热缺血再灌注损伤时肝细胞凋亡的变化

越来越多的研究表明 ,细胞凋亡在肝脏缺血再灌注损伤这一过程中发挥着重要作用。本文探讨肝脏缺血再灌注时细胞凋亡的变化。一、材料与方法1.本实验采用健康雄性Ｗｉｓｔａｒ大鼠 2 4只 ,随机分成 4组 ,每组 6只 :(1)假手术组 :只做大鼠肝脏的游离 ,目的是作为正常对照以排除手术因素对实验结果的影响 ;(2 )对照组 :阻断大鼠肝左叶和中叶的门静脉、肝动脉 6 0ｍｉｎ后 ,立即切取左叶和右叶肝组织标本检测 ;(3)实验组 1:阻断血流操作同对照组 ,恢复血流再灌注 2ｈ后 ,切取左叶和右叶肝组织标本检测 ;(4)实验组 2 :操作同实验组 1,在再灌注 2 …     

缺血后处理对大鼠肝脏缺血再灌注损伤细胞凋亡的影响

目的：探讨缺血后处理对缺血再灌注损伤大鼠肝脏细胞凋亡的影响。方法：建立大鼠局部肝脏缺血再灌注模型．将24只健康雄性Wistar大鼠随机分为假手术(S)、缺血再灌注(IR)、缺血后处理(IPo)3组，以缺血再灌注前反复多次的短暂预再灌注及停灌注作后处理，观察血清肝酶和肝组织中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)水平变化，用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL)及透射电镜观察肝细胞凋亡状态．并行肝组织病理形态学检查。结果：与IR组相比，IPo组血清肝酶水平及肝组织中MDA含量显著降低，而SOD和GSH活性则显著升高；再灌注后可见明显的肝实质细胞凋亡现象，IPo组凋亡指数较IR组显著下降，肝组织病理形态学损伤亦明显减轻。结论：IPo可通过抑制再灌注后氧自由基的过量生成而拮抗肝实质细胞凋亡的发生，从而减轻肝脏缺血再灌注损伤。     

17-β雌二醇对大鼠肝切除肝缺血再灌注损伤肝细胞凋亡和Bcl-2、Bax表达的影响

目的 观察17-β雌二醇预处理对肝切除肝缺血再灌注损伤肝脏组织细胞凋亡及Bcl2、Bax表达的影响,并探讨其肝保护的机制.方法 建立大鼠肝切除肝缺血再灌注损伤模型,75只雄性SD大鼠随机分为3组:假手术组(Sham组)、缺血再灌注组(IR组)和17-β雌二醇预处理组(E2+ IR组).检测各组大鼠再灌注后lh、3h、6h、12 h、24 h肝功能变化.光镜下观察肝组织病理学改变.TUNEL法观察再灌注后12 h大鼠肝细胞凋亡情况、流式细胞学方法测定再灌注后12h肝细胞凋亡率.Western blot法检测再灌注后12 h Bcl-2和Bax的表达情况.结果 与Sham组相比,在IR组各时间点均可见ALT和AST增高,且在再灌注后的12h达到了最高值;病理学检查可见肝细胞肿胀,肝窦变窄,嗜中性粒细胞浸润和片状坏死等变化;在再灌注后12h,凋亡细胞增多及细胞凋亡率明显升高;肝脏组织Bcl 2表达减少,Bax的表达增加.17-β雌二醇预处理组在灌注后各时间点ALT和AST值明显下降,肝脏病理损伤改善;在再灌注后12h,凋亡细胞减少及细胞凋亡率明显降低,肝脏组织Bcl-2表达增加,Bax的表达减少.结论 17-β雌二醇对大鼠肝缺血再灌注损伤有明显的保护作用,其可能通过促进Bcl-2表达及抑制Bax表达,从而抑制肝细胞凋亡.     

