远程预处理通过下调钙网蛋白高表达减轻在体大鼠心肌细胞缺血再灌注损伤

目的:研究远程预处理(remote preconditioning,RP)对在体缺血再灌注心肌的保护作用,探讨钙网蛋白(calreticulin,CRT)在远程预处理心肌细胞缺血再灌注(ischemia reperfusion,IR)损伤的作用.方法:30只成年SD大鼠,随机分成5组(n=6),分别为缺血再灌注组、缺血预处理组、远程预处理Ⅰ组、远程预处理Ⅱ组和假手术组.建立大鼠在体缺血再灌注损伤模型,观察各组缺血再灌注前后心功能变化,并检测再灌注末血清肌钙蛋白T(cardiac troponin T,cTnT)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)的变化以及心肌组织钙网蛋白的表达.结果:远程预处理Ⅰ组心功能、cTnT、MDA、SOD值、CRT的表达与缺血再灌注组无显著差异(P>0.05);缺血预处理组、远程预处理Ⅱ组SOD值均显著高于缺血再灌注组(P<0.05),cTnT、MDA值、CRT的表达均显著低于缺血再灌注组(P<0.05).结论:远程预处理可以模拟缺血预处理的心肌保护作用;远程预处理可能通过下调钙网蛋白高表达减轻在体大鼠心肌细胞缺血再灌注损伤.     

钙卫蛋白在肾脏缺血再灌注损伤大鼠中的表达及其作用

目的:探讨钙卫蛋白(CALP)在肾脏缺血再灌注损伤(IRI)大鼠中的表达及其在炎症反应中的作用。方法:50只雄性SD大鼠随机分为IRI组和假手术组(sham组),每组25只。各组分别于缺血再灌注后6 h、12h、24 h、48 h和72 h观察肾脏病理改变,检测大鼠血清尿素氮(BUN)和肌酐(SCr)水平,ELISA法检测大鼠血清CALP、肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6的含量,免疫组织化学法和Western blot法分别检测大鼠肾脏CALP、Toll样受体4(TLR4)和NF-κB p65蛋白表达水平。结果:IRI组大鼠肾脏呈现不同程度的肾小管上皮细胞和间质损伤,伴炎细胞浸润。血清BUN、SCr、CALP及促炎细胞因子TNF-α、IL-6的含量在各时点均显著高于sham组(P0.05)。CALP、TLR4和NF-κB p65主要表达于肾小管上皮细胞胞质中,在IRI组呈高表达,在各时点各因子的表达与sham组相比均显著增强(P0.05)。结论:肾脏IRI大鼠的血清CALP、TNF-α和IL-6水平升高,CALP、TLR4和NF-κB p65在肾组织中表达增强。CALP在大鼠肾脏IRI的炎症反应中可能起重要作用。     

X线照射增加大鼠心肌对缺血/再灌注损伤的敏感性*

 目的：观察胸部X线放射治疗大鼠对心肌缺血/再灌注损伤的敏感性。方法：用胸部单次照射X线20 Gy构建放射性心脏损伤模型,采用完全随机分组方法将Wistar大鼠分为假损伤组、损伤组、假损伤+假手术组、假损伤+缺血/再灌注组、损伤+假手术组和损伤+缺血/再灌注组,于创伤后2周行心肌缺血/再灌注。通过BL-410生物信号记录分析系统记录各组大鼠左心室发展压（LVDP）、左心室压力上升的最大速率（+dp/dtmax）和左心室压力下降的最大速率（-dp/dtmax）;采用ELISA方法检测大鼠血清心肌肌钙蛋白I（cTnI）和肌酸激酶同工酶（CK-MB）;用BI2000图像分析软件测定心肌梗死面积占总面积的百分比。结果：损伤+缺血/再灌注组离体心功能明显低于假损伤+缺血/再灌注组（P＜0.01）,损伤+缺血/再灌注组血清cTnI和CK-MB水平显著高于假损伤+缺血/再灌注组（P＜0.01）,损伤+缺血/再灌注组心肌梗死面积显著大于假损伤+缺血/再灌注组（P＜0.01）。结论:X线照射增加大鼠心肌对缺血/再灌注损伤的敏感性。     

