BNIP3介导骨骼肌线粒体自噬：不同强度运动的影响

文题释义： BNIP3：隶属于Bcl-2蛋白超家族,是一种线粒体促凋亡蛋白,可与腺病毒转录基因E1B编码的蛋白或Bcl-2蛋白结合。可起到促进细胞凋亡、引起线粒体去极化和自噬,在机体中发挥重要作用。 线粒体自噬：线粒体是细胞的能量工厂,其功能还涉及细胞代谢、信号传导、分化、生长、凋亡和死亡等重要过程。线粒体自噬(mitophagy)是细胞清除损伤或衰老的线粒体,并循环利用其组成元素的过程;与衰老、神经退行性疾病和癌症等诸多生理病理过程密切相关。 背景：不同强度运动对机体可产生不同的影响,运动后骨骼肌的变化也不明确,而运动中机体的生理变化机制更是目前研究的热点。 目的：探讨不同强度运动对大鼠骨骼肌质量的影响以及BNIP3介导的骨骼肌线粒体自噬在维持骨骼肌质量中的作用。 方法：实验方案经北京体育大学动物实验伦理委员会批准。8周龄雄性SD大鼠24只,随机分为对照组、中等强度运动组(5°,15 m/min,1 h,60%VO_2max)和大强度运动组(5°,35 m/min,20 min,85%VO_2max),每组8只,每周运动6次。4周运动后取大鼠比目鱼肌和腓肠肌,称量湿质量,免疫荧光检测肌纤维横截面积,Western Blot检测比目鱼肌和腓肠肌BNIP3、p62和LC3蛋白的表达。 结果与结论：①腓肠肌湿质量大强度运动组和中等强度运动组显著低于对照组(P < 0.01);②比目鱼肌肌纤维横截面积大强度运动组显著小于对照组(P < 0.01);腓肠肌肌纤维横截面积中等强度运动组和大强度运动组均显著大于对照组(P < 0.01);③中等强度运动诱导线粒体自噬增加,表现为BNIP3、LC3-Ⅱ/LC3-Ⅰ值相较对照组表达增加(P < 0.05),而p62较对照组表达下降(P < 0.05);大强度运动组LC3-Ⅱ/LC3-Ⅰ、p62的表达量相较4周中等强度运动分别为上升和下降(P < 0.05),但其BNIP3的表达量却下降(P < 0.05);④结果表明,4周中等强度运动可促进骨骼肌通过BNIP3途径的线粒体自噬清除损伤线粒体,维持骨骼肌功能。4周大强度运动自噬水平更高,但可能对骨骼肌产生不利影响。 ORCID: 0000-0003-3836-3002(于亮) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Swimming exercise effects on the expression of HSP70 and iNOS in hippocampus and prefrontal cortex in combined stress

Exercise could play a beneficial role in stress, but its underlying mechanism especially about heat shock protein 70 (HSP70) and inducible nitric oxide synthase (iNOS) in brain has not been fully clarified. Moreover, few studies have investigated swimming exercise and its effects on the combined stress of both chronic and acute stress. In this study we tried to investigate the role of swimming exercise in combined stress and whether its biological mechanism was related to the HSP70 and iNOS in hippocampus and prefrontal cortex. 32 Wistar rats were enrolled and divided into four groups: control, CUMS, labetalol and exercise. After the animal model of chronic unpredicted mild stress (CUMS) was built in the latter three groups, all the rats were given the novel acute stress of inescapable footshock. The behavioral changes were measured by open field test. Radioimmunoassay (RIA) was adopted to test the change of serum corticosterone (CORT). The expression of HSP70 and iNOS in hippocampus and prefrontal cortex was analyzed by Western blot. The results demonstrated that swimming exercise could not only improve the behavior changes and protect the function of HPA axis stable in CUMS animals exposed to novel acute stress, but also increase the HSP70 expression and decrease the iNOS expression in hippocampus and prefrontal cortex. In conclusion, swimming exercise could play a beneficial role in combined stress by up-regulating HSP70 level and down-regulating iNOS level in brain.     

