目的 探讨以教学微视频结合教学平台"讨论区"互动为基础的在线教学在病理生理学教学中的应用,以评价该教学模式在疫情期间的教学效果,探索后疫情时代深化病理生理学教学改革的可行性.方法 本实验以山东第一医科大学2018级医学影像1、2班和3、4班本科生为实验组和对照组,分别开展以教学微视频结合教学平台"讨论区"互动为基础的在线教学和以直播为基础的在线教学模式.通过分析教学平台数据和问卷星APP问卷调查结果,评价学生理论知识掌握程度和综合能力提高情况.结果 与以直播为基础的在线教学模式比较,以教学微视频结合教学平台"讨论区"互动为基础的在线教学不但加深了学生对所学理论知识的理解和掌握,而且提高了其归纳总结、自主学习能力,分析、解决实际问题和临床思维能力.结论 以教学微视频结合教学平台"讨论区"互动为基础的在线教学优于以直播为基础的在线教学模式,在新冠疫情期间实现了"以学生为中心"的理念,提高了病理生理学教学质量,而且可为后疫情时代开展线上线下混合式教学奠定基础.

The 2 major types of neurodegeneration with brain iron accumulation (NBIA) are the pantothenate kinase type 2 (PANK2)-associated neurodegeneration (PKAN) and NBIA2 or infantile neuroaxonal dystrophy (INAD) due to mutations in the phospholipase A2, group VI (PLA2G6) gene. We have recently demonstrated clinical heterogeneity in patients with mutations in the PLA2G6 gene by identifying a poorly defined subgroup of patients who present late with dystonia and parkinsonism. We report the clinical and genetic features of 7 cases with PLA2G6 mutations. Brain was available in 5 cases with an age of death ranging from 8 to 36 years and showed widespread alpha-synuclein-positive Lewy pathology, which was particularly severe in the neocortex, indicating that the Lewy pathology spread corresponded to Braak stage 6 and was that of the "diffuse neocortical type". In 3 cases there was hyperphosphorylated tau accumulation in both cellular processes as threads and neuronal perikarya as pretangles and neurofibrillary tangles. Later onset cases tended to have less tau involvement but still severe alpha-synuclein pathology. The clinical and neuropathological features clearly represent a link between PLA2G6 and parkinsonian disorders.

Astragaloside Ⅳ (AS-Ⅳ) is one of the main active components extracted from Astragalus membranaceus that exerts an antiatherosclerotic effect. Our study explored the underlying anti-apoptotic effects and the mechanisms of action of AS-Ⅳ in oxidized low-density lipoprotein (oxLDL)-stimulated macrophages and in vulnerable plaques. The results showed that AS-Ⅳ lowered the oxLDL-induced lipid content and reversed the oxLDL-induced reduction in cell viability and elevation in lactate dehydrogenase (LDH) leakage and apoptosis in RAW264.7 macrophages, similar to the effects of 4-phenylbutyric acid (PBA, an ER stress inhibitor). In addition, consistent with the effect exerted by PBA, AS-Ⅳ inhibited oxLDL-triggered ER stress activation by decreasing the level of inositol-requiring enzyme1 phosphorylation and transcription factor 6 nuclear translocation and upregulating the protein and mRNA expression of glucose-regulated protein 78 (GPR78) and C/EBP homologous protein (CHOP). As expected, autophagy activation was induced by AS-IV, evidenced by increased expression of microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ), autophagy-related gene 5, and beclin-1 in macrophages. Furthermore, after pretreatment with 3-methyladenine and beclin-1 small interfering RNA, the inhibitory role played by AS-Ⅳ in oxLDL-induced ER stress-CHOP-mediated macrophage apoptosis was weakened, while its inhibitory effect was further enhanced by rapamycin pretreatment. Moreover, administration of AS-Ⅳ or rapamycin to Apoe-/- mice upregulated LC3-Ⅱ expression and collagen content but decreased CHOP expression, macrophage apoptosis, and lipid areas. Overall, by promoting autophagy, AS-Ⅳ effectively protects macrophages from oxLDL-induced apoptosis mediated by ER stress-CHOP, which may reinforce the stability of atherosclerotic plaques.