脑红蛋白在脊髓缺血再灌注损伤中的表达及其意义

目的:观察大鼠脊髓缺血再灌注损伤(SCI)过程中脑红蛋白(Ngb)的表达变化及其意义。方法:健康成年Wistar大鼠随机分为脊髓压迫缺血再灌注组和假手术对照组。运用原位杂交组织化学和免疫印迹法,检测压迫段脊髓组织中Ngb的表达变化。结果:再灌注2h后,Ngb在压迫段脊髓开始表达上调,24h较为明显,48h达峰值,72h表达减少,与假手术组相比较,具有显著性差异。免疫印迹法的结果和上述结果一致。结论:在脊髓缺血再灌注损伤中Ngb的表达变化与再灌注时间相关,其表达上调是机体内源性神经保护之一。     

再灌注损伤心肌线粒体钙代谢与呼吸功能关系的研究

观察离体缺氧-再灌注兔心肌线粒体钙含量、钙摄取率和不同底物的呼吸耗氧量的变化。结果表明:缺氧组心肌线粒体钙含量轻度增加(3.37±0.25nmol/mg pr,P<0.05),钙摄取率无显著变化,以琥珀酸为底物的呼吸耗氧量代偿性增高(226.0±28.7natoms/min/mg Pr,P<0.05),以谷氨酸加苹果酸为底物时变化不显著;再灌注组心肌线粒体钙含量显著增高(6.10±1.12nmol/mgt pr,P<0.01);钙摄取率和以谷氨酸加苹果酸为底物的呼吸耗氧量显著降低(P<0.01),且与线粒体钙含量呈显著负相关(r=-0.73,P<0.001),提示线粒体钙超载在再灌注损伤线粒体功能损害中起重要作用。     

miRNA-214靶向作用于钙网蛋白保护阿霉素相关大鼠心肌损伤

目的：探讨miRNA-214 对阿霉素相关大鼠心肌损伤的保护作用及其相关机制。方法：SD大鼠随机分为 对照组、阿霉素心肌损伤模型组（ 模型组）和miRNA-214 转染组;用超声心动图检测3 组大鼠心功能相关指 标;用PCR检测3 组大鼠心肌细胞miRNA-214 的表达差异;用免疫印迹检测心肌组织钙网蛋白的表达水平。 结果：miRNA-214 转染组全心质量、左心室质量显著高于模型组,但低于对照组;miRNA-214 转染组全心肥 厚指数、左心室肥厚指数显著低于模型组,但高于对照组。miRNA-214 转染组心肌纤维破坏程度较模型组显 著减轻,肌纤维完整,横纹排列整齐,有少许中性粒细胞浸润与无空泡变性。miRNA-214 转染组大鼠心肌细胞 中miRNA-214 相对表达量显著高于模型组,但低于对照组。miRNA-214 转染组谷丙转氨酶、谷草转氨酶、乳 酸脱氢酶、肌酸激酶、B型利钠肽水平显著低于模型组,但高于对照组。miRNA-214 转染组左心室收缩末期 内径、左心室舒张末期内径显著低于模型组,但高于对照组;miRNA-214 转染组左心室短轴缩短率、左心室 射血分数显著高于模型组,但低于对照组。miRNA-214 转染组钙网蛋白表达量显著低于模型组,高于对照组。 结论：miRNA-214 可通过下调钙网蛋白缓解心肌损伤,有望成为一个新的治疗靶标。     

钙网蛋白与心血管疾病

钙网蛋白是内质网中主要的Ca2 结合分子伴侣,具有调控细胞钙稳态、蛋白质合成与修饰等作用,在生物体内钙网蛋白具有调节细胞凋亡、应激、心血管炎症反应等多种生理和病理生理过程的功能。钙网蛋白在心脏发育、心肌肥大、缺血再灌注和血管生成等过程中的作用是心血管领域的研究热点问题之一。     

