缺血预处理对大鼠缺血再灌注肝组织NF-κB表达、炎症及氧化应激反应的影响

目的：探讨缺血预处理（IP）减轻大鼠肝缺血再灌注（I/R）损伤的作用及机制。
　　方法：将15只雄性SD大鼠随机均分为假手术组、I/R组、IP+I/R组,采用Pringle法制作肝I/R模型（缺血30min+再灌注3h）,IP采用I/R前肝缺血10min+再灌注10min诱导。各组大鼠于再灌注3h后处死取材,行肝组织病理学、血请谷草转氨酶（AST）、谷丙转氨酶（ALT）检测,同时检测肝组织NF-κB蛋白的表达,以及炎性细胞因子IL-1β、TNF-α与氧化应激指标丙二醛（MDA）、髓过氧化物酶（MPO）水平。
　　结果：除假手术组外,I/R组与IP+I/R组大鼠肝组织均出现肝损伤是病理学改变,IP+I/R组的损伤程度明显轻于I/R组;与假手术组比较,I/R组与IP+I/R组大鼠血清AST、ALT水平明显升高,肝组织NF-κB蛋白表达、IL-1β与TNF-α水平、MDA与MPO浓度均明显升高（均P<0.05）,但IP+I/R组的各指标的升高幅度均明显小于I/R组（均P<0.05）。
　　结论：IP减轻大鼠肝I/R损伤的作用与抑制NF-κB活性,从而减轻炎症与氧化应激反应有关。     

牛磺熊去氧胆酸对大鼠肝脏缺血再灌注损伤的保护作用

目的：探讨牛磺熊去氧胆酸（TUDCA）抗大鼠肝脏缺血再灌注（HIRI）损伤的作用及机制。 方法：将20只雄性SD大鼠随机均分为假手术组、TUDCA组、HIRI组、TUDCA+HIRI组,分别行假手术、TUDCA+假手术、HIRI造模,TUDCA+HIRI造模;TUDCA（250 mg/kg）于术前1 h灌胃给予,HIRI造模采用Pringle法（缺血60 min,再灌注12 h）。再灌注12 h后处死各组大鼠取材,观察肝组织病理学改变,检测血清谷丙转氨酶（ALT）水平,TUNEL法检测肝细胞凋亡,Western blot技术检测肝组织内质网应激分子糖调节蛋白78（GRP78）、p-真核细胞翻译起始因子2α（p-eIF2α）和C-EBP同源蛋白（CHOP）的表达。 结果：除假手术组与TUDCA组外,HIRI组与TUDCA+HIRI组大鼠肝组织均出现明显肝损伤病理改变,但TUDCA+HIRI组的损伤程度明显轻于HIRI组。与假手术组比较,HIRI组与TUDCA+HIRI组大鼠血清ALT水平明显升高,肝细胞凋亡和内质网应激分子GRP78、p-eIF2a和CHOP蛋白水平均明显升高（均P<0.05）,但TUDCA+HIRI组各项指标升高幅度均明显低于HIRI组（均P<0.05）;TUDCA组各项指标未见改变（均P>0.05）。 结论：TUDCA有抗大鼠肝HIRI的作用,其机制可能与抑制内质网应激反应有关。     

白藜芦醇对肝脏缺血再灌注损伤的保护作用

摘要：探讨白藜芦醇对肝脏缺血再灌注损伤的防护作用。将雄性SD大鼠随机分为空白对照组、缺血再灌注组（I/R组）、I/R加生理盐水处理组和I/R加白藜芦醇处理组。观察肝脏缺血40 min再灌注1，3，6，12h后血清谷丙转氨酶（ALT）、谷草转氨酶（AST）及肝组织丙二醛（MDA）含量的变化以及肝组织病理学改变。结果示肝脏I/R后血清ALT，AST及肝组织MDA含量均显著升高，肝脏缺血再灌注前用白藜芦醇15 mg/kg者，血清ALT，AST及肝组织MDA含量均明显降低，且肝组织病理学损害明显减轻。结果表明白藜芦醇对肝脏I/R损伤具有保护作用     

