过氧化氢预处理对H9c2心肌细胞氧化应激损伤的保护作用

目的：活性氧介导的氧化损伤是缺血再灌注损伤的重要机制,本研究通过观察H2O2预处理对氧化损伤的H9c2心肌细胞存活率和细胞凋亡的影响,探讨其保护H9c2心肌细胞的作用机制。方法：体外培养H9c2心肌细胞,取对数生长期细胞用于实验研究。建立H2O2预处理抵抗高浓度H：O：诱导的细胞氧化损伤模型,实验分组如下：（1）正常对照组（CTL）;（2）损伤组（INJURY）;（3）预处理组十损伤组（PC）。应用CCK8法检测细胞存活率;试剂盒检测胞内MDA水平和T．sOD活性;Hoechst33258染色观察凋亡形态;Annexin-V／PI双染与流式细胞术检测细胞凋亡率。结果：25vLmol／L的H202预处理90rain能明显地保护H9c2心肌细胞抵抗400μmol／LH2O2诱导的氧化损伤,提高细胞存活率,下调MDA水平,上调SOD活性,抑制细胞凋亡,降低细胞凋亡率。结论：低浓度H2O2预处理能减轻H9c2心肌细胞的氧化损伤,抑制氧化损伤诱导的心肌细胞凋亡,具有很好的抗氧化损伤和抗心肌细胞凋亡的保护作用,其作用机制可能与细胞SOD活性上调有关。H2O2预处理为临床治疗心肌缺血／再灌注损伤提供了一项新策略。     

Protective Effect of Boerhaavia diffusa L. against Mitochondrial Dysfunction in Angiotensin II Induced Hypertrophy in H9c2 Cardiomyoblast Cells

Mitochondrial dysfunction plays a critical role in the development of cardiac hypertrophy and heart failure. So mitochondria are emerging as one of the important druggable targets in the management of cardiac hypertrophy and other associated complications. In the present study, effects of ethanolic extract of Boerhaavia diffusa (BDE), a green leafy vegetable against mitochondrial dysfunction in angiotensin II (Ang II) induced hypertrophy in H9c2 cardiomyoblasts was evaluated. H9c2 cells challenged with Ang II exhibited pathological hypertrophic responses and mitochondrial dysfunction which was evident from increment in cell volume (49.09±1.13%), protein content (55.17±1.19%), LDH leakage (58.74±1.87%), increased intracellular ROS production (26.25±0.91%), mitochondrial superoxide generation (65.06±2.27%), alteration in mitochondrial transmembrane potential (ΔΨm), opening of mitochondrial permeability transition pore (mPTP) and mitochondrial swelling. In addition, activities of mitochondrial respiratory chain complexes (I-IV), aconitase, NADPH oxidase, thioredoxin reductase, oxygen consumption rate and calcium homeostasis were evaluated. Treatment with BDE significantly prevented the generation of intracellular ROS and mitochondrial superoxide radicals and protected the mitochondria by preventing dissipation of ΔΨm, opening of mPTP, mitochondrial swelling and enhanced the activities of respiratory chain complexes and oxygen consumption rate in H9c2 cells. Activities of aconitase and thioredoxin reductase which was lowered (33.77±0.68% & 45.81±0.71% respectively) due to hypertrophy, were increased in BDE treated cells (P≤0.05). Moreover, BDE also reduced the intracellular calcium overload in Ang II treated cells. Overall results revealed the protective effects of B. diffusa against mitochondrial dysfunction in hypertrophy in H9c2 cells and the present findings may shed new light on the therapeutic potential of B. diffusa in addition to its nutraceutical potentials.     

