注射用丹参多酚酸对H2O2诱导的人脐静脉内皮细胞损伤的保护作用

目的 研究注射用丹参多酚酸（SAFI）对过氧化氢（H₂O₂）诱导的人脐静脉内皮细胞（HUVEC）损伤的保护作用及机制。方法 体外培养HUVEC,设置对照组、模型组、SAFI（0.05、0.10、0.20、0.40、0.80 mg/mL）组,对照组及模型组不加药,继续培养24 h,模型组及SAFI组分别加入1 mmol/L H₂O₂作用1 h,对照组不加H₂O₂。CCK-8法检测HUVEC增殖;酶联免疫吸附法（ELISA）测定细胞间黏附因子（ICAM-1）、血管细胞黏附因子（VCAM-1）、丙二醛（MDA）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）的含量;TUNEL染色法观察HUVEC凋亡状态;Western blotting法检测凋亡相关蛋白Bcl-2、Bax的变化。结果 与模型组比较,质量浓度大于0.1 mg/mL的SAFI组细胞存活率显著增加（P<0.05）;质量浓度大于0.2 mg/mL的SAFI组LDH、MDA水平显著降低,SOD水平显著增加（P<0.05）;0.4、0.8 mg/mL的SAFI组ICAM-1、VCAM-1水平显著降低（P<0.05）;TUNEL染色结果显示,0.4、0.8 mg/mL的SAFI显著抑制凋亡;Western blotting结果显示,0.4、0.8 mg/mL的SAFI组Bcl-2蛋白表达显著升高（P<0.05）,Bax蛋白表达显著下降（P<0.05）。结论 SAFI对H₂O₂诱导的HUVEC损伤有保护作用,主要是通过提高SOD含量,降低氧化指标LDH、MDA以及炎症因子ICAM-1、VCAM-1水平,调节凋亡蛋白Bax、Bcl-2的表达发挥作用。     

Protective effect of CPUX1, a progesterone,on hydrogen peroxide‐induced oxidative damage in PC12 cells

The protective effect of CPUX1, a novel progesterone analog, on hydrogen peroxide (H₂O₂)‐induced oxidative damage was investigated in rat pheochromocytoma (PC12) cells. Following the exposure of PC12 cells to H₂O₂, there was a reduction in cell survival and activities of superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) accompanied by increased levels of lactate dehydrogenase (LDH) release, malondialdehyde (MDA) production, and intracellular reactive oxygen species (ROS) and intracellular [Ca²⁺]i levels. Preincubation of cells with CPUX1 prior to H₂O₂ exposure attenuated all these changes mentioned and had a protective effect against H₂O₂‐induced toxicity in PC12 cells, indicating that the compound may have potential therapeutic benefit for CNS disorders influenced by oxidative damage. Drug Dev Res 69: 2008 ©2008 Wiley‐Liss, Inc.     

原花青素对H2O2致PC12细胞氧化损伤作用的保护研究

目的：探讨原花青素对H2O2致PC12细胞氧化损伤的保护作用。方法：使用H2O2造成PC12细胞损伤,MTT法测细胞活性,流式细胞仪检测细胞凋亡率,免疫组化法检测细胞中Bax与Bcl-2的蛋白表达。结果：原花青素能提高H2O2损伤后细胞的活性;能减少细胞凋亡;能够增加Bcl-2蛋白表达,减少Bax蛋白表达。结论：原花青素对H2O2致细胞损伤具有保护作用,能够减少细胞凋亡,其机制可能与其增加Bcl-2蛋白表达,减少Bax蛋白表达有关。     

昆布多糖硫酸酯对H2O2诱导乳鼠心肌细胞损伤的保护作用Δ

目的研究昆布多糖硫酸酯(LAPS)对过氧化氢(H2O2)诱导乳鼠心肌细胞氧化损伤的保护作用。方法分次消化法分离原代培养的乳鼠心肌细胞,建立H2O2诱导心肌细胞损伤模型;四甲基偶氮唑盐(MTT)法检测不同浓度LAPS对心肌细胞的保护作用;试剂盒检测LAPS对细胞乳酸脱氢酶(LDH)、肌酸磷酸激酶(CK)、丙二醛(MDA)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)含量的影响;利用荧光探针二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)检测细胞内活性氧(ROS)含量。结果100μmol/L的H2O2能明显造成心肌细胞损伤,LAPS能改善H2O2所造成的心肌细胞损伤;LAPS能够明显降低的过氧化损伤心肌细胞外液中LDH和CK含量、明显降低过氧化损伤心肌细胞内MDA和活性氧(ROS)的含量、明显提高过氧化损伤心肌细胞内SOD、GSH-Px和CAT的活力。结论 LAPS对心肌细胞的过氧化损伤有保护作用,与其降低或阻断脂质过氧化反应,降低细胞内ROS含量,进而阻断因ROS对细胞造成的损害,及时修复受损细胞有着密切关系。     

