基于NLRP3-IL-1β-NF-κB信号轴研究野菊花总黄酮对 脂多糖诱导HK-2细胞炎性反应及自噬的影响

摘要：目的 探究野菊花总黄酮（TFC）对脂多糖（LPS）诱导HK-2细胞炎性反应及自噬的影响,并初步探究其可 能的作用机制。方法 体外培养人肾小管上皮HK-2细胞,将细胞分为阴性对照（Control）组、LPS处理（LPS）组、TFC 预处理（TFC）组、NLRP3激活预处理（4-MET）组、TFC联合4-MET预处理（TFC+4-MET）组。CCK-8法检测细胞增殖 情况;Hoechst 33342染色观察各组细胞形态变化;免疫印迹法检测各组细胞核苷酸结合域样受体蛋白3（NLRP3）、凋 亡相关斑点样蛋白（Asc）、半胱氨酸天冬氨酸蛋白酶1（caspase-1）、p-IκB、IκB、自噬相关蛋白（Beclin1）、微管相关蛋 白轻链3Ⅱ（LC3Ⅱ）、LC3Ⅰ、蛋白表达情况;ELISA法检测细胞中白细胞介素（IL）-1β、IL-18水平;透射电镜观察细胞 自噬情况。结果 与Control组相比,LPS组细胞增殖率降低,细胞核破碎情况加重,细胞凋亡率升高,NLRP3、Asc、 caspase-1蛋白表达升高、IκB蛋白磷酸化水平升高,IL-1β、IL-18水平升高,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低 （P＜0.05）。与LPS组相比,TFC组细胞增殖率升高,细胞核破碎减轻,细胞凋亡率降低,NLRP3、Asc、caspase-1蛋白 表达降低、IκB蛋白磷酸化水平降低,IL-1β、IL-18水平降低,细胞自噬程度增加,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达升 高;4-MET组细胞增殖率降低,细胞凋亡率升高,细胞核破碎情况加重,NLRP3、Asc、caspase-1蛋白表达升高、IκB蛋 白磷酸化水平升高,IL-1β、IL-18水平升高,细胞自噬程度降低,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低（P＜0.05）。 TFC+4-MET组细胞增殖率、细胞自噬程度、LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达高于4-MET组,细胞核破碎情况减轻, 细胞凋亡率、NLRP3、Asc、caspase-1蛋白表达、IκB蛋白磷酸化、IL-1β、IL-18水平低于4-MET组（P＜0.05）。结论 野菊花总黄酮可能通过抑制NLRP3-IL-1β-NF-κB信号通路活化,进而抑制脂多糖诱导HK-2细胞炎性反应,诱导 细胞自噬     

模拟海拔8000m高原缺氧环境对大鼠脑组织线粒体自噬的影响

目的：研究大型低压氧舱模拟海拔8000 m 高原缺氧环境对大鼠线粒体自噬的影响。方法取30只Wistar 大鼠,随机分为正常、缺氧6 h、12 h、24 h 和48 h 组,每组6只,在大型低压氧舱中模拟海拔8000 m 高原缺氧环境,采集实验大鼠的脑组织进行电镜观察并对线粒体自噬蛋白微管相关蛋白1轻链3（LC3）、Beclin1以及凋亡相关蛋白细胞色素 C（CytC）、Caspase3、Bcl-2相关 X 蛋白（Bax）进行测定。结果电镜结果显示,不同时间缺氧组与正常组相比较,大鼠脑组织空泡增多,细胞间隙增大,细胞膜部分破碎,细胞受损,并且不同缺氧时间自噬发生程度较正常组均增多,缺氧24 h 组自噬程度最强,自噬体数量最多。各缺氧组凋亡相关蛋白 CytC、Caspase3和 Bax 表达水平随着缺氧时间的延长均上调,缺氧24 h 和48 h 组显著高于正常组（ P ﹤0.01）。自噬相关蛋白 LC3和 Beclin1表达随着缺氧时间增加呈现从升高到下降的趋势,其中 LC3在缺氧24 h 组表达程度最高（P ﹤0.05）,Beclin1蛋白在缺氧6 h 组表达升高,缺氧12 h 组表达最高（P ﹤0.01）。结论模拟海拔8000 m 缺氧环境会对大鼠造成损伤,促进凋亡相关蛋白的表达,同时促进了大鼠脑组织线粒体自噬的发生。     