瑞芬太尼对肝硬化大鼠肝脏缺血再灌注损伤的影响

目的 探讨瑞芬太尼对肝硬化大鼠肝脏缺血再灌注损伤的影响.方法 成年健康雄性SD大鼠30只,体重260～300 g,采用随机数字表法,将其随机分为3组(n=10):肝硬化组(C组)、肝硬化+肝缺血再灌注组(I/R组)和瑞芬太尼组(R组).C组、I/R组和R组采用四因素综合法制备大鼠肝硬化模型,I/R组和R组在肝硬化模型制备成功后1周制备大鼠70％肝脏缺血再灌注模型,R组于缺血前10 min开始静脉输注瑞芬太尼1μg·kg-1·min-至再灌注结束.于再灌注4h时取静脉血样和肝组织,测定血清ALT和AST活性、肝细胞Bcl-2和Bax表达及肝细胞凋亡情况,计算细胞凋亡指数,光镜下观察肝组织病理学结果.结果 与C组比较,I/R组血清ALT和AST的活性升高,肝细胞Bcl-2表达下调,Bax表达上调,细胞凋亡指数升高(P＜0.05);与I/R组比较,R组血清ALT和AST的活性降低,肝细胞Bcl-2表达上调,Bax表达下调,细胞凋亡指数降低(P＜0.05).R组肝组织病理学损伤轻于I/R组.结论 瑞芬太尼可减轻肝硬化大鼠肝脏缺血再灌注损伤,其机制与平衡肝细胞Bcl-2与Bax表达而抑制肝细胞凋亡有关.     

p38MAPK信号通路在瑞芬太尼或缺血预处理减轻大鼠肝缺血再灌注损伤中的作用

目的 评价p38丝裂原蛋白激酶(p38MAPK)信号通路在瑞芬太尼或缺血预处理减轻大鼠肝缺血再灌注损伤中的作用.方法 健康SD大鼠l44只,雌雄不拘,体重200 ～ 250 g,采用随机数字表法,将其随机分为6组(n=24):假手术组(S组);肝缺血再灌注组(I/R组)采用动脉夹夹闭左叶和中叶肝蒂30 min,恢复灌注的方法制备大鼠肝缺血再灌注模型;瑞芬太尼组(R组)于缺血前30 min静脉输注瑞芬太尼2μg·kg-1·min-1至再灌注120 min;缺血预处理组(IPC组)于缺血前30 min行缺血5 min,再灌注5 min,重复3次后制备缺血再灌注模型;SB+R组和SB+ IPC组分别于输注瑞芬太尼或缺血预处理前5 min静脉注射p38mAPK特异性抑制剂SB203580 0.2 mg/kg,其余组给予等体积生理盐水.于再灌注30、60、90和120 min时各组分别随机取6只大鼠抽取肝下腔静脉血测定血清ALT和AST活性;采用ELISA法测定TNF-α及IL-1β浓度;随后处死大鼠,取肝组织,采用Western blot法测定磷酸化p38MAPK的表达,观察病理学结果.结果 与S组相比,I/R组各时点血清ALT、AST、TNF-α及IL-1β水平升高(P＜0.05),病理学损伤明显加重;与I/R组相比,RPC组和IPC组血清ALT和AST活性、TNF-α及IL-1β浓度降低,再灌注90 min时磷酸化p38MAPK表达上调(P＜0.05),SB+ RPC组和SB+ IPC组各指标差异无统计学意义(P＞0.05);SB+ RPC组与RPC组,SB+ IPC组和IPC组相比,血清ALT、AST、TNF-α及IL-1β水平升高,再灌注90 min时磷酸化p38MAPK表达下调(P＜0.05),病理学损伤明显加重.结论 瑞芬太尼和缺血预处理可减轻大鼠肝缺血再灌注损伤,其机制可能与激活p38MAPK信号通路抑制炎性反应有关.     

