Andrographolide suppresses NLRP3 inflammasome activation in microglia through induction of parkin-mediated mitophagy in in-vitro and in-vivo models of Parkinson disease

Cellular communication linking microglia activation and dopaminergic neuronal loss play an imperative role in the progression of Parkinson’s disease (PD); however, underlying molecular mechanisms are not precise and require further elucidation. NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation is extensively studied in context to microglial activation and progressive dopaminergic neuronal loss in PD. Several pathophysiological factors such as oxidative stress, mitochondrial dysfunction impaired mitophagy plays a crucial role in activating NLRP3 inflammasome complex. Hence, regulation of microglial activation through mitophagy could be a valuable strategy in controlling microglia mediated neurodegeneration. In this study we have developed a model of inflammasome activation by combining LPS with a mitochondrial complex-I inhibitor MPP⁺. The idea of using MPP⁺ after priming mouse microglia with LPS was to disrupt mitochondria and release reactive oxygen species, which act as Signal 2 in augmenting NLRP3 assembly, thereby releasing potent inflammatory mediators such as active interleukin-1 beta (IL-1β) and IL-18. LPS-MPP⁺ combination was seen to impaired the mitophagy by inhibiting the initial step of autophagosome formation as evidenced by protein expression and confocal imaging data. Treatment with Andrographolide promoted the parkin-dependent autophagic flux formation in microglia; resulting in the removal of defective mitochondria which in turn inhibit NLRP3 inflammasome activation. Additionally, the neuroprotective role of Andrographolide in inhibiting NLRP3 activation together with salvage ATP level via promoting parkin-dependent mitophagy was seen in the substantial nigra par compacta (SNpc) region of mice brain. Furthermore, Andrographolide rescued the dopaminergic neuron loss and improved the behavioural parameters in animal model. Collectively, our results reveal the role of mitophagy in the regulation of NLRP3 inflammasome by removing defective mitochondria. In addition, andrographolide was seen to abate NLRP3 inflammasome activation in microglia and rescue dopaminergic neuron loss.     

四君子汤对脾气虚证模型大鼠股四头肌线粒体自噬和细胞凋亡水平的影响＊

目的 通过观察不同浓度四君子汤治疗脾气虚证模型大鼠股四头肌线粒体自噬及细胞凋亡水平的变化，研究四君子汤治疗“脾气虚四肢不用”的机制。方法 50只雄性SPF级大鼠随机分为对照组（n = 10）和脾气虚证模型组（模型组，n = 40），饮食失节+劳倦法复制脾气虚证模型，造模成功后符合模型评价标准者纳入脾气虚证模型，将其分为盐水组（n = 10），四君子汤治疗低剂量组（低剂量组，n = 10），四君子汤治疗中剂量组（中剂量组，n = 10）和四君子汤治疗高剂量组（高剂量组，n = 10），连续给药15天。治疗结束后测定其抓力并取股四头肌，比色法检测三磷酸腺苷（Adenosine triphosphate，ATP）、线粒体膜电位（Mitochondrial membrane potential，MMP）和过氧化氢（Hydrogen peroxide，H₂O₂）水平；免疫印迹法（Western blot）法检测微管相关蛋白1轻链3-B（LC3 B）、选择性自噬接头蛋白（p62）、Bcl-2、Bax、半胱氨酸天冬氨酸蛋白酶3（Caspase3）、细胞色素c（Cyt-c）、聚腺苷二磷酸-核糖聚合酶（PARP）蛋白表达。结果 与对照组比较，盐水组大鼠抓力、体质量、股四头肌ATP和MMP水平下降（P < 0.05或P < 0.01或P < 0.001），H₂O₂水平升高（P < 0.05）；LC3B-II、Bax、Cyt-c，cleaved-Caspase3、PARP蛋白表达上调（P < 0.05或P < 0.01或P < 0.001），p62、Bcl-2表达下调（均P < 0.001）。与盐水组比较，中/高剂量组大鼠抓力升高（均P < 0.01），体质量未见差异（均P > 0.05）；ATP、MMP水平升高（P < 0.01或P < 0.001）、H₂O₂下降（P < 0.05或P < 0.01）；LC3B-II、Bax、Cyt-c，cleaved-Caspase3、cleaved-PARP表达下调（P < 0.05或P < 0.01或P < 0.001），p62、Bcl-2表达上调（P < 0.05或P < 0.01），低剂量组各指标未见差异（均P > 0.05）。与中剂量组比较，高剂量组H₂O₂水平下降（P < 0.05），Bax表达下调（P < 0.01），其余指标未见差异（均P > 0.05）。结论 四君子汤可能通过抑制过度线粒体自噬，维持适度自噬改善线粒体应激，降低细胞凋亡水平治疗脾气虚四肢不用。     