Long-term Heat Exposure Prevents Hypoxia-Induced Apoptosis in Mouse Fibroblast Cells

Long-term continuous exposure to high ambient temperatures induces complete heat acclimation in humans and animals. However, to date, the effects of long-term exposure to heat stress on cells have not been fully evaluated. In this study, we investigated an adaptive physiological process induced in culture cells by continuous exposure to mild heat stress for 60 days. The results of this investigation provide evidence that after long-term heat acclimation in cells, (1) heat shock protein levels are increased, (2) hypoxia inducible factor-1α (HIF-1α) expression is upregulated, and (3) heat shock-induced and hypoxia-induced apoptoses are attenuated. These results suggest that the hypoxia response pathway is an intrinsic part of the heat acclimation repertoire and that the HIF-1 pathway following long-term heat acclimation induces cells with cross tolerance against hypoxia.     

Activation of AMP-Activated Protein Kinase Contributes to Doxorubicin-Induced Cell Death and Apoptosis in Cultured Myocardial H9c2 Cells

Despite its potent antitumor effect, clinical use of Doxorubicin is limited because of serious side effects including myocardial toxicity. Understanding the cellular mechanism involved in this process in a better manner is beneficial for optimizing Doxorubicin treatment. In the current study, the authors focus on the AMP-activated protein kinase (AMPK) in the said process. In this study, the authors discovered for the first time that Doxorubicin induces AMPK activation in cultured rat embryonic ventricular myocardial H9c2 cells. Reactive oxygen species (ROS)-dependent LKB1 activation serves as the upstream signal for AMPK activation by Doxorubicin. Evidence in support of the activation of AMPK contributing to Doxorubicin-induced H9c2 cell death/apoptosis—probably by modulating multiple downstream signal targets, including regulating JNK, p53, and inhibiting mTORC1—is provided in this article.     

Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate''s protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).     

肌肽通过调节JNK和NF-kappaB通路抑制高糖诱导的心肌细胞凋亡

高血糖症是糖尿病并发心血管疾病的重要危险因素. 高血糖诱导产生的活性氧（ROS）能够引起糖尿病心肌病.我们的前期工作已经证实,肌肽对高糖环境下细胞凋亡具有保护作用,但其机制尚未明确.为研究肌肽对高糖诱导的大鼠心肌细胞凋亡的抑制作用及相关信号机制,以高糖诱导心肌细胞H9c2为模型,采用Brdu-ELISA法检测细胞增殖过程中DNA合成情况,流式细胞术检测细胞凋亡率,免疫印迹实验检测JNK/c-Jun、NF-κB的磷酸化水平,RT-PCR检测TNF-α的mRNA表达. 实验结果显示,肌肽能够提高高糖损伤的H9c2细胞增殖能力,降低心肌细胞凋亡,抑制高糖激活的JNK、c-Jun和NF-κB磷酸化水平及TNFα的mRNA表达. 上述结果表明,肌肽拮抗高糖诱导的心肌细胞凋亡机制与抑制p-JNK /p-c-Jun、p-NF-κB水平和TNF-α表达有关.     

外泌体分泌在1,4-苯醌诱导的HL-60细胞凋亡中起保护作用

慢性苯暴露损害造血系统,可引起再生障碍性贫血,甚至白血病。外泌体(exosomes)是细胞分泌的纳米级膜泡,在许多生理和病理过程中发挥重要作用。然而,苯及其代谢产物对外泌体分泌的影响仍不清楚。本研究旨在观察苯的活性代谢产物1,4-苯醌(1,4-benzoquinone,1,4-BQ)能否引起人早幼粒白血病细胞HL-60外泌体分泌量的变化以及外泌体释放在1,4-BQ诱导的细胞凋亡中的作用。应用不同浓度1,4-BQ处理细胞24 h,超高速离心法提取细胞培养基中的外泌体,结果发现1,4-BQ能促进外泌体分泌,呈剂量反应关系。进一步应用外泌体抑制剂GW4869抑制外泌体分泌,流式细胞仪检测细胞凋亡率、蛋白质免疫印迹法检测抑凋亡蛋白质Bcl-2、凋亡通路关键蛋白质cleaved caspase-9和cleaved caspase-3的表达,探讨外泌体分泌对1,4-BQ所致细胞凋亡的影响。结果显示:与对照组相比,1,4-BQ单独处理组的凋亡率、Bcl-2、cleaved caspase-9和cleaved caspase-3的表达均显著增高(P<0.05),而1,4-BQ+GW4869组的凋亡率及凋亡相关蛋白质的表达均显著高于1,4-BQ单独处理组(P<0.05),表明抑制外泌体分泌可增加1,4-BQ诱导的细胞凋亡。综上表明,1,4-BQ能促进外泌体分泌,并在1,4-BQ诱导的细胞凋亡中起保护作用。本研究为了解苯的毒性效应和毒性机制提供了新的实验证据。     

