大黄素对胃癌细胞增殖、凋亡及ERK1/2-PKM2/P53通路的影响

摘 要 目的：探讨大黄素对胃癌细胞增殖、凋亡及ERK1/2-PKM2/P53通路的影响。 方法: 实验分为MGC803胃癌细胞组、氟尿嘧啶组、大黄素低剂量组、大黄素高剂量组,测定并比较各组细胞癌细胞活力、癌细胞单克隆形成数目、癌细胞凋亡率、穿膜孔数、以及MGC803胃癌细胞ERK1/2、PKM2、P53mRNA、蛋白水平。 结果: 与MGC803细胞组比较,氟尿嘧啶组、大黄素低、高剂量组吸光度（A）值、存活率水平、克隆形成数目、穿膜数、p ERK1/2、p PKM2 mRNA及蛋白表达水平降低,凋亡率、P53 mRNA及蛋白表达水平升高（P<0.05）。与氟尿嘧啶组比较,大黄素低剂量组A值、存活率水平、克隆形成数目、穿膜数、p ERK1/2、p PKM2 mRNA、蛋白升高,凋亡率、P53 mRNA、蛋白表达水平降低（P<0.05）。与大黄素低剂量组比较,大黄素高剂量组A值、存活率、细胞克隆形成数目、穿膜数、P ERK1/2及P PKM2 mRNA及蛋白表达水平明显降低,凋亡率、P53 mRNA及蛋白表达水平明显升高（P<0.05）。 结论: 大黄素可能通过抑制ERK1/2-PKM2通路诱导P53高表达从而抑制胃癌细胞的增殖及迁徙,促进胃癌细胞的凋亡。     

Regulation of lysophosphatidic acid-induced COX-2 expression by ERK1/2 activation in cultured feline esophageal epithelial Cells

Lysophosphatidic acid (LPA), a potent bioactive phospholipid, mediates diverse cellular responses by binding to specific G protein-coupled receptors (GPCRs). We investigated the signaling mechanisms underlying LPA-induced COX-2 expression in primary cultures of feline esophageal epithelial cells. The identity of the cultures was confirmed by immunocytochemistry using a cytokeratin antibody. Western blot analysis revealed a concentration-and time-dependent induction of COX-2 in response to LPA. Of the three major MAPKs, only ERK1/2 was activated by LPA in a time-dependent manner. LPA-induced COX-2 expression was significantly attenuated by the MEK inhibitor, PD98059, but not by the JNK inhibitor, SP600125, or the p38 MAPK inhibitor, SB212090. LPA-induced COX-2 expression was repressed by pertussis toxin, GF109204X, and Ki16425, indicating the involvements of PTX-sensitive G_i/o protein, PKC, and the LPA_1/3 receptor, respectively. Our data suggest that in esophageal epithelial cells, LPA-induced COX-2 expression requires activation of PKC and ERK1/2 downstream of the LPA_1/3 receptor, Understanding the regulation of COX-2 expression induced by LPA in esophageal epithelial cells might provide a new therapeutic strategy for esophageal inflammatory diseases.     

牛磺酸对肺动脉平滑肌中活性氧及ERK1/2通路的影响

目的：探讨牛磺酸（Taurine,Tau）对缺氧诱导的大鼠肺动脉平滑肌细胞（PASMCs）中活性氧（ROS）及ERK1/2通路的影响。方法：原代培养大鼠PASMCs,选用第2-5代用于实验。Tau给药浓度80 mmol·L-1,作用时间24 h。实验分组为：1、常氧组2、常氧＋Tau组3、缺氧组4、缺氧＋Tau组。DCFH-DA荧光探针检测细胞内ROS含量,Westernblot检测磷酸化ERK1/2（p-ERK1/2）的表达。结果：与常氧组比较,常氧＋Tau组中ROS含量没有显著性变化;缺氧组中ROS显著上升,Tau可逆转缺氧诱导的ROS上升。Tau可以逆转缺氧诱导的ERK1/2磷酸化。结论：Tau可维持正常的PASMCs中ROS水平,并且抑制缺氧诱导的ERK1/2磷酸化。     

