麝香保心丸通过抑制p38 MAPK和NF-κB信号传导通路表达对抗高糖诱导的心肌细胞损伤

目的探讨麝香保心丸(SBP)是否通过抑制p38丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和核因子kappa B(nuclear factor kappa B,NF-κB)通路保护H9c2心肌细胞对抗高浓度葡萄糖(高糖,HG)引起的损伤。方法应用35 mmol/L的HG处理H9c2心肌细胞24 h,建立HG诱导的心肌细胞损伤模型;细胞计数试剂盒8测定细胞存活率;Hoechst 33258核染色荧光显微镜照相法测定细胞凋亡;双氯荧光素(2’,7’-dichlorfluorescein-diacetate,DCFH-DA)染色/荧光显微镜照相法测定胞内活性氧(ROS)水平;罗丹明123(Rh123)染色法测定线粒体膜电位(MMP);Western blot法测定p38MAPK和NF-κB p65蛋白表达水平。结果 HG处理H9c2心肌细胞24 h能引起细胞明显的损伤,使细胞存活率降低,凋亡细胞数量和细胞内ROS生成增多,MMP丢失;HG能增加p38 MAPK和NF-κB p65磷酸化水平;SBP预处理能明显抑制HG上调p38 MAPK和NF-κB p65磷酸化水平这一作用;SBP、p38MAPK通路抑制剂SB203580和NF-κB通路抑制剂PDTC均能阻断HG对心肌细胞的上述损伤作用,包括细胞毒性、凋亡、ROS生成增多及MMP丢失等。结论麝香保心丸(SBP)可通过抑制p38 MAPK和NF-κB信号传导通路保护H9c2心肌细胞对抗高糖(HG)引起的损伤。     

硫化氢通过调控瘦素保护心肌细胞对抗高糖引起的损伤

目的探讨瘦素(leptin)在高糖损伤心肌细胞中的作用及硫化氢(H2S)是否通过调控瘦素保护心肌细胞对抗高糖引起的损伤。方法应用细胞计数盒(CCK-8)检测细胞存活率;Hoechst 33258核染色检测凋亡细胞形态及数量的改变,双氯荧光素(DCFH-DA)染色荧光显微镜照相测定胞内活性氧(ROS)水平;罗丹明123(Rh123)染色及荧光显微镜照相测定线粒体膜电位(MMP);Western blot法检测瘦素蛋白的表达。结果 35 mmol/L葡萄糖(高糖)作用H9c2心肌细胞9h可明显地促进瘦素表达,并引起心肌细胞损伤,表现为细胞存活率降低、凋亡细胞数量和ROS生成增多及线粒体膜电位(MMP)丢失。硫化氢钠(NaHS,为H2S的供体)预处理能抑制高糖对心肌细胞瘦素表达的上调作用。NaHS预处理或瘦素拮抗剂均能保护H9c2心肌细胞对抗高糖引起的上述损伤。结论瘦素参与高糖引起的心肌细胞损伤。外源性H2S可通过抑制瘦素表达保护心肌细胞对抗高糖引起的损伤。     

