Activation of phospholipase D involved in both injury and survival in A549 alveolar epithelial cells exposed to H2O2

To determine the role of the phospholipase D (PLD) pathway in injury and survival of alveolar epithelial cells, A549 cells were exposed to H₂O₂ (500 μM) which resulted in time-dependent injury and bi-phasic increase of PLD activity at 5 min and at 3 h, respectively. n-Butanol (0.5%) inhibited PLD activation, attenuated cell injury at 5 min of H₂O₂ exposure, but enhanced injury at 3 h of exposure. This activation was inhibited by treatment with catalase (500 units/ml). Exogenous phosphatidic acid mimicked the effects of PLD activation, and diphenyliodonium (NADPH oxidase inhibitor) reversed the decline in cell viability induced by H₂O₂ exposure. Propranolol (phosphatidic acid phospholydrolase inhibitor) and quinacrine (phospholipase A2 inhibitor) had weak effects on H₂O₂-induced PLD activation but reversed H₂O₂-induced injury. We speculate that PLD activation at the initiation of H₂O₂ exposure predominantly results in NAPDH oxidase activation, which mediates A549 cell injury, but turns to mediating cell survival as the H₂O₂ attack continues, which might be mainly due to the accumulation of intracellular phosphatidic acid.     

Hydrogen Peroxide Reduces Lead-Induced Oxidative Stress to Mouse Brain and Liver

Lead (Pb) intoxication may initiate many disorders in human and animals. This study investigates the role of exogenous hydrogen peroxide (H₂O₂) in inducing mouse tolerance to Pb exposure. Results showed that the simultaneous application of 1.2 μg H₂O₂ per kg body weight efficiently protected mice against the Pb-caused injury, as revealed by decreased growth suppression caused by the Pb stress, increased antioxidative enzyme activity, reduced lipid peroxidation, and the protective effect on the nuclear DNA integrity. To our knowledge, this is the first finding of this sort. R. G. Li and T. T. Li contributed equally to this work.     

过氧化氢导致肠上皮细胞线粒体DNA损伤及其编码蛋白活性变化

目的：探讨过氧化氢（H2O2）对肠上皮细胞线粒体DNA（mtDNA）结构与功能的损伤作用。方法：肠上皮细胞株（SW－480），采用4mmol/L H2O2处理细胞后进行线粒体DNA的提取，对细胞色素C氧化酶（COXⅠ、COXⅡ、COXⅢ）基因进行聚合酶链反应（PCR）扩增，并将PCR产物进行直接测序；同时于该时相点测定酶活性。结果：细胞色素C氧化酶COXⅠ从6803－6848出现大范围的点突变及7027出现点突变（TC），在7329（CG）、7341（TG）、7352（GA）出现点突变，在7337出现缺失突变（缺T）；COXⅡ序列在8219－8260段出现大范围的点突变；H2O2可导致mtDNA突变基因所编码的细胞色素C氧化酶活性明显下降，结论：H2O2可明显损伤mtDNA编码的细胞色素C氧化酶基因，这种损伤作用最终导致其编码的酶蛋白活性明显下降。     

Intracellular pH and contraction of isolated rabbit and cat papillary muscle: effect of superfusate buffering

The influence of external buffering on surface pH (pHs), intracellular pH (pHi) and developed twitch tension was investigated in rabbit and cat papillary muscle. pHs and pHi were measured using single and double-barreled microelectrodes respectively. In 20 mM HEPES buffered solution, steady state pHi is close to that in control CO2/HCO-3 (25 mM HCO-3, 5% CO2) solution. pHs and developed tension also do not differ greatly from their control values. Decreasing the HEPES concentration to 5 mM, at constant external pH, lowers pHs considerably. The surface acidosis is associated with a small intracellular acidification; steady state pHi in 5 mM HEPES is always more acid than that in control CO2/HCO-3. A significant decrease in developed tension is also seen in 5 mM HEPES. Alteration of the superfusion velocity influences pHs only slightly. Stimulation of the muscle at high frequency is shown to increase surface acidification, the extent of which is dependent on the buffer concentration. The conclusion from the present experiments is that in papillary muscle external buffering influences intracellular pH and contraction via its effect on pHs.     

内源性小气体信号分子硫化氢与肺缺血再灌注损伤

硫化氢(H2S)是继一氧化氮和一氧化碳之后新近发现的第三种内源性小气体信号分子,并有实验证实其在神经、消化、泌尿、心血管系统均有重要生理作用。然而,H2S与肺缺血再灌注损伤(LIRI)的研究尚不多见,对于H2S在抗LIRI中的作用知之甚少。现就关于H2S在心血管系统及LIRI中作用的研究趋势予以介绍。     