ERK1/2介导H_2O_2预处理对抗氧化应激损伤的保护作用

目的探讨H2O2预处理能否激活ERK1/2及ERK1/2在H2O2预处理引起的适应性细胞保护中的作用。方法在PC12细胞,建立H2O2预处理对抗高浓度H2O2诱导细胞损伤的实验模型。应用甲氮甲唑蓝(MTT)法检测细胞存活率;碘化丙啶(PI)染色流式细胞术检测细胞凋亡率;免疫印迹法(Western blot)测定ERK1/2蛋白的表达及procaspase-3的表达。结果100μmol.L-1H2O2预处理PC12细胞90min能明显地保护PC12细胞对抗300μmol.L-1H2O2引起的损伤,使细胞存活率增加,细胞凋亡率降低及procaspase-3增多。H2O2预处理对ERK1/2具有明显的激活作用:诱导胞质ERK1/2磷酸化及促进其核转移。在H2O2预处理前30min应用ERK1/2抑制剂UO126(10μmol.L-1)可明显地阻断H2O2预处理的抗细胞毒性及抗细胞凋亡作用。结论H2O2预处理能激活ERK1/2,ERK1/2介导H2O2预处理的适应性细胞保护作用。     

异氟醚可能通过ERK磷酸化增强肾动脉平滑肌收缩

目的实验观察异氟醚对去内皮肾动脉血管平滑肌收缩增强的作用,并观察肾动脉血管平滑肌细胞在异氟醚的作用下细胞内MAPK通路的激活状态,以探讨异氟醚引发肾动脉血管平滑肌收缩增强的可能信号通路。方法①去内皮肾动脉条,3%皂苷处理使膜通透,咖啡因诱除内质网内贮钙,利用10-6钙离子浓度EGTA缓冲液平衡后,应用接近最大的钙离子浓度的EGTA缓冲液使肾动脉条压缩达平衡,加入不同浓度异氟醚EGTA缓冲溶液,观察动脉条张力变化。②1%、3%、5%异氟醚分别作用于培养的肾动脉血管平滑肌细胞,提取细胞内蛋白,Westenblot检测ERK磷酸化的变化。结果异氟醚能使压缩达平衡的肾动脉环张力进一步增强,且与异氟醚浓度呈剂量依赖性。随异氟醚浓度的增加,培养的肾动脉血管平滑肌细胞内ERK1/2(p44/42)磷酸化逐步增强,并随时间增强,在15min后,逐渐下降。结论异氟醚引起的肾动脉血管条收缩增强可能与异氟醚引起MAPK系统活化有关     

ERK1/2的激活参与七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用

目的观察ERK1/2的激活在七氟醚预处理对大鼠海马脑片缺氧无糖损伤保护中的作用。方法采用脑片灌流及电生理技术,细胞外记录海马CA1区的顺向群锋电位(OPS);利用2,3,5-三苯基氯化四氮唑(TTC)染色定量比色方法分析脑片损伤程度。结果用4%七氟醚预处理海马脑片,可延迟OPS的消失时间,提高复氧后OPS的恢复程度和恢复率。以ERK1/2特异性抑制剂PD98059(50μmol.L-1)预处理海马脑片,可以取消七氟醚的作用。单独使用PD98059对OPS无明显影响。七氟醚预处理组组织损伤百分率明显低于其它各组。结论ERK1/2的激活参与了七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用。     

ERK1/2通路在4-AP诱导大鼠肺动脉收缩中的作用

目的探讨细胞外信号调节激酶-1/2(ERK1/2)通路在4-氨基吡啶(4-aminopyridione,4-AP)阻断正常大鼠肺动脉平滑肌细胞(PASMCs)膜上电压依赖性钾通道(KV)所引起的肺动脉收缩中的作用。方法取正常鼠肺动脉制作肺动脉环,分别加入4-AP(KV通道阻断剂),PD98059/U0126+4-AP,比较肺动脉收缩的变化。同时培养肺动脉平滑肌细胞进行Western blot分析4-AP对ERK1/2的影响。结果①在血管环试验中,4-AP引起的肺动脉收缩有浓度依赖性;加入20mmol.L-1PD98059或2μmol.L-1U0126可以抑制4-AP引起的肺动脉收缩。②4-AP可刺激PASMCs ERK1/2蛋白磷酸化;③U0126可抑制4-AP引起的ERK1/2蛋白磷酸化。结论ERK1/2通路参与4-AP阻断正常大鼠肺动脉平滑肌细胞膜上电压依赖性钾通道(KV)引起肺动脉收缩。     