骨骼肌细胞αB-晶状体蛋白基因在大强度离心运动后的表达

背景：骨骼肌含有被称为“分子伴侣”的小热休克蛋白αB-晶状体蛋白,有可能在肌肉运动中具有重要的生理功能。 目的：观察离心运动后骨骼肌细胞αB-晶状体蛋白基因表达。 方法：将成年雄性Wistar大鼠随机分为安静对照组和运动后即刻组,运动后24 h组。安静对照组正常喂养,两个运动组进行一次性大负荷离心运动,分别取对照组腓肠肌和运动组运动后0和24 h的腓肠肌,使用冰冻切片原位杂交法检测αB-晶状体蛋白基因表达情况。 结果与结论：骨骼肌细胞αB-晶状体蛋白基因在对照组呈低水平表达,在运动后即刻和运动后24 h组均呈较高表达 (P < 0.05),αB-晶状体蛋白基因表达杂交信号主要位于肌细胞膜下。结果证实,大强度离心运动可诱导骨骼肌细胞αB-晶状体蛋白基因表达增高。     

长期运动预适应模型大鼠心脏蛋白激酶C和热休克蛋白70的表达

背景：研究表明蛋白激酶C和热休克蛋白70可能参与运动预适应的心脏保护作用。 目的：观察长期运动预适应对大鼠心脏蛋白激酶C和热休克蛋白70的影响及其对心脏保护的作用机制。 方法：将SD大鼠随机分为对照组,力竭运动组和运动预适应组。对照组和力竭运动组大鼠常规饲养3周,运动预适应组大鼠进行3周的间歇性游泳运动建立长期运动预适应动物模型。3周后,力竭运动组和运动预适应组大鼠进行一次性力竭游泳运动。 结果与结论：大鼠力竭运动后,力竭运动组心脏热休克蛋白70表达高于对照组(P < 0.05);先经3周运动预适应再进行力竭运动后,运动预适应组心脏蛋白激酶C和热休克蛋白70的表达高于力竭运动组(P < 0.05)。结果证实,长期运动预适应能激活心脏蛋白激酶C,诱导热休克蛋白70的合成增多,从而发挥其心脏保护作用。     

乌司他丁对中度烧冲复合伤大鼠肺损伤保护作用的研究

目的研究乌司他丁对中度烧冲复合伤大鼠肺组织保护作用及其可能机制。 方法120只雄性SD大鼠按随机数字表法分为4组：假伤＋0.9%氯化钠溶液组(简称SS组,n＝20)、假伤＋乌司他丁组(简称SU组,n＝20)、烧冲复合伤＋0.9%氯化钠溶液组(简称BBS组,n＝40)和烧冲复合伤＋乌司他丁组(简称BBU组,n＝40)。BBS组和BBU组均制成25%TBSA深Ⅱ度烧伤＋中度冲击伤的中度烧冲复合伤模型,SS组与SU组大鼠麻醉后,除不致伤外其他干预方法同烧冲复合伤模型制作。伤后即刻,大鼠尾静脉分别对应给予注射0.9%氯化钠溶液和乌司他丁4万U/kg。各组大鼠分别于伤后6、24 h以腹主动脉抽血法处死(BBS、BBU组每时刻点各随机选取20只)后取肺组织观察病理学变化并进行肺损伤评分,采用ELISA法检测肺组织热休克蛋白70(HSP70)的含量,Western Blot试验测定肺组织中乙酰化组蛋白H3赖氨酸9(Ac-H3K9)相对表达量,干湿重法测定肺组织含水率。 结果伤后6 h,BBS组、BBU组大鼠肺损伤评分分别为(5.82±0.37)、(4.20±0.32)分,肺组织保护性蛋白HSP70含量分别为(205.0±2.3)、(220.0±2.4) pg/mL,Ac-H3K9的相对表达量分别为0.68±0.13、1.45±0.12,肺组织含水率分别为(85.30±2.13)%、(82.70±1.57)%;伤后24 h,BBS组、BBU组大鼠肺损伤评分分别为(8.25±0.42)、(6.11±0.31)分,HSP70含量分别为(221.0±2.4)、(226.0±2.7)pg/mL,Ac-H3K9的相对表达量分别为1.59±0.17、1.78±0.15,肺组织含水率分别为(82.80±1.57)%、(80.20±0.32)%。伤后6、24 h,BBS组与BBU组肺损伤评分比较差异均有统计学意义(t＝6.132、6.791,P＝0.026、0.016),肺组织含水率比较差异均有统计学意义(t＝6.123、5.894,P＝0.034、0.019),伤后6 h,两组HSP70、Ac-H3K9表达水平比较差异均有统计学意义(t＝ 2.795、3.165,P＝0.031、0.028),伤后24 h,两组HSP70、Ac-H3K9表达水平比较差异均无统计学意义(t＝ 2.143、3.154,P值均大于0.05)。伤后6、24 h,同一观测时间点BBS组与BBU组肺损伤评分、HSP70、Ac-H3K9表达水平、肺组织含水率分别与SS组、SU组比较,差异均有统计学意义(P值均小于0.05)。 结论乌司他丁能减轻大鼠烧冲复合伤早期肺组织水肿和病理损害,其机制可能与乌司他丁能增加肺组织保护性蛋白的表达、提高大鼠对损伤的耐受力有关。     