Bcl-2基因修饰的肝细胞移植对肝脏缺血再灌注损伤的影响

目的观察人Bcl-2(hBcl-2)基因修饰的肝细胞移植对肝脏缺血再灌注损伤的影响。方法将包含hBcl-2基因阅读框架(0·7kb)的核苷酸亚克隆至Psectag2A载体,脂质体转染balb/c小鼠胎肝细胞(BNL.CL2),经门静脉移植入balb/c小鼠(2×106细胞)。移植72h后左肝后叶常温下缺血45min后再灌注8h,比较测定凋亡细胞,血清ALT、AST、LDH,以及组织学的改变。结果转染hBcl-2基因的balb/c小鼠在肝脏缺血再灌注后ALT(P<0·05或0·01)、AST(P<0·05)、LDH(P<0·05)、凋亡细胞(P<0·05)均较对照组显著降低,组织学改变减轻。进行缺血再灌注的各组细胞损伤均以细胞凋亡为主(P<0·05或0·01)。结论hBcl-2基因修饰的肝细胞移植对肝脏缺血再灌注损伤具有保护作用,主要通过抑制缺血再灌注后的细胞凋亡。     

大鼠肝脏缺血再灌注时肝细胞凋亡与肿瘤坏死因子-α的关系

目的探讨大鼠肝脏缺血再灌注时肝细胞凋亡与肿瘤坏死因子-α(TNF-α)的关系。方法建立大鼠局部肝脏缺血再灌注模型,将72只健康雄性Wistar大鼠随机分为正常组、假手术组和缺血再灌注组,采用放射免疫法测定再灌注后不同时相中血清TNF-α含量,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL)和透射电镜观察肝细胞凋亡状态。结果再灌注后1、3、6h和24h血清TNF-α含量及肝细胞凋亡指数(HAI)明显高于正常组和假手术组,且均于6h达到高峰,再灌注后各时相TNF-α水平与HAI呈显著正相关。结论肝脏缺血再灌注损伤过程中,TNF-α表达水平异常上调并可能对肝实质细胞凋亡起介导作用。     

肝脏缺血/再灌注后线粒体损伤与促凋亡蛋白的释放

肝脏缺血／再灌注（I／R）损伤是临床上较为常见的组织器官损伤之一，细胞凋亡在肝脏I／R损伤中起到了重要作用。近年来的研究发现，I／R导致线粒体通透性改变，由此引起线粒体膜电位的降低和促凋亡蛋白的释放等一系列变化，在细胞凋亡中发挥关键作用。     

葛根素对大鼠肝缺血再灌注损伤脑组织c-fos和Bcl-2表达的影响

目的：观察肝缺血再灌注损伤时c-fos、Bcl-2与脑细胞凋亡的关系及葛根素对其影响的可能机制。方法：建立肝缺血再灌注损伤动物模型。选健康雄性SD大鼠56只,随机分为对照组、缺血30min组（I组）、缺血30min即刻再灌注组（I/R组）、缺血30min再灌注1h组（I/R1h）、缺血30min再灌注2h组（I/R2h）、30min再灌注4h组（I/R4h）及葛根素预处理组（PUE＋I/R4h组）,每组8只。观缺血察各组肝、脑HE染色;应用免疫组织化学方法测定各组大鼠脑组织c-fos、Bcl-2的表达;应用原位细胞凋亡法测定脑细胞凋亡。结果：I/R2h组、I/R4h组肝组织中散在分布大量炎症细胞,肝细胞明显肿胀,有的呈空泡状变性,肝脏结构紊乱;PUE＋I/R4h组上述改变明显改善。I/R2h、I/R4h组脑组织水肿明显,PUE＋I/R4h组明显改善。与对照组比较,其余各组脑组织c-fos表达均增高﹙P〈0.01）,I/R4h组水平最高,PUE＋I/R4h组较I/R1h组、I/R2h组、I/R4h组明显降低（P〈0.01）。与对照组比较,其余各组脑组织Bcl-2表达增高（P〈0.01）,I/R4h组与I/R2h组差异无统计学意义﹙P〉0.05）,PUE＋I/R4h组较对照组、组表达增多（P〈0.01）,较I/R1h组、I/R2h组、I/R4h组明显降低（P〈0.01）。I组、I/R组细胞I凋亡指数较对照组明显增加（P〈0.01）,随着再灌注时间的延长细胞凋亡指数逐渐增加。PUE＋I/R4h组较I/R2h组、I/R4h组明显降低（P〈0.01）。结论：肝缺血再灌注损伤可引起脑组织的损伤及脑细胞凋亡。随着再灌注时间的延长,脑组织中c-fos表达增高,脑细胞凋亡与c-fos的表达有关。Bcl-2在缺血期发挥了抑凋亡的作用,随着再灌注时间的延长,其作用减弱,脑细胞凋亡指数增加。葛根素可能通过抑制c-fos的表达、增加Bcl-2的表达发挥减轻肝缺血再灌注损伤所致脑细胞凋亡的作用。     

