内质网应激与自噬的交互作用对血管钙化的影响

目的证实内质网应激(ERS)和自噬的交互作用对血管钙化(VC)的影响。方法维生素D3肌注和尼古丁灌胃制备大鼠在体血管钙化模型,取主动脉行茜素红染色和钙含量检测,Western blot检测相关蛋白的表达水平。结果与对照组相比,钙化组大鼠主动脉管壁钙沉积显著增加,血管平滑肌细胞(VSMC)收缩表型标志蛋白SM-22α和Calponin表达显著降低,而成骨细胞样表型标志蛋白骨形态发生蛋白2(BMP-2)和Runt相关转录因子2(RUNX2)表达显著升高,ERS标志蛋白葡萄糖调节蛋白(GRP78)和C/EBP同源蛋白(CHOP)以及自噬标志蛋白轻链3(LC3Ⅱ)和Beclin-1表达显著升高。钙化大鼠应用ERS激动剂衣霉素[10μg/(kg·d)]可进一步增加血管壁钙沉积及BMP-2和RUNX2表达水平,而SM-22α和Calponin表达进一步减少,GRP78和CHOP以及LC3Ⅱ和Beclin-1表达水平进一步增加。钙化大鼠应用自噬抑制剂3-甲基腺嘌呤[10 mg/(kg·d)]可降低LC3Ⅱ和Beclin-1水平,同时GRP78和CHOP表达升高,增加血管壁钙沉积及BMP-2和RUNX2表达水平,降低SM-22α和Calponin表达。结论内质网应激与自噬的交互作用影响血管钙化的发展。     

细胞凋亡和细胞自噬在慢性环孢素A肾毒性中的作用

目的探讨细胞凋亡和细胞自噬在慢性环孢素A(CsA)肾毒性中的作用。方法 Sprague-Dawley大鼠分为两组:对照组:皮下注射橄榄油(1 ml·kg-1·d-1)4 w;慢性CsA肾毒性组:皮下注射CsA(15 mg·kg-1·d-1)4 w。检测两组大鼠的体重和肾功能;三色染色检测肾小管间质纤维化程度;ELISA法检测血、尿8羟基脱氧鸟苷(8-OHdG)水平;原位末端标记法(TUNEL)染色观察细胞凋亡;免疫印迹法检测细胞凋亡调控基因(Bcl-2、Bax、Caspase-3)和细胞自噬体膜型LC3-Ⅱ蛋白的表达;Pearson直线相关分析肾小管间质纤维化程度与细胞凋亡和LC3-Ⅱ蛋白表达的相关关系。结果与对照组相比,毒性组大鼠体重减轻、肾功能低下、血尿8-OHdG水平升高,同时肾毒性组可见明显的肾小管间质纤维化和大量TUNEL阳性细胞。免疫印迹结果表明,毒性组Bax、Caspase-3、LC3-Ⅱ蛋白的表达明显增加,反之,Bcl-2的表达显著减少。直线相关分析提示,肾小管间质纤维化程度与TUNEL阳性细胞数和LC3-Ⅱ蛋白表达呈正向相关。结论细胞凋亡和细胞自噬参与了慢性CsA肾毒性肾小管间质的损伤。     

特发性膜性肾病30例肾小管上皮细胞自噬改变及其临床意义

目的观察特发性膜性肾病(idiopathic membranous nephropathy,IMN)患者肾小管自噬体数量、自噬相关蛋白Beclin-1和LC3的表达,了解肾小管上皮细胞自噬是否参与了IMN肾间质纤维化的进展。方法回顾性总结2015年6月至2016年12月在中国医科大学附属盛京医院经肾活检确诊为IMN患者30例,在透射电子显微镜下观察IMN患者肾组织肾小管上皮细胞自噬体的数量,免疫组化检测自噬相关蛋白Beclin-1和LC3的表达,分析其与24 h尿蛋白定量、肾间质纤维化程度及肾功能等临床相关指标的相关性。结果与正常肾组织相比,IMN组的肾小管上皮细胞自噬体数量、自噬相关蛋白Beclin-1和LC3表达显著增加(t=-13.266,P0.001;t=-5.261,P0.001;t=-2.394,P=0.026);在IMN组中,与无纤维化组相比,有纤维化组自噬小体数量、Beclin1和LC3表达均显著增加(t=-6.04,P0.001;t=-2.795,P=0.014;t=-2.761,P=0.004);在IMN患者中,自噬小体数量、Beclin1和LC3均与患者肾间质纤维化累及面积成正相关(r分别为0.757、0.658和0.638,均P0.001);LC3与24 h尿蛋白定量成负相关(r=-0.463,P=0.01),Beclin1与血清尿素氮(BUN)成正相关(r=0.433,P=0.017),自噬小体数量与患者性别具有一定的相关性(r=-0.472,P=0.008)。结论在IMN患者中,肾小管上皮细胞自噬参与了肾间质纤维化的进展。     