N-乙酰半胱氨酸对抗丙酮醛引起的心肌细胞损伤

 目的: 观察N-乙酰半胱氨酸(N-acetylcysteine,NAC)对抗丙酮醛诱导的H9c2心肌细胞损伤及相关机制。方法: 实验分为正常对照组、丙酮醛损伤组(不同浓度丙酮醛处理)、NAC+丙酮醛组(NAC与丙酮醛共处理)、SP600125预处理+丙酮醛组、NAC组和SP600125组。H9c2心肌细胞常规消化种板,经相应处理24 h后:应用CCK-8法检测心肌细胞的存活率;Western blot法检测H9c2心肌细胞内磷酸化和总的c-Jun氨基端激酶(p-JNK、t-JNK)表达水平;双氯荧光素(DCFH-DA)染色法检测心肌细胞内活性氧(ROS)水平;罗丹明123(Rh123)染色法检测细胞线粒体膜电位(MMP);Hoechst 33258染色法观察H9c2心肌细胞凋亡形态学变化。结果: 与对照组相比,不同浓度的丙酮醛均能够降低H9c2心肌细胞存活率,且呈剂量依赖性(P<0.01),NAC在一定浓度范围内(500~1500μmol/L)可对抗丙酮醛引起心肌细胞损伤(P<0.01),抑制丙酮醛引起细胞内ROS水平升高,对抗丙酮醛引起细胞内MMP降低,抑制丙酮醛诱导细胞内JNK蛋白的磷酸化(P<0.01)。与NAC的细胞保护作用类似,选择性JNK抑制剂SP600125也可抑制丙酮醛诱导的细胞损伤,包括减轻氧化应激、改善线粒体膜电位及抑制细胞凋亡。结论: N-乙酰半胱氨酸能够保护H9c2心肌细胞对抗丙酮醛引起的损伤,其机制可能与其降低细胞内ROS水平、改善MMP、抑制JNK磷酸化和抗凋亡有关。     

依达拉奉通过调控p38MAPK通路抑制阿霉素的心肌毒性

目的探讨新型抗氧化剂依达拉奉（edaravone,EDA）能否通过调控p38丝裂原激活蛋白激酶（mitogen．acti—ratedproteinkinase,MAPK）通路保护H9c2心肌细胞对抗阿霉素（doxombicin,DOX）引起的损伤。方法应用5~mol／LDOX处理H9c2心肌24h以建立心肌毒性损伤模型。CCK一8比色法检测细胞存活率;双氯荧光素（DCFH—DA）染色荧光显微镜照像测定细胞内活性氧（reactiveoxygenspecies,ROX）水平;罗丹明123（rhl23）染色荧光显微镜照像检测线粒体膜电位（mitochondrialmembranepotential,MMP）;Westernblot法测定p38MAPK蛋白表达水平。结果在15～60min的时间范围内,5p．mol／LDOX呈时间依赖性地上调磷酸化（phosphorylated,P）p38MAPK表达。在DOX处理心肌细胞前,应用40~mol／LEDA预处理60min不仅能抑制DOX对p-p38MAPK表达的上调作用,也能抑制DOX引起的心肌细胞损伤,使细胞存活率升高、胞内ROS生成减少及MMP丢失减少。另方面,应用了31~mol／LSB203580（p38MAPK抑制剂）预处理60min也产生类似于上述的EDA对抗DOX心肌毒性的作用。结论EDA可通过抑制p38MAPK通路保护H9c2心肌细胞对抗DOX引起的损伤。     

抑制蛋白激酶D1表达水平对高糖诱导心肌细胞凋亡的影响及作用机制

目的:探讨下调蛋白激酶D1(PKD1)对高糖环境下心肌细胞凋亡的影响及机制。方法:实验分5组:Control组(正常培养的H9c2细胞)、HG组(H9c2细胞高糖条件培养24h)、si-NC组(H9c2细胞转染siRNA control后高糖条件培养24h)、si-PKD1组(H9c2细胞转染PKD1siRNA后高糖条件培养24h)和si-PKD1+SB203580组[H9c2细胞转染PKD1siRNA 12h后加入p38丝裂原活化蛋白激酶(p38MAPK)信号通路抑制剂SB203580,再高糖条件培养24h],用qRT-PCR和Western blot方法检测各组细胞PKD1 mRNA和蛋白表达水平(si-PKD1+SB203580组除外);流式细胞术测定细胞凋亡情况,Western blot测定细胞促凋亡蛋白Bcl-2相关X蛋白(Bax)的表达水平,同时检测细胞中p38MAPK及其磷酸化p-p38MAPK水平。结果:HG组细胞PKD1mRNA和蛋白水平均明显高于Control组(P0.01)。与HG组比较,si-PKD1组细胞中PKD1 mRNA和蛋白表达明显降低(P0.01),细胞凋亡率降低,Bax蛋白表达水平下降,细胞中p-p38MAPK水平降低(P0.01)。与si-PKD1组比较,siPKD1+SB203580组心肌细胞凋亡率进一步降低,细胞中Bax蛋白水平下降,p-p38MAPK、p-p38MAPK/p38MAPK水平降低(P0.01)。结论:高糖诱导心肌细胞PKD1表达,下调PKD1可能通过抑制p38MAPK信号通路激活减少高糖诱导的心肌细胞凋亡。     

