在低氧预处理延迟心肌保护中钙网蛋白表达升高

本文分别在整体实验和细胞培养条件下研究钙网蛋白（calreticulin,CRT）在低氧预处理（hypoxic preconditioning,HPC）延迟心肌保护中的表达及其信号转导机制。（1）整体实验时Wistar大鼠随机分为3组：假手术（sham）组、仅结扎冠状动脉的心肌缺血（myocardial infarction,MI）组和HPC后再结扎冠状动脉的HPC＋MI组,分别于术后24h、14d和28d观察HPC对缺血后心功能和梗死区、危险区面积的影响;采用Western blot检测CRT表达以及p38丝裂素活化蛋白激酶（p38mitogenactivated protein kinase,p38MAPK）、应激活化蛋白激酶（stress-activated protein kinase,SAPK）活性。（2）原代培养Sprague—Dawley乳鼠心肌细胞,随机分为6组：低氧,复氧（hypoxia／reoxygenation,H／R）组、HPC组、HPC＋H,R组、p38MAPK抑制剂SB203580＋HPC＋H／R（SB＋HPC＋H／R）组、SAPK抑制剂SP600125＋HPC＋H瓜（SP＋HPC＋H／R）组和正常对照组;采用台盼蓝排斥实验、乳酸脱氢酶（1actate dehydrogenase,LDH）活性检测及流式细胞仪检测各组细胞损伤情况;采用Western blot检测CRT表达及p38MAPK、SAPK的磷酸化水平。主要结果如下：（1）整体动物实验结果表明,HPC改善缺血对心肌左室压力最大上升,下降速度（＋dp／dtmax）的抑制,限制心肌梗死面积;HPC后CRT表达呈动态变化：术后24h时HPC＋MI组CRT表达增高106％（P〈0．05vsMI组）,以危险区最为显著;14d至28d表达逐步降低。相关分析显示,术后24h时CRT表达量与心功能呈正相关（r=0．9867,P〈0．05）,与梗死面积呈负相关（r=-0．9709,P〈0．05）。（2）细胞培养实验结果表明,HPC可减轻H／R诱导的心肌细胞LDH漏出,增加心肌细胞存活率,降低细胞凋亡;单纯HPC可诱导CRT表达轻度增加（222％,P〈0．05vs对照组）,而损伤性H／R诱导CRT过表达（503％,P〈0．05vs对照组）,HPC可降低H／R诱导CRT表达升高的幅度;p38MAPK活性与HPC诱导的CRT表达呈正相关（r=0．9021,P〈0．05）,而SAPK活性与其呈负相关（r=-0．8211,P〈0．05）。由此得出结论：（1）整体实验中HPC可明显改善缺血后心脏的收缩与舒张功能,限制心肌梗死范围,促进危险区心肌恢复;心肌梗死早期,HPC诱导CRT表达上调,参与心肌保护;（2）细胞培养实验中HPC可诱导CRT适度表达,增强原代培养乳鼠心肌细胞对H／R损伤的抵抗力;p38MAPK可能介导HPC诱导的CRT表达,而SAPK激活可能不利于心肌保护。     

