锌转运蛋白ZIP8 在骨关节炎中的表达及其机制研究

目的:探讨锌转运蛋白ZIP8在骨关节炎患者中的表达及其对软骨细胞生长及基质金属蛋白酶(MMPs)表达的影响。方法:收集20例骨关节炎患者(OA组)和20例非骨关节炎患者(对照组)血清和软骨组织;采用原子吸收分光光度计测定患者血清和软骨组织中锌离子的表达水平;MTT方法检测软骨细胞的生长活力;采用小RNA干扰沉默ZIP8基因的表达;实时荧光定量PCR方法检测ZIP8及金属基质蛋白酶MMP3、MMP9、MMP12和MMP13等基因的m RNA表达水平;蛋白免疫印迹检测ZIP8及MMP3、MMP9、MMP12和MMP13等蛋白的表达水平。结果:OA组的血清和软骨组织中的锌离子浓度明显高于对照组(P0.01)。OA组软骨组织中ZIP8的m RNA(P0.05)和蛋白(P0.01)表达水平显著高于对照组。ZIP8小RNA干扰片段可以有效的沉默ZIP的基因表达(P0.01);沉默ZIP8的表达促进骨关节炎患者来源的软骨细胞的生长(P0.05),并且降低基质金属蛋白酶包括MMP3,MMP9,MMP12和MMP13的表达水平(P0.05)。结论:ZIP8与骨关节炎密切相关,沉默ZIP8的表达可以提高软骨细胞的生长活力,并且抑制基质金属蛋白酶的表达,为骨关节炎的治疗提供了新的靶点。     

Role of autophagy in liver physiology and pathophysiology

Autophagy is a highly conserved intracellular degradation pathway by which bulk cytoplasm and superfluous or damaged organelles are enveloped by double membrane structures termed autophagosomes. The autophagosomes then fuse with lysosomes for degradation of their contents, and the resulting amino acids can then recycle back to the cytosol. Autophagy is normally activated in response to nutrient deprivation and other stressors and occurs in all eukaryotes. In addition to maintaining energy and nutrient balance in the liver, it is now clear that autophagy plays a role in liver protein aggregates related diseases, hepatocyte cell death, steatohepatitis, hepatitis virus infection and hepatocellular carcinoma. In this review, I discuss the recent findings of autophagy with a focus on its role in liver pathophysiology.     

橙皮素抑制NF-κB通路调控软骨细胞炎性退变的实验研究

目的:炎症因子所介导的慢性炎症瀑布反应是引起关节软骨退变的的主要原因。橙皮素具有抗炎、抗氧化应激等作用,研究橙皮素对关节软骨细胞炎症因子表达及相关信号通路的影响可以加深对关节软骨退变的认识,进而为其预防、治疗提供新的参考依据。研究橙皮素对人关节软骨细胞退变的影响,并从炎症角度来探讨其具体的分子机制。方法:体外分离培养人关节软骨细胞,首先采用CCK-8方法检测橙皮素对人关节软骨细胞增殖的抑制作用;运用RT-PCR和western blot研究橙皮素对于脂多糖(LPS)诱发的关节软骨细胞炎症反应和分解代谢的影响,运用Western blot研究橙皮素对于LPS所诱导的NF-κB信号通路的激活的影响。结果:当橙皮素的浓度低于10μM时,对于人关节软骨细胞的生长没有明显的抑制作用;real-time PCR和western blot结果显示,在LPS刺激下,关节软骨细胞中IL-6, TNF-α, MMP9, MMP13的基因表达水平明显升高,而橙皮素可以明显抑制炎症反应的激活;Western blot结果显示在LPS的刺激下,NF-κB信号通路显著激活,IKBα降解,随后P65磷酸化。而在橙皮素预处理组中,IKBα降解减少,P65磷酸化减少,NF-κB信号通路的激活受到了明显的抑制。以上结果均有统计学差异(P0.05)。结论:橙皮素可通过NF-κB信号通路影响人关节软骨细胞炎症反应和分解代谢相关基因的表达,进而降低关节软骨细胞内外的慢性炎症反应,进而延缓老年性关节软骨退变。     

Autophagy paradox and ceramide

Sphingolipid molecules act as bioactive lipid messengers and exert their actions on the regulation of various cellular signaling pathways. Sphingolipids play essential roles in numerous cellular functions, including controlling cell inflammation, proliferation, death, migration, senescence, tumor metastasis and/or autophagy. Dysregulated sphingolipid metabolism has been also implicated in many human cancers. Macroautophagy (referred to here as autophagy) “self-eating” is characterized by nonselective sequestering of cytosolic materials by an isolation membrane, which can be either protective or lethal for cells. Ceramide (Cer), a central molecule of sphingolipid metabolism, has been extensively implicated in the control of autophagy. The increasing evidence suggests that Cer is highly involved in mediating two opposing autophagic pathways, which regulate either cell survival or death, which is referred here as autophagy paradox. However, the underlying mechanism that regulates the autophagy paradox remains unclear. Therefore, this review focuses on recent studies with regard to the regulation of autophagy by Cer and elucidates the roles and mechanisms of action of Cer in controlling autophagy paradox. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

