缺血后处理对脑缺血再灌注损伤后ERK1/2和Akt磷酸化及神经细胞凋亡的影响

目的:观察缺血后处理对大鼠局灶性脑缺血再灌注损伤后ERK1/2和Akt及神经细胞凋亡的影响。方法:成年健康SD大鼠72只,随机分为假手术(sham)组、缺血再灌注(I/R)组、缺血后处理(Postcond)组各24只,应用线栓法建立大脑中动脉闭塞(MCAO)再灌注模型。分别于再灌注10min、30min、6h、24h后留取大脑皮质。Western blot检测再灌注10min、30min、6h后ERK1/2和Akt活性变化;原位末端标记(TUNEL)检测再灌注后24h神经细胞凋亡。结果:Postcond组再灌注10min、30min、6h后ERK1/2和Akt活性高于I/R组(P<0.05);脑缺血再灌注24h后,Postcond组与I/R组比较,TUNEL阳性细胞减少(P<0.05)。结论:缺血后处理可提高大鼠脑缺血再灌注后皮质内ERK1/2和Akt活性,减少神经细胞凋亡。     

Role of tumor necrosis factor-alpha in myocardial dysfunction and apoptosis during hindlimb ischemia and reperfusion

OBJECTIVE: Peripheral vascular surgery involving limb ischemia/reperfusion is associated with tumor necrosis factor-alpha production and an increased risk of cardiac complications. The objective of this study was to investigate the role of tumor necrosis factor-alpha in myocardial apoptosis and dysfunction following hindlimb ischemia/reperfusion. DESIGN: Randomized perspective animal study. SETTING: Research laboratory. SUBJECTS: Adults male tumor necrosis factor-alpha(-/-) and littermate wild-type mice. INTERVENTIONS: Bilateral hindlimb ischemia/reperfusion was induced in wild-type and tumor necrosis factor-alpha(-/-) mice using tourniquet occlusion. After 2 hrs of hindlimb ischemia, the tourniquets were released, allowing reperfusion for 0.5-24 hrs. MEASUREMENTS AND MAIN RESULTS: In wild-type mice, hindlimb ischemia/reperfusion resulted in myocardial depression early during the reperfusion period (p < .05). These effects were temporally correlated with enhanced levels of myocardial and plasma tumor necrosis factor-alpha. All variables were restored to baseline levels by 24 hrs of reperfusion. Myocardial apoptosis, assessed by cell death enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling staining, and caspase-3 activity, was also significantly higher at 6 hrs of reperfusion (p < .05) but returned to baseline levels by 24 hrs. Interestingly, cardiac dysfunction and myocardial apoptosis were abolished in tumor necrosis factor-alpha mice subjected to the same degree of hindlimb ischemia/reperfusion as the wild-type mice. Treatment of etanercept restored cardiac function in wild-type mice. CONCLUSIONS: Tumor necrosis factor-alpha contributes significantly to myocardial dysfunction and apoptosis in hindlimb ischemia/reperfusion. Although a causal link between myocardial apoptosis and cardiac dysfunction is not established, our study does suggest that tumor necrosis factor-alpha may be a potential therapeutic target for cardiac injury in clinical situations involving prolonged remote ischemia/reperfusion.     

大鼠局灶性脑缺血再灌注时c－fos、bcl－2蛋白的动态变化

目的:探讨细胞凋亡在脑缺血再灌注时脑损伤过程中的作用。方法 :采用免疫组化法检测大鼠脑缺血再灌注不同时间内c -fos、bcl -2蛋白表达的水平。结果 :(1)脑缺血再灌注时在缺血侧皮层和基底节区可见c -fos阳性表达 ,并于缺血30min再灌注1h时阳性表达达高峰 ;(2)脑缺血再灌注时缺血侧皮层和基底节区均有bcl-2阳性表达 ,并随再灌注时间不同 ,其阳性表达率亦不同。于缺血2h再灌注3h时阳性表达达高峰。结论 :c -fos和bcl-2参与大鼠脑缺血再灌注时缺血性细胞损伤(包括细胞凋亡)的发生     

Differential activation of extracellular signal regulated kinase isoforms in preconditioning and opioid-induced cardioprotection

