钩藤碱减轻大鼠蛛网膜下腔出血后脑血管痉挛

目的 探讨钩藤碱（Rhy）能否减轻大鼠蛛网膜下腔出血（SAH）后的脑血管痉挛（CVS）及其机制。 方法 成年雄性SD大鼠,采用血管内刺破法建立SAH模型。SAH后立即腹腔注射Rhy,24h后通过磁共振成像（MRI）测量基底动脉(BA)直径。BA经HE染色后,测量BA的直径和血管壁厚度;应用Western blotting和免疫荧光染色检测不同组动物BA的磷酸化p38丝裂原活化蛋白激酶（p-p38MAPK）、p-p53和剪切Caspase-3（cleaved-Caspase-3）蛋白的表达水平。结果 MRI和组织学染色结果均表明,10mg/kgRhy在SAH后可明显增加BA的直径并显著减少血管壁厚度（P<0.05）。Rhy明显降低了SAH后BA的p-p38MAPK、p-p53和cleaved-Caspase-3蛋白的表达水平（P<0.05）。Rhy能够明显减少SAH后BA内皮细胞p-p38MAPK、p-p53和cleaved-Caspase-3等凋亡蛋白的表达。结论 Rhy能通过抑制BA内皮细胞凋亡减轻SAH后脑血管痉挛的严重程度。     

The influence of subarachnoid hemorrhage on neurons: an animal model

Subarachnoid hemorrhage (SAH) has considerable mortality and morbidity, but the pathophysiologic mechanism is not entirely clear. Following SAH, blood or its lysate enters the subarachnoid space. This study examined how blood lysate influences the vulnerable brain following SAH. Heparinized hemolysate was slowly injected into the cisterna magna of 10 female rabbits, while a control group of 10 rabbits received a similar injection of heparinized isotonic sodium chloride solution without hemolysate. The basilar artery and brain tissue were excised after perfusion fixation. The degree of cerebral vasospasm was evaluated by measuring the cross-sectional area of the basilar artery, and brain damage was investigated by TUNEL staining. In the SAH group, the apoptosis index of neuronal cells located at the base of the temporal lobe averaged 26% (range = 3 to 56%), which was significantly higher than the corresponding apoptosis index in the control group (mean 0.5%, range = 0 to 4%, p <0.001). The mean cross-sectional area of the basilar artery in the SAH group did not differ significantly from that in the control group. These results suggest that SAH induces apoptosis of neuronal cells by a mechanism that is independent of cerebral vasospasm.     

Regulation of p38 MAPK phosphorylation inhibits chondrocyte apoptosis in response to heat stress or mechanical stress

Activation of p38 MAPK has been associated with a stress response and with apoptotic processes. However, the function of p38 MAPK in chondrocytes is not clearly understood. In this study, we analyzed the expression of p38 MAPK in chondrocytes and investigated the function of p38 MAPK in response to heat stress and mechanical stress. Chondrocytes were isolated from human cartilage and cultured. Expression of p38 and phosphorylated p38 in cartilage of patients with osteoarthritis (OA) was compared to those in normal cartilage by immunohistochemistry and Western blotting. Human knee chondrocytes were exposed to heat stress or mechanical stress. Normal knee chondrocytes were pre-treated with SB203580 or p38 small interfering RNA (siRNA) before induction of heat stress or mechanical stress. Chondrocyte apoptosis was detected by TUNEL staining and Western blotting of cleaved caspases. OA and normal chondrocytes expressed p38; however, OA chondrocytes showed much higher phosphorylated p38 compared to normal chondrocytes. Heat stress or mechanical stress induced apoptosis and increased phosphorylated p38 in normal chondrocytes. The TUNEL positive cells and expression levels of phosphorylated p38 in response to stress decreased when chondrocytes were incubated with SB203580 or transfected with siRNA against p38. In conclusion, we have demonstrated that heat stress or mechanical stress increased chondrocyte apoptosis via phosphorylation of p38. Stress-induced chondrocyte apoptosis decreased due to inhibition of p38 MAPK activation. In contrast, the phosphorylation of p38 MAPK increased in OA chondrocytes. Our results show that down-regulation of p38 MAPK activation inhibits chondrocyte death induced by heat stress or mechanical stress.     