苦参碱对大鼠肝缺血再灌注损伤的抑制作用及机制

目的:探讨苦参碱对大鼠肝缺血再灌注损伤的抑制作用及机制。方法:大鼠肝缺血再灌注损伤模型采用阻断肝血流60 min再灌注120 min诱导;将40只SD大鼠随机均分为假手术组、肝缺血再灌注损伤模型组(模型组)、低剂量苦参碱(25 mg/kg)预处理+肝缺血再灌注损伤模型组(低剂量苦参碱组)、高剂量苦参碱(50 mg/kg)预处理+肝缺血再灌注损伤模型组(高剂量苦参碱组),低、高剂量苦参碱组在肝缺血前30 min,经门静脉主干注入各自剂量的苦参碱溶液,假手术组与模型组大鼠则以相同的方式注入等体积的生理盐水;120 min再灌注结束后,收集各组大鼠血标本行血清转氨酶、炎症因子水平检测,收集肝组织标本行组织病理学、肝细胞凋亡检测,以及TRAIL、BAX、激活型caspase-3(cleaved caspase-3)蛋白表达检测。结果:组织病理学检测结果显示,除假手术组外,其余各组肝组织均有肝损伤表现,但损伤程度轻重不一(模型组低剂量苦参碱组高剂量苦参碱组)。与假手术组比较,其余各组血清转氨酶、炎症因子水平均明显升高,肝组织中肝细胞凋亡明显增加,TRAIL、BAX、cleaved caspase-3蛋白表达明显上调(均P0.05),但以上指标的变化程度在低、高剂量苦参碱组均明显小于模型组,且在高剂量苦参碱组更为明显(均P0.05)。结论:苦参碱对大鼠肝缺血再灌注损伤具有抑制作用,机制可能与其抑制TRAIL的表达,减少BAX与caspase-3的活化,从而抑制肝细胞凋亡有关。     

高渗氯化钠羟乙基淀粉40注射液高容量血液稀释对大鼠肝脏缺血再灌注损伤的影响

目的 探讨高渗氯化钠羟乙基淀粉40注射液高容量血液稀释对大鼠肝脏缺血再灌注损伤的影响.方法 雄性Wistar大鼠30只,体重300～350 g,随机分为3组(n=10):假手术组(S组)、缺血再灌注组(IR组)和高容量血液稀释组(HH组).S组仅开腹,不阻断血管;IR组阻断肝门静脉和左肝动脉30 min,再灌注2 h;HH组30 min内经尾静脉输注高渗氯化钠羟乙基淀粉40注射液10 ml/kg进行高容量血液稀释,输注完毕后15 min,行肝脏缺血再灌注.再灌注2 h时,下腔静脉取血样,测定血清谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性;取左肝叶组织,光镜下观察病理学结果,采用比色法测定丙二醛(MDA)含量,采用黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性.结果 与S组比较,IR组和HH组血清ALT和AST的活性、肝组织MDA含量升高,肝组织SOD活性降低(P<0.01),肝组织病理学损伤明显;与IR组比较,HH组血清ALT和AST的活性、肝组织MDA含量降低,肝组织SOD活性升高(P<0.01),肝组织病理学损伤减轻.结论 高渗氯化钠羟乙基淀粉40注射液高容量血液稀释可减轻大鼠肝脏缺血再灌注损伤,可能与氧自由基生成减少有关.     

缺血后处理对大鼠肝缺血再灌注时肝细胞线粒体损伤的影响

目的 评价缺血后处理对大鼠肝缺血再灌注时线粒体损伤的影响.方法 雄性SD大鼠30只,体重180～230 g,随机分为3组(n=10):假手术组(S组)、肝缺血再灌注组(IR组)和缺血后处理组(Ipo组).IR组和Ipo组采用阻断肝门60 min再灌注6 h的方法 制备肝缺血再灌注模型,Ipo组缺血60 min时再灌注10 s、缺血10 s,反复6次,进行缺血后处理.于再灌注6 h时取静脉血样,测定血清谷丙转氨酶(ALT)及天门冬氨酸氨基转移酶(AST)活性,然后取肝组织,制备病理切片及分离肝细胞,电镜下观察线粒体超微结构,测定线粒体膜电位及线粒体Na+-K+-ATP酶活性.结果 与S组比较,IR组和Ipo组血清ALT和AST活性升高,线粒体Na+-K+-ATP酶活性及线粒体膜电位降低(P<0.01);与IR组比较,Ipo组血清ALT和AST活性降低,线粒体Na+-K+-ATP酶活性及线粒体膜电位升高(P<0.05或0.01).Ipo组线粒体损伤程度轻于IR组.结论 缺血后处理可减轻大鼠肝缺血再灌注时肝细胞线粒体损伤.     