Stimulation of the delta(1)-opioid receptor has been shown to trigger ischemic preconditioning (IPC). Additionally, myocardial ischemia/reperfusion induces the activation of extracellular signal-regulated kinase (ERK). Therefore, we examined the role of ERK in acute cardioprotection induced by delta(1)-opioid receptor stimulation or IPC. Infarct size (IS) was expressed as a percentage of the area at risk (AAR). Control animals had an IS/AAR of 60.6 +/- 1.8. IPC and delta(1)-opioid receptor stimulation with TAN-67 reduced IS/AAR (8.2 +/- 1.3 and 30.2 +/- 2.4). Inhibition of ERK with the selective MEK-1 antagonist, PD 098059 during IPC or TAN-67 administration significantly reduced cardioprotection (41.5 +/- 6.4 and 63.0 +/- 4.8). Western Blot analysis and subsequent densitometry corroborated these observations. Control, TAN-67-, or IPC-treated hearts were harvested after 0, 5, 15, and 30 min of ischemia or 5, 30, and 60 min of reperfusion and separated into cytosolic and nuclear fractions. Both isoforms of ERK (p44 and p42) rapidly increased to greater levels throughout reperfusion in the nuclear fraction of IPC- and opioid-treated versus control rats, however, this increase was not attenuated by PD 098059. Conversely, the rapid activation of the 44-kDa isoform of ERK after 5 min of reperfusion in the cytosolic fraction was significantly increased in IPC- and opioid-treated hearts versus control, and this increase was abolished by pretreatment with PD 098059. Additionally, p42 was activated in the cytosolic fraction of IPC-treated animals. These results suggest a key role for the 44-kDa isoform of ERK in the cytoplasm during cardioprotection induced by either IPC or stimulation of the delta(1)-opioid receptor.     

Targeted inhibition of the extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy in Asia and Africa. We previously reported that overexpression of extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) and ERK1/2 was detected in HCC, and that their activation was required for liver cancer cell proliferation and survival. In the present study, we determined the efficacy of a specific MEK1/2 inhibitor AZD6244 (ARRAY-142886) in treatment of HCC. Treatment of primary HCC cells with AZD6244 led to growth inhibition, elevation of the cleavage of caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase, but inhibition of ERK1/2 and p90RSK phosphorylation. Studying the protein expression profile of seven HCC xenografts revealed that their growth rate was positively correlated with the levels of phosphorylated MEK. AZD6244, when given p.o. to mice bearing these xenografts, resulted in a dose-dependent inhibition of tumor growth. AZD6244-induced growth suppression was associated with inactivation of ERK1/2 and p90RSK, and up-regulation of activated caspase-3 and caspase-7, and cleaved poly(ADP)ribose polymerase. Our data suggest that the MEK-ERK pathway plays an important role in the growth and survival of liver cancer cells and that the HCC xenograft models are excellent tools for screening preclinical drugs. Targeted inhibition of the MEK-ERK pathway with AZD6244 may represent an alternative approach for the treatment of this disease.     

Cardioprotection by remote ischemic preconditioning exhibits a signaling pattern different from local ischemic preconditioning

Remote ischemic preconditioning (RIPC) and local ischemic preconditioning (IPC) protect the myocardium from subsequent ischemia/reperfusion (I/R) injury. In this study, the protective effects of early RIPC, IPC, and the combination of both (RIPC-IPC) were characterized. Furthermore, the hypothesis was tested that protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs), important mediators of IPC, are activated in RIPC. Infarct size, serum troponin T, and creatine kinase levels were assessed after 4 × 5-min noninvasive RIPC, local IPC, or a combination of both and 35 min of regional ischemia and 120 min of reperfusion. Protein kinase C ε and the MAPKs extracellular signal-regulated MAPK (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK were analyzed by Western blot analysis and activity assays in the myocardium and skeletal muscle immediately after the preconditioning protocol. Remote ischemic preconditioning, IPC, and RIPC-IPC significantly reduced myocardial infarct size (RIPC-I/R: 54% ± 15%; IPC-I/R: 33% ± 15%; RIPC-IPC-I/R: 33% ± 15%; P < 0.05 vs. I/R [76% ± 14%]) and troponin T release (RIPC-I/R: 15.4 ± 6.4 ng/mL; IPC-I/R: 10.9 ± 7.0 ng/mL; RIPC-IPC-I/R: 9.8 ± 5.6 ng/mL; P < 0.05 vs. I/R [27.1 ± 12.0 ng/mL]) after myocardial I/R. Ischemic preconditioning led to an activation of PKCε and ERK 1/2, whereas RIPC did not lead to a translocation of PKCε to the mitochondria or phosphorylation of the MAPKs ERK 1/2, JNK 1/2, and p38 MAPK. Remote ischemic preconditioning did not induce translocation of PKCε to the mitochondria or phosphorylation of MAPKs in the preconditioned muscle tissue. Remote ischemic preconditioning, IPC, and RIPC-IPC exert early protection against myocardial I/R injury. Remote ischemic preconditioning and local IPC exhibit different activation dynamics of signal transducers in the myocardium. The studied PKC-MAPK pathway is likely not involved in the protective effects of RIPC.     