鹿红方对氧糖剥夺模型心脏干细胞自噬和抗凋亡能力的影响

目的探讨鹿红方(LHF)对氧糖剥夺模型心脏干细胞(CSCs)自噬和抗凋亡能力的影响。方法将CSCs随机分为对照组(正常培养)与氧糖剥夺造模组(缺血缺氧)。将造模组分为模型组(不予有效干预措施)、自噬阻断到(3-MA)组(给予自噬阻断剂)、鹿红方200μg/mL组(给予LHF200μg/mL)和LHF200+3-MA组(给予LHF200μg/mL+自噬阻断剂)。各组给予相应干预措施后光镜下观察细胞形态,并采用CCK-8法测定细胞凋亡情况;WesternBlot检测自噬相关蛋白Beclin-1、LC3-Ⅱ的表达;RT-PCR检测自噬相关蛋白Beclin-1、LC3-ⅡmRNA的表达。结果光镜下观察可见:LHF200组较模型组和3-MA组凋亡细胞显著减少,LHF200+3-MA组与LHF200组比较,细胞凋亡增加;CCK-8结果显示:LHF200组与模型组比较,CSCs活细胞OD值显著增高(P=0.006);LHF+3-MA组与LHF200组比较,CSCs活细胞OD值下降(P=0.03);WesternBlot结果显示:LHF200组Beclin-1和LC3-Ⅱ蛋白表达较模型组显著降低(P<0.001),但显著高于3-MA组和LHF200+3-MA组(P<0.001);RT-PCR结果显示:LHF200组Beclin-1和LC3-ⅡmRNA表达较模型组显著降低(P<0.001),但显著高于3-MA组和LHF200+3-MA组(P<0.001)。结论LHF可提高氧糖剥夺模型CSCs的抗凋亡能力,其机制与调节自噬有关。     

外源性睾酮通过诱导自噬致大鼠肾脏损伤

目的 探索外源性睾酮(TP)致大鼠肾脏损伤的作用及可能机制.方法 SD雄性大鼠48只,随机分为6组(每组8只):control组、TP组、TP+氯喹(CQ)组、TP+甘草甜素(Gly)组、CQ(自噬抑制剂)组、Gly〔高迁移率族蛋白(HMG)B1抑制剂〕组.各组大鼠处理21 d后,测量其体重、肾脏湿重、计算肾脏指数;苏木素-伊红(HE)染色观察大鼠肾脏组织病理形态学变化;免疫组化法和Western印迹印迹法分析大鼠肾脏组织自噬相关蛋白LC3B-Ⅱ、Beclin1、P62、HMGB1的定位和表达情况.结果 与control组比较,TP组大鼠肾脏湿重、肾脏指数无明显变化(P>0.05);肾小球毛细血管襻多呈分叶状改变,肾小囊间隙变窄,肾小管上皮细胞水肿,管腔变窄;肾脏组织LC3B-Ⅱ、Beclin1和HMGB1蛋白表达水平上调(P<0.05),联合给予TP与CQ后,仅见少数肾小体血管球呈分叶状改变,肾小管水肿明显减轻;Beclin1、LC3B-Ⅱ和HMGB1蛋白表达水平均下调(P<0.05);TP+Gly组大鼠肾脏组织病理学改变及自噬相关蛋白表达水平结果 与TP+CQ组类似.结论 外源性TP可能通过上调细胞自噬致大鼠肾脏组织损伤的发生,HMGB1可能参与此过程自噬水平的调节.     

PI3K-AKT-mTOR信号通路在大鼠肺性脑病模型神经元自噬中的作用

目的探讨PI3K-AKT-m TOR信号通路在肺性脑病神经元自噬中的作用。方法对36只新生健康SD大鼠乳鼠建立离体培养大鼠皮层神经元,随机分为空白对照组、肺性脑病组、自噬抑制剂3-MA组后进一步建立了大鼠肺性脑病细胞模型,借助该模型运用CCK8法检测神经元活力、MDC染色观察神经元自噬泡数量、Western印迹法检测自噬相关蛋白LC3、Beclin-1及PI3K蛋白表达。结果与空白对照组比较,肺性脑病组神经元活力降低(P0.05);神经元自噬泡数量增多;LC3-Ⅱ/Ⅰ的比值、Beclin-1及磷酸化PI3K表达水平增加(P0.05)。使用自噬抑制剂3-MA预处理后,神经元活力降低更明显、自噬泡数量明显减少;缺氧诱导的自噬水平明显受到抑制,LC3-Ⅱ/Ⅰ的比值、Beclin-1及磷酸化PI3K表达水平明显降低。结论缺氧和二氧化碳潴留的信号使Beclin-1表达增加、LC3-Ⅰ向LC3-Ⅱ转化增加,加速PI3K磷酸化,使PI3K-AKT-m TOR信号通路失活,从而激活自噬信号通路,导致大脑神经元自噬增强。     