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.     

异氟醚预处理对大鼠肝脏缺血再灌注损伤的影响

目的 探讨异氟醚预处理对大鼠肝脏缺血再灌注损伤的影响.方法 成年雄性SD大鼠24只,体重180～220 g,随机分为3组(n=8):假手术组(S组)吸人纯氧30 min,间隔30 min后仅开腹;肝脏缺血再灌注组(IR组)吸入纯氧30 min,间隔30 min后行肝脏缺血60 min,再灌注4 h;异氟醚预处理组(Iso组)吸入1.4%异氟醚30 min,间隔30 min后行肝脏缺血60 min,再灌注4 h.于再灌注4 h时处死大鼠,留取肝脏及腹主动脉血5ml.测定血清谷丙转氨酶(ALT)和谷草转氨酶(AST)浓度,血清及肝组织匀浆上清液中肿瘤坏死因子α(TNF-α)的浓度,肝组织髓过氧化物酶(MPO)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,观察肝组织病理学改变.结果 与S组比较,IR组、Iso组血清ALT、AST和TNF-α水平明显升高,肝组织TNF-α含量升高,肝组织MPO活性升高,MDA含量升高,SOD活性降低(P＜0.05或0.01),肝组织病理损伤明显;与IR组比较,Iso组血清ALT、AST和TNF-α水平降低,肝组织TNF-α含量降低,肝组织MPO活性降低,MDA含量降低,SOD活性升高(P＜0.05或0.01),肝组织病理损伤程度减轻.结论 1.4%异氟醚预处理可明显减轻大鼠肝脏缺血再灌注损伤,其机制可能与抑制TNF-α的释放、减少中性粒细胞在肝组织的浸润有关.     

Ischemic Preconditioning Protects the Pig Liver by Preserving the Mitochondrial Structure and Downregulating Caspase-3 Activity

Summary Background Data: The beneficial effects of ischemic preconditioning (IPC) on hepatic ischemia-reperfusion injury (I/RI) have been described. However, the way in which IPC causes the changes in mitochondrial ultrastructure seen in hepatic I/RI is not well understood. Objective: The objective of the present study was to determine whether IPC protects the liver from changes in mitochondrial structure and caspase 3 activity in the early phase of post-ischemic injury. Methods: A pig model consisting of 90 min of hepatic ischemia and 180 min of reperfusion was employed. Eighteen female pigs were randomly divided into three groups: sham-operated, non-preconditioned, and ischemic preconditioned (10 min ischemia followed by 10 min reperfusion). Serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and thiobarbituric acid reactive substances (TBARS), as well as bile flow, were measured. Liver biopsies were taken after reperfusion for histological, immunohistochemical (anti-caspase 3), and ultrastructural examinations. Results: The IPC procedure increased bile flow (p < 0.01), reduced serum AST level (p < 0.01), and reduced serum concentration of TBARS at 180 min of reperfusion (p = 0.05). Ischemic-preconditioned liver cells had less caspase 3 activity than the non-preconditioning group (p < 0.01), and changes in mitochondrial ultrastructure were reduced (p < 0.01). Conclusion: IPC exerts a powerful protective effect against hepatic I/RI in the early phase of reperfusion, which may be mediated by preservation of mitochondrial structure and inhibition of caspase-3 activity.     