In vivo production of heat shock protein in mouse peritoneal macrophages by administration of lipopolysaccharide.

The in vivo production of heat shock protein was studied by administration of bacterial lipopolysaccharide (LPS) into mice. Heat shock protein 70 was detected in the extract of adherent peritoneal cells from mice injected intraperitoneally with LPS by using the immunoblotting method. The expression of heat shock protein 70 was found 2 days after injection of LPS and reached its peak 4 days after injection. The intraperitoneal injection of LPS induced the expression of heat shock protein 70, whereas its subcutaneous injection did not. The in vivo production of heat shock protein 70 was inhibited by administration of LPS together with quercetin, an inhibitor of accumulation of heat shock protein 70 mRNA. Tumor necrosis factor alpha enhanced LPS-induced heat shock protein production in vivo. There was a decrease of gamma delta T cells in the peritoneal cavity of mice injected intraperitoneally with LPS. It was suggested that bacterial LPS is a stressful agent which induces the in vivo heat shock protein response, and its administration leads to the production of heat shock protein 70 in peritoneal macrophages.     

Expression of autophagy related proteins in invasive lobular carcinoma: comparison to invasive ductal carcinoma

The aim of this study is to compare the expression of autophagy related proteins in invasive lobular carcinoma (ILC) with that of autophagy related proteins in invasive ductal carcinoma (IDC), and to determinate its implication. Tissue microarray containing 114 ILC and 692 IDC was constructed, and immunohistochemistry was performed for autophagy related protein (beclin-1, LC3A, LC3B, p62) and Ki-67. No significant difference in expression of autophagy-related proteins between pleomorphic type (n = 12) and classic type (n = 102) of ILC was observed, whereas ILC and IDC showed distinguished features that tumoral beclin-1, stromal LC3A, tumoral LC3B, tumoral p62 were highly expressed in IDC and tumoral BNIP3 was highly expressed in ILC (P < 0.001). Beclin-1 expression was correlated with ER negativity (P = 0.016) and TNBC type (P = 0.024). BNIP3 expression was correlated with ER positivity (p = 0.040). Using multivariate Cox analysis, shorter overall survival was associated with tumoral beclin-1 positivity (hazard ratio: 21.19, 95% CI: 1.098-409.1, P = 0.043). In conclusion, ILC and IDC showed different expression pattern of autophagy-related proteins in tumor and stroma that demonstrated by higher expression of tumoral beclin-1, stromal LC3A, tumoral LC3B, tumoral p62 in IDC, and higher expression of tumoral BNIP3 in ILC.     