大鼠肝脏保存再灌注时OH与细胞凋亡及Bcl-2蛋白表达的关系

目的 阐明羟自由基（ＯＨ），细胞凋亡及Ｂｃｌ－２蛋白表达在大鼠肝脏不同保存时相再灌注过程中的变化规律及相互关系。方法 以大鼠肝脏离体非循环灌注为模型，用高效液相色谱（ＨＰＬＣ）紫外检测法，测定ＯＨ与水杨酸的生成物二羟苯甲酸（ＤＨＢＡ）含量，用原位末端标记法及电镜观察细胞凋亡，用免疫组织化学法检测Ｂｃｌ－２蛋白表达。结果 随着保存时间延长，ＯＨ水平及细胞凋亡指数（ＡＩ）进行性升高，且两者呈显著正相关     

葛根素对大鼠肝缺血再灌注损伤心肌组织c-fos、Bcl-2表达的影响及意义

目的：研究肝缺血再灌注损伤时c-fos、Bcl-2在心肌组织中的表达与心肌细胞凋亡的关系及葛根素对其影响的可能机制。方法：建立肝缺血再灌注损伤动物模型,选健康雄性SD大鼠32只,每组8只,随机分为对照组、缺血再灌注组（I/R组）、葛根素预处理组（PUE组）和生理盐水预处理组（N组）。应用免疫组织化学方法分别测定各组大鼠心肌组织c-fos、Bcl-2的表达,应用原位细胞凋亡法测定心肌细胞凋亡情况,应用常规HE染色观察各组肝脏及心肌组织形态改变。结果：c-fos、Bcl-2表达及心肌细胞凋亡指数I/R组较对照组明显增加（P〈0.01）;PUE组c-fos表达与I/R组比较明显降低（P〈0.01）,Bcl-2表达及心肌细胞凋亡指数与对照组及I/R组相比,差异均有统计学意义（P〈0.01）;N组与PUE组各指标比较差异均有统计学意义（P〈0.01）。心肌组织HE染色切片观察显示I：/R组心肌细胞部分横纹不清楚,有炎性细胞浸润,间质出血,心肌嗜酸性增强及肌纤维肿胀,心肌水肿;PUE组心肌组织水肿明显减轻。肝组织HE染色切片观察显示I,/R组炎性细胞在肝组织中大量散在分布,肝细胞明显肿胀,有的呈空泡状变性,肝脏结构紊乱;PUE组上述改变明显改善。结论：肝缺血再灌注损伤可引起心肌组织的损伤及心肌细胞凋亡。葛根素可能通过抑制c-fos蛋白表达,增加Bcl-2蛋白表达起到抑制肝缺血再灌注损伤所致心肌细胞凋亡作用。     

间断低氧预适应对大鼠肝切除缺血再灌注肝脏凋亡相关蛋白Bcl-2、Bax表达的影响

目的 观察术前间断低氧预适应对大鼠70％肝切术后缺血再灌注损伤肝脏凋亡相关蛋白Bcl-2和Bax表达的影响。方法 健康清洁级SD大鼠54只,用SPSS软件随机分为3组,每组18只：（1）肝切除组（PH组）,切除肝脏的左叶和中叶（约占总肝重的70％）;（2）缺血再灌注组（IR组）,即在肝门阻断下切除肝脏的左叶和中叶,肝门阻断20 min后开放血流,残余肝脏发生了缺血再灌注过程;（3）间断低氧预适应组（IHP组）,术前1周将大鼠置于氧气体积分数为10％的低氧环境中,每天1h。1周后在肝门阻断下行肝切除术（同IR组）。各组分别于术后12、24、48 h进行取材检测,用全自动生化分析仪检测血清谷丙转氨酶（ALT）、谷草转氨酶（AST）含量,采用免疫组化方法检测残余肝组织Bcl-2、Bax表达情况。结果 在术后各时间点,IR组和IHP组血清ALT和AST水平均显著高于PH组,但IHP组明显低于IR组。与IR组相比,IHP组术后各时间点肝脏Bcl-2蛋白表达显著升高,而Bax蛋白表达显著下降。差异均有统计学意义（P＜0.05）。结论 间断低氧预适应对残余肝脏缺血再灌注损伤具有保护作用,其途径可能是通过促进抗凋亡蛋白Bcl-2表达和抑制促凋亡蛋白Bax表达,来减少肝细胞凋亡。     