p38 MAPK在顺铂诱导肾小管上皮细胞凋亡中的作用

目的:探讨p38 MAPK在顺铂诱导大鼠近端肾小管上皮细胞(RPTC)凋亡中的作用。方法:首先采用Western blot实验检测0、5、10和20μmol/L顺铂处理24 h对细胞凋亡的影响,确定最佳处理剂量;而后采用20μmol/L顺铂联合50 mg/L p38 MAPK抑制剂SB203580刺激RPTC,实验分为对照组、顺铂组及顺铂+SB203580(加入SB203580处理RPTC 1 h后再给予顺铂处理24 h)。采用相差荧光显微镜观察和流式细胞术分析顺铂处理后RPTC的凋亡情况;采用Ac-DEVD-AFC试剂盒检测RPTC裂解液中的caspase活性;Western blot实验检测p38、磷酸化p38、cleaved PARP和cleaved caspase-3等的蛋白水平;pH计检测顺铂处理后RPTC外环境pH值改变。结果:20μmol/L顺铂处理RPTC 24 h,可以明显诱导细胞凋亡;顺铂处理15 min后RPTC中p38 MAPK开始磷酸化并达到高峰。顺铂处理后12.08%的RPTC呈凋亡形态,具有增强的caspase活性,并且cleaved PARP和cleaved caspase-3水平明显升高(P0.05);p38 MAPK抑制剂SB203580可抑制p38的磷酸化,降低RPTC的凋亡率和caspase活性,并减少cleaved PARP和cleaved caspase-3的蛋白水平。同时,SB203580可逆转顺铂诱导的RPTC培养基pH值的改变。结论:p38 MAPK的磷酸化在顺铂诱导的RPTC凋亡中发挥作用。顺铂诱导RPTC凋亡后,可改变细胞外酸性环境,并可被p38 MAPK抑制剂SB203580所抑制。     

p38丝裂原活化蛋白激酶通路介导的早期生长反应基因-1活性与乳腺癌细胞表柔比星耐药性的关系

目的 探讨p38丝裂原活化蛋白激酶(MAPK)通路介导的早期生长反应基因(EGR)-1活性与乳腺癌细胞表柔比星耐药的关系.方法 SB203580(15 μmol/L)干预后,激光共聚焦显微镜观察;流式细胞术、四甲基偶氮唑蓝(MTT)、凝胶电泳迁移率法(EMSA)、RT-PCR及Western blot分别检测耐药MCF-7/Adr及亲本MCF-7细胞内磷酸化p38MAPK蛋白表达、细胞凋亡及细胞内表柔比星浓度、EGR-1蛋白活性改变、细胞对表柔比星敏感性;EGR-1 mRNA、P糖蛋白、磷酸化p53及p38蛋白表达.结果 p38MAPK通路激活的MCF-7/Adr细胞经SB203580(15 μmol/L)干预24和48 h后,(1)MCF-7/Adr细胞(早+晚期)凋亡率分别由(0.54±0.17)%和(0.81±0.16)%提高为(25.36±1.17)%和(38.21±1.25)%,P<0.05,并呈一定时间依赖性;(2)MCF-7/Adr细胞的平均荧光强度分别为(32.45±2.36)及(41.66±3.12),均高于空白对照组及DMSO组MCF-7/Adr细胞的(14.17±1.45)及(16.28±0.63),P<0.01;MCF-7/Adr细胞对表柔比星药物的耐受性显著降低;(3)增加了MCF-7/Adr细胞的EGR-1活性,IC50分别为(21.53±2.17)和(8.77±1.02),低于空白对照组(40.74±2.56);伴随p38MAPK通路活性抑制和EGR-1 mRNA表达增加,磷酸化p53蛋白表达显著上调,而P糖蛋白显著下调.结论 p38MAPK通路与乳腺癌表柔比星耐药密切相关,可能与p38MAPK通路介导的EGR-1表达相关,EGR-1激活抑制了其下游耐药基因转录,从而使表柔比星耐药得以逆转.     