针刺对运动性骨骼肌损伤大鼠骨骼肌线粒体自噬的影响

目的:通过观察针刺对大负荷运动大鼠骨骼肌线粒体自噬相关蛋白表达的影响,探讨针刺在运动性骨骼肌损伤修复中的作用及其机制。方法:将128只成年雄性Sprague-Dawley大鼠随机分为4组:空白对照(control,C;n=8)组、单纯运动(exercise,E;n=40)组、单纯针刺(acupuncture,A;n=40)组和运动针刺(exercise and acupuncture,EA;n=40)组。其中,E和EA组进行1次下坡跑运动,A组和EA组(运动后即刻)施加针刺处理。后3组根据干预后不同时相又分为0 h、12 h、24 h、48 h和72 h亚组(n=8),分别于对应时点分离比目鱼肌进行检测,使用透射电子显微镜观察骨骼肌线粒体超微结构变化;采用ELISA法检测比目鱼肌线粒体定量酶柠檬酸合成酶(CS)的含量变化;应用Western blot法检测骨骼肌PTEN诱导假定激酶1(PINK1)、parkin和微管相关蛋白1轻链3(LC3)的蛋白表达变化。结果:1次大负荷运动后大鼠比目鱼肌线粒体出现明显肿胀和肌膜下积聚等超微结构异常变化,伴有大量自噬体形成;同时CS的含量明显减少(P0.05);线粒体自噬蛋白PINK1、parkin和LC3均出现一过性的表达升高(P0.05)。运动后针刺明显改善了线粒体超微结构的异常变化,减少自噬溶酶体的出现,同时抑制CS的含量减少,下调PINK1、parkin和LC3在线粒体上的表达(P0.05)。结论:1次大负荷运动后骨骼肌线粒体结构和数量受损,通过激活PINK1/parkin途径诱发线粒体自噬的过度发生。大负荷运动后针刺可以缓解骨骼肌线粒体的损伤,其作用机制可能是通过下调线粒体外膜蛋白PINK1表达,抑制其对下游胞浆蛋白parkin的招募,进而影响LC3与线粒体的结合以抑制线粒体自噬过度激活。     

热休克处理骨髓Sca-1+细胞移植治疗小鼠心肌梗死

背景：近年来的研究发现,干细胞可以直接定向分化为成熟心肌细胞或促进其再生,为心肌梗死治疗提供一种全新的治疗策略,但是细胞移植率低使其向心肌细胞分化和执行心肌修复能力下降。 目的：探讨热休克Sca-1+细胞在小鼠心肌梗死治疗中的作用。 方法：磁珠分选骨髓中Sca-1+细胞,对Sca-1+细胞进行热休克处理3 h。建立心肌梗死小鼠动物模型,将其随机分为2组,分别经尾静脉注入1 mL热休克Sca-1+细胞悬液、1 mL非热休克Sca-1+细胞悬液。移植后检测细胞存活率、小鼠心脏功能恢复情况、心肌细胞凋亡数目、心肌纤维化程度以及左心室HSF、HSP70及miR-34a表达。 结果与结论：①热休克Sca-1+细胞移植组小鼠心脏sry基因表达显著高于非热休克Sca-1+细胞移植组。②热休克Sca-1+细胞移植组小鼠射血分数及左心室短轴缩短率显著高于非热休克Sca-1+细胞移植组,左心室的舒张末期内径以及收缩末期内径显著低于非热休克Sca-1+细胞移植组。③热休克Sca-1+细胞移植组小鼠的心脏纤维化程度及心肌细胞凋亡均显著低于非热休克Sca-1+细胞移植组。④热休克Sca-1+细胞移植组小鼠左心室HSF和HSP70表达明显高于非热休克Sca-1+细胞移植组,miR-34a表达明显低于非热休克Sca-1+细胞移植组。结果表明热休克Sca-1+细胞移植能够减少心肌细胞凋亡和心肌梗死面积,改善心脏功能。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

Autophagy in Niemann-Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects

Niemann-Pick C (NPC) disease is an autosomal recessive lipid storage disorder characterized by a disruption of sphingolipid and cholesterol trafficking that produces cognitive impairment, ataxia and death, often in childhood. Most cases are caused by loss of function mutations in the Npc1 gene, which encodes a protein that localizes to late endosomes and functions in lipid sorting and vesicle trafficking. Here, we demonstrate that NPC1-deficient primary human fibroblasts, like npc1(-/-) mice fibroblasts, showed increased autophagy as evidenced by elevated LC3-II levels, numerous autophagic vacuoles and enhanced degradation of long-lived proteins. Autophagy because of NPC1 deficiency was associated with increased expression of Beclin-1 rather than activation of the Akt-mTOR-p70 S6K signaling pathway, and siRNA knockdown of Beclin-1 decreased long-lived protein degradation. Induction of cholesterol trafficking defects in wild-type fibroblasts by treatment with U18666A increased Beclin-1 and LC3-II expression, whereas treatment of NPC1-deficient fibroblasts with sphingolipid-lowering compound NB-DGJ failed to alter the expression of either Beclin-1 or LC3-II. Primary fibroblasts from patients with two other sphingolipid storage diseases, NPC2 deficiency and Sandhoff disease, characterized by sphingolipid trafficking defects also showed elevation in Beclin-1 and LC3-II levels. In contrast, Gaucher disease fibroblasts, which traffic sphingolipids normally, showed wild-type levels of Beclin-1 and LC3-II. Our data define a critical role for Beclin-1 in the activation of autophagy because of NPC1 deficiency, and reveal an unexpected role for lipid trafficking in the regulation of this pathway in patients with several sphingolipid storage diseases.     