Effects of carnosine on prooxidant–antioxidant status in heart tissue,plasma and erythrocytes of rats with isoproterenol-induced myocardial infarction

Rats were injected with isoproterenol (ISO; 110 mg/kg, ip, 2 doses, 24 h interval) to induce acute myocardial infarction (AMI) and were sacrificed 6 and 24 h after the last ISO injection. The heart tissue, plasma and erythrocytes of these rats were evaluated for cardiac markers and oxidative stress parameters. Levels of cardiac troponin T (cTnT) and the activities of creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) in plasma were increased 6 and 24 h after ISO treatment. The levels of malondialdehyde (MDA), diene conjugate (DC), and protein carbonyl (PC) were increased in heart tissue and plasma, while levels of erythrocyte MDA and glutathione (GSH) and plasma ferric reducing antioxidant power (FRAP) were also increased. However, GSH levels and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased in heart tissue of rats with AMI. We also investigated the effects of carnosine (CAR) treatment on these parameters 24 h after the last ISO injection. CAR (250 mg/kg/day; ip) treatments were carried out either 10 days before ISO injection or 2 days concomitant with ISO. Pretreatment with CAR decreased plasma LDH and AST activities and ameliorated cardiac histopathological changes in ISO-treated rats. Cardiac MDA, DC and PC levels decreased, but GSH levels and SOD and GSH-Px activities increased. However, the increases in plasma MDA and PC levels as well as erythrocyte H₂O₂-induced MDA and GSH levels did not change due to CAR pretreatment. In conclusion, our findings indicate that CAR pretreatment may have protective effects on ISO-induced cardiac toxicity by decreasing oxidative stress.     

淫羊藿苷对H2O2诱导的软骨细胞氧化损伤的保护作用及其机制

目的 探究淫羊藿苷（icariin，ICA）对H₂O₂诱导的软骨细胞氧化损伤的保护作用及相关机制。方法 分离SD新生大鼠软骨细胞，随机分为对照组、H₂O₂模型组、ICA低剂量组、ICA中剂量组、ICA高剂量组；采用CCK8法检测各组细胞增殖能力的变化；采用ELISA试剂盒检测各组细胞中活性氧（reactive oxygen，ROS）、超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）、过氧化氢酶（catalase，CAT）以及谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）的表达情况；流式细胞术检测各组细胞周期情况，并计算增殖指数（proliferation index，PI）；Hoechst染色观察各组细胞核凋亡情况；分别采用荧光定量PCR （qRT-PCR）和Western blotting检测凋亡相关因子及Nrf2/HO-1通路的表达情况。结果 与对照组相比，H₂O₂模型组细胞增殖能力降低，ROS、MDA含量升高，SOD、CAT及GSH-Px含量下降，细胞凋亡情况加重；经ICA干预后，软骨细胞的增殖能力上升，ROS、MDA含量下降，SOD、CAT及GSH-Px含量增加，并且ICA能够有效抑制软骨细胞凋亡，上调Nrf2和HO-1蛋白的表达。结论 ICA对H₂O₂诱导的软骨细胞氧化损伤具有保护作用，能够抑制软骨细胞凋亡，其机制跟Nrf2/HO-1信号通路有关。     

Cardioprotective effect of KR-33889, a novel PARP inhibitor,against oxidative stress-induced apoptosis in H9c2 cells and isolated rat hearts