金属硫蛋白在心肌缺血再灌注损伤修复中的抗氧化作用

目的 探讨金属硫蛋白（ＭＴ）在心肌梗塞和缺血再灌注心肌损伤修复中与脂质过氧化的关系。方法 采用大鼠心肌梗塞／缺血再灌注模型,测定手术后２４ｈ和８周心肌组织的ＭＴ及脂质过氧化的中间产物丙二醛（ＭＤＡ）的含量,结果：心肌梗塞和缺血再灌注２４ｈ后ＭＤＡ含量均显著高于对照组（Ｐ〈０．０１）,８周后ＭＤＡ显著降低（Ｐ〈０．０５）,而ＭＴ的含量在术后２４ｈ即升高,８周ＭＴ继续升高,缺血再灌注组ＭＴ达（３１４．     

细胞膜钠钙交换蛋白与心肌缺血/再灌注损伤

20世纪初有研究者注意到降低细胞外钠离子浓度可产生类似于洋地黄类药物的正性肌力作用。60年代，钠钙交换现象和钠钙交换蛋白（sodium-calcium exchanger，NCX）在蛙心肌细胞中首次被描述，随后发现NCX在多个种属、多种脏器普遍存在。1988年实现了该蛋白的部分纯化，1990年由犬心肌细胞首次克隆成功。经过数十年的研究，特别是近年来随着Jiant patch技术的发展，对钠钙交换电流的测量更为精确方便，逐渐认识到此细胞膜交换蛋白与心肌缺血／再灌注损伤密切相关。     

Delayed treatment with nimesulide reduces measures of oxidative stress following global ischemic brain injury in gerbils

Metabolism of arachidonic acid by cyclooxygenase is one of the primary sources of reactive oxygen species in the ischemic brain. Neuronal overexpression of cyclooxygenase-2 has recently been shown to contribute to neurodegeneration following ischemic injury. In the present study, we examined the possibility that the neuroprotective effects of the cyclooxygenase-2 inhibitor nimesulide would depend upon reduction of oxidative stress following cerebral ischemia. Gerbils were subjected to 5 min of transient global cerebral ischemia followed by 48 h of reperfusion and markers of oxidative stress were measured in hippocampus of gerbils receiving vehicle or nimesulide treatment at three different clinically relevant doses (3, 6 or 12 mg/kg). Compared with vehicle, nimesulide significantly (P<0.05) reduced hippocampal glutathione depletion and lipid peroxidation, as assessed by the levels of malondialdehyde (MDA), 4-hydroxy-alkenals (4-HDA) and lipid hydroperoxides levels, even when the treatment was delayed until 6 h after ischemia. Biochemical evidences of nimesulide neuroprotection were supported by histofluorescence findings using the novel marker of neuronal degeneration Fluoro-Jade B. Few Fluoro-Jade B positive cells were seen in CA1 region of hippocampus in ischemic animals treated with nimesulide compared with vehicle. These results suggest that nimesulide may protect neurons by attenuating oxidative stress and reperfusion injury following the ischemic insult with a wide therapeutic window of protection.     

Energy deficiency,calcium overload or oxidative stress: Possible causes of irreversible ischemic myocardial injury

Summary After prolonged ischemia or hypoxia myocardial injury is not reversed but exacerbated by a resupply of the tissue with oxygen and substrates. The mechanism by which reversible ischemic or hypoxic myocardial injury becomes irreversible is not yet understood. It has been debated whether reperfusion injury merely uncovers pre-existing irreversible injury, or is indeed caused by the reperfusion/reoxygenation process. In recent years, three theories have been discussed that relate the onset of irreversibility either to: a critical energy loss; a critical accumulation of cellular calcium; or to the deleterious effects of free radical formation. In certain experimental models for each of these theories favourable results have been obtained. Current research suggests that absolute reversibility thresholds in energy depletion or calcium accumulation in the ischemic or hypoxic cell do not exist. A key role of free radical injury for reperfusion injury must also be questioned. There is, however, evidence that in tissue reversibility of ischemic cardiomyocyte injury is limited by conditions that make calcium-induced hypercontracture upon reoxygenation unavoidable. This occurs when, by hypercontracture, mutual mechanical disruption of the cells destroys the tissue. From isolated cardiomyocytes that are able to metabolically survive hypercontracture it has been observed that these metabolic conditions do not represent the last biological possibility to reverse injury.     