远期缺血预适应激活热休克蛋白27、促红细胞生成素、血红素加氧酶1对肾脏缺血再灌注损伤的影响

背景:缺血再灌注损伤是临床导致急性肾衰竭等其他疾病的重要原因,其机制为多因素、多途径的复杂的病理过程。目的:观察肾脏进行预处理后激活热休克蛋白、促红细胞生成素和血红素加氧酶1对肾脏缺血再灌注损伤的影响。方法:雄性C57BL/6小鼠90只随机分成3组。缺血再灌注组为右肾切除,左肾缺血25min再灌注24h;预适应组为双侧肾脏缺血20min再灌注8d后再进行缺血再灌注。假手术组开腹游离肾蒂。结果与结论:血清肌酐和尿素氮检测预适应组和假手术组明显低于缺血再灌注组(P<0.01);MPO染色发现缺血再灌注组大量中性粒细胞浸润(P<0.01);PAS染色发现预适应组肾组织病理情况轻于缺血再灌注组(P<0.05);TUNEL染色分析结果表明预适应组和假手术组细胞凋亡数明显少于缺血再灌注组(P<0.01);预适应组热休克蛋白27mRNA表达明显高于缺血再灌注和假手术组(P<0.05),热休克蛋白27mRNA于第8天时最强,促红细胞生成素、血红素加氧酶1mRNA在24～48h达到峰值A,然后逐渐下降,第8天后达到峰值B,B>A,并且高于假手术组(P<0.01)。提示远期缺血预适应激活热休克蛋白27、促红细胞生成素、血红素加氧酶1,能减少炎症因子浸润、促进肾小管细胞修复和抑制细胞凋亡从而参与肾脏内源性保护机制。     

七氟烷预处理对脑缺血-再灌注损伤大鼠脑组织热休克蛋白70和27表达的影响

【目的】观察七氟烷对大鼠局灶性脑缺血-再灌注时脑组织热休克蛋白（HSP）70和27表达的影响,以探讨其脑保护的机制。【方法】采用大脑中动脉线栓法建立大鼠局灶性脑缺血-再灌注模型。30只雄性SD大鼠,随机分为假手术组（Sham组）、缺血-再灌注组（I-R组）和七氟烷组（S组）,每组10只。大鼠脑缺血60min,然后进行再灌注。S组在脑缺血前30min吸入氧气＋2．4％七氟烷60rain。Sham组和I-R组吸入氧气。缺血60min,再灌注24h后处死大鼠,取缺血侧顶叶皮层脑组织,采用Western-blot法检测HSP_70和HSP-27蛋白的表达水平。【结果】局灶性脑缺血再灌注后,HSP-70和HSP-27蛋白的表达增加（P〈o．01）,应用七氟烷预处理能显著地促进脑缺血-再灌注后脑组织中HSP-70和HSP-27蛋白的表达（P〈0．01）。【结论】七氟炕能上调大鼠局灶性脑缺血-再灌注时HSP70和HsPL27的表达,这可能是其脑保护作用的部分机制。     

Attenuation of reperfusion lung injury and apoptosis by A2A adenosine receptor activation is associated with modulation of Bcl-2 and Bax expression and activation of extracellular signal-regulated kinases