Oxidative stress plays a critical role in cardiac injury during ischemia/reperfusion (I/R). Despite a potent cardioprotective activity of KR-33889, a novel poly (ADP-ribose) polymerase inhibitor, its underlying mechanism remains unresolved. This study was designed to investigate the protective effects of KR-33889 against oxidative stress-induced apoptosis in rat cardiomyocytes H9c2 cells and isolated rat hearts. H₂O₂ caused severe injury to H9c2 cells, mainly due to apoptosis, as revealed by TUNEL assay. However, KR-33889 pretreatment significantly attenuated H₂O₂-induced apoptosis of H9c2 cells, which was accompanied by decrease in expression of both cleaved caspase-3 and Bax and increase in Bcl-2 expression and the ratio of Bcl-2/Bax. KR-33889 also significantly enhanced the expression of anti-oxidant enzymes including heme oxygenase-1, Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase, thereby inhibiting production of intracellular ROS. Furthermore, KR-33889 reversed H₂O₂-induced decrease in phosphorylation of Akt, GSK-3β, ERK1/2, p38 MAPK, and SAPK/JNK during most H₂O₂ exposure time. In globally ischemic rat hearts, KR-33889 inhibited both I/R-induced decrease in cardiac contractility and apoptosis by increasing Bcl-2, decreasing both cleaved caspase-3 and Bax expression, and enhancing expression of anti-oxidant enzymes. Taken together, these results suggest that KR-33889 may have therapeutic potential to prevent I/R-induced heart injury in ischemic heart diseases mainly by reducing oxidative stress-mediated myocardial apoptosis.     

Protective effects of protopine on hydrogen peroxide-induced oxidative injury of PC12 cells via Ca(2+) antagonism and antioxidant mechanisms

Calcium and lipid peroxidation play important roles in oxidative stress-induced cellular injury and apoptosis, which ultimately cause cell death. In this study we examined whether protopine had a neuroprotection against H₂O₂-induced injury in PC12 cells. Pretreatment of PC12 cells with protopine improved the cell viability, enhanced activities of superoxide dismutase, glutathione peroxidase and catalase, and decreased malondialdehyde level in the H₂O₂ injured cells. Protopine also reversed the increased intracellular Ca²⁺ concentration and the reduced mitochondrial membrane potential caused by H₂O₂ in the cells. Furthermore, protopine was able to inhibit caspase-3 expression and cell apoptosis induced by H₂O₂. In summary, this study demonstrates that protopine is able to relieve H₂O₂-induced oxidative stress and apoptosis in PC12 cells, at least in part, by Ca²⁺ antagonism and antioxidant mechanisms.     

3′,4′,7-Trihydroxyflavone prevents apoptotic cell death in neuronal cells from hydrogen peroxide-induced oxidative stress

In this study, we investigated the mechanisms of 3′,4′,7-trihydroxyflavone (THF) protection of neuronal cells from neuronal cell death induced by the oxidative stress-related neurotoxin hydrogen peroxide (H₂O₂). Pretreatment with THF significantly elevated cell viability, reduced H₂O₂-induced lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) production, glutathione (GSH) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, and mitochondria membrane potential (MMP) loss. Western blot data demonstrated that THF inhibited the H₂O₂-induced up- or down-regulation of cleaved caspase-3, cleaved caspase-9, cleaved poly-ADP-ribose polymerase (PARP), Bax, Bcl-2, and Bcl-xL, and attenuated the H₂O₂-induced release of cytochrome c from the mitochondria to the cytosol. In addition, pretreatment with THF attenuated H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinases (PI3K)/Akt. THF also inhibited nuclear factor-κB (NF-κB) translocation to the nucleus induced by H₂O₂, down-stream of H₂O₂-induced phosphorylation of MAPKs and PI3K/Akt. These data provide the first evidence that THF protects neuronal cells against H₂O₂-induced oxidative stress, possibly through ROS reduction, mitochondria protection, and NF-κB modulation via MAPKs and PI3K/Akt pathways. The neuroprotective effects of THF make it a promising candidate as a therapeutic agent for neurodegenerative diseases.     

灯盏乙素对过氧化氢致PC12细胞损伤的抗氧化作用

目的研究灯盏乙素对过氧化氢(H2O2)诱导PC12细胞损伤的保护作用。方法建立H2O2致PC12细胞损伤模型,倒置相差显微镜下进行一般形态学观察,化学比色法测定乳酸脱氢酶(LDH)释放量及细胞培养液和细胞内丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果100μmol/L H2O2诱导PC12细胞4 h,细胞呈现明显损伤形态,LDH释放量增加,细胞培养液和细胞内MDA含量升高,SOD活性降低。灯盏乙素可明显改善形态学损伤,显著降低LDH释放量、细胞培养液及细胞内MDA含量,提高SOD活性。结论灯盏乙素对H2O2诱导PC12细胞损伤具有保护作用,其作用机制可能与提高PC12细胞的抗氧化能力有关。     