p38MAPK参与千金藤素诱导的心肌细胞凋亡

目的: 探讨千金藤素(CEP)致Sprague-Dawley(SD)乳大鼠心肌细胞的凋亡作用及其信号途径。方法: 应用MTT法检测千金藤素对心肌细胞活性的抑制作用;利用Hoechst 33342染色及Western blotting方法检测凋亡相关信号分子caspase-3,观察CEP致心肌细胞凋亡的作用;采用Western blotting法观测 CEP对有丝分裂原活化蛋白激酶(MAPKs)家族3个主要信号分子c-Jun氨基端激酶(JNK)、细胞外信号调节激酶(ERK)、p38 MAPK磷酸化水平的影响,并利用ERK和p38 MAPK的特异性抑制剂,分别验证两种分子所介导的信号通路在CEP致心肌细胞凋亡中的作用。结果: (1)CEP能够剂量依赖和时间依赖地抑制心肌细胞的活性。(2)CEP作用于心肌细胞,出现细胞核碎裂现象和caspase-3激活。(3)CEP作用下p38 MAPK和ERK磷酸化水平显著增强,JNK的磷酸化状态未发生显著改变。(4)p38 MAPK磷酸化抑制剂SB203580显著减轻CEP对心肌细胞活性的抑制作用;ERK磷酸化抑制剂PD98059不能影响CEP对心肌细胞活性的抑制作用。结论: p38 MAPK参与CEP致心肌细胞凋亡作用。     

血管紧张素-(1-7)通过抑制活性氧激活的环氧合酶-2通路保护心肌细胞对抗高糖引起的损伤

目的探讨活性氧(ROS)激活的环氧合酶-2(COX-2)通路在高糖(HG)损伤H9c2心肌细胞中的作用及血管紧张素-(1-7)[Ang-(1-7)]能否通过调控ROS激活的COX-2通路抑制HG引起的心肌细胞损伤。方法应用Western blot法检测COX-2蛋白的表达;细胞计数盒(CCK-8)测定细胞存活率;Hoechst 33258核染色检测凋亡细胞的形态及数量的变化;DCFH-DA染色荧光显微镜测定细胞内ROS水平;JC-1染色荧光显微镜照相测定线粒体膜电位(MMP)。结果应用1000μmol/L ROS清除剂(N-乙酰半胱氨酸,NAC)或1μmol/L Ang-(1-7)共处理H9c2心肌细胞12 h能显著地抑制HG对COX-2表达的上调作用;35 mmol/L葡萄糖(HG)处理心肌细胞24 h引起明显的损伤作用,使细胞存活率和MMP降低,凋亡细胞数量及细胞内ROS生成增多;Ang-(1-7)或COX-2抑制剂(NS-398)共处理心肌细胞明显地抑制上述HG引起的损伤作用。结论 Ang-(1-7)通过抑制ROS激活COX-2通路保护心肌细胞对抗HG引起的损伤。     