目的 研究大蒜素对氧化低密度脂蛋白(oxidized low density lipoproein,ox-LDL)触发的巨噬细胞焦亡的抑制作用,并探究其可能的分子机制.方法 RAW 264.7巨噬细胞,给予大蒜素(12.5、25、50和100 mg/L)和二亚苯基碘鎓(DPI)5μmol/L预处理1 h,再加入100 mg/L ox-LDL继续孵育24 h.分别采用TUNEL、MTT、ELISA法和相应的试剂盒检测细胞死亡情况和细胞活力、培养基IL-1β和IL-18浓度以及培养基乳酸脱氢酶(lactate dehydrogenase,LDH)和细胞内caspase-1、超氧化物歧化酶(superoxide dismutase,SOD)活性、活性氧(reactive oxygen species,ROS)和丙二醛(malondialdehyde,MDA)水平.随后,应用Western blotting法检测核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)的表达变化.结果 和DPI氧化应激抑制剂相似,大蒜素可以显著抑制ox-LDL触发的巨噬细胞焦亡作用,增加细胞活力,减少ox-LDL刺激引起的TUNEL阳性细胞率以及巨噬细胞LDH漏出,并可减少白介素-1β(interleukin-1β,IL-1β)和白介素-18(interleukin-18,IL-18)的分泌,且呈浓度依赖性;与DPI相似,大蒜素能够显著抑制ox-LDL触发的NLRP3上调和caspase-1活化,并明显抑制ox-LDL介导的氧化应激反应,表现为SOD活性增加、ROS和MDA生成减少.结论 大蒜素可减轻ox-LDL所触发的巨噬细胞焦亡,该作用主要与减弱氧化应激反应从而抑制NLRP3-caspase-1信号通路的活化有关.

从小规模限制性在线课程(small private online course,SPOC)建设、教学实施方案和考核评价体系的优化等方面,探讨线上线下混合式教学模式在病理生理学教学中的应用,以评价其对教学质量的提升作用,为高校混合式教学改革提供参考.以山东第一医科大学2018级临床医学本科12个班631人为教学对象,基于SPOC开展线上-线下混合式教学,每一章内容的教学分为课前、课中和课后三个阶段.通过教学平台数据分析、问卷星App问卷调查以及期末考试成绩分析评价学生理论知识掌握和综合能力提高情况.结果显示,该教学模式不仅加深了学生对所学理论知识的理解和掌握,而且提高了其归纳总结、自主学习能力和分析、解决实际问题和临床思维以及团队合作、语言表达等方面的能力,且在期末时学生对该混合式教学模式的认可度比期中时显著升高(P＜0.01).全部章节开展混合式教学的2018级期末考试成绩高于部分章节开展混合式教学的2017级成绩,且高于开展传统课堂授课的2016级成绩(P＜0.05),差异有显著性.本研究结果表明以高质量教学微视频为核心、临床病例和问题为基础,以线上-线下"零时空"讨论为主要沟通方式,以行之有效的教学实施方案和形成性评价为保障,全程开展的线上-线下混合式教学可将线上知识传授与线下能力提升有机结合,真正实现了"学生为中心"的教育理念.

细胞焦亡(pyroptosis)是一种程序性的死亡方式,活化的天冬半胱氨酸酶-1(caspase-1)可以诱导焦亡的发生,并促进炎症因子IL-1β和IL-18等的释放,且焦亡广泛参与各种慢性疾病的进展,如动脉粥样硬化(atherosclerosis,AS)、2型糖尿病等.有研究表明,自噬的缺陷会刺激细胞焦亡,且会加快AS的进程.本文总结了近几年来有关细胞焦亡及其在AS发展中的作用研究进展,并探讨了细胞焦亡与自噬的相互关系是否可作为防治AS的新靶点.

The present study aimed to investigate the role of D4F, an apolipoprotein A-I mimetic peptide, in macrophage apoptosis induced by the glycated high-density lipoprotein (gly-HDL)-induced endoplasmic reticulum (ER) stress C/EBP homologous protein (CHOP) pathway, and unravel the regulatory role of autophagy in this process. Our results revealed that except for suppressing the accumulation of lipids within RAW264.7 macrophages caused by gly-HDL, D4F inhibited gly-HDL-induced decrease in the cell viability and increase in lactate dehydrogenase leakage and cell apoptosis, which were similar to 4-phenylbutyric acid (PBA, an ER stress inhibitor). Besides, similar to PBA, D4F inhibited gly-HDL-induced ER stress response activation evaluated through the decreased PERK and eIF2α phosphorylation, together with reduced ATF6 nuclear translocation as well as the downregulation of GRP78 and CHOP. Interestingly, D4F facilitated gly-HDL-triggered activation of autophagy, measured as elevated levels of beclin-1, LC3-II, and ATG5 expressions in macrophages. Furthermore, the inhibition effect of D4F on gly-HDL-induced ER stress-CHOP-induced apoptosis of macrophages was restrained after beclin-1 siRNA and 3-methyladenine (3-MA, an inhibitor of autophagy) treatments, while this effect was further reinforced after rapamycin (Rapa, an inducer of autophagy) treatment. Furthermore, administering D4F or Rapa to T2DM mice upregulated LC3-II and attenuated CHOP expression, cell apoptosis, and atherosclerotic lesions. However, the opposite results were obtained when 3-MA was administered to these mice. These results support that D4F effectively protects macrophages against gly-HDL-induced ER stress-CHOP-mediated apoptosis by promoting autophagy.