氨溴索对大鼠肝脏缺血再灌注损伤的保护作用

目的：探讨氨溴索对大鼠肝脏缺血再灌注损伤（I/RI）的保护作用及其机制。方法：18只雄性Wistar大鼠被随机均分为假手术组、肝I/RI模型组（模型组）、氨溴索预处理+肝I/RI模型组（氨溴索预处理组）。肝I/RI模型采用阻断入肝血流30 min后再灌注方法诱导,氨溴索预处理组于缺血前20 min尾静脉注射氨溴索（100 mg/kg）,而模型组给予等体积生理盐水。术后6 h处死大鼠,检测血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）水平和肝组织病理学改变,同时检测肝组织超氧化物岐化酶（SOD）,谷胱甘肽（GSH）,丙二醛（MDA）含量,caspase-3的活化水平。结果：与假手术组比较,模型组与氨溴索预处理组血清ALT和AST水平明显升高（均P<0.05）;肝组织出现明显的病理学改变;肝组织SOD和GSH含量明显下降,而MDA水平明显升高（均P<0.05）;肝组织caspase-3活化水平明显升高（均P<0.05）。与模型组比较,氨溴索预处理组以上各项指标的变化均明显减弱（均P<0.05）。结论：氨溴索预处理能减轻大鼠肝脏I/RI,机制可能与其调控抗氧化和抗凋亡信号通路有关。     

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.     

Pentoxifylline contributes to the hepatic cytoprotective process in rats undergoing hepatic ischemia and reperfusion injury.

AIM: Considerable efforts have been made to find and/or eliminate the underyling causes of hepatic ischemia-reperfusion injury, but many points are still unclear. Pentoxifylline-related cytoprotection is one of these unclear points. Our study tests the effects of pentoxifylline on the hepatic cytoprotective process in an experimental model. MATERIALS AND METHODS: The animals were divided into two groups: (1) placebo-pretreated rats and (2) pentoxifylline-pretreated rats. After pretreatment, all rats underwent the hepatic ischemia-reperfusion procedure which was performed by clamping the hepatoduodenal ligament. To evaluate the liver injury, serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), and liver tissue levels of prostaglandin E(2) (PGE(2)) were measured before ischemia, immediately after ischemia and immediately after reperfusion. RESULTS: Before ischemia and immediately after ischemia, there were no significant differences between ALT and AST levels of groups 1 and 2 (p >0.05). However, at the end of reperfusion, ALT and AST levels of group 2 were significantly decreased when compared with group 1 (p < 0.05 and p < 0.01, respectively). Additionally, tissue levels of PGE(2) that were obtained before ischemia, immediately after ischemia and immediately after reperfusion in group 2 were significantly higher than those of group 1 (p < 0.001). CONCLUSION: Pentoxifylline reduces reperfusion injury of the liver through significantly decreased transaminase levels, and contributes to hepatic cytoprotection by increasing tissue levels of PGE(2) significantly. These effects reflect the role of tissue PGE(2) in pentoxifylline-related hepatoprotection against ischemia-reperfusion injury of the liver.     

缺血后处理减轻肠缺血再灌注引起的肝损伤的机制研究

目的：探讨缺血后处理减轻肠缺血再灌注引起的肝损伤的作用机制,为外科防治缺血再灌注损伤提供策略。方法将36只SD大鼠随机分为假手术组（SO组,仅手术显露肠系膜上动脉）、缺血再灌注组（IR组,阻断肠系膜上动脉60min,再灌注120min）、缺血后处理组（IP组,阻断肠系膜上动脉60min后行3个循环的灌注30s/阻断30s,再持续灌注117min）,每组12只。建立模型2h后采集各组大鼠动、静脉血及部分小肠、肝组织,检测血肿瘤坏死因子α（TNF-α）、白细胞介素10（IL-10）、丙氨酸氨基转氨酶（ALT）、天门冬氨酸氨基转移酶（AST）水平,测定血清及肝组织内丙二醛（MDA）、髓过氧化酶（MPO）水平,光学显微镜下观察小肠及肝脏病理学改变,免疫组织化学法检测肝脏组织中核因子κBp65（NF-κBp65）和缺氧诱导因子1α（HIF-1α）的表达变化。结果与SO组比较,IR组小肠、肝脏病理损伤加重,肝组织NF-κBp65和HIF-1α的表达显著升高,血清和肝组织中MDA、MPO水平及血清TNF-α、IL-10、ALT和AST水平升高;与IR组比较,IP组小肠、肝脏损伤减轻,肝组织NF-κBp65表达下降而HIF-1α的表达显著升高,血清和肝组织中MDA、MPO水平及血清TNF-α、ALT和AST水平均显著下降,血清IL-10水平增加,差异均有统计学意义（P＜0.05）。结论缺血后处理可以促进抗炎因子的激活,抑制NF-κB信号通路调控的炎症级联反应,上调HIF-1α的表达,减轻小肠缺血再灌注引起的肝损伤。     