Manumycin诱导细胞凋亡抑制乳腺癌细胞SK-BR-3活性

目的:研究manumycin对乳腺癌腹腔转移癌细胞株SK-BR-3的抑癌效应及其诱导凋亡。方法:用MTT法检测manumycin对SK-BR-3细胞的抑癌作用。免疫印迹方法检测p38 MAPK蛋白表达。用caspase-3活性检测试剂盒定量检测manumycin诱导细胞凋亡的水平及评估特异性的p38 MAPK抑制剂SB203580对凋亡的影响。结果:经6 μmol/L、18 μmol/L、54 μmol/L manumycin处理SK-BR-3细胞24 h时,其抑制率分别为(7.4±3.9)%、(21.0±4.4)%和(64.7±4.1)%,呈量效关系。其中后2者的细胞活性与对照组比有显著差异(P<0.01)。用药24 h的IC₅₀为42.5 μmol/L。同时此药物可明显增加caspase-3的活性,且这一效应可部分地被p38抑制剂SB203580阻断。免疫印迹结果显示manumycin促进p38的磷酸化。结论:manumycin可通过诱导SK-BR-3细胞凋亡而产生抑癌作用,p38 MAPK是manumycin诱导细胞凋亡的通路之一。     

肝星状细胞增殖与p38丝裂原活化蛋白激酶信号传导通路的关系

背景：肝星状细胞的激活、增殖导致肝纤维化,p38丝裂原活化蛋白激酶信号通路可参与调控细胞增殖。 目的：探讨SB203580作用于乙醛刺激的大鼠肝星状细胞后p38丝裂原活化蛋白激酶活性变化和细胞增殖变化。 方法：体外培养大鼠肝星状细胞株,在乙醛干预的基础上加入不同浓度的p38特异性抑制剂SB203580进行培养,并设置对照。以Western blot检测磷酸化p38 蛋白表达水平变化,MTT比色法检测细胞增殖。 结果与结论：乙醛刺激后大鼠肝星状细胞内磷酸化p38水平增强,细胞增殖明显。使用5,10,20 μmol/L SB203580能明显抑制乙醛刺激的肝星状细胞增殖(P < 0.05),加大浓度至30 μmol/L时,抑制作用更明显(P < 0.01),抑制率为43.9%,而磷酸化p38水平也降低(P < 0.05)。结果证实,抑制p38丝裂原活化蛋白激酶活性可能影响肝星状细胞的增殖。     

Mitofusin-2在大鼠蛛网膜下腔出血后大脑动脉内表达的变化

目的 研究Mitofusin-2(Mfn2)在大鼠蛛网膜下腔出血（SAH）后大脑动脉中表达的变化,寻找Mfn2基因与脑血管痉挛(CVS)之间的关系。 方法 蛛网膜下腔出血模型由刺破颈内动脉的颅内动脉分叉处诱导。146只SD大鼠被随机分为6组：假手术组（sham组）,SAH后24h、48h、72h、7d 和14d各组。计算动物死亡率,测定神经功能学评分及脑水含量。组织学检测观察形态学变化,采用Western blotting及RT-PCR 分别检测SAH后不同时间点的大鼠主要大脑动脉Mfn2蛋白及mRNA 水平的表达变化。 结果 围绕在基底动脉周围的血块随着时间逐渐消失,形态学观察显示SAH 24h组基底动脉出现严重的痉挛。SAH7d组的基底动脉中层无阳性的免疫组织化学阳性染色。Western blotting结果显示,Mfn2蛋白表达 sham组与SAH48h组和SAH72h组相比,均显著增加(P<0.05),SAH 7d出现显著性降低(P<0.05),SAH14d 恢复至 sham 和SAH24h 组水平。而mRNA 水平变化与蛋白水平变化相类似。Mfn2基因参与到SAH后血管的自我调节过程中,表达随着时间增加而增加,并在72h到达高峰,7d时显著下降,14d左右恢复至sham组水平。 结论 Mfn2在SAH后的早期以及迟发型脑血管痉挛中起重要作用,为揭示SAH后脑血管痉挛的机理提供实验依据。     

周期性张应力诱导成纤维细胞凋亡中p38MAPK信号通路的作用

背景：当牙齿受异常咬合力时会导致牙体吸收、牙周组织的大量破坏。 目的：研究牙周膜成纤维细胞在受到周期性张应力刺激后是否发生凋亡及p38MAPK信号通路是否参与该凋亡过程。 方法：取4~7代成纤维细胞,同步化后随机分为对照组、加力组和SB203580组。加力组和SB203580组细胞加载力值为12%表面应变率,加力频率为6个循环/min,即5 s拉伸,5 s松弛。SB203580组细胞在加力前1 h加入终浓度为20 mmol/L的p38MAPK抑制剂SB203580。分别在加力6,12,24 h,取各组细胞,流式细胞仪检测细胞凋亡,RT-PCR检测细胞凋亡基因bax mRNA的表达。 结果与结论：与对照组比较,加力后成纤维细胞凋亡率及bax mRNA表达增加 (P < 0.05),且随着加力时间的延长而增强,12 h达高峰,之后逐渐下降。与加力组比较,SB203580组对应时间点细胞凋亡减少 (P < 0.05),bax mRNA表达降低。说明细胞受到力学刺激会发生凋亡,而丝裂原活化蛋白激酶p38MAPK信号通路参与了该凋亡过程。     

