低氧训练对大鼠骨骼肌HIF-1mRNA表达及其对骨骼肌铁代谢影响的调节机制

发布时间：2018-08-18 11:26

【摘要】：铁参与机体氧的转运、贮存与利用及ATP合成等许多生理过程。血红蛋白和肌红蛋白与氧气的运输和扩散有着密切的关系。运动训练和低氧环境均可改变机体铁状态。低氧训练能发挥缺氧和运动双重刺激，提高机体运载及利用氧的能力。骨骼肌作为运动中利用氧和铁的重要组织，低氧刺激下可引起骨骼肌中铁代谢发生适应性变化。然而国内尚缺乏低氧训练对骨骼肌铁代谢的机制研究，因此本研究采用在体和离体实验结合分析的方法，以阐明低氧训练对骨骼肌铁代谢的影响机制。研究内容与方法：在体实验：选取健康雄性SD大鼠32只，随机分为常氧安静组（normalcontrol，NC）、常氧运动组(normal exercise，NE)、低氧安静组(hypoxia control，HC)、低氧运动组(hypoxia exercise，HE)。每组各8只。常氧安静组为对照组，常氧运动组在常氧下进行运动，强度为从21m/min递增至25m/min，每周增1m/min，1h/d，6天/周，坡度为0°。低氧安静组每天在低氧舱安静8h(氧浓度13.6%,相当于3500m海拔)。低氧运动组在进行完常氧运动后进行低氧暴露。共处理5周。最后一次处理24h后安静常氧时取材，采用原子吸收法检测大鼠骨骼肌总铁含量；应用RT-PCR法分析大鼠骨骼肌低氧诱导因子-1（hypoxiainducible factor1，HIF-1）mRNA相对表达变化；应用Western Blot检测大鼠骨骼肌二价金属离子转运体(divalent metal transporter l, DMT1),转铁蛋白受体1(transferring receptor1, TfR1),膜铁转运蛋白1(ferroportin1, FPN1)的表达的变化离体实验：培养骨骼肌L6细胞系，当90％以上为贴壁细胞即可用于实验。将L6细胞随机分为3组：对照组（0-h hypoxia），12h低氧处理组（12-h hypoxia），24h低氧处理组（24-h hypoxia），每组6个重复。实验组分别放入低氧培养箱中并通入低氧气体（1%02）、37℃、95%-98%相对湿度条件下培养，分别处理12小时和24小时。对照组置于普通培养箱中培养。采用55Fe同位素示踪检测铁摄取与释放；细胞内铁池的测定运用流式细胞仪法； Western blot检测L6细胞铁转运蛋白（HIF-1，DMT1，TfR1，FPN1）表达的变化。研究结果： 1低氧训练对大鼠骨骼肌铁代谢的影响：（1）大鼠骨骼肌总铁含量的结果表明，与NC组相比各组骨骼肌总体铁含量增加（P0.05, P0.01）；而HE组明显高于各组（P0.05）。（2）HC组和HE组大鼠骨骼肌中HIF-1mRNA的表达显著高于NC组和NE组（P0.01）。（3）各组大鼠铁转运蛋白表达的比较结果显示：与NC相比，各组的铁吸收蛋白（DMT1、TfR1）均显著性升高(P0.05,P0.01)，，NE组和HC组的铁释放蛋白(FPN1)明显下降（P0.05,P0.01），HE组明显升高（P0.01）。HE组铁吸收蛋白（DMT1、TfR1）和铁释放蛋白（FPN1）均非常显著高于NE组和HC组(P0.01)。 2不同时间低氧暴露对骨骼肌L6细胞系铁代谢的影响：（1）12h组细胞的铁摄取能力明显高于对照组（P0.05），铁释放能力显著低于对照组（P0.01）。24h组肌细胞铁摄取能力与对照组相比无明显差异（P0.05），铁释放能力显著低于对照组（P0.05）。（2）与0h组相比12h组LIP明显升高（P0.05），24h组LIP非常明显降低（P0.01）。（3）与对照组比较，12h组HIF-1的表达非常显著升高（P0.01），DMT1(IRE)表达显著升高（P0.05），DMT1(non IRE)表达显著升高（P0.05），FPN1表达非常显著降低（P 0.01），TfR1表达无明显变化（P0.05）。与0h组相比24h组HIF-1的表达非常显著升高（P0.01），DMT1(IRE)表达非常显著降低（P0.01），DMT1(non IRE)表达非常显著降低（P0.01），FPN1表达非常显著降低（P0.01），TfR1表达非常显著性升高（P0.01）。研究结论：（1）在体实验显示，大鼠适度运动和单纯的低氧暴露均能增加骨骼肌的铁贮存，而低氧训练在促进骨骼肌铁吸收的同时铁的释放也显著增加，这加强了骨骼肌铁循环利用的能力从而进一步满足机体在运动中对铁的需求。（2）离体实验显示，低氧对骨骼肌铁代谢的影响与时间密切相关，一定时间的低氧暴露可以提高肌细胞的铁吸收能力，降低铁释放，增加细胞内的铁含量，而长时间的低氧暴露会引起细胞铁代谢紊乱。（3）骨骼肌中HIF-1的表达在低氧中对调节骨骼肌铁代谢起到关键作用，是低氧对机体铁代谢影响的重要连接纽带。
[Abstract]:Iron is involved in many physiological processes, such as oxygen transport, storage and utilization, and ATP synthesis. Hemoglobin and myoglobin are closely related to oxygen transport and diffusion. Exercise training and hypoxic environment can change the body's iron status. Hypoxic training can play a dual stimulation of hypoxia and exercise, improve the body's ability to transport and utilize oxygen. Skeletal muscle, as an important tissue of utilizing oxygen and iron during exercise, can cause adaptive changes of iron metabolism in skeletal muscle under hypoxic stimulation. However, there is no study on the mechanism of iron metabolism in skeletal muscle after hypoxic training in China. Therefore, this study uses in vivo and in vitro experiments to clarify the effect of hypoxic training on iron metabolism in skeletal muscle. Influence mechanism.
Research contents and methods:
In vivo experiment: 32 healthy male SD rats were randomly divided into normal control group (NC), normoxic exercise group (NE), hypoxia control group (HC), hypoxia exercise group (HE). Hypoxic exercise group was exposed to hypoxia after normoxic exercise for 5 weeks. Total iron content in skeletal muscle; relative expression of hypoxia inducible factor 1 (HIF-1) mRNA in rat skeletal muscle was analyzed by RT-PCR; divalent metal transporter 1 (DMT1), transferring receptor 1 (TfR1), membrane iron transport was detected by Western Blot. L6 cells were randomly divided into three groups: control group (0-h hypoxia), 12-h hypoxia treatment group (12-h hypoxia), 24-h hypoxia treatment group (24-h hypoxia), 6 replicates in each group. The control group was cultured in a normal incubator. The uptake and release of iron were detected by 55Fe isotope tracer. The iron pool in L6 cells was determined by flow cytometry. The iron transport in L6 cells was detected by Western blot. Changes in expression of white (HIF-1, DMT1, TfR1, FPN1).
Research findings:
1 Effect of hypoxic training on iron metabolism in skeletal muscle of rats: (1) The total iron content in skeletal muscle of rats was higher than that of NC group (P 0.05, P 0.01), while that of HE group was higher than that of each group (P 0.05). (2) The expression of HIF-1 mRNA in skeletal muscle of HC group and HE group was significantly higher than that of NC group and NE group (P 0.01). Compared with NC, the expression of ferritin (DMT1, TfR1) increased significantly (P 0.05, P 0.01), and that of ferritin (FPN1) decreased significantly (P 0.05, P 0.01) in NE and HC groups, and increased significantly (P 0.01) in HE group. Group (P0.01).
2 Effects of hypoxic exposure at different time on iron metabolism of L6 cell line of skeletal muscle: (1) The iron uptake ability of L6 cells in 12h group was significantly higher than that of control group (P 0.05), and the iron release ability was significantly lower than that of control group (P 0.01). The iron uptake ability of L6 cells in 24h group was not significantly different from that of control group (P 0.05), and the iron release ability was significantly lower than that of control group (P 0.05). Compared with the control group, the expression of HIF-1 was significantly increased (P 0.01), the expression of DMT1 (P 0.05), the expression of DMT1 (non-IRE) was significantly increased (P 0.05), the expression of FPN1 was significantly decreased (P 0.01), and the expression of TfR1 was not significantly changed (P 0.05). The expression of HIF-1 was significantly increased (P 0.01), the expression of DMT1 (IRE) was significantly decreased (P 0.01), the expression of DMT1 (non-IRE) was significantly decreased (P 0.01), the expression of FPN1 was significantly decreased (P 0.01), and the expression of TfR1 was significantly increased (P 0.01).
Research conclusions:
(1) In vivo experiments showed that both moderate exercise and pure hypoxic exposure could increase iron storage in skeletal muscle, while hypoxic training promoted iron absorption in skeletal muscle, the release of iron was also significantly increased, which strengthened the ability of iron recycling in skeletal muscle to further meet the body's demand for iron during exercise.
(2) In vitro experiments showed that the effect of hypoxia on iron metabolism of skeletal muscle was closely related to time. Hypoxia exposure for a certain period of time could improve iron absorption capacity of muscle cells, reduce iron release and increase iron content in cells. Long-term hypoxia exposure could cause disorder of iron metabolism.
(3) The expression of HIF-1 in skeletal muscle plays a key role in regulating iron metabolism in skeletal muscle under hypoxia, and is an important link between hypoxia and iron metabolism.
【学位授予单位】：河北师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R87

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