缺血后处理对大鼠肝缺血再灌注时肝细胞线粒体膜通透性转换和膜电位的影响

目的 评价缺血后处理对大鼠肝缺血再灌注时肝细胞线粒体膜通透性转换和膜电位(△Ψm)的影响.方法 成年健康雄性SD大鼠40只,体重220～260 g,采用随机数字表法,将其随机分为5组(n=8):假手术组(S组)、苍术苷+假手术组(A+S组)、缺血再灌注组(IR组)、缺血后处理组(IPO组)和苍术苷+缺血后处理组(A+IPO组).采用阻断肝中叶和左叶60 min,恢复血流灌注6 h的方法 建立大鼠肝缺血再灌注模型.S组和A+S组仅游离肝门,不阻断血管;A+S组关腹前静脉注射苍术苷5 mg/kg;IR组制备肝缺血再灌注模型;IPO组于再灌注前行缺血后处理,再灌注1 min,缺血1 min,反复3次;A+IPO组于再灌注前静脉注射苍术苷5 mg/kg.于缺血前即刻和再灌注6 h时,采集左颈静脉血样,测定血清ALT和AST的活性.再灌注6 h时处死大鼠,取肝左叶组织,观察超微结构和细胞凋亡情况,计算凋亡指数,测定细胞色素c(Cyt c)的表达水平、△Ψm和线粒体通透性转换孔(MPTP)活性.结果 与S组比较,A+S组时血清ALT和AST的活性、凋亡指数、Cyt c表达、△Ψm和MPTP活性差异无统计学意义(P＞0.05),IR组、IPO组和A+IPO组再灌注6 h时血清ALT和AST的活性、凋亡指数升高,Cyt c表达上调,△Ψm降低,MPTP活性升高(P＜0.05);与IR组比较,IPO组血清ALT和AST的活性、凋亡指数降低,Cyt c表达下调,△Ψm升高,MPTP活性降低(P＜0.05),肝组织病理学损伤减轻,A+IPO组各指标差异无统计学意义(P＞0.05);与IPO组比较,A+IPO组血清ALT和AST的活性、凋亡指数升高,Cyt c表达上调,△Ψm降低,MPTP活性升高(P＜0.05),肝组织病理学损伤加重.结论 缺血后处理可抑制肝细胞线粒体膜通透性转换,减少线粒体△Ψm的耗散,从而减轻大鼠肝缺血再灌注损伤.

Abstract：

Objective To investigate the effects of ischemic postconditioning on mitochondrial permeability transition and mitochondrial transmembrane potential(△Ψm)following hepatic ischemia-reperfusion(I/R)in rats.Methods Forty male SD rats weighing 220-260 g were randomly divided into 5 groups with 8 animals in each group:sham operation group(group S);atractyloside+sham operation group(group A+S);I/R group;ischemic postconditioning group(group IPO)and atractyloside+ischemic postconditioning group(group A+IPO).The animals were anesthetized with intramuscular injection of atropine 0.05 mg/kg.Hepatic I/R was produced by occlusion of hepatic blood flow for 60 min followed by 6 h reperfusion.In group A+S,atractyloside 5 mg/kg was injected intravenously before abdomen Was closed.In group IPO,the animals were subjected to 3 cycles of 1 min reperfusion interspersed with 1 min hepatic isehemia at the end of 60 min hepatic ischemia.In group A+IPO,atractyloside 5 mg/kg was injected intravenously before reperfusion. Venous blood samples were collected for determination of serum ALT and AST activities immediately before ischemia and at 6 h of reperfusion. The animals were then sacrificed.Their livers were removed for microscopic examination, detection of apoptosis and determination of cytochrome c (Cyt c) expression, △Ψm and mitochonerial permeability transition pore (MPTP)activity. Apoptosis index (AI) was calculated. Results There was no significant difference in serum ALT and AST activities, AI, Cyt c expression, △Ψm and MPTP activity between S and A + S groups (P＞0.05). Compared with group S, serum ALT and AST activities and AI were significantly increased, Cyt c expression was up-regulated, △Ψm was decreased and MPTP activity was increased in groups I/R, IPO and A+IPO(P＜0.05).Compared with group I/R, serum ALT and AST activities and AI were significantly decreased,Cyt c expression was down-regulated, △Ψm was increased and MPTP activity was decreased in group IPO(P＜0.05), while no significant change was found in group A+IPO(P＞0.05).Compared with group IPO,serum ALT and AST activities and AI were significantly increased, Cyt c expression was up-regulated, △Ψm was decreased and MPTP activity was increased in group A + IPO(P＜ 0.05).Microscopic examination showed that hepatic injury was reduced in group IPO compared with group I/R, while aggravated in group A+ IPO compared with group IPO. Conclusion Ischemic postconditioning can protect liver from I/R injury by attenuating the I/R-induced increase in MPTP opening and decrease in △Ψm in rats.     