低氧预处理通过上调钙网蛋白表达减轻大鼠心肌细胞氧化应激损伤

本文旨在探讨钙网蛋(calreticulin,CRT)是否参与低氧预处理(hypoxic preconditioning,HPC)对心肌细胞氧化应激损伤的保护及其信号转导过程.将原代培养的Sprague.Dawley乳鼠心肌细胞随机分为8组:氧化应激(H2O2)组、短暂低氧(HPC)组、HPC H202组、SB203580(p38 MAPK特异性抑制剂) HPC H2O2组、干扰心肌细胞CRT表达的反义寡核苷酸(antiscnse oligodeoxynucleotides,AS)组、AS H2O2组、AS HPC H202组和对照组,以细胞存活率、乳酸脱氢酶(1actate dehydrogenase,LDH)漏出及流式细胞术检测细胞损伤情况;采用RT-PCR和Western blot分别检测CRT表达和p38MAPK磷酸化水平.结果表明:(1)HPC可减轻氧化应激损伤,与H202组比较,HPC H2O2组细胞存活率增高18.0%,细胞凋亡率和LDH漏出分别降低19.4%和53.0%(均P<0.05);HPC前以SB203580预孵育可消除HPC保护作用,与HPC H202组相比,SB203580 HPC H2O2组细胞凋亡率和LDH漏出分别增高13.1%和96.0%,存活率降低7.3%(均P<0.05);(2)氧化应激明显上调CRT表达(H202组较对照组高7.1倍,P<0.05);HPC也诱导CRT表达上调(HPC组较对照组高2.4倍,P<0.05),但上调程度较H2O2组低59%(P<0.05);即HPC可减轻氧化应激诱导的CRT过表达:(3)AS干扰CRT表达后,HPC保护作用降低,相关性分析显示HPC诱导的CRT适度表达与细胞存活率呈正相关(r=0.8023,P<0.05);(4)HPC前SB203580预孵育可抑制CRT表达上调(分别较HPC H2O2组和HPC组低75%和53%,均P<0.05).上述结果提示,HPC可能通过p38 MAPK信号途径诱导CRT表达上调,减轻心肌细胞氧化应激损伤.     

钙网蛋白参与缺血后处理减轻大鼠骨骼肌缺血/再灌注损伤

本实验分别在整体和细胞水平观察缺血后处理（ischemic postconditioning,I-postC）对骨骼肌缺血／再灌注（ischemia／reperfusion,I／R）损伤的影响,并探讨钙网蛋白（calreticulin,CRT）介导的信号转导机制。（1）整体实验：健康雄性Wistar大鼠48只,无创动脉夹夹闭右侧股动脉4h,松夹再灌注12h或24h建立大鼠右后肢I／R损伤模型,随机分为I／R组、缺血预处理（ischemic preconditioning,IPC）组（5min缺血／5min再灌,3个循环）和I-postC组（1min再灌／1min缺血,3个循环）（n=16）,大鼠左后肢做对照处理。再灌注结束时测定血浆乳酸脱氢酶（1actate dehydrogenase,LDH）活性、骨骼肌湿干重比值（wet／dryweightratio,W／D）;电镜观察骨骼肌超微结构变化：Westernblot检测骨骼肌CRT、钙调神经磷酸酶（calcineurin,CaN）的表达。（2）细胞培养实验：原代培养Sprague-Dawley乳鼠骨骼肌细胞,随机分为6组：正常对照组、缺氧／复氧（hypoxia／reoxygenation,H／R）组、缺氧预处理（hypoxic preconditioning,HPC）组、缺氧后处理（hypoxic postconditioning,H-postC）组、CaN抑制剂环孢素A（cyclosporine,CsA）＋H／R组和CsA＋H-postC组。台盼蓝排斥实验、流式细胞仪检测细胞损伤情况：Westernblot检测骨骼肌细胞CRT和CaN的表达。结果显示：（1）在整体动物实验中,I-postC可显著降低血浆LDH活性和组织水肿,骨骼肌超微结构损伤减轻,无细胞核凋亡现象,与IPC组相比无显著差异。I-postC再灌注12h和24hCRT表达分别较I／R12h和24h组高4．39倍和1．02倍（P〈0．05）,CaN表达分别增高1．96倍和0．63倍（尸〈0．05）。相关分析显示CRT表达与CaN表达呈正相关（r-0．865,P〈0．01）。（2）在细胞培养实验中,H-postC可减轻H／R诱导的骨骼肌细胞凋亡,增加细胞存活率,与HPC组相比无显著差异,CsA可抑制H-postC的保护作用;H-postC可上调CRT和CaN的表达,分别较H／R组增加31．8％（P〈0．05）和6．02％,加入CsA后CaN表达降低44．02％（P〈0．05vsH-postC）。上述整体实验和细胞培养实验结果提示,I-postC与IPC保护作用相似,可显著减轻I／R损伤;CRT上调介导的CaN表达增加可能参与了I-postC的保护作用,抑制CaN表达可降低I-postC的保护作用。     

