抑制或激活泛素-蛋白酶体途径对大鼠肺泡巨噬细胞内质网应激的影响

目的:研究肺泡巨噬细胞(NR8383)不同蛋白酶体激活程度对内质网应激的影响。方法:构建UbG76V-GFP融合蛋白,将含有UbG76V-GFP的质粒导入NR8383细胞,筛选出可稳定表达UbG76V-GFP的细胞系,通过蛋白酶体抑制剂(MG132)、蛋白酶体激活剂(阿霉素)干预蛋白酶体活性。荧光显微镜观察不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时蛋白酶体活性,Western blot及PCR技术检测不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时泛素化蛋白及内质网应激相关基因的表达。结果:在缺氧复氧2 h、4 h、6 h这3个时间点,加入MG132组大鼠肺泡巨噬细胞绿色荧光及泛素化蛋白(Ubiquitin)表达明显降低(P0.05),而PCR及Western blot示内质网应激基因BIP(免疫球蛋白结合蛋白)、XBP-1(X-盒结合蛋白)和CHOP(C/EBP同源蛋白)平均扩增量及蛋白表达量明显增加(P0.05);加入阿霉素组大鼠肺泡巨噬细胞在缺氧复氧2 h、4h、6 h表现出相反的实验结果,绿色荧光及Ubiquitin蛋白相对表达均明显增加(P0.05),而PCR及Western blot示内质网应激基因BIP、XBP-1和CHOP平均扩增量及蛋白表达量明显增加(P0.05)。结论:本实验结果表明活细胞泛素-蛋白酶体活性程度与内质网应激存在紧密联系,外源性增强泛素蛋白酶体活性会抑制内质网应激,外源性减弱泛素蛋白酶体活性会增强内质网应激。     

Mitochondrial nitric oxide inhibits ATP synthesis. Effect of free calcium in rat heart

Summary.  Nitric oxide is a small potentially toxic molecule and a diatomic free radical. We report the interaction of L-arginine, oxygen and calcium with the synthesis of nitric oxide in heart mitochondria. Nitric oxide synthesis is increased in broken rat heart mitochondria compared with intact and permeabilized mitochondria. Intact mitochondria subjected to hypoxia-reoxygenation conditions accumulated nitric oxide that inhibits oxygen consumption and ATP synthesis. ATPase activity is not affected during this augment of nitric oxide. Physiological free calcium concentrations protected mitochondria from the damage caused by the accumulation of nitric oxide. Higher concentrations of the divalent cation increase the damage exerted by nitric oxide. Received April 15, 2002 Accepted June 17, 2002 Published online November 14, 2002 Acknowledgements This work was supported in part by Mexican Grants from CONACYT (to A.S.M. during its sabbatical) and CIC-UMSNH (2.5). Authors' address: Alfredo Saavedra-Molina, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3. C.U., Morelia, Mich. 58030, México, Fax: 52-443-326-5788, E-mail: saavedra@zeus.umich.mx     

SERP1 prevents hypoxia-reoxygenation-induced H9c2 apoptosis through activating JAK2/STAT3 pathway-dependent attenuation of endoplasmic reticulum stress

The endoplasmic reticulum (ER) stress plays an important role in myocardial ischemia/reperfusion (MI/R) injury. SERP1, the stress-associated endoplasmic reticulum protein 1, is involved in regulating ER stress response. However, whether it associates with MI/R injury is not identified. Here, we show that SERP1 is induced in the mouse heart after MI/R injury as well as in H9c2 cells under hypoxia/reoxygenation (H/R) treatment. Additionally, SERP1 overexpression reduces H/R-induced H9c2 apoptosis. Moreover, SERP1 overexpression suppresses H/R-induced ER stress and activates JAK2/STAT3 pathway. Furthermore, JAK2/STAT3 pathway inhibition by the specific inhibitor JSI-124 minimizes the suppressive effect of SERP1 overexpression on H/R-induced ER stress and H9c2 apoptosis. Together, these results uncover the protection of SERP1 against H/R-induced H9c2 apoptosis and further relate it to JAK2/STAT3 pathway-dependent attenuation of ER stress. This study suggests SERP1 as a potential regulator invovled in the pathophysiology of MI/R injury.     

Expression of angiostatin and its related factors in the plasma of newborn pigs with hypoxia and reoxygenation

Little is known about angiostatin and its related factors in the hypoxia-reoxygenation of neonates. In this study we compared the effect of 21% and 100% reoxygenation on temporal changes in the plasma level of these factors in newborn piglets subjected to hypoxia. Newborn piglets were subjected to 2 h hypoxia followed by 1 h of reoxygenation with either 21% or 100% oxygen and observed for 4 days. On day 4 of recovery in 100% hypoxic-reoxygenated group, there were increases in total angiostatin, plasminogen/plasmin and MMP-2 levels, and decreases in VEGF levels (vs. respective baseline levels, all P <0.001), whereas no significant temporal changes were found in the 21% hypoxic-reoxygenated and sham-operated groups. Angiostatin levels correlated positively with the levels of MMP-2 and HIF-1alpha and negatively with VEGF levels in 100% hypoxic-reoxygenated group (all P <0.05). In comparison to 21% oxygen, neonatal resuscitation with 100% oxygen was found to increase the levels anti-angiogenic factors.     

