大鼠局灶性脑缺血预处理后活化转录因子6 mRNA及其蛋白表达的变化

目的观察脑缺血预处理对大鼠脑缺血再灌注后活化转录因子6(ATF6)mRNA及其蛋白表达的影响。方法选择SD大鼠120只,随机分为假手术组、缺血再灌注组、缺血预处理组,每组40只,每组又按照再缺血后12h,1、2、3d分为4个时间点,每个时间点10只。采用二次线栓法制备大鼠局灶性脑缺血预处理模型,实时荧光定量PCR和免疫组织化学法观察再缺血后各个时间点ATF6mRNA及其蛋白的表达变化。结果与假手术组比较,缺血再灌注组ATF6mRNA及其蛋白表达均于缺血再灌注后12h开始明显上升,1d达高峰,随再灌注时间延长其表达逐渐下降,但仍保持较高表达水平(P<0.05,P<0.01);缺血预处理组各时间点ATF6mRNA及其蛋白表达水平较缺血再灌注组明显升高(P<0.05)。结论脑缺血预处理可能通过诱导ATF6表达发挥其神经保护作用。     

缺血预处理对大鼠缺血再灌注后心房肌Cx43和Cx40的影响

目的 探讨缺血预处理对大鼠缺血再灌注后的心房肌缝隙连接蛋白43(Cx43)和缝隙连接蛋白40(Cx40)表达和分布的影响。方法 30只Wistar大鼠随机分为假手术组(或对照组,n=5)：只穿线不结扎左冠状动脉前降支。缺血再灌注组(I/R组,n=5)：给予前降支结扎30 min,再灌注120 min。早期缺血预处理组(IPC组,n=5)：行IPC处理后,余处理同I/R组。延迟IPC组(L-IPC组,n=5)：在IPC处理24h后,余处理同I/R组。早期远程缺血预处理组(RIPC组,n=5)：给予RIPC后,余处理同I/R组。延迟RIPC组(L-RIPC组,n=5)：RIPC处理24h后,余处理同I/R组。测量心房组织Cx40、43的mRNA表达、Cx40、43蛋白表达以及用免疫组化法测定Cx40、43的分布。结果 I/R组Cx43和Cx40在mRNA水平和蛋白水平均明显降低,分布无规律且侧面分布相对增加。而各种IPC方式(IPC、L-IPC、RIPC、L-RIPC)在I/R后,心房Cx43和Cx40mRNA水平和蛋白水平下降不明显,其分布多于心肌细胞闰盘处,仅少量分布于心肌细胞侧面。结论 IPC能维持I/R后的心房肌Cx43和Cx40的较高表达,并维持其空间分布相对稳定。     

缺血预处理对大鼠脑缺血再灌注损伤神经细胞的保护作用

目的探讨大鼠局灶性脑缺血预处理对脑缺血再灌注损伤后神经元的保护作用。方法健康雄性SD大鼠60只,随机分为3组:假手术组、大脑中动脉缺血再灌注(MCAO)组、预处理(BIP)组,每组按照再灌注后12 h、1、2、3 d四个时间点平均分为4个亚组,制备缺血预处理模型,分别用流式细胞术和ELISA法观察脑缺血预处理对缺血再灌注大鼠缺血半暗带神经细胞凋亡率及血清神经元特异性烯醇化酶(NSE)含量的影响。结果大鼠脑缺血再灌注后12 h,MCAO组细胞凋亡发生率及血清中NSE的含量较假手术组显著增加(P<0.01),1 d时达到高峰,以后时间点逐渐下降,但仍高于假手术组(P<0.01);BIP组各个时间点神经元凋亡发生率及血清NSE较MCAO组显著降低(P<0.05,P<0.01)。结论大鼠局灶性脑缺血预处理对脑缺血再灌注神经元损伤有保护作用。     

Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factor-induced P-selectin up-regulation in the rat