p38有丝分裂原激酶活化参与继发性脊髓损伤后神经细胞凋亡的实验研究

探索脊髓损伤(SCI)后p38有丝分裂原激酶(p38MAPK)的表达及其与神经细胞凋亡的关系。采用Allen’s法建立大鼠急性SCI动物模型,用蛋白印迹法和免疫组织化学染色方法检测SCI后p38MAPK和caspase-3表达的变化;采用实时定量PCR法检测SCI后caspase-3 mRNA的表达;用原位末端标记法(TUNEL)检测SCI后神经细胞凋亡。结果表明SCI后总p38MAPK表达无变化,但p38MAPK磷酸化明显增多,于伤后6h达高峰;伤后24hcaspase-3表达明显增加。TUNEL检测显示,伤后6h受损伤的脊髓组织有少许凋亡细胞出现,24h细胞凋亡最明显。鞘内注射p38MAPK抑制剂SB203580,明显减少caspase-3的表达,并减少细胞凋亡。以上结果显示SCI后损伤局部p38MAPK激活诱导了caspase-3基因表达,导致神经细胞凋亡;抑制p38MAPK可以阻止SCI后继发的神经细胞凋亡。     

TPX2通过p38 MAPK信号通路诱导直肠癌HR-8348细胞凋亡

目的:研究下调非洲爪蟾驱动蛋白样蛋白2靶蛋白(TPX2)对直肠癌细胞凋亡的影响及机制。方法:用TPX2小干扰RNA(si RNA)转染直肠癌HR-8348细胞,记为TPX2 si RNA组;以不做转染的细胞作为正常对照(control)组;以转染si RNA阴性对照(si RNA-NC)的细胞作为si RNA-NC组;用p38 MAPK抑制剂处理敲减TPX2表达后的直肠癌HR-8348细胞记为TPX2 si RNA+SB203580组。RT-qPCR和Western blot测定TPX2的表达水平,MTT法测定细胞存活率,流式细胞术测定细胞凋亡,Western blot测定细胞中p38 MAPK、p-p38 MAPK、cleaved caspase-3和Bcl-2的蛋白水平。结果:TPX2 si RNA转染后HR-8348细胞中TPX2的m RNA和蛋白表达水平显著下降(P 0. 05),而转染si RNA-NC对HR-8348细胞中TPX2的m RNA和蛋白水平没有影响。敲减TPX2表达后的直肠癌HR-8348细胞存活率降低,凋亡率升高,细胞中的cleaved caspase-3、p-p38 MAPK/p38 MAPK蛋白水平明显升高,Bcl-2水平水平降低,与control组比较,差异有统计学意义(P 0. 05)。与TPX2 si RNA组相比,TPX2 si RNA+SB203580组的HR-8348细胞凋亡率、cleaved caspase-3水平和p-p38 MAPK/p38 MAPK蛋白水平明显降低,存活率明显升高(P 0. 05)。结论:TPX2表达下调可以通过激活p38 MAPK促进直肠癌HR-8348细胞凋亡。     

蛛网膜下隙出血后脑动脉痉挛形态学研究

采用大鼠蛛网膜下隙出血模型，经光镜和Ｖｉｄａｓ图像分析的方法，对不同时间段的脑动脉平滑肌的横断面积（Ｓ１），内膜的横断面积（Ｓ２），脑动脉壁横断面积（Ｓ３），脑动脉腔横断面积（Ｓ４），Ｖ１，平滑肌的体密度（Ｓ１／Ｓ３）；Ｖ２；内的体密度（Ｓ２／Ｓ３）；Ｄ１；蛛网膜下隙面脑动脉壁厚度；Ｄ２：脑面及动脉壁厚度进行了观察测定发现蛛网膜下隙出血后脑血管痉挛具有双相性，管腔缩窄，管壁增厚，急性脑血管痉挛比迟     