目的 探讨高等医学院校线上教学的现状及存在的问题,为应对新冠肺炎疫情等突发事件及深化教学改革提供依据.方法 采用自制电子调查问卷,利用问卷星平台对山东省多所医学院校共计7000余名医学生进行网络问卷调查,并对问卷进行分析.结果 大部分学生对疫情下的线上教学资源和教学模式表示满意,以各校教师自行录制的SPOC视频资源为基础的教学方式取得的教学效果相对好于以直播和MOOC为基础的教学方式,但是总的教学效果不如传统课堂教学,其原因可能与教师准备不充分,学生压力过大、困难多及网络拥堵、卡顿等因素有关.结论 各高等医学院校有必要从转变教学观念和提升教学能力、优化线上教学资源和教学实施方案、培养学生综合素质和能力等方面深化医学院校教学改革.

以山东第一医科大学2000余名医学生为研究对象,通过问卷星APP平台问卷调查学生对线上教学、线上—线下混合式教学以及传统课堂教学方式的选择意愿,并分析其影响因素,为探寻后疫情时代医学教育改革的方向和策略提供依据.

目的:探讨自噬对糖基化高密度脂蛋白(glycosylated high-density lipoprotein,gly-HDL)所致的血管内皮细胞凋亡的影响及其分子机制.方法:体外培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),分别与100 mg/L HDL和不同浓度(25、50和100 mg/L)gly-HDL共同孵育24 h;另再培养HUVECs给予1μmol/L自噬诱导剂雷帕霉素或2 mmol/L自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)预处理1 h,或5 mg/L抗Toll样受体4(Toll-like receptor 4,TLR4)单克隆中和抗体预处理30 min,再与gly-HDL(100 mg/L)共同孵育24 h.采用MTT法检测细胞活力,Annexin V-FITC/PI双染法检测细胞凋亡情况,试剂盒测定培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,采用Western blot技术检测自噬标志分子beclin-1和微管相关蛋白1轻链3-II(microtu?bule-associated protein 1 light chain 3-II,LC3-II)、内质网应激凋亡途径关键分子caspase-12及TLR4的表达变化,采用激光共聚焦显微镜观测细胞内LC3的变化.结果:经gly-HDL处理的HUVECs活力下降,LDH漏出和细胞凋亡显著增加(P<0.01),且caspase-12被激活(P<0.05);雷帕霉素预处理HUVECs后,gly-HDL对细胞的损伤作用和对caspase-12的活化作用减弱(P<0.05);而3-MA预处理HUVECs后,gly-HDL对细胞的损伤作用和对caspase-12的活化作用则进一步加强(P<0.05).gly-HDL显著上调TLR4的表达,并触发自噬反应,表现为beclin-1和LC3-II表达上调及LC3显著颗粒化,且呈浓度依赖性(P<0.05);而抗TLR4单克隆中和抗体预处理可显著抑制gly-HDL所诱导的beclin-1上调和LC3颗粒化(P<0.01).结论:TLR4介导gly-HDL对HUVECs自噬的诱导作用,而一定程度的自噬可通过抑制caspase-12活化减轻gly-HDL所诱导的HUVECs凋亡.