siRNA沉默过度内质网应激下DⅨ基因在缺血／再灌注肺损伤中的作用

目的：探讨siRNA沉默过度内质网应激（ERS）下C．Jun氨基末端激酶（州K）通路在缺血／再灌注肺凋亡中的作用。方法：采用C57BIM6J小鼠左侧肺原位缺血／再灌注（IVR）损伤模型。实验随机分为7组（／7,=10）,包括假手术组（Sham组）,缺．tk／再灌注组（I／R组）,PBS＋Lipofectamine2000TM转染试剂组（VR＋PBS＋Lipo组）,阴性对照组（VR＋SCR组）,JNK-siRNA~g（VR＋siRNAJNl（1组、I／R＋siRNAJ眦组、I／R＋si时忱JNl。组）。各组实验结束后处死小鼠,留取左肺,分别检测肺湿干重比（W／D）和总肺水含量（TLW）;光镜观察肺组织形态结构变化,并进行肺组织损伤评估（IQA）;RT-PCR、检测JNK和葡萄糖调节蛋白78（GRP78）的基因表达;Westernblot检测磷酸化JNK（p-JNK）和GRP78的蛋白表达;原位缺口末端标记法（TUNEL法）检测肺细胞凋亡指数（AU。结果：与Sham组相比,I／R＋PBS＋Lipo组和SCR组各组W／D、’ILw、IQA、JNK与GRP78mRNA和p-JNK、GRP78蛋白质表达量及AJ均明显升高（P〈0．01）,光镜显示肺组织结构出现明显损伤性变化,且各组两两比较,上述各指标均无统计学差异;与I／R＋PBS＋Lipo组和SCR组相比,JNK-siRNA各组GRP78表达水平无明显变化,余各指标均降低（P〈0．05,P〈0．01）且肺组织损伤减轻。结论：I／R引起肺组织细胞发生过度的ERS,并通过JNK通路引起细胞凋亡,损伤肺组织。     

曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖和凋亡的作用及其机制

观察曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖及凋亡的作用,并探讨其机制。TSA与p38抑制剂（SB203580,3μmol／L）及JNK抑制剂（SP600125,0．5μmol／L）单独或同时处理143B细胞,分别以MTT、台盼蓝染色、流式细胞术和JC—1（测定线粒体跨膜电位）法检,TSA对143B细胞的增殖、存活、周期以及凋亡的影响。应用RT-PCR、Westernblot检测Bax、Bcl．2、p38／JNK表达。结果显示,TsA能够以时间和剂量依赖方式抑制143B细胞增殖,使细胞周期阻滞于GdG。与G2／M期,并能诱导143B细胞凋亡,引起线粒体膜电位降低,促凋亡蛋白Bax表达上调,抑凋亡蛋白Bcl．2表达下调,同时使p38／ⅢK活化增加。p38／JNK4检测剂则能逆转TSA对Bax／Bcl．2的上调及抑制作用。研究结果揭示,TSA可以时间剂量依赖方式抑制143BN胞增殖,阻滞细胞周期,诱导细胞凋亡;其诱导细胞凋亡的机制可能与活化MAPK通路中p38和JNK的活性从而激发线粒体凋亡通路有关。     