辛伐他汀通过抑制p38 MAPK活化降低肝硬化门静脉高压大鼠门静脉压力

 目的:探讨p38 MAPK信号通路在辛伐他汀降低肝硬化门静脉高压症大鼠门静脉压力（PP）中的作用。方法:采用四氯化碳复合因素法构建大鼠肝硬化门静脉高压症模型,成模后将存活大鼠随机分为模型组（n=10）、辛伐他汀治疗组（n=11）和p38 MAPK信号通路抑制剂SB203580处理组（n=10）,后2组分别给予辛伐他汀及SB203580干预处理;另设正常对照组（n=8）。处理结束后检测大鼠PP、肝脏总p38 MAPK蛋白、磷酸化p38 MAPK蛋白、总eNOS蛋白、磷酸化eNOS蛋白表达水平以及肝脏一氧化氮（NO）含量的变化。结果:（1）模型组大鼠PP明显高于正常对照组;辛伐他汀治疗组及SB203580处理组PP均明显低于模型组（P＜0.01）,辛伐他汀治疗组PP明显低于SB203580处理组（P＜0.01）。（2）与正常大鼠相比,模型组大鼠肝脏总p38 MAPK蛋白及总eNOS蛋白表达水平无明显变化（P＞0.05）,而磷酸化p38 MAPK蛋白及磷酸化eNOS蛋白表达水平分别增高与降低（P＜0.01）;辛伐他汀治疗组大鼠肝脏磷酸化p38 MAPK蛋白及磷酸化eNOS蛋白表达水平分别降低与增高（P＜0.01）;SB203580处理组大鼠肝脏磷酸化p38 MAPK蛋白及磷酸化eNOS蛋白表达水平分别降低与增高（P＜0.01）,但磷酸化eNOS蛋白表达水平增高的程度低于辛伐他汀治疗组（P＜0.01）。(3)辛伐他汀治疗组肝脏NO含量［（15.73±1.59） μmol/(g protein)］及SB203580处理组肝脏NO含量［（13.98±1.27) μmol/(g protein)］明显高于模型组［（9.81±1.12） μmol/（g protein）］（P＜0.01）,辛伐他汀治疗组NO含量明显高于SB203580处理组（P＜0.01）。结论: 辛伐他汀降低肝硬化门静脉高压症大鼠门静脉压力可能与其抑制p38 MAPK信号通路的活化有关。     

The role of p38 MAPK in valproic acid induced microglia apoptosis

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, induces apoptosis in microglia, but the underlying mechanism by which microglia apoptosis in response to VPA is not yet known. In this study, we found that the mitochondrial pathway played an important role in VPA-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. In addition, VPA increased the level of phospho-p38 mitogen-activated protein kinase (MAPK), but had no effects on phospho-ERK and phospho-JNK MAPKs. Moreover, p38 inhibitor SB203580 strongly inhibited VPA-induced apoptosis and caspase-3 activation. Taken together, our results clearly demonstrated that VPA could induce apoptosis of microglia via p38 MAPK and mitochondrial apoptosis pathway.     

Sodium salicylate-induced apoptosis of human peripheral blood eosinophils is independent of the activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

BACKGROUND: It has been shown that the inhibition of eosinophilic apoptosis is an important mechanism for the development of blood and tissue eosinophilia in allergic diseases. Considerable attention has recently been focused on the role played by different intracellular kinase cascades in the control of apoptosis. In the present study, we investigated the effect of sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug, on mitogen-activated protein kinases (MAPK) and apoptosis of human eosinophils. METHODS: Human blood eosinophils were purified from buffy coat. NaSal-induced apoptosis of eosinophils was assessed by morphological changes and Annexin-V binding assay. Changes of MAPK activity upon treatment with NaSal were measured by kinase activity assay and Western blot. RESULTS: NaSal could induce apoptosis of human blood eosinophils in a dose- and time-dependent manner. It could also activate c-Jun N-terminal kinase (JNK) and p38 MAPK but not extracellular signal-regulated protein kinase (ERK) activity within 1 h. Pretreatment of eosinophils with p38 MAPK and JNK anti-sense (AS) phosphorothioate oligodeoxynucleotides (ODN) or specific p38 MAPK inhibitor SB 203580 did not have any significant effect on NaSal-induced apoptosis. However, ERK AS ODNs could trigger the apoptosis of normal eosinophils. CONCLUSION: There is no direct relationship between the activation of JNK and p38 MAPK pathways and NaSal-induced apoptosis in human peripheral blood eosinophils.     