Adenosine receptors (AR) and extracellular signal-regulated kinases (ERK) have been implicated in tissue protection and apoptosis regulation during ischemia/reperfusion (I/R) injury. This study tests the hypothesis that reduction of reperfusion lung injury after A2A AR activation is associated with attenuation of apoptosis, modulation of ERK activation, and alterations in antiapoptotic and proapoptotic protein expression (Bcl-2 and Bax, respectively). Experiments were performed in intact-chest, spontaneously breathing cats in which the arterial branch of the left lower lung lobe was occluded for 2 h and reperfused for 3 h (I/R group). Animals were treated with the selective A2A AR agonist ATL313 given 5 min before reperfusion alone or in combination with the selective A2A AR antagonist ZM241385. Western blot analysis showed significant reduction in expression of Bcl-2 and increase in expression of Bax after reperfusion, compared with control lungs. Phosphorylated ERK1/2 levels were also increased after reperfusion. Compared with the I/R group, ATL313 markedly (P < 0.01) attenuated indices of injury and apoptosis including the percentage of injured alveoli, wet-dry weight ratio, myeloperoxidase activity, in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive cells, and caspase 3 activity and expression. Furthermore, compared with reperfused lungs, in ATL313-pretreated lungs, Western blot analysis demonstrated substantial ERK1/2 activation, increased expression of Bcl-2, and attenuated expression of Bax. The protective effects of ATL313 were blocked by pretreatment with ZM241385. In summary, the present study shows that in vivo activation of A2A AR confers protection against reperfusion lung injury. This protection is associated with decreased apoptosis and involves ERK1/2 activation and alterations in antiapoptotic Bcl-2 and proapoptotic Bax proteins.     

Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion

Cardiac mitochondria sustain damage during ischemia and reperfusion, contributing to cell death. The reversible blockade of electron transport during ischemia with amobarbital, an inhibitor at the rotenone site of complex I, protects mitochondria against ischemic damage. Amobarbital treatment immediately before ischemia was used to test the hypothesis that damage to mitochondrial respiration occurs mainly during ischemia and that protection of mitochondria during ischemia leads to decreased cardiac injury with reperfusion. Langendorff-perfused Fischer-344 rat hearts were treated with amobarbital (2.5 mM) or vehicle for 1 min immediately before 25 min of global ischemia. Both groups were reperfused for 30 min without additional treatment. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated after reperfusion. Ischemia and reperfusion decreased state 3 and increased state 4 respiration rate in both SSM and IFM. Amobarbital treatment protected oxidative phosphorylation measured following reperfusion and improved the coupling of respiration. Cytochrome c content measured in SSM and IFM following reperfusion decreased in untreated, but not in amobarbital-treated, hearts. H(2)O(2) release from SSM and IFM isolated from amobarbital-treated hearts during reperfusion was markedly decreased. Amobarbital treatment before ischemia improved recovery of contractile function (percentage of preischemic developed pressure: untreated 51 +/- 4%, n = 12; amobarbital 70 +/- 4%, n = 11, p < 0.01) and substantially reduced infarct size (untreated 32 +/- 2%, n = 7; amobarbital 13 +/- 2%, n = 7, p < 0.01). Thus, mitochondrial damage occurs mainly during ischemia rather than during reperfusion. Reperfusion in the setting of preserved mitochondrial respiratory function attenuates the mitochondrial release of reactive oxygen species, enhances contractile recovery, and decreases myocardial infarct size.     

急性缺血再灌注心肌细胞凋亡及凋亡基因与左室局部功能的实验研究

目的 应变率成像观察犬急性缺血再灌注后左心室局部功能，与细胞凋亡及心肌组织中caspase-3 mRNA和蛋白表达的变化。方法 健康杂种犬30只，分对照组（10只），缺血组（10只），再灌注组（10只）。缺血组于第一对角支1cm以下套扎左冠状动脉前降支30min，再灌注组套扎30min后再灌注120min，对照组游离左冠状动脉前降支不结扎。超声心动图左室收缩期应变率测量左室局部收缩功能，TUNEL法检测缺血区心肌细胞凋亡数，RT-PCR法检测caspase-3 mRNA的表达，免疫组化法测caspase-3蛋白的表达。结果 结扎30min后，左室前壁缺血节段收缩期应变率明显降低（P〈0.01）。并出现收缩后压缩（PSC），再灌注120min后缺血节段的应变率有所恢复，但仍低于对照组（P〈0.05）。缺血组缺血区心肌TUNEL阳性细胞数与对照组比较无显著性差异;再灌注组缺血区心肌TUNEL阳性数明显增加（P〈0.05）。缺血组缺血区caspase-3 mRNA与对照组比较表达增高（P〈0.05），再灌注组进一步增高（P〈0.01）。缺血组缺血区心肌细胞caspase-3表达升高，与对照组比差异有显著意义（P〈0.05），再灌注组caspase-3表达进一步增强（P〈0.01）。结论 急性心肌缺血再灌注促凋亡基因capase-3激活，缺血心肌细胞凋亡可能为其早期改变，应变率成像可以评价缺血早期心肌局部收缩功能。     