针刺通过调节自噬反应对脑卒中大鼠的神经元损伤的影响

目的 探讨针刺通过腺苷酸活化蛋白激酶(AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)、UNC-51样自噬激活激酶(ULK)1/2介导的自噬对脑卒中大鼠神经元的影响。方法 50只SD大鼠随机分为假手术组、模型组、针刺组、3-甲基腺嘌呤(3-MA)组、针刺+5-氨基-4-甲酰胺咪唑核糖核苷酸(AICAR)组，每组10只，除假手术组其他4组采用中动脉栓塞法构建模型，并给予相应治疗。治疗后24 h,对各组大鼠神经功能缺损程度进行评分。结果 与假手术组比较，模型组治疗后神经功能缺损评分、海马组织AMPK、磷酸化ULK1(p-ULK1)/ULK1、微管相关蛋白1轻链3(LC3)B/A、Beclin-1表达升高，mTOR表达降低(P<0.05);与模型组比较，针刺组和3-MA组大鼠神经元损伤、自噬减轻，神经功能缺损评分、海马组织AMPK、p-ULK1/ULK1、LC3B/A、Beclin-1表达降低，mTOR表达升高(P<0.05);针刺+AICAR组治疗后神经功能缺损评分、海马组织AMPK、p-ULK1/ULK1、LC3B/A、Beclin-1表达明显高于针刺组(P<0.05),...     

外源性雄激素通过诱导自噬促大鼠睾丸萎缩

目的探讨外源性雄激素对大鼠睾丸萎缩的影响及其可能机制。方法 48只SD雄性大鼠随机分成6组,每组8只:对照组(control)、雄激素(丙酸睾丸酮,TP)组、雄激素+氯喹(TP+CQ)组、雄激素+甘草甜素(TP+Gly)组、氯喹(CQ,自噬抑制剂)组、甘草甜素〔Gly,高迁移率族蛋白(HMGB) 1抑制剂〕组。各组分别处理21 d后,测量体重、睾丸湿重、睾丸指数;采用HE染色法观察睾丸组织病理学改变; Western印迹法大鼠睾丸组织Beclin-1、LC3B-Ⅱ、p62、HMGB1蛋白表达情况。结果给予大鼠TP干预21 d后,大鼠睾丸湿重、睾丸指数均显著降低(P<0. 01);睾丸生精小管发生萎缩,各级生精细胞排列紊乱、层数减少,未见成熟的精子;睾丸组织Beclin-1、LC3B-Ⅱ和HMGB1蛋白表达均显著上调(P<0. 05),p62蛋白表达无明显变化(P>0. 05)。联合给予TP和CQ后,大鼠睾丸指数显著增加(P<0. 05);生精小管和各级生精细胞损伤明显改善,可见到成熟的精子;睾丸组织Beclin-1、LC3B-Ⅱ和HMGB1蛋白表达水平均显著下调(P<0. 05),p62蛋白表达仍无明显变化(P>0. 05)。联合给予TP和Gly后,各观测指标变化与联合给予TP和CQ后结果类似,且两组各指标均无明显差异(P>0. 05)。结论外源性雄激素可能通过上调自噬水平引发大鼠睾丸萎缩病理过程发生,此过程中HMGB1可能调节自噬水平改变。     

基于PI3K/AKT/mTOR信号通路激活自噬通路研究荜茇改善小鼠肺纤维化的机制

目的 研究荜茇是否可通过调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路诱导自噬改善小鼠肺纤维化。方法 将50只小鼠随机分为5组即空白组、博来霉素模型组(BLM组)、低剂量组(LD组)、高剂量组(HD组)、雷帕霉素组(RAPA组)。空白组予等容积生理盐水,其余小鼠通过气管内滴注博来霉素(5mg/kg)诱导建立PF模型,造模2周后开始灌胃给药,LD和HD组给予荜茇(5g/kg、10 g/kg)灌胃,RAPA组予自噬诱导剂雷帕霉素(5 mg/kg),连续给药4周。使用Anires2005系统测量小鼠肺功能即用力肺活量(FVC)和动态顺应性(Cdyn),并检测羟脯氨酸(HYP)含量水平。苏木素-伊红(HE)和马松(Masson)染色观察小鼠肺组织纤维化,免疫组化法和RT-qPCR测定α-平滑肌肌动蛋白(α-SMA)和转化生长因子β1(TGF-β1)表达水平,蛋白免疫印迹法(Western Blot)检测巨噬效应蛋白(Beclin-1)、自噬相关蛋白1轻链3Ⅱ(LC3Ⅱ)蛋白表达水平,并检测通路相关蛋白p-PI3K、p-AKT、p-mTOR相...     