Extracellular heat shock proteins (eHSP70) in exercise: Possible targets outside the immune system and their role for neurodegenerative disorders treatment

The intracellular heat shock protein 70kDa (iHSP70) is a universal marker of stress protein whose expression is induced by different cell stressors, such as heat, metabolite deprivation, redox imbalances and also during physical exercise. The activation of the iHSP70 is sine qua non for the promotion of tissue repair, since the expression of this chaperone confers cytoprotection and also exerts anti-inflammatory effects. On the other hand, exercise also induces the appearance of HSP70 in the extracellular medium (eHSP70) but, so far, the eHSP70 function has been mainly attributed to the activation of the immune system, seeming to perform an opposite function from the iHSP70. Since a moderate intensity exercise bout induces a general anti-inflammatory response even in the presence of an elevated eHSP70, this protein could carry out other functions rather than immune activation. Because exercise generates heat and metabolic challenges (especially on glucose metabolism) we suggests that the motoneurons, a very active (possibly one of the most stressed cells during exercise) and also very sensitive cells to heat and glucose metabolism imbalances, could be the major sites for the eHSP70 function. Due to the importance of the iHSP70 for repair and stress adaptation, this protein must be present in abundance on the site of stress and, because of its intrinsic inability response to stress [low heat shock factor 1 (HSF-1) activation] and the structure of the motoneurons (very long cells), the iHSP70, produced on the very far nucleus, is not appropriately transported through the axon to the axon terminal, were it is required. Then, during the exercise, the released eHSP70 can be internalized by the motoneurons and act as intracellular chaperons, protecting this cell against oxidative damage, protein denaturation and many others. Since a decreased iHSP70 expression capacity is associated with neurodegeneration diseases (such as Parkinson, polyglutamine, Amyotrophic lateral sclerosis, Alzheimer's, Huntington's and many others), the understanding of the physiological function of the extracellular HSP70 could be helpful on the treatment of neurodegenerative and other neuronal diseases. Besides that, it could explain some of the beneficial effects of the pharmacological HSP70 activators and also the beneficial effects of the exercise among neuronal cells during neurodegenerative-inducing diseases.     

Is exercise beneficial to the inflammatory bowel diseases? An implication of heat shock proteins

Inflammatory bowel diseases (IBD) cause the intestines to become inflamed (red and swollen) and typically include Crohn's disease and ulcerative colitis. Heat shock protein (Hsp)70, which exhibits greater expression in the intestines of patients with IBD, may act to protect the intestine against this inflammatory insult. However, most procedures for eliciting this protective response, such as heating and/or pharmacological interventions are non-physiological and can have serious side-effects. Exercise is a biologically relevant means of inducing protective heat shock proteins in the myocardium and other organs but it has yet to be studied in the bowel. We herein hypothesize that (1) exercise will be beneficial in reducing the occurrence of IBD and suppressing intestine inflammatory injuries, (2) exercise will provide protection through induction of anti-inflammatory Hsps. Further studies using both genetically manipulated animal models and animals undergoing exercise are needed to determine the efficacy of exercise and the role of Hsps in inhibiting or treating inflammatory bowel disease.     

Long-term resistance exercise-induced muscular hypertrophy is associated with autophagy modulation in rats

Elevation of anabolism and concurrent suppression of catabolism are critical metabolic adaptations for muscular hypertrophy in response to resistance exercise (RE). Here, we investigated if RE-induced muscular hypertrophy is acquired by modulating a critical catabolic process autophagy. Male Wistar Hannover rats (14 weeks old) were randomly assigned to either sedentary control (SC, n = 10) or resistance exercise (RE, n = 10). RE elicited significant hypertrophy of flexor digitorum profundus (FDP) muscles in parallel with enhancement in anabolic signaling pathways (phosphorylation of AKT, mTOR, and p70S6K). Importantly, RE-treated FDP muscle exhibited a significant decline in autophagy evidenced by diminished phosphorylation levels of AMPK, a decrease in LC3-II/LC3-I ratio, an increase in p62 level, and a decline in active form of lysosomal protease CATHEPSIN L in the absence of alterations of key autophagy proteins: ULK1 phosphorylation, BECLIN1, and BNIP3. Our study suggests that RE-induced hypertrophy is achieved by potentiating anabolism and restricting autophagy-induced catabolism.     

