Name: activating autophagy by aerobic exercise in mice
Uploaded: 2017-02-03T14:00:00.000+00:00
Duration: 8 min 44 s
Description: Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

所有涉及动物的程序进行根据西北大学机构动物护理和使用委员会（IACUC）批准的指导进行。 1.鼠标型号<ol><li>使用8-12周龄小鼠的运动训练。以检测体内行使诱导自噬，用于成像研究和C57BL / 6小鼠进行生化分析的GFP-LC3转基因小鼠（C57BL / 6背景）。 </li></ol> 2.运动诱发的自噬<ol><li> 跑步机设置-强制运动 <ol><li>适应和培养在10°上坡开跑步机小鼠2天。在第1天，锻炼小鼠5分钟以8米/分，并在第2天，运动5分钟将小鼠在8米/分钟，随后另外5分钟，在10米/分钟10。鼓励小鼠温柔的手轻推运行。 </li><li>在第 3天，让小鼠经历对10°上坡跑步机，starti运行90分钟的单一回合以10米/分钟的速度纳克。 40分钟后，增加跑步机的速度以分钟1米/分钟，每10的速度，总共30分钟，然后增加的速度的速度1m /分钟，每5分钟，直到其达到17，米/分钟，总共的运动时间90分钟1070米转动的距离。 </li><li>在低强度范围（1.2赫兹）设置电刺激，鼓励老鼠跑，以避免重复一脚惊世骇俗。 </li><li>对于组织收集，通过安乐死吸入异氟醚小鼠，颈椎脱位运动后即刻。其他核准安乐死方法也可以使用，如CO 2或其他麻醉剂的IP注射。自噬的水平似乎没有由安乐死方法的影响。 </li></ol></li><li> 跑轮设置-自由运动 <ol><li>放置一个笼子内的鼠标运行轮（11.4厘米直径的）用单容纳鼠标。 </li><li>验证使用自行车里程表的运行能力。成立[每车轮的完全旋转距离（毫米）]车轮参数在358（11.4 *π）。测量后24小时运行距离。 </li><li>让鼠标自愿运行2周运行的轮子。对于组织收集，牺牲在上午鼠标的运行期。 </li></ol></li></ol> 3.自噬通量评价<ol><li>为了测量静止和运动的条件下的自噬通量，治疗的小鼠与自噬抑制剂氯喹三天比较它们用PBS处理的小鼠。 <ol><li>溶解氯喹在PBS（5毫克/毫升），以及腹膜内的剂量为50mg / kg /天的连续三天注射喹到小鼠。牺牲小鼠和最后一次注射后收集组织3小时。 </li><li>为行使小鼠以50mg / kg /天的剂量与氯喹预处理他们连续两天。在第三天，注入小鼠与氯喹的相同浓度，和后90分钟让他们在跑步机上另外90分钟运行。 </li><li>安乐死通过异氟烷吸入将小鼠，随后通过颈椎脱位。收集组织运行后，使氯的总温育时间是一样的对照（静止）小鼠（3小时）。 注:另外，用氯喹单次注射，以确定在小鼠组织中自噬通量。 </li></ol></li></ol> 4.组织收获和固定<ol><li>在7.4的最终pH在PBS:在此之前组织准备收获的磷酸盐缓冲液（PBS）和新鲜的4％多聚甲醛（必要的个人防护装备PFA，慎用）。储存在4℃下两种解决方案。 </li><li>填写30毫升注射器15 - 20毫升4％PFA的（对于GFP-LC3的小鼠）。连接到一个20G的针头导管注射器。 </li><li>的运动后，通过异氟烷吸入安乐死的小鼠，随后通过颈椎脱位。 </li><li>将动物上的BA清洁的工作面上CK。剪切和通过切割膜片暴露心脏打开胸腔。 </li><li>作一小切口对肝脏，让血出来，缓缓注入一共有15毫升4-％PFA进入经导管心脏的右心室，直到肺和肝脏变成完全苍白。立即灌注鼠标运动后，使用注射泵以90毫升/小时的恒定流速。 </li><li>灌注后，取出针，并收集感兴趣的组织（ 如大脑11和骨骼肌）。对于骨骼肌，解剖股外侧。简要地说，拉腿的皮肤向后暴露的肌肉，本地化股四头肌12和剖析出股外侧肌，这是外部肌肉附连到股骨的上部。 </li><li>为进一步固定和脱水，放置的GFP-LC3的小鼠的组织在4％PFA 24小时，在4℃下，然后将它们转移到15％的蔗糖的PBS在4℃过夜，或直到组织定居，然后至30％蔗糖的PBS在4℃下过夜或更长的储存空间。