E1A激活基因阻遏子过表达抑制体外人血管平滑肌细胞凋亡

为探讨E1A激活基因阻遏子（cellular repressor of E1A-stimulated genes,CREG）对人血管平滑肌细胞（vascular smooth muscl ecells,VSMCs）凋亡的影响及调控机制,应用正、反义重组逆转录病毒表达载体pLNCX,（＋）／CREG及pLXSN（-）／CREG制备稳定感染人胸廓内动脉平滑肌细胞克隆株（human internal thoracic artery-Shenyang,HITASY）细胞模型,观察CREG蛋白过表达及表达抑制对平滑肌细胞凋亡的影响。荧光显微镜下观察DAPI染色后凋亡细胞核形态,AnnexinV／PI流式细胞术检测细胞凋亡率,RT-PCR技术分析凋亡相关基因caspase-9mRNA的表达,蛋白质印迹法分析p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38MAPK）、磷酸化p38MAPK（phosphorylated p38 mitogen-activated proteinkinase,P-p38 MAPK）的表达变化。研究结果显示,CREG蛋白过表达明显抑制血清饥饿诱导的HITASY细胞凋亡的发生;同时细胞中p38MAPK、P-p38MAPK的表达增加。相反,抑制CREG蛋白表达则引起正常血清培养状态下VSMCs的自发凋亡明显增加,同时细胞内p38MAPK、P-p38MAPK表达显著下降。进一步研究发现,预先应用特异性抑制剂SB203580阻断p38MAPK信号转导通路后,CREG蛋白过表达引起的细胞凋亡抑制作用被明显减弱,血清饥饿后CREG蛋白过表达引起的HITASY细胞凋亡现象明显增加。上述结果提示,CREG蛋白过表达可以抑制体外培养的VSMCs凋亡,p38MAPK信号转导通路可能介导CREG蛋白对VSMCs凋亡的抑制作用。     

p38 MAPK介导高糖诱导的肾小管上皮细胞向间充质细胞转变

本文旨在观察p38MAPK与高糖诱导的肾小管上皮细胞向间充质细胞转变之间的关系。将雄性Sprague—Dawley（SD）大鼠随机分为对照组、糖尿病组、胰岛素治疗组,用免疫组织化学、Western blot检测p38MAPK和磷酸化p38MAPK（P—p38MAPK）蛋白表达。采用机械分离和酶消化获取SD大鼠肾小管节段,进行肾小管上皮细胞培养,将肾小管上皮细胞分为对照组、高渗组（20mmol／L D—mannitol）、高糖组（20mmol／L D—glucose）和SB202190（p38MAPK特异性抑制剂）＋高糖组,处理72h后收集细胞,用免疫细胞化学检测α-平滑肌肌动蛋白（α—smooth muscleactin,α-SMA）、p-p38MAPK和Snaill蛋白表达,Western blot检测p38MAPK、p-p38MAPK、Snaill、转化生长因子β1（transforming growth factor—β1,TGF-β1）、α-SMA和E-cadherin的表达,RT-PCR检测α-SMA和E-cadherin mRNA的表达。体内和体外结果均显示,高糖状态激活了p38MAPK,这种活化作用在体内可因胰岛素控制血糖而被消除,在体外可被p38MAPK特异性抑制剂SB202190显著抑制;高糖组α-SMA蛋白和mRNA在原代培养肾小管上皮细胞的表达较对照组分别增加12倍和8倍（P〈0．01）,SB202190处理组其表达则较高糖组分别减少67％和50％（P〈0．01）。SB202190不影响TGF—β1蛋白表达,但下调Snaill蛋白表达,并部分恢复高糖组E—cadherin蛋白和mRNA的表达。上述结果提示,p38MAPK可能通过转录因子Snaill介导高糖诱导的肾小管上皮细胞向间充质细胞转变。     

UVB 诱导凋亡时MAPK 和Mst1/caspase-3 信号通路调节 H2B 磷酸化作用

凋亡是真核细胞执行的高度协调的程序性自杀机制. 细胞凋亡时, 组蛋白的修饰与核
内事件有关. 尤其H2B 被Mst1 激酶磷酸化后, 参与调节核内凋亡事件染色质凝聚作用. 本研究
发现, UVB诱导细胞凋亡时, H2B发生磷酸化作用, 并且受MAPK家族(ERK1/2, JNK1/2 和p38),
Mst1 和caspase-3 信号通路调控. UVB能够以时间依赖方式激活MAPK家族激酶, 进而介导H2B
磷酸化, 但是H2B 乙酰化作用不受影响. 分别阻断ERK1/2, JNK1/2 或p38 任何一种激酶, 均能
抑制H2B 磷酸化作用. 而且, UVB 也能激活caspase-3, 活化的caspase-3 激活下游Mst1. 受到激
活的Mst1 直接磷酸化H2B, 导致染色质凝聚. 但是caspase-3 和Mst1 信号通路被完全阻断时, 只
能部分抑制H2B 磷酸化作用, 同时MAPK 家族激酶的活化不受影响. 因此, 细胞在受到UVB
诱导发生凋亡时, MAPK 和caspase-3/Mst1 信号途径分别独立调节H2B 磷酸化和染色质凝聚
作用.     