外周血白细胞热休克蛋白70 mRNA在太极拳运动后的表达

背景：大部分研究显示,太极拳运动有望通过改善机体免疫功能而延缓衰老,但其机制不明。 目的：观察太极拳运动后外周血白细胞热休克蛋白70 mRNA的表达情况。 方法：在健康中老年女性学练太极拳前、练习3个月和6个月时对其外周血白细胞热休克蛋白70 mRNA表达进行实时定量PCR 检测。 结果与结论：经过3个月的太极拳运动干预,中老年妇女外周血白细胞热休克蛋白70 mRNA的表达量与干预前比略增加,但没有明显差异,但干预6个月后表达量下降明显,与干预前和干预3个月的表达量相比差异均有显著性意义。结果初步表明热休克蛋白mRNA表达的改变反映了太极拳健身的效果,而热休克蛋白的分子伴侣作用是太极拳健身效果的可能机制。     

红景天干预可改善大强度运动小鼠骨骼肌细胞线粒体自噬及融合-分裂等功能

文题释义： 自噬：指从粗面内质网的无核糖体附着区脱落的双层膜包裹部分胞质和细胞内需降解的细胞器、蛋白质等成分形成自噬体(autophagosome),并与溶酶体融合形成自噬溶酶体,降解其所包裹的内容物,以实现细胞本身的代谢需要和某些细胞器的更新。自噬广泛存在于人体的正常细胞和恶性肿瘤细胞中。 线粒体融合-分裂：线粒体是一种高度动态变化的细胞器,在细胞中不断融合与分裂,形成紧密连接的线粒体网络。这种融合与分裂的变化主要通过在线粒体融合、分裂蛋白的精确控制下,线粒体可在不断变化的生理环境中做出迅速准确的反应,这对于线粒体的遗传以及维持其功能至关重要。 背景：超负荷量的运动会引起体内氧化活性物质大量堆积从而损害骨骼肌细胞,而线粒体在运动过程能量代谢中具有关键作用。研究表明,红景天可以减少肌组织脂质过氧化水平,保护受损内皮细胞。 目的：探讨红景天通过调节线粒体功能改善大强度运动小鼠骨骼肌细胞的功能。 方法：实验方案经西安石油大学伦理委员会批准。选择40只SPF级BALB/c小鼠,根据干预措施的不同,将小鼠分为空白对照组、单纯运动组、红景天对照组、红景天运动干预组。①空白对照组：不进行运动;②单纯运动组：采用生理盐水灌胃后,进行大强度运动;③红景天对照组：将红景天与生理盐水悬浊液灌胃,不运动;④红景天运动干预组：红景天干预措施同红景天对照组,运动方案同单纯运动组。以上各组干预措施均为每天1次,连续28 d。观察小鼠的体质量、前肢握力、力竭时间;Western Blot检测骨骼肌锰超氧化物歧化酶蛋白、p53蛋白、线粒体起源、自噬启动相关蛋白表达;RT-qPCR检测骨骼肌Mfn-1、Mfn-2、Opa-1、Drp-1、fis-1 mRNA表达。 结果与结论：①从第2周开始,单纯运动组小鼠前肢握力显著低于其他3组(P < 0.05),但空白对照组、红景天对照组与红景天运动干预组之间,小鼠前肢握力始终无明显差异(P > 0.05);②第3,4周时,单纯运动组小鼠负重游泳训练力竭时间均显著短于红景天运动干预组(P < 0.05);③单纯运动组小鼠骨骼肌细胞内的锰超氧化物歧化酶、p53蛋白表达显著高于其他组小鼠(P < 0.05),红景天运动干预组小鼠骨骼肌细胞内的锰超氧化物歧化酶、p53蛋白表达显著高于红景天对照组(P < 0.05);④与空白对照组相比,单纯运动组小鼠骨骼肌内PGC-1α、LC3-Ⅱ/LC3-Ⅰ水平明显升高,Atg7、P62水平显著下降(均P < 0.05),与红景天对照组相比,红景天运动干预组PGC-1α、LC3-Ⅱ/LC3-Ⅰ水平明显升高,Atg7、P62水平显著下降(均P < 0.05);⑤与空白对照组相比,单纯运动组的融合基因表达下降,分裂基因Drp-1 mRNA表达上升(P < 0.05);红景天运动干预组的融合基因表达也呈下降趋势,Drp-1 mRNA表达水平呈上升趋势,但差异无显著性意义(P > 0.05);⑥结果说明,红景天可显著提高大强度运动量小鼠的运动耐力,这可能与改善了骨骼肌线粒体自噬、起源及线粒体融合-分裂有关。 ORCID: 0000-0002-4143-1195(曹海信) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