Previous studies indicate that ischemic preconditioning protects against lung injury resulting from hepatic ischemia-reperfusion (I/R) through inhibition of tumor necrosis factor (TNF) release from Kupffer cells. The present study investigated whether this effect is limited to the lung or is a generalized systemic response and explores the molecular mechanisms involved. Hepatic I/R led to an increase in neutrophil accumulation in liver, lung, and splanchnic organs. Although preconditioning did not modify neutrophil infiltration in liver during reperfusion, it conferred protection against hepatic injury associated with I/R. In remote organs, preconditioning abrogated the increase in P-selectin up-regulation, preventing neutrophil infiltration and thus reducing the oxidative stress and microvascular disorders following hepatic I/R in these organs. Administration of Abs against P-selectin or TNF previous to ischemia had the same effects as preconditioning. The effects of preconditioning on the blockade of P-selectin up-regulation probably results from inhibition of systemic TNF release from Kupffer cells. Supplementation of TNF abolished the benefits of preconditioning, whereas the injurious effects of TNF were prevented by previous blockade of P-selectin. The results of the present study suggest that ischemic preconditioning protects the liver against I/R injury by a mechanism independent of adhesion molecule expression and neutrophil accumulation. In remote organs, however, hepatic preconditioning prevents inflammatory damage by reducing the systemic TNF release from the liver and thus preventing P-selectin up-regulation.     

Effects of ischemic preconditioning on cyclinD1 expression during early ischemic reperfusion in rats

AIM: To observe the effect of ischemic preconditioning on cyclinD1 expression in rat liver cells during early ischemic reperfusion. METHODS: Fifty-four SD rats were randomly divided into ischemic preconditioning group (IP), ischemia/ reperfusion group (IR) and sham operation group (SO). The IP and IR groups were further divided into four sub-groups (n - 6). Sham operation group (SO) served as the control group (n = 6). A model of partial liver ischemia/reperfusion was used, in which rats were subjected to liver ischemia for 60 min prior to reperfusion. The animals in the IP group underwent ischemic preconditioning twice for 5 min each time prior to the ischemia/reperfusion challenge. After 0, 1, 2, and 4 h of reperfusion, serum and liver tissue in each group were collected to detect the level of serum ALT, liver histopathology and expression of cyclinDi mRNA and protein. Flow cytometry was used to detect cell cycle as the quantity indicator of cell regeneration. RESULTS: Compared with IR group, IP group showed a significantly lower ALT level in 1 h to 4 h sub-groups (P < 0.05). Proliferation index(PI) indicated by the S-phase and G2/M-phase ratio [(S G2/M)/(G0/G1 S G2/M)] was significantly increased in IP group at 0 and 1 h (26.44±7.60% vs 18.56±6.40%,41.87±7.27% vs 20.25±6.70%, P < 0.05). Meanwhile, cyclinDi protein expression could be detected in IP group. But in IR group, cyclinDi protein expression occurred 2 h after reperfusion. The expression of cyclinDi mRNA increased significantly in IP group at 0 and 1 h (0.568±0.112 vs 0.274±0.069, 0.762±0.164 vs 0.348±0.093, P < 0.05). CONCLUSION: Ischemic preconditioning can protect liver cells against ischemia/reperfusion injury, which may be related to cell proliferation and expression of cyclinD1 during early ischemic reperfusion.     

