A complement-dependent balance between hepatic ischemia/reperfusion injury and liver regeneration in mice

Massive liver resection and small-for-size liver transplantation pose a therapeutic challenge, due to increased susceptibility of the remnant/graft to ischemia reperfusion injury (IRI) and impaired regeneration. We investigated the dual role of complement in IRI versus regeneration in mice. Complement component 3 (C3) deficiency and complement inhibition with complement receptor 2–complement receptor 1–related protein y (CR2-Crry, an inhibitor of C3 activation) provided protection from hepatic IRI, and while C3 deficiency also impaired liver regeneration following partial hepatectomy (PHx), the effect of CR2-Crry in this context was dose dependent. In a combined model of IRI and PHx, either C3 deficiency or high-dose CR2-Crry resulted in steatosis, severe hepatic injury, and high mortality, whereas low-dose CR2-Crry was protective and actually increased hepatic proliferative responses relative to control mice. Reconstitution experiments revealed an important role for the C3a degradation product acylation-stimulating protein (ASP) in the balance between inflammation/injury versus regeneration. Furthermore, liver regeneration was dependent on the putative ASP receptor, C5L2. Several potential mechanisms of hepatoprotection and recovery were identified in mice treated with low-dose CR2-Crry, including enhanced IL-6 expression and STAT3 activation, reduced hepatic ATP depletion, and attenuated oxidative stress. These data indicate that a threshold of complement activation, involving ASP and C5L2, promotes liver regeneration and suggest a balance between complement-dependent injury and regeneration.     

抗氧化剂在降低肝缺血再灌注损伤中的作用

肝移植、肝部分切除、缺血性休克等往往引起临床上肝脏缺血再灌注损伤,在这些过程中一个显著的特征就是氧化应激反应的发生并伴有内源性抗氧化剂的缺失.机体内复杂的抗氧化系统包括细胞内的酶类或非酶类的自由基清除剂以及饮食中的物质等.采用抗氧化剂治疗可以降低肝缺血再灌注损伤,关且当某些外科策略如缺血预处理不能实施时,药物治疗就显示出独特的优势.近年来,在肝缺血再灌注损伤治疗上有一些新的发展,如超氧化物歧化酶衍生物、含巯基化合物、选择性一氧化氮合酶抑制剂的出现及基因治疗等,多在一定程度上降低了肝缺血再灌注损伤的程度.     

吗啡对大鼠肝脏缺血再灌注损伤的影响

目的:缺血再灌注是肝脏手术和肝移植术中不可避免的病理过程,吗啡对于心肌缺血再灌注损伤以及对抗心肌细胞凋亡等方面已有了很大的研究进展.观察吗啡对大鼠肝缺血再灌注损伤的影响,并分析其可能作用途径.方法:实验于2006-08/2007-03在辽宁医学院附属医院外科实验室完成,动物实验方法符合动物伦理学要求.①实验分组及方法:选用Wistar大鼠60只,按随机数字表法分成4组(n=15),正常对照组、假手术组、缺血再灌注组及缺血再灌注 吗啡干预组,缺血再灌注组建立大鼠肝脏缺血再灌注损伤模型;缺血再灌注 吗啡组于缺血再灌注前给予0.3 mg/kg盐酸吗啡注射液腹腔内注射.②实验评估:于再灌注90 min后,各组大鼠经左心室取血,测定血浆中谷丙转氨酶、谷草转氨酶活性;取肝脏组织,应用流式细胞仪检测肝细胞凋亡率,应用免疫组化法检测半胱氨酸蛋白酶3的表达变化.结果:60只大鼠均进入结果分析.①血浆谷丙转氨酶、谷草转氨酶活性:缺血再灌注组显著高于正常对照组和假手术组(P＜0.01),吗啡 缺血再灌注组显著低于缺血再灌注组(P＜0.01).②肝细胞凋亡率:缺血再灌注组显著高于正常对照组和假手术组(P＜0.01),吗啡 缺血再灌注组显著低于缺血再灌注组(P＜0.01).③肝脏组织半胱氨酸蛋白酶3的表达:缺血再灌注组显著高于正常对照组和假手术组(P＜0.01),吗啡 缺血再灌注组显著低于缺血再灌注组(P＜0.01).结论:吗啡可抑制缺血再灌注损伤后肝细胞的凋亡,减轻肝脏的损伤,从而起到保护肝脏的作用.     