利用CRISPR/Cas-9技术抑制热休克蛋白Gp96的表达对小鼠酒精性肝纤维化的影响

目的 探讨热休克蛋白Gp96对小鼠酒精性肝纤维化的影响。 方法 健康雄性C57BL/6 J小鼠220只,随机分为4组：正常对照组(n=10),生理盐水+酒精诱导纤维化组(n=70)。尾静脉注射CRISPR表达质粒Gp96-sgRNA3+酒精诱导肝纤维化组（n=70）,腹腔注射核因子κB（NF-κB）抑制剂PDTC+酒精诱导肝纤维化组（n=70）。于酒精诱导第8周眼球取血后处死各组小鼠,测定各组小鼠血清谷草转氨酶（AST）活性,HE染色检测各组小鼠肝脏病理变化,天狼猩红染色检测各组小鼠肝纤维化情况,过碘酸-雪夫(PAS)染色检测各组小鼠肝糖原变化情况;免疫印迹法检测小鼠肝脏Gp96和转化生长因子β1(TGF-β1)的表达变化。 结果 与正常对照组相比,其余3组AST酶活性显著上升,肝纤维化加重,糖原显著减少（P<0.01）;与生理盐水+酒精组相比,注射质粒Gp96-sgRNA3+酒精组和注射NF-κB抑制剂+酒精组AST上升更为明显,肝纤维化更严重,糖原减少更多,Gp96表达显著下降,TGF-β1表达显著增加（P<0.01或 P<0.05）。 结论 尾静脉注射CRISPR表达质粒Gp96-sgRNA3显著抑制了小鼠肝脏Gp96的表达,促进了小鼠酒精性肝纤维化程度,NF-κB信号通路对Gp96的表达起到一定的调控作用。     

MRTF-A诱导自噬从而缓解脑缺血再灌注大鼠皮层神经细胞损伤

目的:研究心肌素(myocardin)相关转录因子A(MRTF-A)对脑缺血再灌注大鼠皮层神经细胞损伤的影响。方法:90只SD大鼠随机分为3组:假手术组、缺血再灌注模型组和MRTF-A实验组。采用慢病毒注射及大脑中动脉栓塞法处理动物。Longa’s 5分法进行神经功能评分;2,3,5-氯化三苯基四氮唑(TTC)染色评价脑梗死体积;透射电镜(TEM)观察自噬溶酶体形成;Western blot检测MRTF-A、自噬相关蛋白(LC3-I、LC3-II和beclin-1)及凋亡相关蛋白(Bcl-2和Bax)的表达水平;TUNEL联合免疫荧光法检测神经细胞凋亡率与LC3蛋白表达分布的相关性。结果:假手术组、模型组和实验组的神经功能评分分别为0、2.57±0.20和1.71±0.18,脑梗死体积百分率分别为0、(31.67±2.73)%和(12.00±1.16)%;与模型组相比,实验组的神经功能评分和梗死体积百分率均显著降低(P<0.01)。TEM结果显示,模型组神经细胞核膜结构不完整,细胞器肿胀变形,伴随有自噬溶酶体形成;实验组神经细胞损伤较模型组有所缓解,自噬溶酶体形成增多。Western blot结果显示,造模后Bcl-2/Bax比值显著下降(P<0.01);与模型组相比,实验组LC3-II/LC-I和Bcl-2/Bax比值及beclin-1和MRTF-A蛋白表达量均显著升高(P<0.05或P<0.01)。与假手术组[(3.31±2.72)%]相比,模型组神经细胞凋亡率[(57.02±11.04)%]明显升高(P<0.01);高表达MRTF-A后,实验组神经细胞凋亡率[(42.10±5.20)%]较模型组明显降低(P<0.05)。假手术组的LC3蛋白分布均匀呈弥散状;与模型组对比,实验组的LC3蛋白荧光强度显著上升(P<0.05),在神经细胞胞浆中聚集明显,呈斑片状。结论:脑缺血再灌注损伤诱导大鼠皮层神经细胞凋亡和轻微自噬反应;在皮层神经细胞中上调MRTF-A表达后,MRTF-A可通过增强自噬水平有效抑制脑缺血再灌注过程中皮层神经细胞损伤。     