目的:探讨乔松素对内质网应激(ERS)诱导剂衣霉素(TM)所致人脐静脉内皮细胞(HUVECs)凋亡的抑制作用及其机制.方法:体外培养HUVECs,分别给予6.25、12.5和25 mg/L乔松素预处理1 h,再加入TM(10 mg/L)继续培养24 h.分别采用MTT法和Annexin V-FITC/碘化丙啶(PI)双染法检测细胞活力和细胞凋亡情况;采用试剂盒测定培养液中乳酸脱氢酶(LDH)活性,并采用Hoechst 33342/PI双染法检测细胞膜损伤情况;采用West?ern blot法检测ERS标志分子葡萄糖调节蛋白78(GRP78)和ERS凋亡途径关键蛋白C/EBP同源蛋白(CHOP)的表达情况,以及蛋白激酶R样内质网激酶(PERK)和真核翻译起始因子2α(eIF2α)的磷酸化水平;免疫荧光细胞化学法检测活化转录因子6(ATF6)的核转位情况.结果:乔松素(12.5和25 mg/L)预处理显著抑制TM所诱导的细胞活力降低、LDH漏出、PI阳性细胞率和凋亡率增加(P<0.05或P<0.01).TM显著上调GRP78和CHOP的蛋白表达水平,且促进其上游调控蛋白PERK和eIF2α磷酸化及ATF6核转位,而乔松素显著拮抗TM所致的上述变化,并呈剂量依赖性(P<0.05或P<0.01).结论:乔松素可减轻ERS诱导的HUVECs凋亡,其机制可能与抑制ATF6/PERK-CHOP信号通路的活化有关.

新冠肺炎疫情防控期间高校医学专业在线教学面临巨大挑战.利用问卷星APP平台对山东第一医科大学2700余名医学生进行问卷调查与分析,从学习资源和方式、教学效果和面临的困难等方面了解在线教学现状,并提出疫情期间及后疫情时代在线教学的应对策略.

Patients with acute lung injury (ALI) have increased levels of pro-inflammatory mediators, which impair endothelial progenitor cell (EPC) function. Increasing the number of EPC and alleviating EPC dysfunction induced by pro-inflammatory mediators play important roles in suppressing ALI development. Because the high density lipoprotein reverse-D-4F (Rev-D4F) improves EPC function, we hypothesized that it might repair lipopolysaccharide (LPS)-induced lung damage by improving EPC numbers and function in an LPS-induced ALI mouse model. LPS was used to induce ALI in mice, and then the mice received intraperitoneal injections of Rev-D4F. Immunohistochemical staining, flow cytometry, MTT, transwell, and western blotting were used to assess the effect of Rev-D4F on repairment of lung impairment, and improvement of EPC numbers and function, as well as the signaling pathways involved. Rev-D4F inhibits LPS-induced pulmonary edema and decreases plasma levels of the pro-inflammatory mediators TNF-α and ET-1 in ALI mice. Rev-D4F inhibited infiltration of red and white blood cells into the interstitial space, reduced lung injury-induced inflammation, and restored injured pulmonary capillary endothelial cells. In addition, Rev-D4F increased numbers of circulating EPC, stimulated EPC differentiation, and improved EPC function impaired by LPS. Rev-D4F also acted via a PI3-kinase-dependent mechanism to restore levels of phospho-AKT, eNOS, and phospho-eNOS suppressed by LPS. These findings indicate that Rev-D4F has an important vasculoprotective role in ALI by improving the EPC numbers and functions, and Rev-D4F reverses LPS-induced EPC dysfuncion partially through PI3K/AKT/eNOS signaling pathway.

目的 研究大蒜素对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的血管内皮细胞凋亡的抑制作用,并探讨其可能的分子机制.方法 体外培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),给予大蒜素(12.5、25和50 mg/L)、4-苯丁酸(4-phenylbutyric acid,PBA,4 mmol/L)或抗凝集素样氧化低密度脂蛋白受体-1(lectin-like oxidized low density lipoprotein receptor-1,LOX-1)单克隆抗体(4 mg/L)预处理1 h,再加入ox-LDL(100 mg/L)继续培养24 h.分别采用MTT法和Annexin V-FITC/PI双染法检测细胞活力和细胞凋亡;试剂盒检测培养基乳酸脱氢酶(lactic dehydrogenase,LDH)和细胞内caspase-3活性.Western blot法测定LOX-1及内质网应激(endoplasmic reticulum stress,ERS)关键分子双链RNA依赖的蛋白激酶样ER激酶(double-stranded RNA-activated protein kinase-like ER kinase,PERK)磷酸化水平和促凋亡蛋白caspase-12表达变化.结果 与ERS抑制剂PBA相似,大蒜素显著减轻ox-LDL所诱导的HUVECs损伤,表现为细胞活力增加(P<0.05或P<0.01),LDH漏出、凋亡率和caspase-3活性降低(P<0.05或P<0.01).大蒜素明显抑制ox-LDL所致的LOX-1上调(P<0.05或P<0.01).另外,与PBA相似,大蒜素明显抑制ox-LDL所诱导的PERK磷酸化和caspase-12活化(P<0.05或P<0.01).抗LOX-1单抗可抑制ox-LDL所致的caspase-12活化(P<0.05).结论 大蒜素可减轻ox-LDL所致的HUVECs凋亡,其机制可能与抑制LOX-1上调继而减轻ERS-caspase-12途径活化有关.