p38 MAPK- Pim-3通路可能介导预处理对抗心肌细胞缺氧/复氧损伤

目的： 研究MAPK通路在原癌基因Pim-3抗心肌急性缺氧复氧损伤中的作用。方法：采用原代培养新生大鼠的心肌细胞,随机分为4组：正常对照组（control）、缺氧复氧组(A/R)、缺氧预适应组(APC+A/R)、阻断剂组。在缺氧预处理前分别用终浓度为10 μmol/L SB203850(p38 MAPK阻断剂)、U0126（ERK1/2阻断剂）、SP600125（SAPK/JNK阻断剂）与细胞孵育30 min。实验结束后测定MAPKs通路中ERK1/2、JNK、p38 MAPK 磷酸化蛋白表达水平及Pim-3蛋白的表达水平,同时检测培养液中乳酸脱氢酶(LDH) 活性、四唑盐（MTT）比色试验测定细胞存活率、TUNEL法检测细胞凋亡。结果： SB203850、U0126、SP600125能分别取消由APC或A/R所诱导ERK1/2、JNK、p38 MAPK的磷酸化水平的升高;由APC所诱导的Pim-3表达的升高在p38 MAPK通路被阻断后明显下调(P<0.01),并且心肌细胞LDH值升高,细胞存活率则下降,心肌细胞的凋亡指数升高。结论： p38 MAPK的激活可上调原癌基因Pim-3的表达,从而可能对心肌细胞起到保护作用。     

敲低Txnip抑制高糖诱导的人肾小管细胞系凋亡

目的观察敲低Txnip对高糖诱导人肾小管上皮细胞凋亡的影响。方法将体外培养人肾小管上皮细胞分为正常糖组、高糖组、高糖+质粒载体对照组及高糖+shRNA组。采用原位末端转移酶标记技术(TUNEL)检测细胞凋亡;流式细胞术检测细胞ROS水平;Western blot检测caspase-3、cleaved caspase-3、BAX、BCL-2、P38 MAPK、P-P38 MAPK及细胞色素c的表达。结果与正常糖组相比,高糖组肾小管上皮细胞ROS产生和细胞凋亡明显增加(P0.01),cleaved caspase-3和P-P38 MAPK表达增高,BAX/BCL-2比率明显升高以及细胞色素c易位(P0.05)。敲低Txnip能够显著抑制高糖诱导的肾小管上皮细胞凋亡和ROS产生,下调cleaved caspase-3、和P-P38MAPK的表达,减少BAX/BCL-2比率和细胞色素c易位(P0.05)。结论敲低Txnip能够抑制高糖诱导的人肾小管上皮细胞凋亡可能是通过减少ROS产生,保护线粒体功能,抑制P38MAPK信号通路激活而实现的。     

硫化氢通过抑制坏死性凋亡对抗高糖引起的H9c2心肌细胞损伤

 目的: 探讨硫化氢(hydrogen sulfide,H₂S)能否通过调控坏死性凋亡(necroptosis)对抗高糖(HG)引起的H9c2心肌细胞损伤。方法: 应用Western blot法检测心肌细胞内能反映坏死性凋亡的RIP3蛋白和cleaved caspase-3蛋白的水平;细胞计数盒测定心肌细胞存活率;双氯荧光素染色荧光显微镜照相法检测细胞内活性氧簇(reactive oxygen species,ROS)水平;罗丹明123染色荧光显微镜照相法测定线粒体膜电位(mitochondrial membrane potential,MMP);Hoechst 33258核染色荧光显微镜照相法测定凋亡细胞的数量。结果: 应用HG(35 mmol/L葡萄糖)处理H9c2心肌细胞3 h、6 h、9 h、12 h和24 h均能明显地上调RIP3蛋白的表达水平,其中24 h时RIP3蛋白水平增加最明显。400μmol/L硫氢化钠(NaHS;为H₂S的供体)预处理或坏死性凋亡的特异性阻断剂necrostatin-1(Nec-1;100μmol/L)共处理心肌细胞均能明显地抑制HG对RIP3蛋白表达的上调作用。此外,NaHS预处理或Nec-1共处理心肌细胞均显著地抑制HG引起的心肌细胞损伤,使细胞存活率升高,ROS生成及MMP丢失减少。另一方面,400μmol/L NaHS预处理心肌细胞能使凋亡细胞数量及cleaved caspase-3表达明显减少。结论: H₂S可通过抑制坏死性凋亡保护心肌细胞,对抗高糖引起的损伤。     

Identification of caspase-independent PKCepsilon-JNK/p38 MAPK signaling module in response to metabolic inhibition in H9c2 cells