梓醇对缺糖缺氧诱导PC12细胞损伤的保护作用研究

目的探讨梓醇对抗缺糖缺氧诱导的PCI2细胞损伤作用。方法梓醇预处理PCI2细胞,加入含连二亚硫酸钠的无糖Earle’s液制备损伤模型,检测细胞存活率,乳酸脱氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶、丙二醛水平,检测细胞内游离Ca2＋浓度。结果与模型组比较,梓醇可剂量依赖性的提高细胞存活率（P〈0．01）,减少乳酸脱氢酶漏出率（P〈0．05或〈0．01）,提高超氧化物歧化酶、谷胱甘肽过氧化物酶活性,拮抗丙二醛水平升高,降低细胞内游离Ca2＋浓度（P〈0．05或〈0．01）。结论梓醇对缺糖缺氧诱导的PCI2细胞损伤具有保护作用,作用机制可能与其清除自由基和抑制钙超载有关。     

Protective effects of prostaglandin E1 on human umbilical vein endothelial cell injury induced by hydrogen peroxide

Aim:

To investigate the protective effects of prostaglandin E₁ (PGE₁) against H₂O₂-induced oxidative damage on human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were pretreated with PGE₁ (0.25, 0.50, and 1.00 μmol/L) for 24 h and exposed to H₂O₂ (200 μmol/L) for 12 h, and cell viability was measured by the MTT assay. LDH, NO, SOD, GSH-Px, MDA, ROS, and apoptotic percentage were determined. eNOS expression was measured by Western blotting and real-time PCR.

Results:

PGE₁ (0.25−1.00 μmol/L) was able to markedly restore the viability of HUVECs under oxidative stress, and scavenged intracellular reactive oxygen species induced by H₂O₂. PGE₁ also suppressed the production of lipid peroxides, such as MDA, restored the activities of endogenous antioxidants including SOD and GSH-Px, and inhibited cell apoptosis. In addition, PGE₁ significantly increased NO content, eNOS protein, and mRNA expression.

Conclusion:

PGE₁ effectively protected endothelial cells against oxidative stress induced by H₂O₂, an activity that might depend on the up-regulation of NO expression.     

荠苧黄酮对低压低氧小鼠心肌组织损伤的改善作用与机制研究

目的:研究荠苧黄酮对低压低氧小鼠心肌组织损伤的改善作用与机制.方法:将60只小鼠随机分为正常对照组、缺氧模型组、芦丁组和荠苧黄酮组,连续灌胃(ig)给药5 d,最后1次给药后,在模拟海拔8 000 m 环境停留12h,测定血清中肌酸激酶(creatine kinase,CK)、乳酸脱氢酶(lactic dehydrog...     

美洲大蠊提取物对H2O2诱导的PC12细胞氧化损伤的保护作用

目的:探讨美洲大蠊提取物(PAS840)对大鼠肾上腺嗜铬细胞瘤(PC12)细胞氧化损伤模型的保护作用及机制。方法:采用H₂O₂刺激PC12细胞建立神经细胞氧化损伤模型,实验分为正常组(Con)、模型组(Mod)、PAS840低中高剂量组(20,50,125 μg·mL^-1的PAS840培养基溶液进行处理),采用倒置显微镜观察细胞形态并采用CCK-8法检测各组细胞存活率,生化试剂盒检测各组超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、乳酸脱氢酶(LDH)、谷胱甘肽(GSH)和丙二醛(MDA)的水平;DCFH-DA荧光探针检测各组活性氧簇(ROS)的水平;流式细胞术检测各组细胞凋亡率;JC-1法染色检测各组细胞线粒体膜电位(MMP);RT-qPCR检测各组Nrf2/HO-1通路因子(Nrf2、Keap1、HO-1和NQO1)、凋亡因子(Bcl-2、Bax和Caspase-3)、炎症因子(TNF-α、IL-1β和IL-6)、乙酰胆碱酯酶(AchE)和过氧化氢酶(CAT) mRNA的表达水平;Western blot法检测各组Nrf2、HO-1、Bcl-2、Bax和Caspase-3蛋白的表达水平。结果:PAS840可显著提高氧化损伤细胞的存活率、MMP及SOD、GSH-Px和GSH的水平,降低LDH、MDA和ROS的水平;显著降低Keap1、TNF-α、IL-1β、IL-6和AchE mRNA表达的同时,显著增加CAT和NQO1 mRNA的表达,显著降低Nrf2、Bax和Caspase-3的mRNA及其蛋白表达,显著增加HO-1和Bcl-2的mRNA及其蛋白表达。结论:PAS840可以抑制H₂O₂诱导的PC12细胞凋亡,减轻炎症,其机制可能与降低ROS、调控Nrf2/HO-1通路因子减轻细胞的氧化损伤程度有关。     