Cytoprotective effect of two synthetic enhancer substances, (-)-BPAP and (-)-deprenyl, on human brain capillary endothelial cells and rat PC12 cells

Enhancer regulation is a new control mechanism in the brain [Knoll, J., 2003. Enhancer regulation/endogenous and synthetic enhancer compounds: a neurochemical concept of the innate and acquired drives. Neurochemical Research 28(8), 1275-1297]. Enhancer substances exert their effect in bi-modal form with a highly characteristic dose-dependency. Two bell-shaped concentration curves have been published. The one in ultra low concentration range (fM) specific form of enhancer regulation and the other at high concentration (100 microM) non-specific form of enhancer regulation. Catecholaminergic neurons proved to be enhancer-sensitive cells. Since rat PC12 cells and human brain endothelial cells (HBEC) work under catecholaminergic influence, it was reasonable to expect that both the specific and non-specific form of the enhancer regulation might be detectable in these cells. We tested this possibility on these cultured cells under normoxia and hypoxia-reoxygenation. After 1 h hypoxia produced by Argon gas and 0, 2, 4, and 20 h reoxygenation the cell loss was calculated by propidiumiodide assay and the cell activity was investigated by Alamar Blue assay colorimetric measurement. The percentages of living and necrotic cells were expressed after propidiumiodide staining. Broad scale concentrations of the two compounds (1 fM-100 microM) were added to the culture strait after the oxygen deprivation. (-)-BPAP and (-)-deprenyl, due to their enhancer effect, exerted a significant cytoprotective effect on both HBECs and PC12 cells. In harmony with Knoll's publications we were able to demonstrate by the aid of (-)-BPAP and (-)-deprenyl that both HBEC and PC12 are enhancer-sensitive cells. We detected the specific form of the enhancer regulation in the ultra low concentration range (fM-pM) and also the non-specific form of the enhancer regulation was visible (mM-microM).     

Mitophagy and mitochondrial integrity in cardiac ischemia-reperfusion injury

Ischemia-reperfusion injury (IR injury), produced by initial interruption and subsequent restoration of organ blood flow, is an important clinical dilemma accompanied by various cardiac reperfusion strategies following acute myocardial infarction (AMI). Although the restored blood flow is necessary for oxygen and nutrient supply, reperfusion often results in pathological sequelae leading to elevated ischemic damage. Among various theories postulated for IR injury including vascular leakage, oxidative stress, leukocyte entrapment, inflammation and apoptosis, mitochondrial dysfunction plays an essential role in mediating pathophysiological processes with recent evidence depicting a pivotal role for impaired mitophagy in mitochondrial injury. Given the critical role for mitophagy in mitochondrial quality control and the recent reports supporting a tie between mitophagy and IR injury, this review will revisit the contemporary understanding of mitophagy in the regulation of cardiac homeostasis and update recent progresses with regards to mitophagy and cardiac IR injury. We hope to establish a role for mitophagy as a potential therapeutic target in the management of IR injury.     

Ghrelin对缺氧复氧状态下脂肪间充质干细胞保护作用的研究

目的：探讨缺氧复氧损伤环境下Ghrelin对脂肪来源的间充质干细胞（AD-MSCs）的保护作用,以寻求AD-MSCs心肌内移植的有利因素。方法：采用胶原酶消化法分离小鼠AD-MSCs,流式细胞术鉴定其标志。建立缺氧/复氧细胞模型,分3组：①对照组;②缺氧/复氧组（H/R）;③H/R＋Ghrelin（浓度分别为10-9、10-8、10-7mol/L）干预组。MTT法测定各组细胞增殖,TUNEL法检测细胞凋亡。结果：流式细胞术结果显示AD-MSCs CD44及CD90阳性,CD34、CD45阴性。AD-MSCs MTT分析显示在缺氧环境中,Ghrelin相比于单纯H/R组能够显著促进AD-MSCs的存活与增殖,并抑制其凋亡（P〈0.05）。结论：Ghrelin可以明显提高缺氧复氧环境下AD-MSCs的生存与增殖,抑制缺氧诱导的凋亡发生,有望为心肌梗死的干细胞移植治疗创造新的有利因素。     

Regulation of the rate of synthesis of nitric oxide by Mg(2+) and hypoxia. Studies in rat heart mitochondria

Summary.  In isolated rat heart mitochondria, L-arginine is oxidized by a nitric oxide synthase (mtNOS) achieving maximal rates at 1 mM L-arginine. The NOS inhibitor N^G-nitro-L-arginine methyl ester (NAME) inhibits the increase in NO production. Extramitochondrial free magnesium inhibited NOS production by 59% at 3.2 mM. The mitochondrial free Mg²⁺ concentration increased to different extents in the presence of L-arginine (29%), the NO donor (S-nitroso-N-acetylpenicillamine) (105%) or the NOS inhibitors L-NAME (48%) or N^G-nitro-L-arginine methyl ester, N^G-monomethyl-L-arginine (L-NMMA) (53%). Under hypoxic conditions, mtNOS activity was inhibited by Mg²⁺ by up to 50% after 30 min of incubation. Reoxygenation restored the activity of the mtNOS to pre-hypoxia levels. The results suggest that in heart mitochondria there is an interaction between Mg²⁺ levels and mtNOS activity which in turn is modified by hypoxia and reoxygenation. Received April 2, 2001 Accepted September 21, 2001     

神经元缺氧复氧损伤时氧自由基的毒性作用及其机制

在原代分离培养Ｗｉｓｔａｒ乳鼠大脑皮质神经元上研究了缺氧复氧损伤（Ｈ／Ｒ）对神经细胞乳酸脱氢酶（ＬＤＨ）,漏出率,死亡率和脂质过氧化物含量的影响,并选用一氧化氮（ＮＯ）合酶抑制剂Ｌ－ＮＧ－硝基－精氨酸（Ｌ－ＮＮＡ）巯基供体Ｎ－乙酰半胱氨酸（ＮＡＣ）和超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）三种自由基清除剂进行预保护等方法来探讨机制。结果表明 Ｈ／Ｒ损伤引起ＬＤＨ漏出率,细胞死亡率和脂过氧化物含量极显著