蛋白酶激活受体2对大鼠缺血预处理心肌NF-κBp65和C-myc表达的影响

目的 探讨蛋白酶激活受体2（PAR-2）预处理对缺血预处理（IPC）大鼠心肌细胞核因子（NF-κBp65）和凋亡相关基因C-myc表达的影响及其机制.方法 60只雄性SD大鼠随机分为正常对照组、缺血再灌注组（I/R）、缺血预处理组（IPC）和PAR-2低剂量组（0.5 mg/kg）、高剂量组（3.0 mg/kg）.应用Langendroff离体灌流装置,采用完全停灌复灌的方法制作离体大鼠心肌缺血再灌注模型,采用Western blot法检测大鼠心肌组织NF-κBp65和C-myc的表达水平.结果 与正常对照组比较,I/R组心肌损害程度增加,NF-κBp65和C-myc表达显著增加（83.33%比8.33%,P＜0.01）;与I/R组比较,IPC组NF-κBp65和C-myc表达显著降低（58.33%比83.33%,P＜0.05）;与IPC组比较,PAR-2低剂量组NF-κBp65和C-myc表达明显降低（41.67%比58.33%,P＜0.05）;PAR-2高剂量组与低剂量组比较,NF-κBp65和C-myc表达差异有统计学意义（25.00%比41.67%,P＜0.01）.结论 PAR-2预处理具有明显降低大鼠缺血再灌注后心肌细胞凋亡的作用,能进一步抑制缺血预处理心肌细胞凋亡.其机制可能与PAR-2预处理抑制NF-κBp65和C-myc参与的细胞凋亡有关.     

大鼠局灶性脑缺血预处理后生长停滞与DNA损害可诱导基因34表达的变化

目的观察脑缺血预处理(brain ischemia preconditioning,BIP)对大鼠脑缺血再灌注后,生长停滞与DNA损害可诱导基因34(GADD34)mRNA及蛋白表达的影响。方法健康雄性SD大鼠120只,随机分为假手术组、脑缺血组、BIP组,每组40只,后2组行大脑中动脉栓塞法制模后,各组于再缺血后12 h、1、2和3 d 4个时间点观察,每个时间点10只。采用2次线栓法制备大鼠局灶性脑缺血预处理模型,用原位杂交法检测GADD34 mRNA表达,western blot法观察各组GADD34蛋白表达。结果与假手术组比较,脑缺血组12 h GADD34 mRNA及蛋白表达均达高峰,随再灌注时间延长,其表达逐渐下降,但1、2和3 d时仍高于假手术组(P<0.05,P<0.01);BIP组GADD34 mRNA及蛋白表达各时间点均较脑缺血组明显升高(P<0.05,P<0.01)。结论 BIP可诱导GADD34mRNA及蛋白的表达。     

缺血后处理对大鼠心室肌细胞内向整流钾电流和动作电位的影响

目的探讨缺血后处理(IPC)对大鼠心室肌细胞内向整流钾电流(IK1)和动作电位的影响及机制。方法实验分IPC组、缺血/再灌注(I/R)组、单纯灌流(SP)组,利用大鼠心脏建立离体灌注模型,采用胶原酶酶解法分离得到大鼠单心室肌细胞,采用膜片钳全细胞技术分别记录三组心室肌细胞电流和动作电位的变化。结果在-120mV和-30mV刺激电压水平,IK1电流密度:IPC组低于I/R组,I/R组高于SP组(P均<0.05),IPC组同SP组无差异(P>0.05)。与SP组和IPC组比较,I/R组静息电位明显升高(P<0.05);而动作电位幅度、20%动作电位时程、50%动作电位时程、90%动作电位时程均明显下降(P<0.05)。而IPC组各值与SP组无差异(P>0.05)。结论IPC可以降低大鼠缺血/再灌注心肌细胞IK1,延长缺血/再灌注心肌细胞APD。     