鼠肝缺血再灌注后肺组织ERK1/2通路活性变化及褪黑素的影响

目的探讨大鼠全肝缺血再灌注后肺组织损伤、修复过程中ERK1/2信号通路活性的动态变化与意义,以及褪黑素对该信号通路的影响。方法本实验将分成两部分完成。第一部分,36只SD大鼠随机分成6组（n=6）：①缺血前组;②再灌注0.5 h组;③再灌注3 h组;④再灌注6 h组;⑤再灌注12 h组;⑥再灌注24 h组。阻断肝门30 min后开放血流,建立大鼠全肝缺血再灌注模型。分别于缺血前5 min、再灌注0.5 h、3 h、6 h、12 h、24 h处死动物取肺,检测ERK1/2和p-ERK1/2表达、细胞凋亡、PCNA表达及肺组织病理学变化,观察肝缺血再灌注后肺组织ERK1/2信号通路活性的动态变化及其与细胞增殖、凋亡的关系。第二部分,将12只SD大鼠随机分成2组（n=6）：褪黑素组与基质对照组,依上述方法制备全肝缺血再灌注模型,分别于全肝缺血前15 min和再灌注前10 min静脉注射0.5%褪黑素溶液10 mg/kg或相同剂量的溶剂,于再灌注3 h处死动物取肺,检测前述相同参数,观察褪黑素的作用。结果 （1）与缺血前相比,全肝缺血再灌注后肺组织p-ERK/ERK比值与PCNA阳性指数均于再灌注0.5 h显著降低,此后逐渐增高;细胞凋亡指数则于再灌注0.5 h显著增高,后逐渐上升,再灌注6 h达最高,后又逐渐下降;肺组织病理于再灌注0.5 h即出现损伤改变,后逐渐加重,再灌注3 h最重,此后逐渐恢复。（2）双变量相关分析显示：p-ERK/ERK比值与PCNA阳性指数成正相关（r=0.85,P〈0.01）;与凋亡指数呈负相关（r=-0.38,P〈0.05）。（3）褪黑素组与基质对照组比较,p-ERK/ERK比值显著增加,细胞凋亡指数显著降低,病理学改变减轻,但PCNA阳性指数无明显变化。结论全肝缺血再灌注后肺组织损伤、修复过程中,ERK1/2信号通路活性呈先抑制后激活的变化方式,该通路可能通过抑制凋亡、促进增殖在损伤肺组织自身修复过程中发挥积极作用。褪黑素可激活ERK1/2信号通路,并可能由此减轻肝缺血再灌注后肺损伤。     

酸性成纤维细胞生长因子对大鼠缺血/再灌注损伤肠上皮细胞丝裂素活化蛋白激酶的影响

目的 观察外源性酸性成纤维细胞生长因子（aFGF）对大鼠缺血／再灌注（I／R）损伤后肠上皮细胞丝裂素活化蛋白激酶（MAPK）活性的影响，并探讨其与肠上皮细胞增殖能力变化的关系。方法 采用大鼠肠系膜上动脉夹闭法制备缺血45min后再灌注动物模型。132只Wistar大鼠随机分为假手术组、I／R组、aFGF处理组（再灌注即刻颈静脉注射aFGF 4μg）及细胞外信号调节蛋白激酶（ERK1／2）阻断剂PD98059预处理组（缺血前尾静脉注射PD98059 7．5μg，再灌注即刻静脉注射aFGF 4μg）。检测缺血前，I／R15、30min及1、2、6、12和24h时肠上皮细胞增殖能力和MAPK活性。结果 aFGF处理后肠上皮细胞增殖明显增加，同时MAPK活性显著增高。用p44／p42MAPK（ERK1／2）阻断剂PD98059可抑制ERK1／2的活性，使肠上皮细胞增殖减少。结论 aFGF可促进I／R损伤后肠上皮细胞增殖，并可能与其激活肠上皮MAPK有关。     

解偶联蛋白-2在大鼠缺血预适应心肌中的表达

目的：探讨解偶联蛋白-2（UCP2）在心肌缺血预适应（IPC）心肌保护中的作用。方法：采取结扎左冠状动脉的方法复制大鼠心肌缺血再灌注模型。IPC组行3次缺血5min，再灌注10min的预处理。缺血再灌注（IR）组与IPC组行30min缺血及120min再灌注；对照组不结扎左冠状动脉。电镜观察心肌超微结构。据Rainio评分标准进行心肌超微结构损伤程度的半定量分析，随机选取20个低倍视野，计算平均心肌细胞凋亡数。采用RT—PCR和Western印迹法检测心肌中UCP2的表达。结果：IPC组Rainio评分和心肌细胞凋亡率均低于IR组（氏0．05），IPC组的UCP2 mRNA和蛋白表达水平均较IR组明显增加（P〈0．01）。结论：IPC可减轻心肌超微结构损伤程度和减少细胞凋亡。IPC可诱导UCP2表达，提示UCP2可能参与了IPC的心肌保护作用。     