外源性H2S在大鼠心肌缺血再灌注损伤中的作用

目的探索新型气体信号分子硫化氢(H2S)抗大鼠心肌缺血再灌注损伤的作用。方法Wistar大鼠行冠状动脉左前降支结扎30min再松开灌注2h建立心肌缺血再灌注模型,在结扎前10min分别经股静脉给予生理盐水、30μmol/L、40μmol/L、50μmol/LNaHS溶液。通过经颈动脉插管至左心室监测不同浓度的外源性H2S对缺血再灌注大鼠血流动力学的影响,Real-TimePCR法分析心肌组织中凋亡相关基因bax、bcl-2、sur-vivin(生存素)的mRNA表达量的变化。结果外源性H2S对缺血再灌注大鼠的血流动力学指标有不同程度的改善,并且能够下调凋亡促进因子bax的mRNA表达,上调survivin的mRNA表达,对凋亡抑制基因bcl-2的mRNA表达没有明显影响。结论外源性H2S可以改善缺血再灌注损伤大鼠心肌的血流动力学,影响心肌细胞中凋亡相关基因的表达。     

镉和镍恶性转化16HBE上调基因eIF3 p36的克隆与序列

目的分析镉和镍恶性转化16HBE（人支气管上皮细胞）成瘤细胞株中翻译起始因子eIF3 p36基因序列的变化情况，探讨镉、镍重金属的致癌分子机制。方法取正常对照16HBE细胞、氯化镉（CAC12）和结晶型硫化镍（NiS）恶性转化的16HBE细胞，用有限稀释法分别建立单细胞克隆株，通过逆转录聚合酶链反应（RT—PCR）获得目的基因翻译起始因子eIF3 p36的eDNA，将目的基因产物连接到T载体中，转化、扩增重组质粒。提取大肠杆菌中的重组质粒进行DNA测序。将三组DNA测序所得到的基因序列进行对比分析。结果镉、镍转化组各细胞株（各10株）翻译起始因子的eIF3 p36 cDNA测序结果与正常对照细胞（10株）比较未发现基因的突变位点；所有镉、镍转化细胞和正常对照细胞株eIF3 p36 cDNA序列与Genbank数据库进行同源性分析，相似性比值均为100％。结论实验中镉、镍恶性转化16HBE细胞中翻译起始因子eIF3 p36上调未见基因序列改变，提示在镉、镍致癌中可能涉及到表遗传机制。     

胱硫醚-β-合成酶/硫化氢在非酒精性脂肪肝中的变化及意义

目的：观察脂肪肝中CBS/H2S的变化及其意义。方法用高脂饮食喂养SD 大鼠,建立非酒精性脂肪性肝模型,6周后处死动物,测其血清中的硫化氢,肝酶和血脂变化情况。取肝脏进行病理检查,ELISA法检测肝脏CBS及亚甲基蓝法测H2S的含量。结果模型组血清中TG、TC较对照组明显增加[（2.28±0.51）vs（1.18±0.85）]、[（4.5±1.25）vs （1.61±0.33）],（P<0.05）、H2S明显减少[（16.12±7.21）vs（20.91±10.30）],（P<0.05）;S-腺苷蛋氨酸干预组血清中TG、TC较模型组明显好转[（1.31±0.62）vs（2.28±0.51）]、[（3.2±0.66）vs（4.5±1.25）],（P<0.05）、H2S明显增加[（32.3±3.52）vs（16.12±7.21）],（P<0.01）;羟基胺干预组血清中TG、TC、H2S较模型组显著减少[（3.87±2.63）vs（2.28±0.51）]、[（5.0±1.01）vs（4.5±1.25）]、[（10.3±1.63）vs（16.12±7.21）],（P<0.01）。模型中CBS含量较对照组明显减少[（1.92±0.31）vs（4.12±0.21）],（P<0.05）、过氧化脂质明显增加[（78.2±7.56）vs（28.2±11.9）],（P<0.05）;与模型组比较S-腺苷蛋氨酸干预组CBS含量增加,过氧化脂质减少[CBS ：（2.96±0.78）、过氧化脂质：（34.2±3.68）],羟基胺干预组则CBS减少（1.32±0.17）、过氧化脂质增加（90.13±16.0）。肝脏脂肪变性与硫化氢的量呈反比。结论细胞膜损伤导致CBS外泄可能是非酒精性脂肪肝硫化氢减少的主要原因之一;激活CBS增加硫化氢可以改善肝内脂肪积聚。     

Electro-acupuncture protects against hypoxic–ischemic brain-damaged immature rat via hydrogen sulfide as a possible mediator

We investigated whether hydrogen sulfide (H₂S) may be a mediator of electro-acupuncture (EA) stimulation treatment for hypoxic–ischemic brain-damage (HIBD). We studied a HIBD 7-day-old rat model with 4 types of treatments: (1) 14 sessions of EA; (2) hydroxylamine (HA), an inhibitor of cystathionine-β-synthase (CBS), the key enzyme of H₂S generation; (3) both EA and HA; or (4) no treatment. Sham-treated rats with or without EA were also studied. Regional cerebral blood flow (rCBF) was monitored before, during and after EA at different periods of treatment (d1, 7 and 14 sessions). We evaluated motor function, H₂S levels and CBS expression in the cerebral cortex and prepared cerebral pathomorphological images after 14 sessions of treatment. EA stimulation could increase local blood circulation and improve motor function in HIBD rats. HIBD significantly increased H₂S levels of brain tissue as compared with sham treatment, and EA treatment could decrease the H₂S generation. Rats with HIBD receiving both EA and HA therapy showed greatly recovered motor function and brain morphology. H₂S might be a mediator of EA treatment of HIBD in rats.