保持在黑暗中尽可能样品，因为它们可能对强光敏感。 </li><li>对于免疫印迹分析单元从直接C57BL / 6小鼠在液氮冷冻组织，并将其存储在-80℃。 </li></ol> 5. GFP-LC3泪点的影像学分析<ol><li> 组织流程 <ol><li>预治疗在包埋介质中的组织（预先固定在PFA和存储在30％的蔗糖），例如用于在标记的小培养皿几分钟"最佳切削温度的化合物"（OCT）。 </li><li>在含有足够新鲜OCT以覆盖组织的标记cryomold转移组织。东方朝向cryomold底部的切片表面（对于股截面外侧肌和矢状切面为半大脑）。避免产生气泡。 </li><li> FRE通过将cryomold在填充有干冰带盖泡沫冷却器几分钟EZE样品，直到在OCT变为白色。裹个体样品中的标记的薄片，它们密封在塑料袋中，并把它们存储在-80℃下过夜或更长时间。 </li><li>使用低温恒温器在厚度为10微米的切割样品部分，并且安装在幻灯片的节。存储载玻片在-20℃，并总是从光保持滑动距离只要有可能。 </li></ol></li><li> 幻灯片准备 <ol><li>从冰箱中取出载玻片并在室温下解冻它们。覆盖带安装媒体用DAPI一滴的组织切片。在上面放置适当大小的盖玻片，并避免形成气泡。 </li><li>用指甲油密封盖玻片的边缘，允许指甲油在室温下黑暗中干燥，然后在一个不透光的容器中的载玻片储存在4℃下，或用GFP-LC3泪点的成像捕获进行。 </li></ol></li><li> &lt;GFP的STRONG&gt;量化 - 通过荧光显微镜LC3泪点<ol><li>执行荧光显微镜，捕捉在相同条件下对所有样品的照片（曝光和设置）。的GFP，使用525纳米的发射波长;对于DAPI，使用490纳米。获得每使用股外侧一个60X物镜和大脑的额脑皮层区样品至少十个图像。 </li><li>量化在2500微米2进行统计分析每个图像中的组织区域，失明的实验组的GFP-LC3泪点的数目。计算从各样品的10个图像的平均数目，用"对象计数"的自动测量功能。对于股设置外侧肌是:阈值L = 10，H = 200; 2X流畅; 1X干净，并为脑额叶皮层设置:阈值L = 11，H = 200; 2X流畅; 1X干净。 </li></ol></li></ol> 6. Western印迹分析自噬标记<ol><li> &lt;STRONG&gt;组织过程<ol><li>制备裂解缓冲液:50mM的Tris-HCl; 150毫摩尔NaCl; 1毫摩尔EDTA; 1％的Triton X-100;蛋白酶抑制剂和磷酸酶抑制剂（新鲜添加）。 </li><li> -80℃储存除去C57BL / 6小鼠的组织。切开肌肉组织切成小块之前用以下方法刀片。添加800微升（对于半脑）或500微升（对于股外侧）冷的裂解缓冲液对样品的。与中速均化器均质化，在4℃下的样品。 </li><li>在4℃下用旋转另外的30分钟的均化的混合物充分溶解到1小时。 </li><li>降速匀浆在12,000×g离心10分钟，在4℃，弃沉淀，并回收上清在新管中。 </li></ol></li><li> 自噬分析 <ol><li>确定根据制造商的说明使用BCA蛋白测定试剂盒组织裂解液的蛋白质浓度。规范化每个样品以相同的浓度。 </li><li>在1与2×Laemmli样品缓冲液合并样品:1的比例，煮沸，在95℃，5 - 10分钟，并与自噬免疫印迹分析标记物13，14进行，例如P62（抗p62的抗体， 1:500稀释）与LC3（抗LC3抗体，1:500稀释度）。煮沸样品加入样品缓冲液后立即，如在冰上再温育可以生成LC3的多个退化频带。 </li><li>量化使用ImageJ通过光密度分析p62蛋白和LC3频段，并且归一化值到相应肌动蛋白条带。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

自噬是分解代谢过程提供能量和细胞质组件或受损细胞器溶酶体降解减少细胞毒性。自噬研究重要的是要了解细胞稳态的调节和应激反应的机制。新的模式和方法不断涌现的研究领域15，研究如何吞噬受损有助于众多病理过程16,17。 营养缺乏（饥饿）和药理学诱导常用的方法来诱导自噬在体外和体?...