缺血预处理和Caspase抑制剂对缺血再灌注损伤保护作用的比较

目的　比较缺血预处理和Caspase抑制剂治疗对大鼠肝缺血再灌注的保护作用及其机制。方法　SD大鼠随机分为 6组 :( 1)缺血再灌注1 (IR1 )组 ;( 2 )IR2 组 ;( 3 )缺血预处理1 (IP1 )组 ;( 4 )IP2 组 ;( 5 )Caspase抑制剂治疗1 (T1 )组 ;( 6)T2 组。比较各组大鼠 70 %肝脏 60min或 12 0min缺血 ,在再灌注 3h时的肝组织Caspase 3活性 ,肝细胞凋亡率和血清AST和ALT水平 ,及实验动物 7d存活率。结果　在 60min缺血及 3h再灌注时间 ,IP1 组和T1 组的保护作用相同 ,在 12 0min缺血及 3h再灌注时 ,T2 组对Caspase活性和肝细胞凋亡的抑制优于IP2 组 (P <0 .0 1) ,但两者的AST和ALT水平及动物 7d存活率均无显著性差异。结论　缺血预处理和Caspase抑制剂治疗对鼠肝缺血再灌注损伤都有保护作用 ,两者的保护效果无显著性差异。缺血预处理对缺血再灌注损伤的保护更简便、经济、安全 ,临床应用前景十分广阔。     

缺血预处理减轻大鼠肝缺血再灌注损伤的免疫机制研究

目的探讨大鼠肝缺血再灌注损伤(HIRI)的免疫机制和缺血预处理(IPC)的保护作用。 方法80只大鼠被随机分为假手术组(A组)、肝门阻断20 min组(B组)、30 min组(C组)、40 min组(D组)以及肝门阻断30 min前预处理组(E组),每组再分为再灌注2 h亚组和24 h亚组,各8只。检测再灌注后2 h、24 h的血丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、白细胞介素10、12(IL-10、IL-12)以及外周血T淋巴细胞亚群的水平,观察再灌注后2 h、24 h时的存活率及肝脏病理情况。 结果随着肝门阻断时间的延长,ALT、AST显著升高,肝内炎症细胞浸润增加,24 h存活率逐渐降低。D组再灌注2 h时,CD8+ T淋巴细胞显著升高,CD4+/CD8+比值下降,调节性T淋巴细胞显著减少,血清IL-10显著降低,而IL-12水平显著升高。再灌注后24 h,B组大鼠各项指标逐步恢复至假手术组水平,而D组大鼠CD4+T淋巴细胞、CD4+/CD8+比值尤其是Treg显著升高,且IL-10水平显著升高,IL-12水平明显降低。与C组相比,E组阻断30 min后无一例死亡,再灌注2 h的ALT、AST水平显著降低,Treg和IL-10水平显著升高,IL-12水平明显降低,再灌注24 h后各项指标恢复并接近假手术组水平,肝脏病理损伤较轻。 结论肝缺血再灌注引起肝脏损伤甚至死亡,可能与诱导T淋巴细胞尤其是Treg和细胞因子的紊乱有关。缺血预处理可以增加再灌注早期的Treg细胞,有效纠正免疫紊乱,减轻损伤。