This study was designed to explore the inductive effect of glycated high‐density lipoprotein (gly‐HDL) on endoplasmic reticulum (ER) stress‐C/EBP homologous protein (CHOP)‐mediated macrophage apoptosis and its relationship with autophagy. Our results showed that gly‐HDL caused macrophage apoptosis with concomitant activation of ER stress pathway, including nuclear translocation of activating transcription factor 6, phosphorylation of protein kinase‐like ER kinase (PERK) and eukaryotic translation initiation factor 2α, and CHOP up‐regulation, which were inhibited by 4‐phenylbutyric acid (PBA, an ER stress inhibitor) and the gene silencing of PERK and CHOP. Similar data were obtained from macrophages treated by HDL isolated from diabetic patients. Gly‐HDL induced macrophage autophagy as assessed by up‐regulation of beclin‐1, autophagy‐related gene 5 and microtubule‐associated protein one light chain 3‐II, which were depressed by PBA and PERK siRNA. Gly‐HDL‐induced apoptosis, PERK phosphorylation and CHOP up‐regulation were suppressed by rapamycin (an autophagy inducer), whereas aggravated by 3‐methyladenine (an autophagy inhibitor) and beclin‐1 siRNA. Administration of diabetic apoE−/− mice with rapamycin attenuated MOMA‐2 and CHOP up‐regulation and apoptosis in atherosclerotic lesions. These data indicate that gly‐HDL may induce macrophage apoptosis through activating ER stress‐CHOP pathway and ER stress mediates gly‐HDL‐induced autophagy, which in turn protects macrophages against apoptosis by alleviating CHOP pathway.

背景近年来越来越多的研究证据表明,伴有巨噬细胞浸润和极化的局部和全身炎症反应是导致动脉粥样硬化斑块不稳定的主要原因,因此减少巨噬细胞源性泡沫细胞形成、抑制巨噬细胞炎性反应对提高斑块稳定性及防治心脑血管疾病具有重要意义.目的 探讨色素上皮衍生因子(PEDF)对氧化低密度脂蛋白(Ox-LDL)诱导的巨噬细胞炎性反应的影响.方法 本实验于2018年1—8月完成.实验分组前将巨噬细胞分为空白对照组及A、B、C、D组,A、B、C、D组细胞分别给予终浓度为100、200、400、800 ng/ml的PEDF处理24 h,进行细胞毒性试验.细胞毒性试验完成后将巨噬细胞随机分为实验对照组、炎性反应组、低浓度组、中浓度组、高浓度组.炎性反应组细胞加Ox-LDL处理24 h诱导炎性反应;低浓度组、中浓度组、高浓度组分别加终浓度为100、200、400 ng/ml的PEDF处理24 h,之后加Ox-LDL处理24 h诱导炎性反应.采用CCK-8法检测细胞活力,采用Western blot法检测细胞内白介素1(IL-1)、单核细胞趋化因子1(MCP-1)蛋白表达,采用酶联免疫吸附试验(ELISA)检测细胞外IL-1、MCP-1蛋白表达,采用Annexin V-FITC/PI双染法流式细胞术检测细胞凋亡率.结果(1)D组细胞活力低于空白对照组(P<0.05),A、B、C组细胞活力与空白对照组比较,差异无统计学意义(P>0.05);PEDF适宜干预浓度为100、200、400 ng/ml.(2)炎性反应组、低浓度组、中浓度组、高浓度组细胞活力低于实验对照组,中浓度组、高浓度组细胞活力低于炎性反应组(P<0.05).(3)炎性反应组、低浓度组、中浓度组、高浓度组细胞内外IL-1蛋白相对表达量高于实验对照组,低浓度组、中浓度组、高浓度组细胞内外IL-1蛋白相对表达量低于炎性反应组(P<0.05);炎性反应组、低浓度组、中浓度组、高浓度组细胞内外MCP-1蛋白相对表达量高于实验对照组,中浓度组、高浓度组细胞内外MCP-1蛋白相对表达量低于炎性反应组(P<0.05).(4)炎性反应组、低浓度组、中浓度组、高浓度组细胞凋亡率高于实验对照组,中浓度组、高浓度组细胞凋亡率高于炎性反应组(P<0.05).结论终浓度为200、400 ng/ml的PEDF能有效降低巨噬细胞活力,下调IL-1和MCP-1蛋白表达,促进巨噬细胞凋亡,进而抑制Ox-LDL诱导的巨噬细胞炎性反应.