N-乙酰半胱氨酸对小鼠肝部分缺血/再灌注后肝肺损伤的保护作用

目的 研究抗氧化剂N-乙酰半胱氨酸（NAC）对肝脏部分缺血/再灌注（I/R）后肝、肺损伤的保护作用及对Toll样受体2/4（TLR2/4）表达的影响。方法 以BALB/c小鼠复制肝脏部分I/R损伤模型,将小鼠随机分为假手术组、I/R组和NAC干预组（NAC组）,每组10只。于再灌注后1h和3h取门静脉血测定血清肿瘤坏死因子-α（TNF-α）浓度,取眼动脉血测定血浆丙氨酸转氨酶（ALT）水平;同时取受损肝、肺组织行逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法（Western blotting）观察TLR2/4表达的变化。结果I/R后受损肝叶和肺组织内出现TLR2/4 mRNA和蛋白的高表达,NAC干预可抑制其表达水平（P〈0.05或P〈0.01）。I/R损伤后血中TNF-α和ALT水平均较假手术组明显升高;NAC干预后各时间点TNF-α和ALT均较I/R组明显下降（P〈0.05或P〈0.01）。结论 小鼠肝部分I/R后,TLR2/4不仅在受损肝组织内有表达,在远隔器官肺内也有表达。NAC可通过调整氧化还原状态,抑制再灌注后TLR2/4的活化和细胞因子TNF-α的表达,从而减轻肝、肺组织的损伤。     

潘生丁预处理对大鼠肝缺血/再灌注损伤的保护作用

目的探讨潘生丁预处理对肝缺血／再灌注损伤的保护作用。方法 SD大鼠30只，随机分为假手术组、缺血／再灌注组及潘生丁组，每组10只。常温下制备大鼠肝缺血／再灌注损伤模型，潘生丁组于缺血前30min经门静脉给予潘生丁10mg／kg加生理盐水至0．5ml，假手术组和缺血／再灌注组注人等量生理盐水，用小号无损伤钳阻断肝门45min后恢复血流灌注，并于1h后取门静脉血测定血清丙氨酸转氨酶（ALT）、乳酸脱氢酶（LDH）、肿瘤坏死因子-α（TNF-Ⅱ）及内皮素-1（ET-1），同时取肝组织行病理组织学检查及腺苷酸含量测定。结果缺血／再灌注组血清ALT、LDH、TNF-α及ET-1均明显高于假手术组，潘生丁组则明显低于缺血／再灌注组（P均〈O．01）。缺血／再灌注组肝组织中腺苷酸含量明显低于假手术组，潘生丁组则明显高于缺血／再灌注组（P均〈O．01）。潘生丁组肝组织病理组织学改变明显轻于缺血／再灌注组，并接近假手术组。结论潘生丁预处理对肝缺血／再灌注损伤具有保护作用。     

自噬与缺血再灌注损伤

自噬是存在于真核细胞生物中的一种细胞自我吞噬的现象。在生理状态下,可以维持机体内环境的稳定。在多种疾病的发生过程中,自噬也发挥重要作用。实验证明在缺血再灌注损伤的整个过程中,均有自噬的参与,但在缺血阶段和再灌注阶段,自噬有可能发挥不同的作用。在缺血阶段,自噬发挥保护细胞的作用,而在再灌注阶段,自噬可能会进一步加重细胞损伤甚至导致细胞死亡。     

Effects of pentoxifylline and coenzyme Q10 in hepatic ischemia/reperfusion injury

OBJECTIVES: We examined whether pentoxifylline (PTX) and coenzyme Q10 (Q) pretreatment affect ischemia-reperfusion damage in the rat liver. DESIGN AND METHODS: Twenty minutes of reflow following 30 min of ischemia was performed. Before the experiment, rats were treated PTX 50 mg/kg, IP or PTX 50 mg/kg IP + Q10 mg/kg, intragastric, or untreated. Rats were divided into four groups: control (C), ischemia-reperfusion (IR), PTX-treated (P), and Q+PTX-treated (QP) groups. Hepatic glutathione (GSH) and malondialdehyde (MDA) levels and catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and reductase (GSSGR) activities were measured. RESULTS: In IR group GSH levels decreased (p<0.01), conversely MDA levels increased (p<0.01). PTX pretreatment did not affect GSH and MDA values, but Q+PTX pretreatment improved of those (p<0.01). It was shown that catalase and GSH-Px activities increased during ischemia-reperfusion (p<0.01, both of), but PTX pretreatment did not significantly ameliorate those activities. GSSGR activity was higher in IR group than in basal levels (p<0.01). The decrease GSSGR activity that was observed in P group was not significant compared to IR group. During ischemia/reperfusion also SOD activity increased as compared with controls (p<0.05). In PTX-treated group, SOD activity was significantly higher than control and ischemia/reperfusion groups (p<0.01, both of). Q+PTX treatment ameliorated those enzyme activities to the control values. CONCLUSIONS: Short-term hepatic ischemia-reperfusion diminished GSH, increased MDA levels and induced some antioxidant enzyme activities. Q+PTX pretreatment was useful in hepatic ischemia-reperfusion injury, but treatment of PTX alone did not cause beneficial effect in the present study.     