脑源性神经营养因子可减轻 H9c2 心肌细胞缺氧/复氧损伤

目的 探讨脑源性神经营养因子（BDNF）预处理对 H9c2 心肌细胞缺氧/复氧（H/R）损伤的影响及其作用机制。方法 体外培养 H9c2 心肌细胞并以缺氧 （95%N2+5%CO2） 培养 4 h 后复氧 （95%O2+5%CO2） 培养 12 h 建立 H/R 模型, 并分为 Control 组、 H/R 组、 不同浓度 （1、 10、 100 μg/L） BDNF 预处理后 H/R 组、 酪氨酸蛋白激酶受体 B （TrkB）inhibitor 组 （同时加入 100 μg/L BDNF 和 1∶1 000 的 TrkB inhibitor 预处理后 H/R 组）。利用 MTT 方法检测各组心肌细胞的细胞存活率; 并测定各组心肌细胞 H/R 损伤后乳酸脱氢酶（LDH）、 肌酸激酶（CK）、 丙二醛（MDA）、 超氧化物歧化酶（SOD）含量及活性; 流式细胞术测定各组心肌细胞的凋亡率; Western-blot 检测 TrkB、 Bcl-2、 Bax 蛋白表达。结果 与 Control 组相比, H/R 组细胞存活率明显下降, LDH、 CK 和 MDA 含量升高, SOD 活性降低, 细胞凋亡率升高, 抗凋亡 Bcl-2 蛋白表达下降, 促凋亡 Bax 蛋白表达升高（均 P < 0.05）。与 H/R 组相比, 不同浓度 BDNF 预处理H9c2 心肌细胞后 H/R, 各组细胞存活率均明显上升, CK、 LDH、 MDA 含量逐渐下降, SOD 活性升高, 细胞凋亡率下降, TrkB 和 Bcl-2 蛋白表达升高, 而 Bax 蛋白表达降低, 但 BDNF 的作用均受到 TrkB inhibitor 的抑制。结论 BDNF预处理能够提高 H9c2 心肌细胞 H/R 损伤的细胞存活率, 通过降低细胞凋亡率和提升细胞抗氧化能力而发挥保护作用, 并与 BDNF-TrkB 信号通路有关。     

Protective effect of polypeptides from larva of housefly (Musca domestica) on hydrogen peroxide-induced oxidative damage in HepG2 cells

Housefly (Musca domestica) is an important medical insect and its larva is an ideal high protein food source. We isolated from housefly larvae the polypeptides hydrolyzed by neutral protease (PHNP), and investigated the protective effect of PHNP on hydrogen peroxide (H₂O₂)-induced oxidative damage in HepG2 cells. Cells exposed to H₂O₂ showed a marked decrease in proliferation and intracellular superoxide dismutase (SOD) activity, and a significant increase in reactive oxygen species (ROS) level and malondialdehyde (MDA) content. H₂O₂ also caused apoptosis and mitochondrial dysfunction including mitochondrial fragmentation and the loss of mitochondrial membrane potential. Pretreatment with PHNP at concentrations of 2.5, 5, 10 μg/mL blocked these H₂O₂-induced cellular events in a dose-dependent manner. The effect of PHNP at 10 μg/mL is equal to that of ascorbic acid at 10 μM. In summary, PHNP has a protective effect against H₂O₂-induced oxidative injury in cells due to its ability to decrease intracellular ROS and elevate antioxidant enzyme activities.     