重组nkx2.5腺病毒抑制过氧化氢诱导的H9c2细胞凋亡

为研究心脏发育关键基因nkx2.5的功能及应用价值,构建Ad-Nkx2.5重组腺病毒,并检测nkx2.5过表达拮抗氧化应激损伤的效应及机制。采用AdEasy腺病毒表达系统构建Ad-Nkx2.5重组腺病毒,建立H2O2诱导H9c2心肌细胞凋亡模型,分别用Ad-Nkx2.5重组病毒或对照病毒感染细胞,采用Hoechst33342染色观察细胞形态变化、MTT法检测细胞存活率,免疫印迹检测caspase-3活化、细胞色素C的胞浆含量。并通过Real-timePCR检测凋亡相关基因bcl-2和bax表达。结果发现,nkx2.5过表达促进H9c2细胞存活,抑制H2O2诱导的caspase-3活化及线粒体细胞色素C的释放。Nkx2.5过表达上调bcl-2表达,显著下调H2O2诱导的bax表达。并发现H2O2对Nkx2.5核定位无明显影响。结果显示重组腺病毒介导的Nkx2.5过表达可通过调控凋亡相关基因表达,抑制线粒体凋亡途径,保护心肌细胞抗氧化损伤。     

Resveratrol for protection against statin toxicity in C2C12 and H9c2 cells

Statins are among the most commonly prescribed drugs for the treatment of high blood cholesterol. Myotoxicity of statins in certain individuals is often a severe side effect leading to withdrawal. Using C2C12 and H9c2 cells, both exhibiting characteristics of skeletal muscle cells, we addressed whether resveratrol (RSV) can prevent statin toxicity. Statins decreased cell viability in a dose and time‐dependent manner. Among the five statins tested, atorvastatin, simvastatin, lovastatin, pravastatin, and fluvastatin, simvastatin is the most toxic one. Simvastatin at 10 µM caused about 65% loss of metabolic activity as measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays in C2C12 cells or H9c2 cells. Inhibition of metabolic activity correlates with an increase in caspase activity. RSV was found to protect H9c2 cells from simvastatin‐induced activation of caspase‐3/7. However, such protection was not found in C2C12 cells. This cell type‐dependent effect of RSV adds to the complexity in muscle cell toxicity of statins.     

Vitamin C Attenuates Sodium Fluoride-Induced Mitochondrial Oxidative Stress and Apoptosis via Sirt1-SOD2 Pathway in F9 Cells

Biological Trace Element Research - The thioredoxin-like (Rdx) family proteins contain four selenoproteins (selenoprotein H, SELENOH; selenoprotein T, SELENOT; selenoprotein V, SELENOV;...     