心肌缺血预处理延迟保护作用的实验研究

本实验旨在探讨心肌缺血预处理延迟保护的作用机制。采用大鼠心脏缺血预处理模型,检测缺血预处理后即刻、１２、２４、４８和７２ｈ各时相点再行心肌缺血１ｈ复灌１２ｈ时心肌细胞间粘附分子（ＩＣＡＭ－１）的表达及心肌梗塞范围、超氧化物歧化酶（ＳＯＤ）含量及白细胞（ＰＭＮｓ）浸润数的变化。结果显示,与单纯缺血再灌注组比较,预处理后２４～７２ｈＩＣＡＭ－１表达明显减少,ＳＯＤ含量增高,ＰＭＮ浸润数减少,心肌梗塞范围缩小（Ｐ＜０．０５）,以４８ｈ最显著。本实验表明,预处理后２４～７２ｈ心肌对再次长时间缺血／再灌注的损伤有保护作用,其产生可能与ＩＣＡＭ－１表达降低所介导的ＰＭＮｓ浸润减少及ＳＯＤ含量增加有关     

Remote ischemic preconditioning protects liver ischemia-reperfusion injury by regulating eNOS-NO pathway and liver microRNA expres-sions in fatty liver rats

BACKGROUND: Ischemic preconditioning (IPC) is a strategy to reduce ischemia-reperfusion (I/R) injury. The protective effect of remote ischemic preconditioning (RIPC) on liver I/R injury is not clear. This study aimed to investigate the roles of RIPC in liver I/R in fatty liver rats and the involvement of en-dothelial nitric oxide synthase-nitric oxide (eNOS-NO) path-way and microRNA expressions in this process. METHODS: A total of 32 fatty rats were randomly divided into the sham group, I/R group, RIPC group and RIPC+I/R group. Serum alanine aminotransferase (ALT), aspartate ami-notransferase (AST) and nitric oxide (NO) were measured. Hematoxylin-eosin staining was used to observe histological changes of liver tissues, TUNEL to detect hepatocyte apoptosis, and immunohistochemistry assay to detect heat shock protein 70 (HSP70) expression. Western blotting was used to detect liver inducible NOS (iNOS) and eNOS protein levels and real-time quantitative polymerase chain reaction to detect miR-34a, miR-122 and miR-27b expressions. RESULTS: Compared with the sham and RIPC groups, serum ALT, AST and iNOS in liver tissue were significantly higher in other two groups,while serum NO and eNOS in liver tissue were lower, and varying degrees of edema, degeneration and in-flammatory cell infiltration were found. Cell apoptosis num-ber was slightly lower in the RIPC+I/R group than that in I/R group. Compared with the sham group, HSP70 expressions were significantly increased in other three groups (all P<0.05). Compared with the sham and RIPC groups, elevated miR-34a expressions were found in I/R and RIPC+I/R groups (P<0.05). MiR-122 and miR-27b were found significantly decreased in I/R and RIPC+I/R groups compared with the sham and RIPC groups (all P<0.05). CONCLUSION: RIPC can reduce fatty liver I/R injury by affect-ing the eNOS-NO pathway and liver microRNA expressions.     

Blockade of P-selectin does not affect reperfusion injury in hamsters subjected to glutathione inhibition

P-selectin antibody has been shown to prevent microvascular damage after ischemia reperfusion (I/R). We investigated whether the treatment with anti-P-selectin would attenuate the decrease in capillary perfusion after glutathione (GSH) inhibition in hamster cheek pouch microcirculation subjected to I/R. Animals were treated for 3 days with l-buthionine-[S,R]-sulfoximine (BSO) to inhibit GSH synthesis. P-selectin expression was determined by using an in situ immunofluorescence method in the microvessels. Ischemia was induced by clamping the cheek pouch for 30 min followed by 30 min of reperfusion. Changes in capillary perfusion, RBC velocity, and leukocyte and platelet adhesion on microvessels were measured after I/R. Hamsters subjected to I/R showed increased leukocyte and platelet adhesion as well as decreased capillary perfusion. The anti-P-selectin group showed a significant P-selectin expression, that occurs at the venular bifurcations within 15-30 min of reperfusion, as well as no increase in leukocyte and platelet adhesion on microvessels. BSO partially prevented P-selectin expression but the decrease in capillary perfusion and the increase in both platelet and leukocyte adhesion in microvessels were greater. GSH significantly prevented P-selectin expression as well as capillary perfusion decrease after I/R. In conclusion, GSH inhibition blunted the protective effects of anti-P-selectin treatment with marked leukocyte adhesion on postcapillary venules and platelet-endothelial cell interactions in arterioles and venules and decreased capillary perfusion at reperfusion, thus suggesting that the mechanism of I/R injury is not critically dependent on P-selectin.     