高压氧对永久性大脑中动脉栓塞大鼠心肾组织自噬活性的影响

目的 :观察永久性大脑中动脉栓塞(permanent middle cerebral artery occlusion,p MCAO)模型大鼠心、肾组织内自噬标志物微管相关蛋白轻链3(LC3)和P62的变化情况,探讨高压氧对心、肾组织自噬的影响及意义。方法:雄性Sprague-Dawley大鼠共75只,随机分为对照组、p MCAO组和高压氧组(p MCAO后给予高压氧处理);每组按造模后6 h和5 d 2个时间点处死大鼠并分成2个亚组。通过改良神经功能缺陷评分(m NSS)评估大鼠神经功能损伤情况,利用2,3,5-氯化三苯基四氮唑(TTC)法测定大鼠脑梗死面积,蛋白质印迹法(Western blotting)检测心、肾组织蛋白表达情况,实时荧光定量(real time)PCR检测组织内基因水平。结果:高压氧干预可有效减少脑缺血损伤后梗死面积,有助于神经功能恢复。除了高压氧6 h亚组的肾组织外,各组心、肾组织LC3和P62的蛋白表达水平变化基本与基因水平相符,且LC3与P62蛋白表达变化趋势基本一致。在大鼠心、肾组织中,脑缺血6 h后LC3-Ⅱ/LC3-Ⅰ和P62表达升高,高压氧处理后下降(P     

自噬在支气管哮喘发病机制中的作用

自噬是一种防御和应激调控机制.细胞可以通过自噬调节其生物活性及功能.近年来研究发现,自噬参与代谢性疾病、神经退行性病变、感染、免疫和肿瘤等疾病的发生发展.支气管哮喘是一种具有明显家族聚集倾向的多基因遗传性疾病,它的发生受遗传因素和环境因素的双重影响.自噬主要在固有免疫及获得性免疫、病原体感染、遗传因素等支气管哮喘发病机制中起重要作用.     

Interplay of autophagy and innate immunity in Crohn’s disease: A key immunobiologic feature

Crohn’s disease representing a clinical phenotype of inflammatory bowel disease is a polygenic immune disorder with complex multifactor etiology. Recent genome-wide association studies of susceptibility loci have highlighted on the importance of the autophagy pathway, which previously had not been implicated in disease pathology. Autophagy represents an evolutionarily highly conserved multi-step process of cellular self-digestion due to sequestration of excessive, damaged, or aged proteins and intracellular organelles in double-membranous vesicles of autophagosomes, terminally self-digested in lysosomes. Autophagy is deeply involved in regulation of cell development and differentiation, survival and senescence, and it also fundamentally affects the inflammatory pathways, as well as the innate and adaptive arms of immune responses. Autophagy is mainly activated due to sensors of the innate immunity, i.e., by pattern recognition receptor signaling. The interplay of genes regulating immune functions is strongly influenced by the environment, especially gut resident microbiota. The basic challenge for intestinal immune recognition is the requirement of a simultaneous delicate balance between tolerance and responsiveness towards microbes. On the basis of autophagy-related risk genetic polymorphisms (ATG16L1, IRGM , NOD2 , XBP1 ) impaired sensing and handling of intracellular bacteria by innate immunity, closely interrelated with the autophagic and unfolded protein pathways seem to be the most relevant immunobiologic events. Autophagy is now widely considered as a key regulator mechanism with the capacity to integrate several aspects of Crohn’s disease pathogenesis. In this review, recent advances in the exciting crosstalk of susceptibility coding variants-related autophagy and innate immunity are discussed.     

Rapamycin Enhances HBV Production by Inducing Cellular Autophagy

Background:

Some reports revealed that rapamycin could reactivate HBV infection. However, the mechanism has not been clearly explained.

Objectives:

In this report, we studied the mechanism by which rapamycin enhances HBV replication and expression by inducing cellular autophagy.