组蛋白赖氨酸甲基转移酶2D调控H9c2细胞中HIF-1α/VEGF通路

组蛋白赖氨酸甲基转移酶2D (histone-lysine N-methyltransferase 2D, KMT2D) 作为主要的组蛋白3第4位赖氨酸 (H3K4) 甲基转移酶,在调控胚胎发育、组织分化、代谢和肿瘤抑制方面发挥重要作用。在小鼠体内,敲除Kmt2d会导致严重的心脏发育缺陷最终造成胚胎期死亡。低氧诱导因子-1α (hypoxia-inducible factor 1α, HIF-1α) 作为调节细胞应对低氧的关键转录因子,能够调控多种下游基因转录。有相关研究揭示,表观遗传调控者能够调节HIF-1α的稳定性和活性。同样,作为表观遗传调控者的组蛋白甲基转移酶KMT2D是否参与低氧条件下HIF-1α对下游基因的调控,目前仍未知。在本研究中,观察在Kmt2d正常或缺乏的情况下,心肌细胞H9c2对低氧环境的应答反应。结果显示,与常氧条件相比,低氧状态下HIF-1α、组蛋白乙酰化酶P300、KMT2D及其介导的H3K4一甲基化 (H3K4 mono-methylation, H3K4me1)的蛋白质水平增加 (P<0.05);HIF-1α下游基因血管内皮生长因子 (vascular endothelial growth factor, Vegf) 的mRNA表达水平明显上调 (P<0.01)。染色质免疫共沉淀实验 (chromatin immunoprecipitation assay, ChIP-qPCR) 检测结果显示,H3K4me1和组蛋白3第27位赖氨酸乙酰化 (histone 3 lysine 27 acetylation, H3K27ac) 在Vegf基因启动子区域的结合丰度明显增加 (P<0.05)。低氧条件下沉默Kmt2d之后,H3K4me1蛋白水平和Vegf的mRNA表达下降 (P<0.05)。本研究表明,低氧条件下KMT2D参与调控HIF-1α和下游基因Vegf的表达。     

组蛋白赖氨酸甲基转移酶2D调控H9c2细胞中HIF-1α/VEGF通路

组蛋白赖氨酸甲基转移酶2D (histone-lysine N-methyltransferase 2D, KMT2D) 作为主要的组蛋白3第4位赖氨酸 (H3K4) 甲基转移酶,在调控胚胎发育、组织分化、代谢和肿瘤抑制方面发挥重要作用。在小鼠体内,敲除Kmt2d会导致严重的心脏发育缺陷最终造成胚胎期死亡。低氧诱导因子-1α (hypoxia-inducible factor 1α, HIF-1α) 作为调节细胞应对低氧的关键转录因子,能够调控多种下游基因转录。有相关研究揭示,表观遗传调控者能够调节HIF-1α的稳定性和活性。同样,作为表观遗传调控者的组蛋白甲基转移酶KMT2D是否参与低氧条件下HIF-1α对下游基因的调控,目前仍未知。在本研究中,观察在Kmt2d正常或缺乏的情况下,心肌细胞H9c2对低氧环境的应答反应。结果显示,与常氧条件相比,低氧状态下HIF-1α、组蛋白乙酰化酶P300、KMT2D及其介导的H3K4一甲基化 (H3K4 mono-methylation, H3K4me1)的蛋白质水平增加 (P<0.05);HIF-1α下游基因血管内皮生长因子 (vascular endothelial growth factor, Vegf) 的mRNA表达水平明显上调 (P<0.01)。染色质免疫共沉淀实验 (chromatin immunoprecipitation assay, ChIP-qPCR) 检测结果显示,H3K4me1和组蛋白3第27位赖氨酸乙酰化 (histone 3 lysine 27 acetylation, H3K27ac) 在Vegf基因启动子区域的结合丰度明显增加 (P<0.05)。低氧条件下沉默Kmt2d之后,H3K4me1蛋白水平和Vegf的mRNA表达下降 (P<0.05)。本研究表明,低氧条件下KMT2D参与调控HIF-1α和下游基因Vegf的表达。     

Protection of Neuroblastoma Neuro2A Cells from Hypoxia-Induced Apoptosis by Cyclic Phosphatidic Acid (cPA)

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl₂) to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A) cells with CoCl₂ induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl₂-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl₂. Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA₁, LPA₂, and LPA₆, which are G-protein coupled receptors that can be activated by cPA. To date, LPA₁ and LPA₂ have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA₁ and LPA₂ on cPA-induced neuroprotective functions, Ki16425, a selective LPA₁ and LPA₃ antagonist, was adopted to know the LPA₁ function and siRNA was used to knockdown the expression of LPA₂. On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl₂-induced hypoxia damage is mediated via LPA₂.     