Caspase-3在roscovitine诱发PC12细胞凋亡中发挥重要作用

我们已证实周期蛋白激酶（cyclin-dependent kinases）cdk2、cdc2和cdk5抑制剂roscovitine诱导PC12细胞凋亡。本实验应用caspase-3免疫细胞化学与hoechst 33342荧光化学双标、MTT比色法细胞活性测定和Western blot方法,研究了caspase-3在roscovitine所致PC12细胞凋亡中的作用。结果显示,roscovitine（50μmol／L）处理PC12细胞12h,细胞核染色质凝缩及核碎片形成,同时胞浆中出现caspase-3阳性标志,caspase-3阳性细胞占细胞总数的42％。非特异性caspases抑制剂Z-VAD-FMK（50μmol／L）和caspase-3特异性抑制剂Z-DEVD-FMK（100μmol／L）可部分降低roscovitine所致的细胞死亡,使细胞存活率分别由29．03％（roscovitine）增至58．06％（Z-VAD-FMK＋roscovitine）和45．16％（Z-DEVD-FMK＋roscovitine）：用单克隆non-erythroid α-spectrin抗体检测roscovitine处理组细胞匀浆提取液,表明caspase-3裂解的特异性spectfin 120kDa蛋白产物较对照组显著增加。提示细胞凋亡成分caspases参与roscovitine所敛的细胞凋亡,其中caspase-3发挥重要作用。     

缺血后处理对肺缺血／再灌注损伤的保护作用及其机制

目的：探讨缺血后处理（聃）是否通过抑制P38丝裂原活化蛋白激酶（P38MAPK）活化来减轻再灌注损伤肺细胞的凋亡。方法：雄性SD大鼠40只,随机分成5组（n=8）,即对照组（C组）、肺缺血／再灌注组（I／R组）、肺缺血／再灌注＋缺血后处理组（IPO组）、缺血后处理＋溶剂对照组（D组）、缺血后处理＋SB203580组（SB组）。各组分别于再灌注2h留取左肺组织,检测肺组织湿／干重比（W／D）和总肺含水量（TLW）;光镜观察肺组织形态学结构改变并进行肺组织损伤定量评估（IQA）;原住末端标记法（TUNEL）检测肺细胞凋亡情况并计算凋亡指数（AI）;RT-PCR和免疫组化法测定Bax、Bcl-2基因和蛋白的表达。结果：与C组相比,I／R组W／D、TLW、IQA和AI均显著升高（P〈0．05,P〈0．01）,肺组织结构发生明显损伤;Bcl-2、Bcl-2／Bax基因及蛋白表达明显降低,Bax基因及蛋白表达明显升高（P〈0．05,P〈0．01）;IPO组、D组、SB组与I／R组相比,w／D、TLW、IQA和AI均显著降低（P〈0．05,P〈0．01）,肺组织结构损伤情况有所改善;Bcl-2、Bcl-2／Bax基因及蛋白表达明显升高,Bax基因及蛋白表达明显降低（P〈0．05,P〈0．01）;D组与IPO组比较各项指标均无明显差异（均P〉0．05）;SB组与IPO组相比,肺组织W／D、TLW、IQA和AI均显著降低（P〈0．05,P〈0．01）,肺组织结构未见明显损伤;Bcl-2、Bcl-2／Bax基因及蛋白表达明显升高,Bax基因及蛋白表达明显降低（P〈0．05,P〈0．01）。结论：I／R通过激活P38MAPK导致大鼠肺泡结构严重破坏,肺内细胞大量凋亡;IPO可能是通过抑制P38MAPK通路的激活而减轻L／R损伤。     