Cardioprotective effect of the Hibiscus rosa sinensis flowers in an oxidative stress model of myocardial ischemic reperfusion injury in rat

Background  

The present study investigates the cardioprotective effects of Hibiscus rosa sinensis in myocardial ischemic reperfusion injury, particularly in terms of its antioxidant effects.     

高交感活性诱导的大鼠心肌损伤模型中氧化应激的受体调控机制*

 目的：研究高交感活性诱发大鼠心肌损伤的氧化应激受体调控机制。方法：Sprague-Dawley (SD)大鼠随机分为对照组、模型组、普萘洛尔（Pro) 组、哌唑嗪(Praz)组、普萘洛尔+哌唑嗪 (Pro+Praz) 组、维生素E(VE)组及普萘洛尔+哌唑嗪+维生素E (Pro+Praz+VE) 组,除对照组外其余各组均腹腔注射去甲肾上腺素(NE) 复制高交感活性引起的心肌损伤模型,同时灌胃给予相应药物,连续给药16 d,期间监测各组动物体重的变化。16 d后进行心室重构指标（心指数和羟脯氨酸含量）、病理组织学检查、氧化/抗氧化指标（MDA、SOD、CAT、GSH-Px和T-AOC）和能量代谢指标（Na+-K+ATPase和Ca2+-Mg2+ATPase）分析。结果：从第9天开始,模型组动物体重与对照组的比较差异有统计学意义（P<0.05）,心指数和左心室肥厚明显增加,氧化/抗氧化和能量代谢障碍;Pro、Praz、Pro+Praz和VE各组均出现不同程度的动物体重、心指数、左心室肥厚和氧化/抗氧化失衡的改善;Pro、Praz和Pro+Praz能明显升高左心室Na+-K+ATPase和Ca2+-Mg2+ATPase的活性,Pro+Praz作用最明显（P<0.05）。结论：肾上腺素受体依赖是高交感活性诱导心肌氧化应激损伤的重要途径。     

Pyruvate attenuates cardiac dysfunction and oxidative stress in isoproterenol-induced cardiotoxicity

Pyruvate, a potent endogenous antioxidant and an important metabolic fuel is essential for the cardiac function and tissue defense mechanism. The present study was evaluated to investigate whether pyruvate attenuates the development of cardiotoxicity in isoproterenol (ISO)-induced myocardial infarction by assessing hemodynamic, biochemical and histopathological parameters. Subcutaneous injection of ISO (85 mg/kg) administered for 2 days at an interval of 24 h was used for induction of cardiotoxicity. ISO administration significantly decreased arterial pressure indices, heart rate, contractility {(+)LVdP/dt} and relaxation {(?)LVdP/dt} and increased left ventricular end-diastolic pressure. In addition, a significant reduction in activities of myocardial creatine phosphokinase-MB, lactate dehydrogenase, superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione levels along with increase in thiobarbituric acid reactive substances were also observed following ISO administration. However, pretreatment with pyruvate (0.25, 0.5 and 1.0 g/kg, p.o.) favorably modulated all most every studied parameters in ISO-induced myocardial injury. Furthermore, protective effect of pyruvate was confirmed by histopathological studies. Rats pretreated only with pyruvate did not produce significant change in hemodynamic, biochemical and histopathological parameters. Pyruvate at 0.50 and 1.0 g/kg doses was found to exert optimal cardioprotective effect against ISO-induced myocardial infarction. The results of our study suggest that pyruvate possessing antioxidant activity has a significant cardioprotective effect against ISO-induced myocardial injury.     