异丙酚对大鼠前脑缺血/再灌注后海马Bcl-2表达的影响

大量文献证实异丙酚具有脑保护作用[1],其具体作用机制还未完全阐明。抗凋亡基因bcl-2在对抗细胞凋亡中起重要作用,其高度表达可减少脑缺血时神经元的凋亡[2]。本实验通过脑室内注射异丙酚,采用免疫组化(IH)及半定量逆转录-聚合酶链反应(RT-PCR)方法检测大鼠前脑缺血再灌注后海     

Neutrophil depletion reduces myocardial apoptosis and attenuates NFkappaB activation/TNFalpha release after ischemia and reperfusion

BACKGROUND: This study tested the hypothesis that depletion of neutrophils (PMNs) reduces myocardial apoptosis via reducing oxidant generation and inhibiting NFkappaB-mediated signaling pathways after ischemia/reperfusion. METHODS: Anesthetized rats were randomly divided into one of four groups: Control: 30 min ischemia and 3 h of reperfusion; PMN depletion: anti-PMN serum was injected 6 h before ischemia; N-acetylcysteine (NAC): NAC was given twice before ischemia and at reperfusion. Sham: the ligature was placed without coronary occlusion. Apoptosis was detected by TUNEL staining and DNA fragmentation. PMN accumulation was studied by immunohistochemical staining. Levels of TNF-alpha, IL-6, and caspase-3 were detected by Elisa kits. Expression in NFkappaB, Bcl-2, and Bax was assessed by Western blotting analysis. RESULTS: Relative to Control, depletion of PMNs or NAC treatment reduced levels of plasma TNFalpha (567 +/- 130* and 231 +/- 72* versus 1994 +/- 447 pg/ml) and IL-6 (791 +/- 473* and 666 +/- 300* versus 3724 +/- 1233, pg/ml), accompanying a reduction in PMN accumulation (12 +/- 1* and 13 +/- 0.6* versus 20 +/- 1 mm2 myocardium) in ischemic myocardium. Both groups showed a reduction in expression of nuclear NFkappaB relative to Control (62 +/- 9* and 67 +/- 8* versus 124 +/- 16 arb.u), consistent with reduced NFkappaB binding activity. The number of apoptotic cells (%) in area at risk myocardium was comparably reduced in anti-PMN and NAC groups relative to Control (12 +/- 1* and 14 +/- 0.9* versus 20 +/- 1), consistent with reduced appearance of DNA ladders. Furthermore, activated caspase-3 was significantly reduced and Bcl-2 was increased relative to Control. No difference in all parameters measured was detected during the course of experiment in the Sham group. CONCLUSION: These data suggest that the oxidants generated from activated PMNs after ischemia/reperfusion trigger myocardial apoptosis, which is further supported by an anti-oxidant therapy with NAC, potentially mediated by enhanced NFkappaB-TNFalpha signaling pathway, activated caspase-3 and down-regulated Bcl-2. *P < 0.05 versus Control.     