自噬在脑缺血性损伤中的作用

自噬是一种细胞内成分自我降解的过程,包括自噬的诱导、自噬体膜的形成、自噬体的形成、自噬体与溶酶体的融合以及内容物的降解。自噬若适度激活,则促进细胞存活;若过度激活,则可加重细胞损伤,甚至导致细胞裂解和促进细胞死亡。该文主要从自噬的形成过程及分子机制、脑缺血后自噬的诱发因素、自噬参与脑缺血的信号转导通路及自噬在脑缺血损伤中的作用进行综述,为进一步研究自噬与脑缺血性损伤及其药物调控提供参考。     

气道上皮细胞紧密连接在小鼠哮喘中的病理学改变

摘要： 目的 探讨小鼠支气管哮喘 （哮喘） 中气道上皮紧密连接的病理学改变。方法 10 只小鼠随机分为对照组和哮喘组, 每组 5 只。哮喘组于第 0、 7 天腹腔注射鸡卵清蛋白 （OVA） 溶液, 在第 14、 15、 16 天以雾化的 OVA 熏诱小鼠, 1 h/次、 1 次/d; 对照组用 PBS 溶液替代 OVA; 2 组小鼠在第 18 天回收肺组织, 采用免疫荧光染色法评价小鼠哮喘模型的有效性, 荧光定量 PCR检测紧密连接相关蛋白基因Claudin 1、 Claudin 3、 Claudin 4、 Claudin 5、 Claudin 7、 Clau⁃ din 10在肺组织中的表达。结果 哮喘组 Claudin 3、 Claudin 4、 Claudin 10 mRNA 水平较对照组低 （P＜0.05）, 而 Clau⁃ din 1、 Claudin 5、 Claudin 7 mRNA 水平与对照组比较差异无统计学意义 （P＞0.05）。结论 哮喘病理过程中气道上皮细胞紧密连接松散, 提示气道上皮屏障破坏。     

血管紧张素Ⅱ与血管平滑肌细胞中自噬水平的关系及机制研究

目的研究血管紧张素 Ⅱ(Ang Ⅱ)是否能够引起血管平滑肌细胞( VSMC)自噬,探讨其相关作用机制。方法以 VSMC为研究对象,设立组别分别为空白组、 Ang Ⅱ组、自噬抑制剂( 3?MA)组、自噬促进剂雷帕霉素( Rap)组、血管紧张素 Ⅱ(AT1)受体抑制剂( ARB)组、 3?MA+Ang Ⅱ组、 Rap+Ang Ⅱ组、 ARB+Ang Ⅱ组,采用四唑盐( MTT)比色法检测细胞存活率,蛋白质免疫印迹法检测自噬相关蛋白脂化和膜相关蛋白( LC3 Ⅱ)表达的变化,免疫荧光法检测自噬小体数目的变化。结果实验中空白组吸光度为( 0.159±0.001)Ang Ⅱ组吸光度为( 0.151±0.003),相比空白组,加入 Ang Ⅱ药物对细胞存活率变化不明显,差异无统计学意义(P＞0.05)。蛋白,质免疫印迹结果显示加入 Ang Ⅱ促进 VSMC的 LC3?Ⅱ表达量增多,呈现时间依赖性、浓度依赖性,表明 Ang Ⅱ促进 VSMC自噬发生。 3?MA对 Ang Ⅱ(10-7 mol/L,24 h)引起的 LC?3Ⅱ表达量增多起到抑制作用, Rap对 AngⅡ(10-7 mol/L,24 h)引起的 LC?3Ⅱ表达量增多起到叠加作用。 ARB对 Ang Ⅱ(10-7 mol/L,24 h)引起的 LC?3Ⅱ表达量增多起到抑制作用,表明自噬是由 Ang Ⅱ通过 ATI受体引起的。结论 Ang Ⅱ通过 AT1受体引起血管平滑肌细胞发生自噬。     