Reactive Oxygen Species Mediate ET-1-Induced Activation of ERK1/2 Signaling in Cultured Feline Esophageal Smooth Muscle Cells

Reactive oxygen species (ROS) have been shown to play a critical role in propagating the signals of several growth factors, peptide hormones, and cytokines, such as epidermal growth factor, insulin, and interleukin-1. We investigated a possible role for ROS generation in mediating the action of ET-1 on activation of ERK1/2 in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated by 10nM ET-1; activation of ERK was examined by western blot analysis with phospho-specific antibodies of ERKs. ET-1 induced ERK1/2 phosphorylation in a dose- and time- dependent manner. ERK1/2 activation by ET-1 reached the maximal levels at 5min showing slight activation up to 20min, and then slowly declined. It was confirmed that the activation of ERK1/2 was reduced by MEK inhibitor PD98059. We observed the dose-dependent inhibitory effect of diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the ET-1-enhanced ERK1/2 phosphorylation in ESMC. Pretreatment of ESMC with N-acetylcysteine, a ROS scavenger, also attenuated the ET-1-induced ERK1/2 activation. In addition, DPI significantly inhibited the ET-1- induced ROS production when ROS was measured as a function of DCF fluorescence. The results suggest that ROS might be critical mediators of the ET-1-induced ERK1/2 signaling events in ESMC.     

ERK介导姜黄素抑制人脑胶质瘤细胞U251侵袭性

目的 探讨姜黄素对人脑胶质瘤细胞U251侵袭性的影响及ERK/MAPK信号通路在此过程中的作用。方法 将人脑胶质瘤细胞U251分为对照组和药物组,对照组以含10％小牛血清的DMEM培养基常规培养,药物组用16 μmol·L-1姜黄素（本实验先前的研究结果显示,姜黄素作用于U251细胞48 h的半数抑制剂量为16 μmol·L-1）处理。 Transwell侵袭小室模型法检测细胞侵袭性的变化;免疫细胞化学法检测基质金属蛋白酶－9（MMP-9）蛋白表达变化;免疫印迹（Western blot）法检测细胞外调节蛋白激酶（ERK）和磷酸化细胞外调节蛋白激酶（pERK）表达变化。结果 Transwell侵袭小室模型法显示,在每个高倍镜视野下,对照组穿过微孔膜的细胞数平均值为160个,药物组为42个。免疫细胞化学法显示姜黄素作用后MMP-9蛋白阳性细胞百分率由76％降低至40％。Western blot法显示,16 μmol·L-1姜黄素分别作用于U251细胞0、24、48和96 h,ERK蛋白水平无明显改变,pERK水平与ERK水平比值(pERK/ERK) 分别为0.48、0.42、0.35和0.22。结论 姜黄素在体外可有效抑制胶质瘤U251细胞的侵袭性,其机制可能是通过抑制异常激活的ERK信号通路来实现的。     

家兔颈动脉粥样硬化狭窄简捷模型建立

目的探讨建立简捷、经济实用、稳定可靠的适于颈动脉粥样硬化性狭窄(CASS)的外科治疗研究的家兔CASS动物模型条件。方法新西兰白兔21只,采用3％过氧化氢液灌注动物的双侧颈总动脉,造成氧化应激损伤,然后以特定高脂饲料喂养动物不同时间。评价各组动物血管狭窄程度和病理改变特点。结果8周组和12周组中重度狭窄达到80％,个别血管出现闭塞。4周组轻度狭窄亦达到60％。病理检查证实高脂饲料喂养8周时,动脉的粥样硬化病理改变已属于纤维班快期。结论 按本实验方法,氧化应激损伤后喂养8周的动物颈动脉狭窄程度和病理改变程度已符合CASS外科治疗实验研究需要。     