To understand the molecular mechanism of ischemia-induced cardiac myocyte cell death, H9c2 cells were studied by chemical hypoxia (CH), using metabolic inhibition buffer. CH suppressed the activities of caspase-3, -8, and -9. c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were activated, whereas extracellular regulated kinase (ERK) was inactivated. Only protein kinase Cepsilon (PKCepsilon) among PKC isotypes was translocated to the membrane fraction implying its activation. Moreover, the administration of PKCepsilon inhibitor suppressed the phosphorylations of JNK/p38 MAPK and reduced CH-induced cell death. An administration of JNK/p38 MAPK inhibitors also decreased CH-induced cell deaths, implying JNK/p38 MAPK's causative roles in the deaths. Collectively, this study identified a novel caspase-independent PKCepsilon-JNK/p38 MAPK signaling module induced by CH in cardiac myocytes. Our data show that the PKCepsilon-JNK/p38 MAPK signaling module contributes to CH-induced H9c2 cell death. This contrasts with previous notions, i.e., PKCepsilon's protective effect against ischemic death. Thus our data suggest that PKCepsilon can mediate alternative signals, i.e., beneficiary or deleterious signals, depending on the cell type, intensity, and/or type of injury.     

Role of PKC-delta during hypoxia in heart-derived H9c2 cells

In the present study, we investigated the role of protein kinase C (PKC) isoforms during hypoxia in heart-derived H9c2 cells. Hypoxia caused a rapid translocation of PKC-delta from soluble to particulate fraction and a downregulation of PKC-epsilon and PKC-zeta, whereas PKC-alpha and PKC-beta I remained unaltered. When H9c2 cells were pretreated with PKC-delta inhibitor rottlerin (3 microM), hypoxia-induced apoptotic and necrotic cell death were significantly increased. Hypoxic insult also caused an activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK with no change in c-Jun NH(2)-terminal protein kinase (JNK) phosphorylation. Hypoxia-induced cell death was increased by treatment with ERK1/2 inhibitor U0126 (10 microM), but attenuated by p38 MAPK inhibitor SB202190 (10 microM). Treatment with rottlerin completely blocked the hypoxia-induced ERK phosphorylation, whereas it significantly increased p38 MAPK phosphorylation. The hypoxia-induced translocation of PKC-delta was not altered by U0126 and/or SB202190. From these results, it is suggested that hypoxia causes a rapid translocation of PKC-delta and subsequently ERK activation and p38 inactivation, rendering H9c2 cells resistant to hypoxia-induced cell death.     

KR-31378 protects cardiac H9c2 cells from chemical hypoxia-induced cell death via inhibition of JNK/p38 MAPK activation

Using a metabolic inhibition buffer as an ischemic model, we show here that KR-31378, a cardioselective ATP-sensitive potassium channel opener, protects H9c2 cells from chemical hypoxia (CH)-induced cell death. Our previous study showed that CH downregulated caspase activities, but led to differential activation of mitogen-activated protein kinases (MAPKs) in H9c2 cells. The repression of CH-induced c-jun N-terminal kinase (JNK)/p38 MAPK activation resulted in partial protection against CH- induced cell death, implying JNK/p38 MAPK's causative role in CH-induced cell death. This study furthers that research and examines if KR-31378's protective effect came from modulating MAPK activity and/or caspase activity in H9c2 cells. Although KR-31378 did not restore downregulated caspase-3 activity, it did block the activation of JNK and p38 MAPK in a dose-dependent manner. Extracellular signal-regulated kinase activity was not recovered by KR-31378 treatment. CH-induced reactive oxygen species (ROS) generation was suppressed by KR-31378. Thus our results indicate that the cardioprotective effect of KR-31378 in CH is due, at least in part, to the differential inhibition of MAPKs.     