Materials and Methods:

HepG2.2.15 cells were treated with rapamycin to induce autophagy. Autophagosomes were observed by fluorescence microscopy and transmission electron microscopy. Autophagy marker protein LC3-Ⅱ/LC3-Ⅰwas detected by Western blotting. HBV DNA and mRNA were determined by real time PCR and Southern blotting. HBsAg was evaluated by ELISA.

Results:

In HepG2.2.15 cells, HBV DNA and HBsAg increased when host cells were treated with rapamycin and the effect was reversed by autophagy inhibitor, 3-methyladenine (3-MA).

Conclusions:

These results indicated a potential explanation for reactivation of HBV infection when patients with hepatitis receive rapamycin.     

New mechanisms of metformin action: Focusing on mitochondria and the gut

The most well‐known mechanism of metformin action, one of the most commonly prescribed antidiabetic drugs, is adenosine monophosphate‐activated protein kinase activation; however, recent investigations have shown that adenosine monophosphate‐activated protein kinase‐independent pathways can explain some of metformin's beneficial metabolic effects as well as undesirable side‐effects. Such novel pathways include induction of mitochondrial stress, inhibition of mitochondrial shuttles, alteration of intestinal microbiota, suppression of glucagon signaling, activation of autophagy, attenuation of inflammasome activation, induction of incretin receptors and reduction of terminal endoplasmic reticulum stress. Together, these studies have broadened our understanding of the mechanisms of antidiabetic agents as well as the pathogenic mechanism of diabetes itself. The results of such investigations might help to identify new target molecules and pathways for treatment of diabetes and metabolic syndrome, and could also have broad implications in diseases other than diabetes. Accordingly, new antidiabetic drugs with better efficacy and fewer adverse effects will likely result from these studies.     

细胞自噬在支气管哮喘中的研究进展与展望

自噬是一种溶酶体降解途径,它对于细胞的生存、分化、发展及内环境的稳定起到重要作用.近年来备受关注,但国内外对细胞自噬的研究多集中在肿瘤、神经肌肉、以及免疫系统疾病的研究,少有其在支气管哮喘(简称哮喘)的研究,本文主要对近几年细胞自噬在肺部疾病及哮喘中的研究进展进行综述,从遗传多态性,免疫应答,气道重塑及纤维化三方面,探讨自噬在哮喘发病过程中可能的机制.     

Rapamycin and bafilomycin A1 alter autophagy and megakaryopoiesis

Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents. Genetic deletion of autophagy mediator Atg7 in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Here, we examined the hypothesis that optimal autophagy is essential for megakaryopoiesis and thrombopoiesis by altering autophagy using pharmacological approaches. When autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed a significant decrease in high ploidy megakaryocytes, a reduction of CD41 and CD61 co-expressing cells, and less proplatelet or platelet formation. Additionally, reduced cell size was shown in megakaryocytes derived from rapamycin, but not bafilomycin A1-treated mouse fetal liver cells. However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, and ploidy in Atg7^flox/flox PF4-Cre mice with megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7. Therefore, our findings suggest that either induction or inhibition of autophagy in the early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis.     

自噬在肺部疾病中的研究进展

自噬作为一种维持细胞内环境稳定的重要机制,近年来成为一个研究热点。在受到氧化应激、饥饿诱导、炎症刺激等损伤因素作用后,细胞可以通过激活自噬完成对受损蛋白或细胞器的清除,从而维持细胞的稳态。但是自噬的过度激活也会导致细胞功能受损或自噬性死亡。自噬在肺部疾病发病机制所起的调控作用更加复杂,其中一些疾病的发病机制尚不明确,随着自噬与其关系研究的进一步深入,为临床治疗提供了一个新的思路。本文着重讨论自噬在一些常见肺部疾病发病机制中的作用及其临床运用前景。     

自噬与肝脏脂代谢

细胞自噬是利用溶酶体降解受损的细胞器、未折叠蛋白来维持细胞内稳态,在机体生长、发育和衰老中均起重要作用.研究表明,自噬可能与肝脏脂肪合成及分解相关.固醇调节元件结合蛋白通路、转录因子、激素与营养因素可能会影响自噬,从而改变脂代谢.     

人参皂苷Rg1对阿尔茨海默病大鼠模型脑片神经元自噬小体相关蛋白表达的影响

目的:探讨人参皂苷Rg1对阿尔茨海默病(AD)大鼠模型脑片神经元自噬小体相关蛋白表达的影响及其分子机制。方法:选取6周龄SD大鼠断头取脑制备海马脑片,将海马脑片随机分为空白对照组、模型组及低、中、高浓度Rg1组,每组10张脑片。模型组人工脑脊液中加入Aβ 1-42(终浓度5 μmol/L)处理2 h造模,低...