Manumycin通过诱导细胞凋亡抑制人胰腺导管癌细胞Panc-1活性

目的：观察manumycin对人胰腺导管癌细胞Panc-1的抑制效应，并探讨其诱导细胞凋亡是否经p38MAPK介导。 方法： 用MTT法检测manumycin对Panc-1细胞的抑癌作用。用caspase-3活性检测试剂盒定量检测manumycin诱导细胞凋亡的水平及评估特异性的p38MAPK抑制剂SB203580对它的影响。 结果： 经manumycin(6 μmol/L、18 μmol/L、54 μmol/L)处理Panc-1细胞24 h,对Panc-1细胞生长具有明显的抑制作用，其抑制率分别为8.9%、21.9%和67.0%，其中后二者的细胞活性与对照组相比有显著差异(P<0.01)，呈量效关系。用药24 h的IC50为34.7 μmol/L。同时，此药物可明显增加caspase-3的活性，且这一效应可部分地被p38抑制剂SB203580阻断。 结论： Manumycin可通过诱导Panc-1细胞凋亡而产生抑癌作用，p38MAPK是manumycin诱导细胞凋亡的通路之一。     

Morphological changes of cerebral arteries in a canine double hemorrhage model

Cerebral vasospasm is a major cause of morbidity and mortality in patients suffering from subarachnoid hemorrhage (SAH). Despite numerous studies, the pathogenesis of this deadly disorder is not clearly understood. Alterations in endothelial cells are a distinct morphological feature of cerebral vasospasm and some recent studies suggest that apoptosis might play a role in the cells' death. The goal of the present study is to examine the time course of apoptosis in endothelial cells of spastic cerebral arteries following experimental subarachnoid hemorrhage. Fifteen dogs were used in the present study. Twelve of them were divided into three groups (four per group) and subjected to a double-hemorrhage method of SAH. Following SAH, groups were sacrificed respectively on days 3, 5, and 7. Three dogs served as controls without blood injection. The basilar arteries were studied with the transmission electron microscopy and with angiography. Angiographic vasospasm began on day 3 and peaked on day 7. In morphologic studies, control dogs did not demonstrate apoptotic-like changes in endothelial cells of the basilar arteries. Beginning with day 3, apoptotic-like changes were noted in endothelial cells and consisted of condensation of peripheral nuclear chromatin, blebbing of the cell membrane, and condensation of the cytoplasm. Such changes progressed with time and were maximally developed by day 7. This is the first study that demonstrates the time course of apoptotic-like changes in the endothelial cells in the vasospastic basilar artery. Apoptosis might play an important role in the pathogenesis of vasospasm.     

HYPOXIA INDUCES APOPTOSIS BY CASPASE ACTIVATION ACCOMPANYING CYTOCHROME C RELEASE FROM MITOCHONDRIA IN MC3T3E1 OSTEOBLASTS. p38 MAPK IS RELATED IN HYPOXIA-INDUCED APOPTOSIS

The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, western blot analysis was performed using anticaspase-3 or -6 antibody. The 10-kDa protein, corresponding to the active products of caspase-3 and the 10-kDA protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which processing of the full length form of caspase-3 and -6 was evident. With a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we have observed that pre-treatment with SB203580, a selective p38 MAP kinase (p38 MAPK) inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203580 suppressed caspase-3 and -6-like protease activity by hypoxia up to 50%. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAP kinase -JNK/SAPK or ERK- was not or modestly activated. These results suggest that p38 MAPK positively regulates hypoxia-induced apoptosis in MC3T3E1 osteoblasts.     

HYPOXIA INDUCES APOPTOSIS BY CASPASE ACTIVATION ACCOMPANYING CYTOCHROME C RELEASE FROM MITOCHONDRIA IN MC3T3E1 OSTEOBLASTS. p38 MAPK IS RELATED IN HYPOXIA-INDUCED APOPTOSIS

The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, western blot analysis was performed using anticaspase-3 or -6 antibody. The 10-kDa protein, corresponding to the active products of caspase-3 and the 10-kDA protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which processing of the full length form of caspase-3 and -6 was evident. With a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we have observed that pre-treatment with SB203580, a selective p38 MAP kinase (p38 MAPK) inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203580 suppressed caspase-3 and -6-like protease activity by hypoxia up to 50%. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAP kinase -JNK/SAPK or ERK- was not or modestly activated. These results suggest that p38 MAPK positively regulates hypoxia-induced apoptosis in MC3T3E1 osteoblasts.