Modulation of liver oxidant-antioxidant system by ischemic preconditioning during ischemia／reperfusion injury in rats

AIM: To investigate effects of ischemic pre-conditioning on the liver endogenous oxidant-antioxidant system during ischemia/reperfusion injury. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into sham-operated (Sham), ischemia/ reperfusion (I/R), ischemic pre-conditioning plus ischemia/ reperfusion (IPC) groups. Serum ALT, AST and hyaluronic acid levels were assayed and pathologic alterations observed. Liver malondialdehyde (MDA) contents, endogenous antioxidant enzymes, superoxidase dismutase (SOD), catalase (CAT), gultathionine peroxidase (GSH-Px) activities, neutrophils accumulation marker, myeloperoxidase (MPO) activities were measured respectively. RESULTS: Compared with I/R group, sinusoidal endothelial cells as well as hepatocytes damages, as assessed biochemically and histochemically, were improved significantly in IPC group; neutrophils infiltration was also markedly reduced. In IPC group, liver peroxidation, as measured by MDA contents, was significantly decreased when compared with I/R group; endogenous antioxidant enzymes, SOD, CAT and GSH-Px activities were markedly higher than that in I/R group. CONCLUSION: Ischemic pre-conditioning exerts protective effects on both hepatic sinusoidal endothelial cells and hepatocytes during liver I/R injury. Its mechanisms may involve dimunition of neutrophils infiltration and modulation of the imbalance of endogenous oxidant-antioxidant system in the organism.     

不同时期无创肢体缺血预适应对急性心肌梗死大鼠的保护作用

目的:探讨急性心肌梗死前、再灌注前及再灌注时等不同时期,无创性肢体缺血预适应在减轻大鼠缺血/再灌注损伤中的作用。方法:所有大鼠随机分为4组(每组10只):心肌缺血/再灌注损伤组(A组)、急性心肌梗死前肢体缺血预适应组(B组)、再灌注前肢体缺血预适应组(C组)和再灌注初期肢体缺血预适应组(D组)。观察各组心电、心肌缺血范围(AAR)、心肌梗死范围(IA)、心肌梗死部位质量与左心室质量(LV)的比值(IA/LV)、梗死范围与缺血范围的比值(IA/AAR)、CK-MB值。结果:与A组相比,B、C、D 3组ST段抬高幅度、CK-MB值、IA/LV和IA/AAR比值均显著降低(P<0.01)。结论:无创性肢体缺血预适应在急性心肌梗死前、再灌注前及再灌注时的各个时期应用,都能明显降低大鼠心肌缺血/再灌注时的ST段抬高幅度,明显降低心肌酶水平,明显缩小心肌坏死面积。     

β结合素与大鼠心肌缺血再灌注损伤的作用机制

目的：研究大鼠心肌缺血、缺血再灌注、无创性缺血预处理以及氯化锂干预后心肌细胞β结合素的阳性表达率及细胞凋亡指数的变化,以探讨β结合素与心肌缺血、缺血再灌注的关系以及可能机制。方法将入选的60只大鼠随机分为假手术对照组（C ）、缺血组（I ）、缺血再灌注组（IR ）、氯化锂药物预处理组（PPC ）。以TUNEL方法评估各组大鼠心肌细胞凋亡情况,以免疫组化方法测定各组大鼠心肌细胞中β结合素的表达,并计算各组大鼠的心肌细胞凋亡指数及β结合素。结果与C组比较,I、IR、PPC四组凋亡指数均升高（ P＜0.05）,各组心肌细胞凋亡指数比较的结果为：IR组＞I组＞PPC组＞C组（各组两两比较均 P＜0.05）;各组心肌细胞β-cat阳性表达率比较的结果为：PPC组＞C组＞I组＞IR组。β-cat阳性表达率与凋亡指数呈负相关,R＝-0.90。结论β-cat阳性表达率与凋亡指数呈负相关,提示β-cat可以减少心肌缺血、缺血再灌注引起的细胞凋亡。     