Pramipexole protects against H2O2-induced PC12 cell death

Pramipexole, a novel non-ergot dopamine (DA) agonist, has been successfully applied to the treatment of Parkinson’s disease (PD). Although the specific cause of PD remains unknown, recent studies have provided evidence that oxidative stress plays a role in the parthenogenesis of the disease. In the present study, we examined the effect of pramipexole on hydrogen peroxide (H₂O₂, 100 μM)-induced PC12 cell death, and the intracellular mechanism of this effect. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay revealed that pretreatment of PC12 cells with pramipexole (1–100 μM) resulted in significant protection against H₂O₂-induced cell death in a concentration-dependent manner. The protective effect of pramipexole was not affected by pretreatment with the DA receptor antagonists sulpiride, spiperone or domperidone, suggesting that the effect of pramipexole is not mediated by DA receptors. In PC12 cells, pramipexole inhibited H₂O₂-induced lactate dehydrogenase (LDH) leakage, as well as H₂O₂-induced cytochrome c release and caspase-3 activation with the resultant apoptosis. It was also observed in PC12 cells that H₂O₂ stimulated phosphorylation of mitogen-activated protein (MAP) kinases, i.e., extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun NH₂-terminal kinase (JNK) and p38 MAP kinase. Pramipexole inhibited H₂O₂-induced JNK and p38 MAP kinase, but not ERK1/2 phosphorylation. Furthermore, in these cells experiments with a fluorescent probe, 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, revealed that pramipexole, the JNK inhibitor SP600125 and the p38 MAP kinase inhibitor SB203580 inhibited the generation of H₂O₂-induced reactive oxygen species. Caspase inhibitors Z-DEVD-FMK and Z-IETD-FMK, as well as SP600125 and SB203580, inhibited H₂O₂-induced PC12 cell death to a similar extent as pramipexole. These results suggest that pramipexole exerts a protective effect against oxidative stress-induced PC12 cell death in part through an inhibition of JNK and p38 MAP kinase.     

Ghrelin protects H9c2 cells from hydrogen peroxide-induced apoptosis through NF-κB and mitochondria-mediated signaling

Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. Herein we investigate the protective effects of ghrelin in H₂O₂-induced apoptosis of H9c2 cells, as well as the possible molecular mechanisms involved. To study apoptosis, the cells were assessed by morphologic examination, MTS assay, Annexin V–propidium iodide dual staining and TUNEL analysis. Intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential were also measured. To investigate the underlying molecular mechanisms, the expression of Bcl-2, Bax, active caspase-9 and NF-κB were assessed by Western blotting, and caspase-3 activity was determined by a colorimetric activity assay kit. After stimulation with H₂O₂ for 18 h, H9c2 cells viability decreased significantly; a large fraction of cells underwent apoptosis. We observed a dose-dependent rescue of H9c2 cells from H₂O₂-induced apoptosis in the presence of different ghrelin concentrations. Preincubation with ghrelin also restored the ROS and mitochondrial membrane potential levels that had been altered by H₂O₂ treatment. Moreover, ghrelin decreased H₂O₂-induced Bax production and caspase-9 activation, and increased Bcl-2 levels. NF-κB phosphorylation was also significantly inhibited by ghrelin in H₂O₂-treated cells. Caspase-3 activation was suppressed by ghrelin in H₂O₂-treated H9c2 cells in a dose-dependent manner. In summary, ghrelin protects H9c2 cells from oxidative stress-induced apoptosis through downregulation of Bax expression, caspase-9 activation and NF-κB phosphorylation, and upregulation of Bcl-2 expression. Caspase-3 activation was also reduced in a dose-dependent manner. These data suggest that ghrelin might protect against cardiovascular disease by protecting the mitochondria.     

Safflor yellow A protects neonatal rat cardiomyocytes against anoxia/reoxygenation injury in vitro

Aim:

To investigate the effects of safflor yellow A (SYA), a flavonoid extracted from Carthamus tinctorius L, on cultured rat cardiomyocytes exposed to anoxia/reoxygenation (A/R).

Methods:

Primary cultured neonatal rat cardiomyocytes were exposed to anoxia for 3 h followed by reoxygenation for 6 h. The cell viability was measured using MTT assay. The releases of lactate dehydrogenase (LDH) and creatine kinase (CK), level of malondialdehyde (MDA), and activities of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were analyzed. Hoechst 33258 staining and changes in Bcl-2/Bax ratio and caspase 3 activity were used to examine A/R-induced apoptosis.

Results:

The A/R exposure markedly decreased the viability of cardiomyocytes, suppressed the activities of SOD, GSH, CAT and GSH-Px, and Bcl-2 protein expression. Meanwhile, the A/R exposure markedly increased the release of LDH and CK, and MDA production in the cardiomyocytes, and increased the rate of apoptosis, caspase 3 activity, Bax protein expression. Pretreatment with SYA (40, 60 and 80 nmol/L) concentration-dependently blocked the A/R-induced changes in the cardiomyocytes. Pretreatment of the cardiomyocytes with the antioxidant N-acetylcysteine (NAC, 200 μmol/L) produced protective effects that were comparable to those caused by SYA (80 nmol/L).

Conclusion:

SYA protects cultured rat cardiomyocytes against A/R injury, maybe via inhibiting cellular oxidative stress and apoptosis.