Cyclic arginine-glycine-aspartate attenuates acute lung injury in mice after intestinal ischemia/reperfusion

Introduction

Intestinal ischemia is a critical problem resulting in multiple organ failure and high mortality of 60 to 80%. Acute lung injury (ALI) is a common complication after intestinal ischemia/reperfusion (I/R) injuries and contributes to the high mortality rate. Moreover, activated neutrophil infiltration into the lungs is known to play a significant role in the progression of ALI. Integrin-mediated interaction is involved in neutrophil transmigration. Synthetic peptides containing an arginine-glycine-aspartate sequence compete with adhesive proteins and inhibit integrin-mediated interaction and signaling. Thus, we hypothesized that the administration of a cyclic arginine-glycine-aspartate peptide (cRGD) inhibited neutrophil infiltration and provided protection against ALI induced by intestinal I/R.

Methods

Ischemia in adult male C57BL/6 mice was induced by fastening the superior mesenteric artery with 4-0 suture. Forty-five minutes later, the vascular suture was released to allow reperfusion. cRGD (5 mg/kg body weight) or normal saline (vehicle) was administered by intraperitoneal injection 1 hour prior to ischemia. Blood, gut, and lung tissues were collected 4 hours after reperfusion for various measurements.

Results

Intestinal I/R caused severe widespread injury to the gut and lungs. Treatment with cRGD improved the integrity of microscopic structures in the gut and lungs, as judged by histological examination. Intestinal I/R induced the expression of β₁, β₂ and β₃ integrins, intercellular adhesion molecule-1, and fibronectin. cRGD significantly inhibited myeloperoxidase activity in the gut and lungs, as well as neutrophils and macrophages infiltrating the lungs. cRGD reduced the levels of TNF-α and IL-6 in serum, in addition to IL-6 and macrophage inflammatory protein-2 in the gut and lungs. Furthermore, the number of TUNEL-staining cells and levels of cleaved caspase-3 in the lungs were significantly lowered in the cRGD-treated mice in comparison with the vehicle mice.

Conclusions

Treatment with cRGD effectively protected ALI and gut injury, lowered neutrophil infiltration, suppressed inflammation, and inhibited lung apoptosis after intestinal I/R. Thus, there is potential for developing cRGD as a treatment for patients suffering from ALI caused by intestinal I/R.     

Active site inhibited factor VIIa attenuates myocardial ischemia/reperfusion injury in mice

Summary.  Background:  Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. Objectives:  To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. Methods:  We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. Results:  ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IκBα upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-κB (NF-κB) mediated cell signaling. Levels of nuclear activated NF-κB and proteins influenced by the NF-κB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). Conclusions:  We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-κB mechanism.     

BALB/c小鼠肾缺血再灌注损伤模型的建立与评价

背景:对于一些药物研究,小鼠是理想的造模工具,但由于小鼠耐受性相对较差,肾脏及肾蒂小且难于寻找,容易增加实验误差,导致造模失败.目的:探讨BALB/c小鼠肾缺血再灌注损伤模型的建立方法,评价肾脏缺血时间对肾缺血再灌注损伤的影响.方法:采用微型动脉夹夹闭小鼠双侧肾蒂的方法建立雄性BALB/c小鼠肾缺血再灌注损伤模型,根据肾缺血时间不同分为 0 min组(对照组)、30 min组、35 min组、45 min组,肾再灌注后24 h观察肾功能和肾脏病理组织学的变化,比较不同的肾脏缺血时间对上述指标的影响;观察45 min组小鼠肾缺血再灌注损伤后的生存率.结果与结论:模型成功率95.9%,与对照组相比,肾缺血30 min组、35 min组和45 min组再灌注后24 h血清肌酐、尿素氮和肾脏病理组织学评分均升高,肾缺血45 min组生存率明显下降,差异均有显著性意义(P < 0.05).结果提示,应用微型动脉夹夹闭小鼠双侧肾蒂的方法可制备稳定肾缺血再灌注损伤模型,雄性小鼠肾缺血35～45 min是造模较为理想的肾缺血时间,所得模型效果满意.     