自噬是一种进化上保守的降解途径，这是响应于各种胁迫条件如饥饿，缺氧1,2诱导。在自噬，双膜囊，称为自噬体，包括不必要的或损坏的亚细胞成分，并将它们运送到溶酶体降解3。基底自噬是在许多疾病，包括神经变性，肿瘤和2型糖尿病4，5，6被牵连于细胞功能和生物体的发展，和受损基底自噬是必不可少的。 最知名的生理自噬诱导剂是饥饿。然而，它有两个主要的限制。首先，饥饿需要很长的时间，以有效地诱导自噬在动物中， 例如 ，在小鼠中食物限制的48小时在大多数器官。第二，饥饿勉强诱导脑自噬，由于大脑中的相对稳定的营养供给。实际上，它也难以通过小分子诱导剂来检测自噬诱导，因为许多药物不能通过血脑屏障。因此，为了更好地分析自噬活化在疾病的发病机制的功能，我们最近发现，运动是诱导自噬在时间7，8，9短时间内更有效的生理方法。与饥饿相比，自噬是有效地跑步机上跑步一样快，30分钟所致。因此，运动是一种方便和有效的生理方法来研究细胞自噬机制在调解医疗福利和预防疾病。 有用于检测自噬活动的，包括LC3和p62的几种蛋白质的标志物。 LC3（微管相关蛋白1A / 1B-光速c海恩3）是胞质蛋白（LC3-I形式）是，在自噬诱导缀合的PE（磷脂酰乙醇胺）。 PE-脂化LC3（LC3-II的形式）被募集到autophagosomal膜，可用于当用GFP标记的可视化自噬。其从细胞溶质向点状在显微镜下自噬体结构易位是自噬诱导的指示。 P62是自噬底物（如泛素化蛋白质）货物的受体，并且被并入自噬体为好。因为蛋白质是与自噬体沿溶酶体降解，其水平可用于测量自噬通量。在这里，我们展示了如何使用这些标志通过有氧运动，包括强迫运动（跑步机）和自愿运动（跑步轮）引起不同的鼠标组织量化自噬。相同的过程也可以治疗其它诱导物后施加到自噬的体内测量。

<table><tbody><tr><td>Treadmill</td><td>Columbus Instruments</td><td>150-RM Exer 3/6</td><td></td></tr><tr><td>Mouse running wheel</td><td>Super Pet</td><td>100079365</td><td>diameter 11.4 cm</td></tr><tr><td>Odometer</td><td>Bell</td><td>DASHBOARD 100</td><td></td></tr><tr><td>Syringe pump</td><td>KD Scientific</td><td>KDS100</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>Model: inverted microscope ECLIPSE</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1850UV</td><td></td></tr><tr><td>Homogenizer</td><td>IKA</td><td>003737001 / Model: T10 Basic S1</td><td></td></tr><tr><td>Chloroquine</td><td>CAYMAN CHEMICAL COMPANY</td><td>14194</td><td></td></tr><tr><td>Parafolmaldehye</td><td>SIGMA-ALDRICH</td><td>P6148</td><td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer.</td></tr><tr><td>Mounting media</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>p62 antibody</td><td>BD Biosciences</td><td>610833</td><td></td></tr><tr><td>LC3 antibody</td><td>Novus Biologicals</td><td>NB100-2220</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad Laboratories</td><td>161-0737</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr></tbody></table>

activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

本协议描述两种不同的方法来诱导自噬通过有氧运动小鼠组织:共90分钟由驯化两天进行一个多车道的跑步机锻炼强迫的;两个星期自愿行使对单只装一个用来跑轮。在每个运动方案，我们可以通过测量各个器官荧光显微镜和Western blot分析自噬通量。 我们用表达GFP标记的LC3当记者通过系统的锻炼1，监测自噬的转...

Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

Activating Autophagy by Aerobic Exercise in Mice

自噬活化是在预防许多疾病有益。一种生理方法的诱导自噬体内是体育锻炼。在这里，我们将展示如何通过有氧运动来激活自噬和在小鼠体内测量细胞自噬水平。

小鼠中自噬激活有氧运动

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

activating-autophagy-by-aerobic-exercise-in-mice

Research

JoVE Journal

Biology

11.8K 次观看.  Northwestern University. 自噬活化是在预防许多疾病有益。一种生理方法的诱导自噬体内是体育锻炼。在这里，我们将展示如何通过有氧运动来激活自噬和在小鼠体内测量细胞自噬水平。 

视频: Activating Autophagy by Aerobic Exercise in Mice

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

小鼠运动耐力的评估没有一个冲击电网的使用:另一种强迫运动

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

科研

行为学

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

小鼠低血压运动的下位强度及血管功能评估

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

发育生物学

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

免疫与感染

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

老年小鼠短期高强度间隔训练与跑步机评价

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

生物学

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 &#181;M) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Based on in vitro experiments, this study revealed the mechanism of crocetin in repairing oxidative stress damage of cardiomyocytes by influencing mitophagy, in which the PINK1/Parkin signaling pathway plays an important role.

通过PINK1 / Parkin途径介导的线粒体自噬 保护 H9c2心肌细胞免受藏红花素氧化应激

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and ...

医学

探索 T2DM 运动干预：小鼠静态训练装置

Static Strength Training Method for Type 2 Diabetic Mice

The treatment of type 2 diabetes mellitus (T2DM) is a major difficulty in improving patient health. Exercise is one of the main interventions for T2DM. Static strength training is one of the key forms of traditional sports in China. Research shows that static strength training is an effective clinical method for T2DM intervention, but there is no experimental device suitable for static training in mice. One of the difficulties in moving from clinical to basic research is to design appropriate experimental devices. In order to further study the mechanism of static training intervention in T2DM, a simple method for making a static training device for mice is introduced in this paper. This device has the advantages of simple operation, cheap material, and high feasibility. Previous studies conducted under this protocol have shown that static training can effectively reduce blood glucose levels and improve the mitochondrial function of skeletal muscle cells in T2DM mice. The purpose of introducing this device is to promote research on the mechanism of traditional exercise in the intervention of T2DM and to lay a foundation for the quantitative intervention of exercise.

作者聚焦：通过小鼠静态运动训练推进糖尿病研究

This protocol provides a simple method of making static training equipment for mice. The device maintains the muscle isometric contraction of the limbs of mice so as to verify the intervention effect of traditional exercise on type 2 diabetes (T2DM) and provides new exercise therapy for the clinical treatment of T2DM.

2型糖尿病小鼠静态力量训练方法

The treatment of type 2 diabetes mellitus (T2DM) is a major difficulty in improving patient health. Exercise is one of the main interventions for T2DM. ...

小鼠中自噬激活有氧运动

摘要

探索更多视频

此视频中的章节

小鼠运动耐力的评估没有一个冲击电网的使用:另一种强迫运动

小鼠低血压运动的下位强度及血管功能评估

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

老年小鼠短期高强度间隔训练与跑步机评价

肌肉干细胞中自噬的原位免疫荧光染色

通过PINK1 / Parkin途径介导的线粒体自噬保护 H9c2心肌细胞免受藏红花素氧化应激

2型糖尿病小鼠静态力量训练方法

2型糖尿病小鼠静态力量训练方法

2型糖尿病小鼠静态力量训练方法

小鼠中自噬激活有氧运动

摘要

探索更多视频

此视频中的章节

小鼠运动耐力的评估没有一个冲击电网的使用:另一种强迫运动

小鼠低血压运动的下位强度及血管功能评估

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

老年小鼠短期高强度间隔训练与跑步机评价

肌肉干细胞中自噬的原位免疫荧光染色

通过PINK1 / Parkin途径介导的线粒体自噬 保护 H9c2心肌细胞免受藏红花素氧化应激

2型糖尿病小鼠静态力量训练方法

2型糖尿病小鼠静态力量训练方法

2型糖尿病小鼠静态力量训练方法

通过PINK1 / Parkin途径介导的线粒体自噬保护 H9c2心肌细胞免受藏红花素氧化应激