细胞自噬在心血管疾病发病过程中的作用

心血管疾病是一类危害心脏和血管功能的疾病,其发病是一个复杂的病理生理过程。细胞自噬（ autophagy）是一种广泛存在于真核细胞、进化上高度保守的细胞降解过程。饥饿、缺血、氧化应激等均可诱导其发生。自噬在细胞的生长、代谢、死亡等过程中起至关重要的作用,也参与了多种疾病的发生。近期发现,自噬与心血管疾病的发生、发展密切相关。正常的细胞自噬对心肌细胞有保护作用,自噬不足或自噬过度则可促发疾病或加重病变,本文对细胞自噬在心血管疾病发病中的作用做一综述。     

自噬激活与抗肿瘤药物的作用

自噬是一种在正常细胞和病态细胞中普遍存在的生理机制。某些肿瘤细胞中自噬活动低下与肿瘤的发生有一定的关系。抗肿瘤药物可以诱导细胞产生自噬,并参与了自噬的分子调控,同时它也可能导致细胞凋亡。自噬在抗肿瘤药物中作用与给药浓度及细胞的类型等因素有关。抗肿瘤药物引起的细胞自噬对肿瘤细胞产生正负两方面的影响,将自噬途径作为抗肿瘤药物的靶点有着广阔的前景。     

自噬介导mTOR抑制剂抗肿瘤作用的研究进展

目的:为研发基于哺乳动物雷帕霉素靶蛋白(mTOR)信号通路调控肿瘤细胞自噬的新型抗肿瘤药物提供参考。方法:根据文献,对细胞自噬在肿瘤细胞生长中的作用、mTOR信号对细胞自噬的调节及自噬介导mTOR抑制剂的抗肿瘤作用等方面进行综述。结果:在营养不充分条件下细胞自噬作用可导致肿瘤细胞对化疗或放射治疗耐受;在肿瘤初期自噬限制肿瘤形成,在肿瘤后期自噬则为肿瘤细胞提供能量,促进细胞存活。mTOR的复合体mTOR复合体1(mTORC1)信号参与自噬体的诱导及形成,起负性调控细胞自噬的作用;在营养缺失下,mTOR调节细胞自噬是通过引发mTORC1失活而完成,且mTOR主要作用于自噬的起始阶段。抑制mTOR活性,诱导产生细胞自噬,促使细胞自噬性死亡,不仅能降低毒蛋白压力,也能起到抗肿瘤作用。结论:激活mTOR及其上游信号分子能诱导细胞自噬的产生,mTOR抑制剂及上游分子抑制剂(如磷脂酰肌醇3激酶抑制剂和蛋白激酶B抑制剂)具有明显的抗肿瘤作用。以mTOR为靶标调控细胞自噬形成,在抗肿瘤研究方面取得了较快的进展。进一步明确mTOR通路与细胞自噬关联的分子机制,可为寻找与研发新型靶向抗肿瘤药物提供新的思路。     

土槿皮乙酸对人肺癌A146细胞的自噬作用以及凋亡与自噬的关系

目的土槿皮乙酸是从松科植物金钱松的根皮或近根树皮中分离的二萜酸,具有抗肿瘤、抗生育等作用。本研究主要阐述了土槿皮乙酸能够诱导人肺癌细胞(A146)发生自噬从而抑制细胞生长的机制及在A146细胞中自噬与凋亡的关系。方法采用了形态学观察、四甲基偶氮唑蓝(MTT)分析法、MDC流式细胞术分析、MDC荧光染色法、乳酸脱氢酶LDH活力测试法、免疫沉淀法、蛋白质印迹法等方法考察了土槿皮乙酸通过自噬抑制A146细胞生长以及该细胞中自噬与凋亡的关系。结果研究发现,4μmol/L土槿皮乙酸作用A146细胞0、12、24、36、48 h后,细胞数目减少,MDC染色有荧光亮绿色颗粒出现,流式细胞仪分析显示土槿皮乙酸增加MDC阳性率。而且4μmol/L土槿皮乙酸作用后自噬相关蛋白Beclin 1的表达增高,LC3从Ⅰ型转化为Ⅱ型,说明土槿皮乙酸诱导A146细胞自噬,自噬在抑制A146细胞生长中起到了关键的作用。另外,加入自噬抑制剂3-MA与土槿皮乙酸联用之后,凋亡小体明显增加,凋亡率上升,自噬率下降,自噬相关蛋白Beclin 1和LC3表达降低,说明在A146细胞中,自噬是对抗凋亡的。结论土槿皮乙酸诱导A146细胞自噬,自噬在抑制A146细胞生长中起到了关键的作用。     

