforming growth factor-beta(1) (TGF-beta(1)) and basic fibroblast growth factor (bFGF), has been found to increase in rat skeletal muscle after a single exercise bout. In addition, acute hypoxia augments the VEGF mRNA response to exercise, which suggests that, if VEGF is important in muscle angiogenesis, hypoxic training might produce greater capillary growth than normoxic training. Therefore, we examined the effects of exercise training (treadmill running at the same absolute intensity) in normoxia and hypoxia (inspired O(2) fraction = 0.12) on rat skeletal muscle capillarity and on resting and postexercise gene expression of VEGF, its major receptors (flt-1 and flk-1), TGF-beta(1), and bFGF. Normoxic training did not alter basal or exercise-induced VEGF mRNA levels but produced a modest twofold increase in bFGF mRNA (P < 0.05). Rats trained in hypoxia exhibited an attenuated VEGF mRNA response to exercise (1.8-fold compared 3.4-fold with normoxic training; P < 0.05), absent TGF-beta(1) and flt-1 mRNA responses to exercise, and an approximately threefold (P < 0.05) decrease in bFGF mRNA levels. flk-1 mRNA levels were not significantly altered by either normoxic or hypoxic training. An increase in skeletal muscle capillarity was observed only in hypoxically trained rats. These data show that, whereas training in hypoxia potentiates the adaptive angiogenic response of skeletal muscle to a given absolute intensity of exercise, this was not evident in the gene expression of VEGF or its receptors when assessed at the end of training.     

低氧与铁代谢相关蛋白

氧和铁这两种元素对生命活动十分重要. 低氧诱导因子(hypoxia-inducible factors, HIFs)作为转录因子,参与一系列靶基因的表达调控以适应低氧. 铁参与 DNA合成、氧气运输、代谢反应等多种细胞活动,过量游离铁会通过Haber-Weiss或 Fenton反应产生毒性自由基. 细胞通过与铁吸收、存储和利用有关的多种铁代谢相 关蛋白之间的协同作用来维持铁稳态. 与铁稳态相关的一些基因是HIFs的靶基因或 者间接受低氧调控,包括转铁蛋白、转铁蛋白受体、二价金属转运体1、铁调素、膜 铁转运蛋白、血浆铜蓝蛋白、铁蛋白等,而胞内铁浓度的改变能影响HIFs的表达. 本文就低氧与铁代谢相关蛋白的关系,尤其是低氧对铁代谢相关蛋白的调节作一综 述.     

Iron homeostasis is maintained in the brain,but not the liver,following mild hypoxia

Abstract

Alterations in iron metabolism or oxidative damage in response to hypoxic incidents have been examined following re-oxygenation of the hypoxic tissue. To understand the consequences of decreased tissue oxygen on iron load, metal-catalyzed redox activity and oxidative modifications in isolation from re-oxygenation, the present study exposed mice to either normoxia, or mild hypoxia (380 Torr; ～10% normobaric oxygen) where the tissue was not allowed to re-oxygenate prior to examination. Brain, liver and skeletal muscle were examined for Fe³⁺ load, metal-catalyzed redox activity and oxidative modifications to proteins (N^?-(carboxymethyl)lysine), lipids (4-hydroxynonenal pyrrole) and nucleic acids (8-hydroxyguanosine). Hypoxia induced a 43% increase in the iron content of the liver (P < 0.001) as determined by ICP-MS and a 3.8-fold increase in Fe³⁺ load (P < 0.001) as determined by Perl's stain. There was a corresponding 2-fold increase in metal-catalyzed redox activity (P < 0.01) in the liver, but no change in the expression of oxidative markers. In contrast, non-significant increases in Fe³⁺ and metal-catalyzed redox activity were observed in the cerebral cortex, and molecular and granular layers of the hippocampus and cerebellum. Interestingly, hypoxia significantly decreased oxidative modifications to proteins and lipids, but not nucleic acids in most brain regions examined. In addition, hypoxia did not alter the Fe content of skeletal muscle, or the contents of Zn, Cu, Ni or Mn in liver, skeletal muscle, cerebral cortex or hippocampus. Together, these results indicate that there is a tighter regulation of iron metabolism in the brain than the liver, which limits the redistribution of Fe³⁺ following hypoxia.