BALB/c小鼠肾缺血再灌注损伤模型的建立与评价

背景：对于一些药物研究,小鼠是理想的造模工具,但由于小鼠耐受性相对较差,肾脏及肾蒂小且难于寻找,容易增加实验误差,导致造模失败。目的：探讨BALB/c小鼠肾缺血再灌注损伤模型的建立方法,评价肾脏缺血时间对肾缺血再灌注损伤的影响。方法：采用微型动脉夹夹闭小鼠双侧肾蒂的方法建立雄性BALB/c小鼠肾缺血再灌注损伤模型,根据肾缺血时间不同分为0min组（对照组）、30min组、35min组、45min组,肾再灌注后24h观察肾功能和肾脏病理组织学的变化,比较不同的肾脏缺血时间对上述指标的影响;观察45min组小鼠肾缺血再灌注损伤后的生存率。结果与结论：模型成功率95.9%,与对照组相比,肾缺血30min组、35min组和45min组再灌注后24h血清肌酐、尿素氮和肾脏病理组织学评分均升高,肾缺血45min组生存率明显下降,差异均有显著性意义（P〈0.05）。结果提示,应用微型动脉夹夹闭小鼠双侧肾蒂的方法可制备稳定肾缺血再灌注损伤模型,雄性小鼠肾缺血35～45min是造模较为理想的肾缺血时间,所得模型效果满意。     

自由基与心肌缺血/再灌注损伤

大量的研究证明自由基（FR）与心肌缺血再灌注损伤这一心血管疾病的发生、发展均有密切关系。本文对FR的产生、心肌缺血／再灌注损伤、FR在心肌缺血与缺血／再灌注损伤中的影响以及心肌缺彬再灌注损伤中FR的消除与临床意义进行归纳和总结，为心血管疾病的防治和深入研究提供参考。     

miRNA－92a在缺血再灌注肾损伤中的表达变化

目的观察miRNA-92a在缺血再灌注（I／R）损伤4h、24h后小鼠肾组织的表达变化,探讨肾脏缺血损伤后血管生成的可能机制。方法采用双侧夹闭小鼠肾蒂的方法制备急性缺血再灌注肾损伤模型,术后灌注24h后收集血清和。肾脏标本,分别检测肾功能及观察肾组织病理学改变。实时定量逆转录聚合酶链反应（real—time RT-PCR）的方法检测小鼠I／R损伤4h和24h组中miRNA-92a的表达水平。结果（1）采用血清肌酐（Scr）和尿素氮（UN）评估肾功能,肾脏I／R 24h组与假手术组（sham）比较有显著性差异（P〈0．05）;HE染色观察肾组织病理改变明显,肾缺血再灌注模型成功。（2）而I／R4h和24h后,miRNA-92a的表达上调,其上调倍数分别为3．23±0．74．1．53±0．33（P〈0．05）。结论小鼠I／R损伤中miRNA-92a表达显著上调,miRNA-92a可能参与缺血再灌注肾组织损伤的血管再生的调控过程。     

miRNA－92a在缺血再灌注肾损伤中的表达变化

目的 观察miRNA-92a在缺血再灌注(I/R)损伤4h、24h后小鼠肾组织的表达变化,探讨肾脏缺血损伤后血管生成的可能机制.方法 采用双侧夹闭小鼠肾蒂的方法 制备急性缺血再灌注肾损伤模型,术后灌注24h后收集血清和肾脏标本,分别检测肾功能及观察肾组织病理学改变.实时定量逆转录聚合酶链反应(real-time RT-PCR)的方法 检测小鼠I/R损伤4h和24h组中miRNA-92a的表达水平.结果 (1)采用血清肌酐(Scr)和尿素氮(UN)评估肾功能,肾脏I/R 24h组与假手术组(sham)比较有显著性差异(P<0.05);HE染色观察肾组织病理改变明显,肾缺血再灌注模型成功.(2)而I/R4h和24h后,miRNA-92a的表达上调,其上调倍数分别为3.23±0.74,1.53±0.33 (P<0.05).结论 小鼠I/R损伤中miRNA-92a表达显著上调,miRNA-92a可能参与缺血再灌注肾组织损伤的血管再生的调控过程.     