目的:研究糖尿病患者低密度脂蛋白(low density lipoprotein from diabetes mellitus patients,DM-LDL)对小鼠巨噬细胞内质网应激(endoplasmic reticulum stress,ERS)凋亡途径关键分子caspase-12的影响,以探讨其对鼠巨噬细胞凋亡的诱导作用及机制.方法:体外培养小鼠巨噬细胞RAW264.7,给予DM-LDL(25、50和100 mg/L)处理24 h;分别以正常人来源的低密度脂蛋白(normal low density lipoprotein,n-LDL;50 mg/L)和ERS诱导剂衣霉素(tunicamycin,TM;4 mg/L)处理24 h的巨噬细胞作为阴性和阳性对照组;另外以ERS抑制剂4-苯基丁酸(4-phenylbutyric acid,PBA;5 mmol/L)预处理细胞1 h,然后给予DM-LDL(100 mg/L)处理24 h.分别采用MTT法和Annexin V-FITC/PI双染法检测细胞活力和凋亡情况,乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒测定培养液中LDH的活性,Western blot法检测caspase-12蛋白的表达.结果:与TM相似,DM-LDL明显导致细胞活力下降、LDH释放和细胞凋亡率增加(P<0.05),同时显著增强caspase-12的活性,在50和100 mg/L浓度时作用更明显(P<0.01).PBA预处理可抑制DM-LDL所诱导的巨噬细胞活力下降、LDH释放和凋亡增加(P<0.05),同时抑制DM-LDL所致的caspase-12活化(P<0.05).结论:DM-LDL可导致小鼠巨噬细胞RAW264.7的凋亡,其机制可能与活化caspase-12途径有关.

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 μmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1β (IL-1β) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner. Additionally, AGE-alb promoted IL-1β and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1β and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.

目的:研究氢分子对氧化型低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导巨噬细胞凋亡的影响,并探讨可能的分子机制.方法:体外培养鼠源RAW264.7巨噬细胞,处理前更换为饱和含氢培养基,分别给予3-甲基腺嘌呤(3-methyladenine,3-MA;5 mmol/L)和雷帕霉素(rapamycin,Rap;3μmol/L)预处理1 h,再加入ox-LDL(100 mg/L)继续培养24 h.分别采用MTT法和Annexin V-FITC双染法检测细胞活力和凋亡情况,试剂盒测定培养基中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,Western blot法检测自噬标志分子beclin-1和内质网应激相关促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein,CHOP)表达的变化,激光共聚焦显微镜观测细胞内微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达变化.结果:氢分子显著抑制ox-LDL诱导的RAW264.7巨噬细胞活力降低、LDH漏出增加、细胞凋亡及CHOP表达上调;ox-LDL诱导巨噬细胞自噬反应,表现为beclin-1表达上调,LC3颗粒化聚集,而氢分子可进一步促进ox-LDL对细胞自噬的诱导作用,且氢分子的这种促进作用可被自噬抑制剂3-MA拮抗,而被自噬诱导剂Rap增强(P<0.01).另外,氢分子对ox-LDL所致的巨噬细胞凋亡、细胞活力降低及CHOP上调的抑制作用也可被3-MA拮抗,而被Rap促进.在体外培养的人源THP-1巨噬细胞中也观察到类似的结果,即氢分子不仅可抑制ox-LDL诱导的细胞凋亡和CHOP上调,也可使beclin-1表达进一步上调(P<0.01).结论:氢分子可通过下调CHOP表达抑制ox-LDL诱导的巨噬细胞凋亡,其上游机制可能是通过激活自噬实现的.

糖尿病是世界范围内的流行病,与动脉粥样硬化(As)等心血管疾病密切相关.长期高糖环境导致糖尿病患者体内蛋白质、脂肪酸等大分子物质经一系列复杂的非酶糖基化反应形成糖基化终末产物(AGE).而糖化白蛋白(GA)是AGE的最主要形式,通过与糖基化终末产物受体(RAGE)结合引起氧化应激、炎症、内质网应激等反应,促进动脉粥样硬化进展.因此,深入探究GA致As机制对降低糖尿病患者心血管事件发生率、延缓疾病进程有重大意义.目前,现有相关研究已部分揭示其具体机制,本文就GA在As发生发展中的作用作一综述.