MFN1介导的线粒体融合在心肌细胞凋亡中的作用研究

目的:探讨线粒体融合关键蛋白MFN1介导的线粒体融合在调控心肌细胞凋亡中的作用。方法:通过si RNA降低体外培养H9C2心肌细胞中MFN1的表达后,采用Western blot检测线粒体细胞色素c(Cyto c)释放及其下游凋亡效应分子Caspase9与Caspase3活性,流式细胞术检测细胞内活性氧(ROS)的产生情况,流式细胞术检测细胞凋亡的情况。结果:干扰MFN1可显著促进H9C2心肌细胞内细胞色素c由线粒体释放至胞浆,促进Caspase9与Caspase3的激活,增加细胞内活性氧ROS产生并提高细胞凋亡率(均P0.05)。结论:MFN1介导的线粒体融合可保护心肌细胞凋亡,其机制可能与抑制ROS产生与细胞色素C释放有关。     

紫草乌中生物碱成分及其对H9c2细胞毒性研究

为了解我国特有药用乌头种紫草乌(Aconitum episcopale)的生物碱类化学成分及其对大鼠心脏细胞H9c2的细胞毒性,从紫草乌中分离出10个C19-二萜生物碱,根据理化性质和波谱数据,其结构分别鉴定为滇乌碱(1)、粗茎乌头碱甲(2)、黄草乌碱丙(3)、黄草乌碱甲(4)、丽乌亭(5)、猎鹰乌头碱(6)、14-O-乙酰萨卡可尼亭(7)、萨卡可尼亭(8)、查斯曼宁(9)和展毛黄草乌碱A (10)。化合物5、6和10为首次从该植物中分离得到。化合物1～4、6～7和9～10对大鼠心脏细胞H9c2增殖的半数抑制浓度为163.8～341.6μg/m L,表明紫草乌中的生物碱对H9c2细胞具有毒性作用。     

Flavonoids from Malus hupehensis and their cardioprotective effects against doxorubicin-induced toxicity in H9c2 cells

Three biflavonoid glycosides along with 12 known flavonoids, were isolated from leaves of Malus hupehensis. The complete structures of two of the compounds were established from analysis of MS, NMR spectroscopic and CD data, as well as DFT CD calculations they were determined to be atropisomeric along a central biaryl axis. The antioxidant activities and protective effects of the compounds against doxorubicin-induced cardiomyopathy in H9c2 cells were also investigated. Amongst all of the isolated compounds, quercetin was the most active radical scavenger with EC₅₀ values of 3.2 μM and 17.8 μM by the DPPH and ABTS⁺ methods, respectively. The results indicated that three of the flavanoids also had a strong protective influence against doxorubicin-induced cell death with EC₅₀ values of 8.3, 5.2 and 7.6 μM, respectively.     

Flavonoids from Dracocephalum tanguticum and their cardioprotective effects against doxorubicin-induced toxicity in H9c2 cells

Two new flavonoids, ladanetin-6-O-β-(6″-O-acetyl)glucoside (1) and pedalitin-3'-O-β-glucoside (2), together with 15 known compounds (3-17), were isolated from the whole plants of Dracocephalum tanguticum. Their structures were established on the basis of extensive spectroscopic (IR, MS, 2D NMR) data analysis and by the comparison with spectroscopic data reported in the literature. Antioxidant capacities of the isolated substances were determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrous ions, and ABTS(·)(+) radical in vitro assay, and their cytoprotective activities were also tested on doxorubicin (DOX)-induced toxicity in H9c2 cardiomyocytes. Among all the tested compounds, luteolin-7-O-β-D-glucopyranoside (7) exhibited both strong antioxidative effect and high protective activity against DOX-induced toxicity. Further investigation found 7 could decrease DOX-induced death of H9c2 cell, reduce LDH and CK level, and inhibit the elevated intracellular concentration of ROS and [Ca(2+)](i). The preliminary structure-activity relationships (SAR) of these compounds revealed the Δ(2,3) double bond on C-ring and 3',4'-di-OHs on B-ring with a flavone skeleton such as luteolin and its derivatives, were necessary for their cardioprotective effects.