Prevention of ischemia/reperfusion injury by hepatic targeting of nitric oxide in mice

Macromolecular nitric oxide (NO) donors possessing the ability to target a specific type of liver cells were developed for delivering NO to the liver. Six NO molecules were covalently bound to mannosylated (Man) or galactosylated (Gal) bovine serum albumin (BSA) through an S-nitrosothiol linkage to obtain Man-poly SNO-BSA and Gal-poly SNO-BSA, respectively. The carrier parts of Man-poly SNO-BSA and Gal-poly SNO-BSA predominantly accumulated in the liver after intravenous injection in mice. In an ischemia/reperfusion injury mouse model, in which hepatic injury was induced by occluding the portal vein for 15 min followed by a 6 h reperfusion, the elevation of plasma alanine aminotransferase and aspartate aminotransferase levels was significantly inhibited by a bolus intravenous injection of Man-poly SNO-BSA or Gal-poly SNO-BSA, just before the start of reperfusion. In marked contrast, S-nitroso-N-acetyl penicillamine and NO-conjugated BSA, two classical S-nitrosothiols, had no statistically significant effects on the serum levels of the markers. The released NO in mouse liver was detected by electron spin resonance spectrometry only in the liver of mice receiving Man-poly SNO-BSA or Gal-poly-SNO-BSA. These findings indicate that Man-poly SNO-BSA and Gal-poly SNO-BSA are promising compounds for preventing hepatic ischemia/reperfusion injury by delivering pharmacologically active NO to the liver.     

灌注速度对移植肝脏缺血再灌注的损伤

背景：近年来有学者研究发现供体灌注的压力可直接影响移植物的能量代谢从而影响其活力,适当的灌注压力能明显提高供体的质量。目的：观察不同灌注速度对大鼠移植肝脏再灌注损伤的影响。方法：采用改良的Kamada双袖套法建立SD→SD原位肝移植模型。供体肝脏获取时分别以50,100,150,200mL/h进行灌注。检测移植后外周血清肿瘤坏死因子α和谷丙转氨酶水平,观察肝脏组织病理学改变和肝脏组织内皮源性一氧化氮合酶的表达变化。结果与结论：与低灌注速度相比,供体肝脏制备过程中200mL/h的灌注速度导致了更加明显的肝脏病理形态学改变,肝细胞变性、肝血窦扩张和炎细胞浸润也更加明显。术后24h肝功能的检测也发现,150,200mL/h灌注速度组外周血谷丙转氨酶活性、肿瘤坏死因子水平明显高于50,100mL/h灌注速度组（P〈0.05,P〈0.01）,内皮源性一氧化氮合酶表达明显低于50,100mL/h灌注速度组（P〈0.01）,100mL/h灌注速度后,随着灌注速度的增加肝功能损伤也明显加重。证实适当的灌注压力和速度是获得高质量供体的保障,能够减轻移植后肝功能损伤,改善受体预后,在大鼠肝移植供体制备过程中100mL/h是适宜的灌注速度。     

灌注速度对移植肝脏缺血再灌注的损伤

背景:近年来有学者研究发现供体灌注的压力可直接影响移植物的能量代谢从而影响其活力,适当的灌注压力能明显提高供体的质量.目的:观察不同灌注速度对大鼠移植肝脏再灌注损伤的影响.方法:采用改良的Kamada双袖套法建立SD→SD原位肝移植模型.供体肝脏获取时分别以50,100,150,200 mL/h进行灌注.检测移植后外周血清肿瘤坏死因子α和谷丙转氨酶水平,观察肝脏组织病理学改变和肝脏组织内皮源性一氧化氮合酶的表达变化.结果与结论:与低灌注速度相比,供体肝脏制备过程中200 mL/h的灌注速度导致了更加明显的肝脏病理形态学改变,肝细胞变性、肝血窦扩张和炎细胞浸润也更加明显.术后24 h肝功能的检测也发现,150,200 mL/h灌注速度组外周血谷丙转氨酶活性、肿瘤坏死因子水平明显高于50,100 mL/h灌注速度组(P < 0.05,P < 0.01),内皮源性一氧化氮合酶表达明显低于50,100 mL/h灌注速度组(P < 0.01),100 mL/h灌注速度后,随着灌注速度的增加肝功能损伤也明显加重.证实适当的灌注压力和速度是获得高质量供体的保障,能够减轻移植后肝功能损伤,改善受体预后,在大鼠肝移植供体制备过程中 100 mL/h是适宜的灌注速度.     