Stress-activated protein kinases mediate cell migration in human airway epithelial cells

Airway epithelial cell (AEC) repair immediately after injury requires coordinated cell spreading and migration at the site of injury. Stress-activated protein kinases such as p38 MAPK and c-Jun N-terminal Protein Kinase (JNK) modulate several responses to cell stress and injury, but their role in AEC migration is not clear. We examined migration in confluent 16HBE14o(-) human AEC lines and in primary AEC grown on collagen-IV. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Inhibitors of either p38 extracellular signal-regulated kinase (ERK)1/2 (PD98059), mitogen-activated protein kinase (MAPK) (SB203580), or JNK (SP600125) could block cell migration substantially. Inhibiting JNK but not p38 MAPK or ERK1/2 blocked extension of cells into the wound region from the original line of injury. Initial migration was associated with phosphorylation of ERK, p38 MAPK, and JNK within 5-15 min. The downstream effector of p38, heat shock protein 27, also was phosphorylated rapidly after injury; phosphorylation could be blocked by prior treatment with SB203580 but not SP600125. The downstream effector of JNK, c-Jun, likewise was phosphorylated rapidly after injury and could be blocked by inhibiting JNK. Our data demonstrate that p38 MAPK, JNK, and ERK1/2 participate in the early stages of AEC migration.     

Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells

It has been known that ethanol causes neuronal cell death through oxidative stress. Ethanol itself and reactive oxygen species (ROS) produced by ethanol modulate intracellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades. This study was conducted to examine the impact of ethanol on MAPK signaling in HT22 cells. Ethanol (100 and 400 mM) caused activation of ERK, p38 MAPK, and JNK. ERK activation occurred in early time and p38 MAPK activation was evident when ERK activation was diminished. Specific inhibitor of p38 MAPK (SB203580) protected HT22 cells against ethanol, which was accompanied by an inhibition of ROS accumulation. However, inhibitors of ERK (U0126) and JNK (SP600125) had no effects on ethanol-induced neuronal cell death when they are treated with ethanol for 24 h. These results suggest that p38 MAPK may have important roles in ROS accumulation during ethanol-induced oxidative stress in HT22 cells.     

ATP敏感性钾通道的开放通过抑制TLR4/NF-κB通路对抗高糖引起的H9c2心肌细胞损伤和炎症

目的:探讨开放ATP敏感性钾通道(K_(ATP)通道)能否抑制Toll样受体4(TLR4)/核因子-κB(NF-κB)通路对抗高糖(HG)引起的H9c2心肌细胞损伤和炎症。方法:应用Western blot测定TLR4和NF-κB p65的蛋白水平;应用ELISA法检测细胞培养液中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的水平;采用细胞计数试剂盒8(CCK-8)测定心肌细胞存活率;罗丹明123染色荧光显微镜照相法测定线粒体膜电位(MMP);双氯荧光素染色荧光显微镜照相法测定细胞内活性氧簇(ROS)水平;Hoechst 33258核染色荧光显微镜照相法检测凋亡细胞数量。结果:H9c2心肌细胞经HG(35 mmol/L葡萄糖)处理24 h,胞内TLR4和磷酸化NF-κB p65(p-NF-κB p65)的蛋白水平明显增加,100μmol/L K_(ATP)通道开放剂二氮嗪(DZ)预处理30 min可抑制HG对TLR4和p-NF-κB p65蛋白水平的上调作用;此外,30μmol/L TAK-242(TLR4抑制剂)和HG共处理心肌细胞24 h也可减轻HG对p-NF-κB p65的上调作用。另一方面,100μmol/L DZ预处理有明确的心肌保护作用,可抑制HG引起的细胞毒性、炎症反应、线粒体损伤、氧化应激和细胞凋亡,使细胞存活率升高,并减少IL-1β和TNF-α分泌水平、MMP丢失、ROS生成及凋亡细胞数量;而30μmol/L TAK-242或100μmol/L PDTC(NF-κB抑制剂)共处理心肌细胞24 h也可发挥和DZ相类似的作用,能抑制HG引起的上述损伤和炎症反应。结论:开放K_(ATP)通道可通过抑制TLR4/NF-κB通路对抗HG引起的H9c2肌细胞损伤和炎症。