Exacerbation of ischemia-induced amyloid-β generation by diabetes is associated with autophagy activation in mice brain

To evaluate effect of diabetes on transient ischemia-induced brain damage and autophagy activity, streptozotocin (STZ)-induced diabetic mellitus (DM) mice were subjected to transient common carotid artery occlusion (CCAO) operation. After the operation, immunohistochemistry and transmission electron microscopy (EM) were performed to investigate the astrocytes activation, amyloid-β protein (Aβ) expression and accumulation of autophagy-like vacuoles containing electron-dense material (avd); and hallmarks of autophagy, the microtubule-associated protein light chain 3 (LC3)-II, was detected by western blot analysis. The results showed that DM amplified stroke-induced astrocytes activation and Aβ generation. Western blot analysis showed that LC3-II conjugate was drastically up-regulated at early stages post ischemia and it last for at least 72 h in DM mice brain. DM mice demonstrated increased baseline level of LC3-II as comparing to normal mice; DM also amplified stroke-induced LC3-II level. Under EM, avd was most markedly accumulated in neurons of DM mice brain after ischemia. Immunofluorescence double-staining showed that most Aβ and autophagosomes co-localized. Therefore, our results suggested that exacerbation of ischemia-induced Aβ generation by diabetes might be associated with autophagy activation in mice brain, and modulating neuronal autophagy might be a new therapeutic strategy to depress the risk of development of dementia in diabetic patients with stroke.     

PI3K/Akt/mTOR信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用

 目的：探讨磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在巨噬细胞自体吞噬以及动脉粥样硬化斑块不稳定中的作用。方法：利用Akt抑制剂康士得（20 μmol/L）、mTOR抑制剂雷帕霉素（10 nmol/L）及mTOR-siRNA（30 nmol/L）体外处理小鼠RAW 264.7 巨噬细胞株 48 h后,透射电镜观察巨噬细胞自噬体的变化,细胞免疫荧光法及Western blotting法检测微管相关蛋白LC3-II表达,实时荧光定量qRT-PCR和Western blotting法检测Akt、mTOR及自噬相关蛋白Beclin 1的表达,ELISA检测巨噬细胞分泌炎症因子水平。体内实验中, 24只雄性新西兰兔给予球囊损伤+ 1%胆固醇喂养8周,然后随机分为对照组、康士得（1.0 mg·kg-1·d-1）组和雷帕霉素（0.5 mg·kg-1·d-1）组,每组8只,干预4周。血管内超声（IVUS）检测斑块的影像学特征,透射电镜观察斑块中巨噬细胞超微结构的改变,免疫荧光法检测微管相关蛋白LC3-II表达,免疫组织化学法检测巨噬细胞Akt和mTOR的蛋白表达。 结果：与对照组比较,康士得、雷帕霉素及mTOR-siRNA干预巨噬细胞后,透射电镜下观察到自噬体明显增多,微管相关蛋白LC3-II和自噬相关蛋白Beclin 1的表达水平明显上调,而Akt及mTOR 的mRNA及蛋白表达水平明显减少,巨噬细胞分泌的IL-10明显降低,而IFN-γ的分泌显著增加。体内实验： IVUS显示,与对照组比较,康士得组及雷帕霉素组的外弹性膜面积（EEMA）、斑块面积（PA）及斑块负荷（PB）明显减少,透射电镜下观察到巨噬细胞中自噬体增加,组织免疫荧光法示LC3-II明显增加,HE染色显示斑块纤维帽的厚度明显增加,内、中膜厚度显著减低,组织免疫组化染色显示巨噬细胞RAM-11及p-mTOR染色显著减少。结论：选择性抑制PI3K/Akt/mTOR信号通路能诱导巨噬细胞自噬,减少斑块巨噬细胞的浸润, 抑制炎症反应进而稳定动脉粥样硬化易损斑块。