脊髓再灌注损伤后细胞间黏附分子-1和白细胞介素-1β的表达

目的探讨细胞间黏附分子-1(intercellularadhesionmolecul-1,ICAM-1)及其调节因子内源性白细胞介素-1β(Interleukin-1β,IL-1β)的表达在脊髓缺血-再灌注损伤中作用机制。方法采用反转录-聚合酶链反应、免疫组化及免疫荧光激光共聚焦扫描显微镜技术检测脊髓缺血再灌注损伤后血管内皮ICAM-1mRNA和IL/1βmRNA表达量。结果正常组和单纯缺血组不引起细胞因子和黏附分子表达量的增加。而再灌注后缺血区细胞因子、黏附分子的表达及多形核白细胞的浸润先后发生了改变。再灌注4h,IL-1βmRNA的表达首先升高,约为对照组的2倍,各组灰度比率分别为再灌组0.94±0.12,缺血组0.52±0.11,正常组0.51±0.10,再灌各组与单纯缺血组相比较有差异显著性意义(P<0.01)。再灌注4h,微血管内皮细胞ICAM-1的表达开始增加,并持续到12h。激光共聚焦扫描显微镜对单位血管面积上ICAM-1荧光强度的定量检测结果为正常组(180.0±32.0)V,单纯缺血组(164.2±2.0)V,再灌4h组为(316.9±26.0)V,再灌6h组(361.4±18.0)V,再灌12h组(406.0±23.0)V,再灌各组与单纯缺血组相比较有差异显著性意义(P<0.01)。结论再灌注损伤后脊髓内炎症反应是导致血脊髓屏障损害的重要分子基础,在继发性脊髓损伤过程中起到重要作用。     

PKCbeta regulates ischemia/reperfusion injury in the lung

Activation of PKCbetaII is associated with the response to ischemia/reperfusion (I/R), though its role, either pathogenic or protective, has not been determined. In a murine model of single-lung I/R, evidence linking PKCbeta to maladaptive responses is shown in the following studies. Homozygous PKCbeta-null mice and WT mice fed the PKCbeta inhibitor ruboxistaurin subjected to I/R displayed increased survival compared with controls. In PKCbeta-null mice, phosphorylation of extracellular signal-regulated protein kinase-1 and -2 (ERK1/2), JNK, and p38 MAPK was suppressed in I/R. Expression of the immediate early gene, early growth response-1 (Egr-1), and its downstream target genes was significantly increased in WT mice in I/R, particularly in mononuclear phagocytes (MPs), whereas this expression was attenuated in PKCbeta-null mice or WT mice fed ruboxistaurin. In vitro, hypoxia/reoxygenation-mediated induction of Egr-1 in MPs was suppressed by inhibition of PKCbeta, ERK1/2, and JNK, but not by inhibition of p38 MAPK. These findings elucidate key roles for PKCbetaII activation in I/R by coordinated activation of MAPKs (ERK1/2, JNK) and Egr-1.     

Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice

The ubiquitin-proteasome system degrades most intracellular proteins, including misfolded proteins. Proteasome functional insufficiency (PFI) has been observed in proteinopathies, such as desmin-related cardiomyopathy, and implicated in many common diseases, including dilated cardiomyopathy and ischemic heart disease. However, the pathogenic role of PFI has not been established. Here we created inducible Tg mice with cardiomyocyte-restricted overexpression of proteasome 28 subunit α (CR-PA28αOE) to investigate whether upregulation of the 11S proteasome enhances the proteolytic function of the proteasome in mice and, if so, whether the enhancement can rescue a bona fide proteinopathy and protect against ischemia/reperfusion (I/R) injury. We found that CR-PA28αOE did not alter the homeostasis of normal proteins and cardiac function, but did facilitate the degradation of a surrogate misfolded protein in the heart. By breeding mice with CR-PA28αOE with mice representing a well-established model of desmin-related cardiomyopathy, we demonstrated that CR-PA28αOE markedly reduced aberrant protein aggregation. Cardiac hypertrophy was decreased, and the lifespan of the animals was increased. Furthermore, PA28α knockdown promoted, whereas PA28α overexpression attenuated, accumulation of the mutant protein associated with desmin-related cardiomyopathy in cultured cardiomyocytes. Moreover, CR-PA28αOE limited infarct size and prevented postreperfusion cardiac dysfunction in mice with myocardial I/R injury. We therefore conclude that benign enhancement of cardiac proteasome proteolytic function can be achieved by CR-PA28αOE and that PFI plays a major pathogenic role in cardiac proteinopathy and myocardial I/R injury.