姜黄素预处理对大鼠缺血再灌注肾小管上皮细胞凋亡的影响

目的：建立大鼠。肾脏缺血再灌注损伤（IRI）模型,观察姜黄素预处理对大鼠肾脏缺血再灌注肾小管上皮细胞凋亡的影响。方法：36只SD雄性大鼠随机分为3组,分别为假手术（Sham）组、肾脏缺血再灌注模型（IR）组、姜黄素预处理（CUR）组,每组12只。CUR组在缺血前2h给予姜黄素100mg／kg剂量溶于0．1％二甲基亚砜1ml中,注入腹腔。24小时后沿原切口进入,切除左。肾。肾组织用4％多聚甲醛固定24h,常规石蜡包埋切片。采用TUNEL法检测各组缺血肾小管上皮细胞凋亡。结果：与Sham组相比,IR组肾小管上皮细胞凋亡明显增加。与IR组比较,CUR组肾小管上皮细胞凋亡减少（P＜0．05）。结论：姜黄素预处理可减轻肾脏IRI的肾小管上皮细胞凋亡。     

Ischemic postconditioning inhibits apoptosis after renal ischemia/reperfusion injury in rat.

Ischemic postconditioning is a phenomenon that intermittent interruptions of blood flow in the early phase of reperfusion can protect organ from ischemia/reperfusion (I/R) injury. In the present study, we investigated whether the protective effect of ischemic postconditioning was associated with modulation of apoptosis after renal I/R injury. Rats were subjected to 45 min of renal ischemia, both with and without treatment with ischemic postconditioning. Serum urea nitrogen and creatinine levels, phosphorylation of Akt and ERK1/2 and apoptosis were compared after renal injury. Our data showed that ischemic postconditioning attenuated the renal dysfunction and cell apoptosis induced by I/R and increased phosphorylation of Akt and ERK1/2. The results indicated that ischemic postconditioning decreased apoptosis and improved renal function. This protective effect may be related with the levels of Akt and ERK1/2 activation. These findings may have major implications in the treatment of renal transplantation.     

Role of P-selectin expression in hepatic ischemia and reperfusion injury

Background. Researchers have shown that reperfusion of ischemic tissues initiates a complex series of reactions that paradoxically injure tissues. Although several mechanisms have been proposed to explain the pathobiology of ischemic/reperfusion (I/R) injury, much attention has focused on adhesion molecules. Our research is intended to show the kinetics of P-selectin in the liver in response to I/R injury.Methods. Left-lobar hepatic ischemia was induced for 30 min in 35 C57BL-6 mice and 20 P-selectin-deficient (K-O) mice. P-selectin expression was measured in these mice at 20 min, 2, 5, 12 and 24 h reperfusion times, as well as in control and sham animals. The animals were injected with radio-labeled P-selectin monoclonal antibody and the organs were harvested for counts/g tissue, expressed as the percentage injected dose. Serum liver enzymes were measured and pathological sections of ischemic and control livers were performed. The unpaired t -test was used for statistical analysis.Results. P-selectin expression showed two peaks in this animal model. The first peak was at 20 min and the second peak at 5 h of reperfusion (p<0.001). We documented an 8-fold increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels 10 h following I/R injury. Pathological specimens showed periportal necrosis consistent with an ischemic event. P-selectin K-O mice showed no up-regulation as a separate control group, and the liver enzymes were significantly lower than the wild-type mice at 10 h (p<0.001).Conclusion. P-selectin has a bimodal expression following hepatic I/R injury. The first peak is attributed to the Weibel–Palade bodies and the second to new translational P-selectin. We noted no difference in the up-regulation of P-selectin in the ischemic and non-ischemic liver lobes in the same animal.     

Effect of apigenin on apoptosis induced by renal ischemia/reperfusion injury in vivo and in vitro

Objectives: This study aims to investigate the effects and molecular mechanisms of apigenin (ApI) on renal ischemia/reperfusion (I/R) injury in vivo and in vitro.

Methods: In vivo, the left renal artery was clamped for 45?min and the right kidney was removed to study renal I/R injury on Sprague-Dawley (SD) rats. ApI was injected at 60?min before renal ischemia. In vitro, renal tubular epithelial cells (HK-2) were pretreated with or without ApI (20 uM) for 60?min and then treated with hypoxia/reoxygenation (H/R). Renal function, histology, cells apoptosis, and cell viability were tested. Furthermore, the potential molecular mechanisms were assessed.