肾透明细胞癌中氧化应激蛋白的表达

目的:探讨人肾透明细胞癌细胞株RLC-310与人正常肾近曲小管上皮细胞株HK-2,肾癌及其癌旁组织中氧化应激蛋白表达的差异。方法:体外培养RLC-310和HK-2,采用PF-2D蛋白分级分离系统分离细胞总蛋白,选取差异蛋白组分进行毛细管LC-ESI-MS/MS分析,结合蛋白质数据库检索对差异蛋白进行鉴定,并对代表性氧化应激差异蛋白采用免疫组织化学方法进行验证。结果:两个细胞株经串联质谱分析共鉴定出12个氧化应激差异表达蛋白,分别是peroxiredox-in-1(PRX-1)、peroxiredoxin-6(PRX-6)、superoxide dismutase[Cu-Zn]SOD1、glutathione peroxidase 1、catalase、glutathionesynthetase、glutathione S-transferase Pi(GSTPi)、thioredoxin、热休克蛋白10(heat shock protein,HSP10)、HSP 60、HSP 70和HSP 90。其中PRX-6、HSP 60、GSTPi三种代表性差异蛋白在人肾透明细胞癌细胞株RLC-310和人正常肾近曲小管上皮细胞株HK-2中均有表达,前者的表达水平较后者显著升高(P<0.05),这三种蛋白在肾癌组织中表达也较癌旁组织显著升高(P<0.05)。结论:人肾透明细胞癌中存在抗氧化应激蛋白的差异表达,这些氧化应激蛋白的异常表达在阻止肾癌细胞氧化损伤中起一定作用。     

Expression of SIRT1 and oxidative stress in diabetic dry eye

Objective: To explore the expression of SIRT1 with oxidative stress and observe physiological and pathological changes in the corneas as well as the association between SIRT1 and oxidative stress of diabetic dry eyes in mice. Method: Forty-eight C57BL/6Jdb/db mice at eight weeks of age were divided randomly into two groups: the diabetic dry eye group and the diabetic group. An additional forty-eight C57BL/6J mice at eight weeks of age were divided randomly into two groups: the dry eye group and the control group. Every mouse in the dry eye groups (diabetic and normal) was injected with scopolamine hydrobromide three times daily, combined with low humidity to establish a dry eye model. After the intervention, phenol red cotton string tests and corneal fluorescein staining were performed. In addition, HE staining and immunofluorescence were done. Expression of SIRT1 in the cornea was examined by real-time PCR and Western Blot and expression of FOXO3 and MnSOD proteins was detected by Western Blot. Results: At one, four, and eight weeks post intervention, all of the groups except the controls showed significant decreases in tear production and increases in the corneal fluorescein stain (P<0.05 vs control). Between the experimental groups, the diabetic dry eye group had the least tear production and the highest corneal fluorescein stain score (P<0.05). As the disease progressed, all of the experimental groups showed obviously pathological changes in HE staining, particularly the diabetic dry eye group. In the 1^st and 4^th week, the expression of SIRT1, FOXO3, and MnSOD were significantly higher in the diabetic DE and DM groups but lower in the DE group compared to the controls (P<0.05). In the 8^th week, the expression of SIRT1, FOXO3, and MnSOD was significantly down-regulated in the diabetic DE group and the DM group (P<0.05). Immunofluorescence showed similar results. Conclusion: In the condition of diabetic dry eye, tear production declined markedly coupled with seriously wounded corneal epithelium. Oxidative stress in the cornea was enhanced significantly and the expression of SIRT1 was decreased.     

Depletion of ATP and oxidative stress underlie zinc-induced cell injury

The mechanisms of cell injury resulting in a special type of cell death combining the features of apoptosis and necrosis were examined in Hep-2 cells exposed to 300 microM zinc sulfate during 24 h. Acute exposure to zinc induced a rapid rise in metallothionein levels and increased oxidative stress occurring in the absence of a significant early ATP depletion. Accentuated ATP loss and elevated levels of superoxide at later treatment intervals (12 h and longer) were present along with increased DNA damage. Manipulation with ATP production and inhibition of NADPH oxidase had a positive effect on zinc-related increase in oxidative stress and influenced the observed type of cell death. These results suggest that Hep-2 cells acutely exposed to zinc increase intracellular labile zinc stores and over express metalothioneins. Elevated production of peroxides in zinc-treated cells is at later treatment intervals accompanied by an increase in superoxide levels, possibly by activation of NADPH oxidase, DNA damage and severe ATP loss. Prevention of critical ATP depletion and, in particular, inhibition of oxidative stress attenuates zinc-mediated cell injury and stimulates apoptosis-like phenotype in exposed cells.