Hypoxic preconditioning protects microvascular endothelial cells against hypoxia/reoxygenation injury by attenuating endoplasmic reticulum stress

Endothelial cells (ECs) are directly exposed to hypoxia and contribute to injury during myocardial ischemia/reperfusion. Hypoxic preconditioning (HPC) protects ECs against hypoxia injury. This study aimed to explore whether HPC attenuates hypoxia/reoxygenation (H/R) injury by suppressing excessive endoplasmic reticulum stress (ERS) in cultured microvascular ECs (MVECs) from rat heart. MVECs injury was measured by lactate dehydrogenase (LDH) leakage, cytoskeleton destruction, and apoptosis. Expression of glucose regulating protein 78 (GRP78) and C/EBP homologous protein (CHOP), activation of caspase-12 (pro-apoptosis factors) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) were detected by western blot analysis. HPC attenuated H/R-induced LDH leakage, cytoskeleton destruction, and cell apoptosis, as shown by flow cytometry, Bax/Bcl-2 ratio, caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. HPC suppressed H/R-induced ERS, as shown by a decrease in expression of GRP78 and CHOP, and caspase-12 activation. HPC enhanced p38 MAPK phosphorylation but decreased that of protein kinase R-like ER kinase (PERK, upstream regulator of CHOP). SB202190 (an inhibitor of p38 MAPK) abolished HPC-induced cytoprotection, downregulation of GRP78 and CHOP, and activation of caspase-12, as well as PERK phosphorylation. HPC may protect MVECs against H/R injury by suppressing CHOP-dependent apoptosis through p38 MAPK mediated downregulation of PERK activation.     

Calreticulin induces delayed cardioprotection through mitogen-activated protein kinases

Hypoxic preconditioning (HPC) attenuates tissue injury caused by ischemia/reperfusion. The protective mechanisms of HPC involve up-regulation of the protective proteins and mitigation of cellular calcium overload. Calreticulin (CRT), a Ca(2+)-binding chaperone, plays an important role in regulating calcium homeostasis and folding of proteins. The role of CRT in cardioprotection of HPC and the pathways determining CRT expression during HPC are not clear. In this work, 2-DE and MALDI-MS were employed to analyze CRT differential expression in cardiomyocytes subjected to transient hypoxia. Western blotting analysis was used to detect the CRT expression and activities of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal kinase (JNK) in myocardium subjected to ischemia with and without HPC and sham operation. The hearts from HPC group were more resistant to sustained ischemia and had much stronger phosphorylation of p38 MAPK, with a reduced phosphorylation of JNK, than controls. The CRT expression was positively correlated with the phosphorylation of p38 MAPK and negatively correlated with the level of JNK phosphorylation. Furthermore, inhibition of the p38 MAPK with SB202190 abolished, while inhibition of the JNK with SP600125 enhanced the CRT up-regulation in cardiomyocytes induced by HPC. The results indicate that HPC up-regulates CRT expression through the MAPK signaling pathways.     

The Roles of p38 MAPK/MSK1 Signaling Pathway in the Neuroprotection of Hypoxic Postconditioning Against Transient Global Cerebral Ischemia in Adult Rats

Postconditioning has regenerated interest as a mechanical intervention against cerebral ischemia/reperfusion injury, but its molecular mechanisms remain unknown. We previously reported that hypoxic postconditioning (HPC) ameliorated neuronal death induced by transient global cerebral ischemia (tGCI) in hippocampal CA1 subregion of adult rats. This study tested the hypothesis that p38-mitogen-activated protein kinase (p38 MAPK)/mitogen- and stress-response kinase 1 (MSK1) signaling pathway plays a role in the HPC-induced neuroprotection. Male Wistar rats were subjected to 10 min ischemia induced by applying the four-vessel occlusion method. HPC with 120 min was applied at 24 h after reperfusion. Immunohistochemistry and Western blot were used to detect the expression of phosphorylation of p38 MAPK and MSK1, as well as cleaved caspase-3. We found that HPC induced a significant increase of phosphorylated p38 MAPK and MSK1 in neurons of hippocampal CA1 region and a significant decrease in glial cells after tGCI as well. Furthermore, HPC attenuated caspase-3 cleavation triggered by tGCI in CA1 region. Moreover, p38 MAPK inhibition by SB203580 significantly decreased the phosphorylation of MSK1, increased cleaved caspase-3 expression, and abolished the neuroprotection of HPC. These findings suggested that p38 MAPK/MSK1 signaling axis contributed to HPC-mediated neuroprotection against tGCI, at least in part, by regulating the activation of caspase-3.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.     

缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。     

Characterization of apoptosis signal transduction pathways in HL-5 cardiomyocytes exposed to ischemia/reperfusion oxidative stress model

During ischemia/reperfusion (I/R), cardiomyocytes are exposed to sudden lack of nutrients and successively to radical oxygen species (ROS). In the present study, we used the HL-5 cardiac atrial myocyte cell line exposed to serum/glucose depletion added or not in H(2)O(2) to mimic ROS during ischemia, then replaced in their standard culture medium to simulate reperfusion. We investigated the effects of serum/glucose depletion combined or not to ROS exposure on AKT and MAP kinases activation to address the role of each event with respect to apoptosis. We demonstrate that serum/glucose depletion per se did not induce apoptosis when compared to ROS exposure. In particular, ROS recruited p38MAPK and JNK pathways. SB202190 preventing p38MAPK activity, partially protected HL-5 from apoptosis while blocking JNK, thanks to JNKI, further enhanced apoptosis. Blocking phosphatidylinositol (PI) 3-kinase with LY294002 or ERKs with U0126 was without consequence on apoptosis. Finally, BCL-2 and BCL-X(L/S) expression levels were analyzed in cells exposed to 1 h ischemia followed by 12-h reperfusion in the presence or not of SB202190; BCL-2, but not BCL-X(L/S), expression was decreased in ROS treated cells but SB202190 failed to restore BCL-2 level. Our data suggest that p38MAPK activation primarily mediates ROS-induced apoptosis while concomitant JNK activation would represent a scavenger pathway for cells trying to escape apoptosis.     

USP49 inhibits ischemia–reperfusion-induced cell viability suppression and apoptosis in human AC16 cardiomyocytes through DUSP1–JNK1/2 signaling

Dual-specificity protein phosphatases (DUSP) also known as mitogen-activated protein kinase (MAPK) phosphatases (MKPs) can dephosphorylate MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38. DUSP1-mediated JNK dephosphorylation has been found to play an antiapoptotic role against cardiac ischemia–reperfusion (I/R) injury. However, the regulation of DUSP1–JNK pathway remains unclear. In the current study, ubiquitin-specific peptidase 49 (USP49) expression in human AC16 cardiomyocytes following I/R injury was measured by real-time polymerase chain reaction and western blot analysis. Cell viability, apoptosis, the Bax, Bcl-2, and DUSP1 expression, and the activity of MAPKs in AC16 cardiomyocytes following indicated treatment was measured by CCK-8, flow cytometry, and western blot analysis. The direct interaction between USP49 and DUSP1 was measured by coimmunoprecipitation and ubiquitination analysis. The effect of USP49 on apoptosis and JNK activity in rat cardiomyocytes following I/R injury was also measured by TUNEL and western blot analysis. Here, we found that USP49 expression was time-dependently increased in AC16 cardiomyocytes following I/R. I/R-induced cell apoptosis and JNK1/2 activation both in in vivo and in vitro reversed by USP49 overexpression in AC16 cardiomyocytes. Inhibiting JNK1/2 activation significantly inhibited USP49 knockdown-induced the cell viability inhibition, apoptosis and the JNK1/2 activation in AC16 cardiomyocytes. Moreover, USP49 positively regulated DUSP1 expression through deubiquitinating DUSP1. Overall, our findings establish USP49 as a novel regulator of DUSP1–JNK1/2 signaling pathway with a protective role in cardiac I/R injury.