Results: ApI pretreatment could significantly alleviated the renal function and the pathological damage as well as cells apoptosis after I/R injury. Meanwhile, ApI treatment protects H/R induced HK-2 cell apoptosis in vitro. The results of Western blot showed that ApI significantly increased the expressions of B-cell lymphoma 2 (Bcl-2) and phosphor-AKt (p-AKt), Phosphoinositide 3-kinase (PI3K), while down-regulated the expressions of Caspase3 and Bax induced by H/R injury.

Conclusions: ApI pretreatment can protect renal function against I/R injury and prevent renal tubular cells from apoptosis in vivo and in vitro which might through PI3K/Akt mediated mitochondria-dependent apoptosis signaling pathway.     

Role of leukotrienes on hepatic ischemia/reperfusion injury in rats

BACKGROUND: Leukotrienes (LT), composed of cysteinyl LT (cLT; LTC(4), LTD(4), and LTE(4)) and LTB(4), are potent lipid mediators enhancing the vascular permeability and recruitment of neutrophils, which are common features of hepatic ischemia/reperfusion (I/R) injury. The aim of this study was to investigate whether LT can mediate the liver and lung injuries following hepatic I/R. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to 90 min of partial hepatic ischemia followed by 3, 12, and 24 h of reperfusion. In the hepatic and pulmonary tissues, LT content and the mRNA expression of LT-synthesis enzymes, 5-lypoxygenase (5-LO), LTC(4) synthase (LTC(4)-S), and LTA(4) hydrolase (LTA(4)-H) were measured. Tissue injuries were assessed by plasma ALT, histological examination, and wet-to-dry tissue weight ratios. RESULTS: The cLT content in the hepatic tissue after 12 and 24 h reperfusion was increased 4- to 5-fold compared to controls and this was accompanied by the enhancement of hepatic edema and plasma ALT elevation. There were no significant changes in the mRNA expression of LT-synthesis enzymes in both tissues. LTB(4) levels were not increased despite a significant neutrophil infiltration in both tissues. CONCLUSIONS: These data suggest that cLT are generated in the liver during the reperfusion period and may contribute to the development of hepatic edema and exert cytotoxicity. Factors other than LTB(4) may contribute to neutrophil infiltration.     

Endothelin-1 levels in portal venous blood in relation to hepatic tissue microcirculation disturbance and hepatic cell injury after ischemia/reperfusion

This study was conducted to clarify the role of endothelin-1 in the portal vein after hepatic ischemia/reperfusion and to ascertain whether it is related to hepatic microcirculation disturbance. Using a canine ischemic liver model, the portal and systemic endothelin-1 levels were measured before ischemia, then after 1h and 2h of reperfusion, and comparatively evaluated with the serum levels of GOT and lactic dehydrogenase (LDH). As an indicator of liver tissue microcirculation, tissue blood flow volume (TBF) was also measured in the site subjected to ischemia. The animals were divided into: group 1, which received ischemia for 30 min; group 2, which received ischemia for 60 min; and group 3, which received a sequence repeated four times of 15 min ischemia and 10 min reperfusion. The portal endothelin-1 level became significantly elevated after reperfusion compared to that before ischemia in all groups, being significantly higher in group 2 than in the other groups. The systemic endothelin-1 level also increased after reperfusion; significantly in group 2. The portal endothelin-1 level was generally higher than the systemic level, which again was statistically significant in group 2. After 2h of reperfusion, a significant positive correlation was found between the portal endothelin-1 level and serum LDH, whereas a significant negative correlation was found between the portal endothelin-1 level and TBF. The finding that the portal endothelin-1 level became elevated after hepatic ischemia/reperfusion suggests that it probably plays an essential role in hepatic ischemia/ reperfusion injury by adversely influencing tissue microcirculation.     

异丙酚对大鼠脑缺血再灌注后脑细胞凋亡和Bcl-2、Caspase-3蛋白表达的影响

脑缺血再灌注后脑损伤的发病机制较复杂,其所致病理损伤包括细胞坏死和凋亡。细胞凋亡与脑缺血再灌注损伤关系密切,是神经元迟发性死亡的主要形式。研究提示与凋亡密切相关的Bcl-2、Caspase-3蛋白等参与了这种迟发性死亡,它们表达的量决定了细胞的生存。本实验采用流式细胞仪、