Re-emphasizing early Alzheimer’s disease pathology starting in select entorhinal neurons,with a special focus on mitophagy

The entorhinal-hippocampal system contains distinct networks subserving declarative memory. This system is selectively vulnerable to changes of ageing and pathological processes. The entorhinal cortex (EC) is a pivotal component of this memory system since it serves as the interface between the neocortex and the hippocampus. EC is heavily affected by the proteinopathies of Alzheimer’s disease (AD). These appear in a stereotypical spatiotemporal manner and include increased levels of intracellular amyloid-beta Aβ (iAβ), parenchymal deposition of Aβ plaques, and neurofibrillary tangles (NFTs) containing abnormally processed Tau. Increased levels of iAβ and the formation of NFTs are seen very early on in a population of neurons belonging to EC layer II (EC LII), and recent evidence leads us to believe that this population is made up of highly energy-demanding reelin-positive (RE+) projection neurons. Mitochondria are fundamental to the energy supply, metabolism, and plasticity of neurons. Evidence from AD postmortem brain tissues supports the notion that mitochondrial dysfunction is one of the initial pathological events in AD, and this is likely to take place in the vulnerable RE + EC LII neurons. Here we review and discuss these notions, anchored to the anatomy of AD, and formulate a hypothesis attempting to explain the vulnerability of RE + EC LII neurons to the formation of NFTs. We attempt to link impaired mitochondrial clearance to iAβ and signaling involving both apolipoprotein 4 and reelin, and argue for their relevance to the formation of NFTs specifically in RE + EC LII neurons during the prodromal stages of AD. We believe future studies on these interactions holds promise to advance our understanding of AD etiology and provide new ideas for drug development.     

Epithelial Injury and Dysfunction in the Pathogenesis of Idiopathic PulmonaryFibrosis

Idiopathic pulmonary fibrosis is a disease of older adults leading to progressive dyspnea and reduced exercise capacity, typically resulting in death within 3-5years of diagnosis. Underlying genetic susceptibility combined with environmental insults is proposed to trigger a chronic wound repair response, leading to activation of the fibrotic cascade. Perturbations in several molecular pathways mediate vulnerability of the alveolar epithelium to injurious agents, including the unfolded protein response, autophagy, mitophagy, and cellular senescence. These cellular responses are intricately intertwined and link genetic susceptibility to the progressive fibrotic phenotype. Ongoing studies investigating these pathways in type II alveolar epithelial cells show promise for identifying new targeted interventions that could prevent or halt the progression of IPF.     

线粒体自噬在褪黑素抗缺血再灌注损伤中的作用

目的初步探讨线粒体自体吞噬在褪黑素抗缺血再灌注损伤中的作用。方法体外培养N2a细胞,模拟缺血再灌注,加入褪黑素(melatonin,Mel),用DNA琼脂糖凝胶电泳和Caspase3活性测定分析细胞凋亡情况,并采用激光共聚焦分析线粒体自噬现象。结果(1)Mel能抑制缺血再灌注介导的N2a细胞caspase3的活性,并减轻N2a细胞DNA的片段化;(2)N2a细胞缺血90min再灌注12h,未观察到线粒体自噬现象,而加入Mel的N2a有大量的线粒体自噬现象。结论线粒体自噬可能是褪黑素抗缺血再灌注损伤的机制之一。     