尼卡地平联合异丙酚预处理对沙鼠缺血后再灌注神经细胞凋亡的影响

目的　探讨尼卡地平联合异丙酚预处理对缺血后再灌注神经细胞凋亡的影响 ,并检测经尼卡地平和异丙酚联合预处理后的沙土鼠脑组织中抑凋亡基因Bcl 2的表达。方法　采用夹闭沙土鼠双侧颈总动脉造成脑缺血模型。 5 0只健康成年沙土鼠随机分成五组 ,每组各 1 0只 :假手术对照组 (A组 ) ;脑缺血对照组 (B组 ) ,全脑缺血 5min后再灌注 72h ;尼卡地平预处理组 (C组 ) ;异丙酚预处理组 (D组 ) ;尼卡地平联合异丙酚预处理组 (E组 )。C、D、E三组缺血前 30min分别静脉给予尼卡地平 0 2mg/kg、腹腔注射异丙酚 1 0 0mg/kg以及两者同时联合处理 ,缺血 5min后分别再灌注 72h。实验终末 ,断头取脑 ,石蜡切片 ,采用DNA末端标记技术 (TUNEL法 )观察各组脑细胞凋亡的变化情况并采用免疫组化染色评定Bcl 2蛋白反应强度。结果　E组的凋亡细胞百分数明显低于其他各组 ,而Bcl 2蛋白免疫反应强度高于其它各组(P <0 0 5 )。结论　短暂性脑缺血再灌注后有细胞凋亡发生 ,脑缺血前经尼卡地平联合异丙酚预处理后 ,能明显减少细胞凋亡的发生 ,此与Bcl 2蛋白表达增强有关。     

Hepatocyte apoptosis is enhanced after ischemia/reperfusion in the steatotic liver

Liver steatosis is associated with organ dysfunction after hepatic resection and transplantation which may be caused by hepatic ischemia/reperfusion injury. The aim of the current study was to determine the precise mechanism leading to hepatocyte apoptosis after steatotic liver ischemia/reperfusion. Using a murine model of partial hepatic ischemia for 90 min, we examined the levels and pathway of apoptosis, and the peroxynitrite expression, serum alanine aminotransferase levels, and liver histology 1 and 4 h after reperfusion. In the steatotic liver, the peroxynitrite expression increased after ischemia/reperfusion. Significant hepatocyte apoptosis in the steatotic liver was seen after reperfusion, caused by upregulation of cleaved caspases 9 and 3, but not caspase 8. Serum alanine aminotransferase levels were elevated and histological examination revealed severe liver injury in the steatotic liver 4 h after reperfusion. In mice treated with aminoguanidine, ischemia/reperfusion-induced increases in serum alanine aminotransferase levels and apoptosis were significantly reduced in steatotic liver compared with mice treated with phosphate buffered saline. Survival of mice with steatotic livers significantly improved by treatment with aminoguanidine. Our data suggested that the steatotic liver is vulnerable to hepatic ischemia/reperfusion, leading to significant hepatocyte apoptosis by the mitochondrial permeability transition, and thereby resulting in organ dysfunction.     

Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis

The Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathway regulates diverse cellular processes such as proliferation, differentiation, and apoptosis and is implicated as an important contributor to the pathogenesis of cardiac hypertrophy and heart failure. To examine the in vivo role of Raf-1 in the heart, we generated cardiac muscle-specific Raf-1-knockout (Raf CKO) mice with Cre-loxP-mediated recombination. The mice demonstrated left ventricular systolic dysfunction and heart dilatation without cardiac hypertrophy or lethality. The Raf CKO mice showed a significant increase in the number of apoptotic cardiomyocytes. The expression level and activation of MEK1/2 or ERK showed no difference, but the kinase activity of apoptosis signal-regulating kinase 1 (ASK1), JNK, or p38 increased significantly compared with that in controls. The ablation of ASK1 rescued heart dysfunction and dilatation as well as cardiac fibrosis. These results indicate that Raf-1 promotes cardiomyocyte survival through a MEK/ERK-independent mechanism.