自噬介导肿瘤发展的现状

细胞自噬几乎存在所有的细胞中,是维持细胞内稳态的基本机制,是细胞成分降解和回收运用的根本.在外界刺激下,细胞受损触发自噬性溶酶体形成,降解受损的细胞器和变性蛋白质,为细胞再生、修复提供营养物质和能量,对细胞的破坏起一种防御作用.随着学者对自噬溶酶体的进一步研究,发现有多种自噬性相关基因通过各种信号通路参与调控细胞自噬....     

Asthma entities

Asthma is a syndrome characterized by variable airflow limitation, airway hyperresponsiveness and airway inflammation. Considering asthma in aggregate, it is clear that a number of distinct mechanisms underlie the development of this disorder. In some patients, the mechanistic distinctions can be clearly drawn, and important therapeutic insights can be gained. In other patients, several mechanisms may coexist, or it may be impossible to separate them with current methods and technology. To distinguish subsets of asthma is more than an academic exercise. For both clinicians and asthma researchers, it is valuable to distinguish asthma subtypes as clearly as possible. Clinicians strive to prescribe the most effective, most safe, and most cost effective therapy possible, and understanding asthma subsets and their underlying mechanistic differences can substantively facilitate achieving that objective. Asthma research is often limited by significant, and sometimes dramatic intersubject variability. It is likely that at least some of that variability may arise from the (unrecognized) mechanistic heterogeneity of asthma. Better definition and selection of more homogeneous subsets of asthma may then lead to greater statistical power, and more definitive conclusions from asthma investigations.     

Autophagy in vascular endothelial cells

The importance of autophagy in cardiovascular physiology and cardiovascular disease is increasingly recognized; however, the precise biological effects and underlying mechanisms of autophagy in the cardiovascular system are still poorly understood. In the last few years, the effects of autophagy in endothelial cells have attracted great interests. This article provides a summary of our current knowledge on the regulatory factors, signalling mechanisms, and functional outcomes of autophagy in endothelial cells. It is suggested that in most situations, induction of an autophagic response has cytoprotective effects. The beneficial effects of autophagy in endothelial cells are likely to be context‐dependent, since autophagy may also contribute to cell death under certain circumstances. In addition to regulating endothelial cell survival or death, autophagy is also involved in modulating other important functions, such as nitric oxide production, angiogenesis and haemostasis/thrombosis. The mounting data will help us draw a clear picture of the roles of autophagy in endothelial cell biology and dysfunction. Given the pivotal role of endothelial dysfunction in the pathogenesis of vascular disease, disruptions of autophagy in endothelial cells are likely to have significant contributions. This is supported by some preliminary ex vivo data indicating that compromised autophagic functions may be important in the development of endothelial dysfunctions associated with diabetes and ageing.     

Autophagy & Phagocytosis in Neurological Disorders and their Possible Cross-talk

Autophagy and phagocytosis are two important endogenous lysosomal dependent clearing systems in the organism. In some neurological disorders, excessive autophagy or dysfunctional phagocytosis has been shown to contribute to brain injury. Recent studies have revealed that there are underlying interactions between these two processes. However, different studies show inconsistent results for the contribution of autophagy to the phagocytic process in diverse phagocytes and relatively little is known about the link between them especially in the brain. It is critical to understand the role that autophagy plays in phagocytic process in order to promote the clearance of endogenous and exogenous detrimental materials. In this review, we highlight the studies focusing on phagocytosis and autophagy occurring in the brain and summarizing the possible regulatory roles of autophagy in the process of phagocytosis. Balancing the roles of autophagy and phagocytosis may be a promising therapeutic strategy for the treatment of some neurological diseases in the future.     