Autophagy in liver diseases

Autophagy is the liver cell energy recycling system regulating a variety of homeostatic mechanisms.Damaged organelles,lipids and proteins are degraded in the lysosomes and their elements are re-used by the cell.Investigations on autophagy have led to the award of two Nobel Prizes and a health of important reports.In this review we describe the fundamental functions of autophagy in the liver including new data on the regulation of autophagy.Moreover we emphasize the fact that autophagy acts like a two edge sword in many occasions with the most prominent paradigm being its involvement in the initiation and progress of hepatocellular carcinoma.We also focused to the implication of autophagy and its specialized forms of lipophagy and mitophagy in the pathogenesis of various liver diseases.We analyzed autophagy not only in well studied diseases,like alcoholic and nonalcoholic fatty liver and liver fibrosis but also in viral hepatitis,biliary diseases,autoimmune hepatitis and rare diseases including inherited metabolic diseases and also acetaminophene hepatotoxicity.We also stressed the different consequences that activation or impairment of autophagy may have in hepatocytes as opposed to Kupffer cells,sinusoidal endothelial cells or hepatic stellate cells.Finally,we analyzed the limited clinical data compared to the extensive experimental evidence and the possible future therapeutic interventions based on autophagy manipulation.     

低氧复合运动对大鼠骨骼肌线粒体含量的影响

 目的：探讨低氧复合运动对线粒体含量的影响及线粒体生物合成和自噬在其中的作用。方法：雄性SD大鼠随机分为常氧对照（NC）组、常氧运动（NT）组、低氧对照（HC）组和低氧复合运动（HT）组。低氧干预为常压低氧帐篷,11.3%氧浓度持续暴露4周。运动干预为跑台训练（5°,15 m/min）,60 min/d,每周5 d,共4周。JC-1荧光探针检测线粒体膜电位;萤光素酶法检测线粒体ATP合成能力;Western blotting检测骨骼肌过氧化物酶体增殖物激活受体γ辅激活因子1（PGC-1α）、线粒体转录因子A（Tfam）、Bcl-2/腺病毒E1B 19kD相互作用蛋白3（Bnip3）、苄氯素1（beclin-1）、细胞色素C氧化酶亚基IV（COXIV）和电压依赖性阴离子通道1（VDAC-1）蛋白表达量。结果：HC组与NC组比较,线粒体膜电位、ATP合成能力及COXIV、VDAC-1、PGC-1α和Tfam蛋白表达显著降低（P<005或P<001）,Bnip3和beclin-1蛋白表达显著升高（P<005）。HT组与HC组比较,线粒体膜电位、ATP合成能力及COXIV、VDAC-1、PGC-1α、Tfam、Bnip3和beclin-1蛋白表达均显著升高（P<005或P<001）。结论：慢性低氧暴露提高了线粒体自噬但抑制了线粒体生物合成,导致线粒体含量减少。低氧复合运动促进低氧状态下骨骼肌线粒体自噬,并促进线粒体生物合成,从而提高线粒体含量及功能。     

Activation of Autophagy,Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals,Protects Hepatocytes From Copper-Induced Apoptosis

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线粒体自噬对结直肠癌的影响

线粒体是细胞新陈代谢和生长发育过程中重要的细胞器,能够为细胞的生命活动提供能量和底物.当细胞处于恶劣环境时,线粒体通过自噬清除不需要或损伤的线粒体来减轻负荷,补充营养物质,适应恶劣的环境.线粒体自噬对维持细胞生存,抵御环境压力,衰老和凋亡过程起着重要作用.线粒体自噬的异常将导致多种疾病,如:神经退行性变、心脏病、糖尿病以及肿瘤等.随着人们生活习惯的改变,结直肠癌的发病率和死亡率逐年增加,成为中国最常见的恶性肿瘤之一,结直肠癌与线粒体自噬的研究也成为该领域的研究热点.明确线粒体自噬对结直肠癌的作用,有助于进一步了解结直肠癌的发病机制,并为结直肠癌的诊断和治疗提供新的方向和思路.因此,本文将着重阐述线粒体自噬的发生机制及其与结直肠癌的关系.