右美托咪定神经保护作用的自噬机制研究

摘要 目的：探讨右美托咪定发挥神经保护作用的细胞自噬和线粒体自噬机制。方法：通过对SH-SY5Y细胞进行氧糖剥夺再灌注模拟全脑的缺血再灌注损伤,将细胞随机分为7组：（1）C组：对照组;（2）OGD/R组：氧糖剥夺再灌注损伤组;（3）DEX组：右美托咪定组;（4）3MA组：3-甲基腺嘌呤组;（5）D+3MA组;（6）RAPA组：雷帕霉素组;（7）D+RAPA组。结果：与OGD/R组相比,DEX组、3MA组、D+3MA组的细胞活性、电镜下完整线粒体的数量、自噬体数量明显好于OGD/R组（P<0.05）;RAPA组与OGD/R组相比上述指标无明显差异（P>0.05）;而RAPA中加入右美托咪定以后,可以部分逆转RAPA的作用,细胞活性增加,完整线粒体数量增加,自噬体数量减少（P<0.05）。免疫印迹结果显示,与OGD/R组相比,DEX组、3MA组、D+3MA组LC3II/LC3I、Beclin 1表达减少,BCL-2、P62、TOM20的表达增加,RAPA组各种自噬蛋白的表达与OGD/R组相比没有统计学意义,当应用右美托咪定之后逆转了各种蛋白的表达（P<0.05）。结论：右美托咪定通过减少过度的细胞自噬和线粒体自噬发挥神经保护作用。     

Microplate live cell assay system for early events in mechanotransduction

For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5(')-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.     

Parkin介导的线粒体自噬对1型糖尿病急性心肌梗塞早期损伤的保护作用

目的:观察线粒体自噬在急性心梗(MI)早期的变化及对1型糖尿病(DM)小鼠心肌急性缺血损伤的影响。方法:将100只健康雄性C57BL/6小鼠随机分为5组,对照+假手术组(CS组);1型糖尿病+假手术组(DS组);对照+心肌梗死组(CMI组);1型糖尿病+心肌梗死组(DMI组);1型糖尿病+心肌梗死组+Parkin腺病毒过表达组(DMIPO组),每组20只。检测和比较各组小鼠的心脏功能,心肌梗死面积,心肌细胞凋亡,自噬小体含量以及心肌组织中Parkin和LC3的表达量变化。结果:与CS组相比,CMI组自噬小体含量增多,LC3II的表达量上调,Parkin的表达量明显上调(P0.05)。与CMI组比,DMI组小鼠心功能下降加剧,心梗面积增大,心肌细胞凋亡数量明显增加(P0.05),自噬水平未见明显升高。DMIPO组较DMI组自噬水平升高,心肌梗死面积减小(P0.05),心肌细胞凋亡数量减少(P0.05),心功能改善。结论:1型糖尿病通过抑制Parkin介导的心肌线粒体自噬增加心肌急性缺血损伤易感性,上调Parkin的表达可以减轻1型糖尿病时急性缺血性心肌损伤。     

Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL,FAS, and Caspase 3

Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as an anabolic effect on chondrocytes through significantly reversing Dex-induced chondrocytes apoptosis. This study may contribute to further investigating the clinical application of LF on OA.     

Liraglutide alleviates H2O2-induced retinal ganglion cells injury by inhibiting autophagy through mitochondrial pathways

Retinal ganglion cells (RGCs), which exist in the inner retina, are the retinal neurons which can be damaged in the early stage of diabetic retinopathy (DR). Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, exerts biological functions by binding the receptor (GLP-1R), the expression of which in RGC-5 cells was first shown by our team in 2012. It was reported that liraglutide prevented retinal neurodegeneration in diabetic subjects. However, the involvement of mechanisms such as autophagy and mitochondrial balance in liraglutide-induced retinal protection is unknown. Here, we aimed to investigate the protective effects of liraglutide and explore the potential mechanisms of liraglutide-induced retinal RGC protection. RGC-5 cells were treated with H₂O₂ and/or liraglutide. Cell viability was detected with the CCK-8 kit. The axon marker GAP43, autophagy and mitophagy indicators LC3A/B, Beclin-1, p62, Parkin, BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) and the key regulator of mitochondrial biogenesis PGC-1α were examined via western blot analysis. Autophagy was also evaluated using the ImageXpress Micro XLS system and transmission electron microscopy (TEM). Reactive oxygen species (ROS), mitochondrial membrane potential and fluorescent staining for mitochondria were also measured using the ImageXpress Micro XLS system. Our results showed that pretreatment with liraglutide significantly prevented H₂O₂-induced cell viability decline, mitochondrial morphological deterioration and induction of autophagy, which appeared as increased expression of LC3 II/I and Beclin-1, along with p62 degradation. Moreover, liraglutide suppressed the H₂O₂-induced decline in GAP43 expression, thus protecting cells. However, rapamycin induced autophagy and blocked the protective process. Liraglutide also provided mitochondrial protection and appeared to alleviate H₂O₂-induced ROS overproduction and a decline in mitochondrial membrane potential, partially by promoting mitochondrial generation and attenuating mitophagy. In conclusion, liraglutide attenuates H₂O₂ induced RGC-5 cell injury by inhibiting autophagy through maintaining a balance between mitochondrial biogenesis and mitophagy.