Neuronal Autophagy: Characteristic Features and Roles in Neuronal Pathophysiology

Autophagy is an important degradative pathway that eliminates misfolded proteins and damaged organelles from cells. Autophagy is crucial for neuronal homeostasis and function. A lack of or deficiency in autophagy leads to the accumulation of protein aggregates, which are associated with several neurodegenerative diseases. Compared with non-neuronal cells, neurons exhibit rapid autophagic flux because damaged organelles or protein aggregates cannot be diluted in post-mitotic cells; because of this, these cells exhibit characteristic features of autophagy, such as compartment-specific autophagy, which depends on polarized structures and rapid autophagy flux. In addition, neurons exhibit compartment-specific autophagy, which depends on polarized structures. Neuronal autophagy may have additional physiological roles other than amino acid recycling. In this review, we focus on the characteristics and regulatory factors of neuronal autophagy. We also describe intracellular selective autophagy in neurons and its association with neurodegenerative diseases.     

Recent advances in targeting autophagy in cancer

Autophagy is a cellular homeostasis mechanism that fuels the proliferation and survival of advanced cancers by degrading and recycling organelles and proteins. Preclinical studies have identified that within an established tumor, tumor cell autophagy and host cell autophagy conspire to support tumor growth. A growing body of evidence suggests that autophagy inhibition can augment the efficacy of chemotherapy, targeted therapy, or immunotherapy to enhance tumor shrinkage. First-generation autophagy inhibition trials in cancer using the lysosomal inhibitor hydroxychloroquine (HCQ) have produced mixed results but have guided the way for the development of more potent and specific autophagy inhibitors in clinical trials. In this review, we will discuss the role of autophagy in cancer, newly discovered molecular mechanisms of the autophagy pathway, the effects of autophagy modulation in cancer and host cells, and novel autophagy inhibitors that are entering clinical trials.     

Toluene diisocyanate-induced inflammation and airway remodeling involves autophagy in human bronchial epithelial cells

Toluene-diisocyanate (TDI) is one of the main causes of occupational asthma. To study the role of autophagy in TDI-induced airway inflammation and airway remodeling in bronchial airway epithelial (16HBE) cells. We treated 16HBE cells with TDI-human serum albumin (TDI-HSA) conjugate to observe reactive oxygen species (ROS) release, autophagy activation, airway inflammation and airway remodeling. 3-Methyladenine (3-MA) and Rapamycin (Rapa) intervention were used to explore the effects of autophagy on inflammatory response and protein expression related to airway remodeling in 16HBE cells treated with TDI-HSA. Experimental results suggested that various concentrations of TDI-HSA (0, 40, 80 and 120 μg/mL) increased the release of ROS and the expression of Nrf2, activated autophagy and increased the expression of AMPK, Beclin-1, LC3 and decreased the expression of p62, promoted the levels of IL-5, IL-6 and IL-8 in 16HBE cells. Results also showed that E-cadherin expression decreased but an increase was observed in α-SMA and MMP-9 in the TDI-HSA group. The treatment of TDI-HSA combined with Rapa aggravated the above reaction whereas the inverse was true for TDI-HSA combined with 3-MA. These results indicated that autophagy is involved in TDI-induced airway inflammation and airway remodeling as a positive regulatory mechanism, inhibiting autophagy can significantly alleviate the TDI-induced inflammatory response and attenuate airway remodeling protein expression in 16HBE cells.     