大鼠心肌再灌注时心肌细胞凋亡,Fas基因表达及缺血预处理对？…

目的 探讨大鼠心肌缺血后不同再灌注时相心肌细胞凋亡,Ｆａｓ基因表达变化及血预处理的影响,方法 １０８只大鼠随分成假手术组（假手术２４小时）缺血３０分钟再灌注６,１２,２４,４８小时组及缺血预处理（ＩＰＣ）组（结扎冠脉５分钟,再灌注５分钟,重复３次后再行结扎冠脉３０分钟,再灌注６小时）口号档端标记法（ＴＵＮＥＬ）标记凋亡细胞,以Ｓ－Ｐ免疫组化及逆转录聚合酶链反应（ＲＴ－ＰＣＲ）法分别检测Ｆａｓ基因蛋     

Ischemic preconditioning enhances regenerative capacity of hepatocytes in long-term ischemically damaged rat livers

BACKGROUND AND AIMS: Ischemic preconditioning (IPC) protects tissues against ischemia and reperfusion (I/R) injury. The aim of this study was to examine the impact of IPC on protection and regeneration of hepatocytes after prolonged I/R injury. METHODS: A rat model of segmental (70%) hepatic ischemia was used to determine the effect of 10-min IPC preceding 40, 60, 90, or 120 min of liver ischemia. The effect was assessed by comparing cytolysis markers and necrotic areas of the liver, as well as the regenerative capacity of hepatocytes using the proliferating cell nuclear antigen labeling index (PCNA-LI) and weight of the ischemic liver lobe. Protein kinase B/Akt (Akt) and caspase-9 were investigated immunohistochemically to determine the effect of IPC on activation of survival and anti-apoptotic signals. RESULTS: In the model of 40 min I/R, which resulted in focal necrosis of the liver, IPC significantly protected against I/R injury by reducing the area of focal necrosis, level of PCNA-LI and immunoreactivities to Akt and caspase-9. In contrast, IPC did not prevent ischemic damage in the 90- and 120-min ischemic model with massive liver necrosis. However, IPC enhanced the regenerative capacity of the remaining hepatocytes with higher levels of PCNA-LI, number of Akt-positive cells and mean weight of the liver lobe postoperatively than in the controls. CONCLUSIONS: In a model of focal necrosis of the liver, IPC protected hepatocytes against I/R injury. In addition, in a model of massive necrosis, IPC maintained the regenerative capacity of the remaining hepatocytes by enhancing the survival signals.     

预处理对肝脏再灌注损伤大鼠肝组织、血液中NO和ET的影响及意义

目的　探讨预处理对肝脏缺血再灌注损伤大鼠肝组织和血液中一氧化氮 (NO)和内皮素 (ET)含量的影响及意义。方法　建立肝脏 70 %缺血再灌注损伤大鼠模型 ,分为对照组、缺血组、缺血预处理组、L -精氨酸组(L - arg)、Nω-硝基 - N -精氨酸甲酯 (L - NAME)组 ,观察各组肝功能变化 ,检测肝组织和血清中 NO和 ET及透明质酸 (HA)水平。结果　预处理可减轻 NO水平的下降和血浆 ET的升高 ,防止肝功酶的升高 (P<0 .0 5 )。结论　预处理可诱导缺血再灌注损伤大鼠 NO产生增加、ET产生减少 ,进而改善其微循环 ,减少再灌注损伤。