Defective autophagy in fibroblasts may contribute to fibrogenesis in autoimmune processes

Fibrosis may represent the final step induced by autoimmune mechanism(s). This may be due to the excess in fibroblast recruitment, activation and differentiation in myofibroblasts. These events may be triggered by cytokines, chemokines and growth factors released by lymphocytes or macrophages. Autophagy is an essential conserved homeostatic process that has long been appreciated for cell adaptation to nutrient deprivation. Autophagy is also recognized as an important component of both innate and acquired immunity to pathogens. Recently, dysregulation of autophagy in haematopoietic cells has been suggested to amplify the autoimmune responses. On the other hand, it is possible that defective autophagy in non-haematopoietic cells contributes to the progression to fibrosis. In fibroblasts some alterations in the metabolic pathways and pharmacological data suggest that a defective autophagy could contribute to excess in the production of extracellular matrix by altering the turnover of protein such as collagen. Our goal in this review is to describe the current knowledge on the role of autophagy in the development of fibrotic autoimmune diseases. Further studies could confirm whether agents modulating autophagy may be used in the treatment of these autoimmune diseases.     

Reactive oxygen species‐dependent JNK downregulated olaquindox‐induced autophagy in HepG2 cells

Autophagy plays an important role in response to intracellular and extracellular stress to sustain cell survival. However, dysregulated or excessive autophagy may lead to cell death, known as “type II programmed cell death,” and it is closely associated with apoptosis. In our previous study, we proposed that olaquindox induced apoptosis of HepG2 cells through a caspase‐9 dependent mitochondrial pathway. In this study, we investigated autophagy induced by olaquindox and explored the crosstalk between apoptosis and autophagy in olaquindox‐treated HepG2 cells. Olaquindox‐induced autophagy was demonstrated by the accumulation of monodansylcadervarine, as well as elevated expression of autophagy‐related MAP‐LC3 and Beclin 1 proteins. The autophagy inhibitor 3‐methyladenine significantly increased the apoptotic rate induced by olaquindox, which was correlated with increased ratio of Bax/Bcl‐2. The further studies showed that olaquindox increased the levels of reactive oxygen species (ROS), and antioxidant N‐acetyl‐L ‐cysteine (NAC) effectively blocked the accumulation of ROS but failed to block autophagy. Moreover, olaquindox induced the activation of c‐Jun N‐terminal protein kinase (JNK), and JNK inhibitor SP600125 failed to block autophagy. Instead, olaquindox‐induced autophagy was enhanced by NAC or SP600125. Meanwhile, JNK activation was remarkably blocked by NAC, indicating that ROS may be the upstream signaling molecules of JNK activation and involved in the negative regulation of olaquindox‐induced autophagy. These results suggest that olaquindox induces autophagy in HepG2 cells and that olaquindox‐induced apoptosis can be enhanced by 3‐methyladenine. Olaquindox‐induced autophagy in HepG2 cells is upregulated by Beclin 1 but downregulated by ROS‐dependent JNK. Copyright © 2014 John Wiley & Sons, Ltd.     

自噬信号通路与心血管疾病的研究进展

自噬是指自噬溶酶体将自身的细胞器分解并回收利用的一种细胞降解过程,广泛存在于机体组织中。在正常情况下,自噬活动水平在维持细胞的稳态和功能中发挥着重要作用;过量或不足的自噬通量水平都可能导致疾病的发生。在本文中,我们讨论了自噬的概念分类以及自噬信号通路的研究进展,并阐述了自噬对心血管疾病的影响。     

Application and interpretation of current autophagy inhibitors and activators

Autophagy is the major intracellular degradation system, by which cytoplasmic materials are delivered to and degraded in the lysosome. As a quality control mechanism for cytoplasmic proteins and organelles, autophagy plays important roles in a variety of human diseases, including neurodegenerative diseases, cancer, cardiovascular disease, diabetes and infectious and inflammatory diseases. The discovery of ATG genes and the dissection of the signaling pathways involved in regulating autophagy have greatly enriched our knowledge on the occurrence and development of this lysosomal degradation pathway. In addition to its role in degradation, autophagy may also promote a type of programmed cell death that is different from apoptosis, termed type II programmed cell death. Owing to the dual roles of autophagy in cell death and the specificity of diseases, the exact mechanisms of autophagy in various diseases require more investigation. The application of autophagy inhibitors and activators will help us understand the regulation of autophagy in human diseases, and provide insight into the use of autophagy-targeted drugs. In this review, we summarize the latest research on autophagy inhibitors and activators and discuss the possibility of their application in human disease therapy.