Paeonol attenuates heart failure induced by transverse aortic constriction via ERK1/2 signalling

ContextPaeonol (PAE) is the main phytochemical from Cortex Moutan. Its main pharmacological effects are anti-inflammatory and antioxidant, but its cardioprotective effect is unclear.ObjectiveThe study investigates the effects and underlying mechanisms of PAE on transverse aortic constriction (TAC)-induced heart failure (HF) in mice.Materials and methodsC57BL/6 mice were randomly divided into five groups: sham, TAC, PAE10 (TAC + PAE 10 mg/kg), PAE20 (TAC + PAE 20 mg/kg) and PAE 50 (TAC + PAE 50 mg/kg). Paeonol was intragastrically administered to mice for 4 weeks. Mice were anaesthetized with pentobarbital sodium and underwent cardiac echocardiography using echocardiography system. Serum levels of atrial natriuretic peptide (ANP), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). Myocardial apoptosis was detected with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining. Haematoxylin–eosin (H&E) and Masson’s staining were used for histopathological evaluation. Western and quantitative real-time PCR (qRT-PCR) were performed to detect levels of apoptosis and fibrosis-related proteins.ResultsEchocardiography showed PAE improved cardiac function (LVEF: TAC, 52.3±6.8%; PAE20, 65.8±3.6%; PAE50, 71.4±2.5%) and H&E staining showed PAE alleviated myocardial injury (TAC: 1170.3 ± 134.6 μm²; PAE50: 576.0 ± 53.5 μm²). Western and qRT-PCR results showed that PAE down-regulated the levels of ANP, BNP and α-MHC. In addition, TUNEL and western results showed PAE significantly inhibited apoptosis. Masson and western results showed PAE inhibited cardiac hypertrophy. Western results showed the ERK1/2/JNK pathway could be inhibited by PAE.Discussion and conclusionsPaeonol regulates ERK1/2/JNK to improve cardiac function, which provides theoretical support for the extensive clinical treatment of HF.     

敌百虫经ERK1/2通路影响类固醇激素的合成

目的初步探讨细胞外信号调节激酶(ERK1/2)通路在敌百虫抑制小鼠睾丸间质细胞瘤细胞(MLTC-1)类固醇激素合成中的作用。方法运用放射免疫法检测不同浓度敌百虫及加入ERK1/2通路抑制剂UO126后对MLTC-1细胞孕酮合成的影响;采用蛋白质印迹技术(Western-blot)检测敌百虫对ERK1/2磷酸化表达的影响。结果敌百虫显著抑制MLTC-1细胞孕酮的合成,并且随敌百虫剂量的增加,孕酮合成量呈下降趋势;当同时加入UO126后,随敌百虫染毒剂量的升高孕酮合成量的下降趋势更为明显;敌百虫与UO126一样能明显抑制ERK1/2的磷酸化,而对总ERK1/2表达无明显影响。结论在本试验条件下,敌百虫能明显抑制MLTC-1细胞的孕酮合成,其机制可能是通过ERK1/2通路。     

Signaling mechanisms of sphingosine 1-phosphate-induced ERK1/2 activation in cultured feline esophageal smooth muscle cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid, stored and released from activated platelets, macrophages, and other mammalian cells. We previously reported that S1P induces esophageal smooth muscle contraction in freshly isolated intact cells. Here, we measured S1P-induced ERK1/2 activation and upstream signaling in cultured feline esophageal smooth muscle cells. Activation of ERK1/2 by S1P peaked at 5 min, was sustained up to 30 min, and was blocked by PTX. In contrast, S1P did not activate p38 MAPK or JNK. PTX inhibited S1P-induced ERK1/2 activation. We then used phospholipase inhibitors, DEDA for PLA₂, U73122 for PLC, and ρCMB for PLD, to determine that ERK1/2 activation was downstream of PLC activation. The PKC inhibitors, GF109203X and chelerythrine, also suppressed ERK1/2 activation. Whereas the PTK inhibitor, genistein, partially inhibited ERK1/2 activation, the EGFR tyrosine kinase inhibitor, tyrphostin 51, had no effect. Taken together, S1P-induced ERK1/2 activation in cultured ESMCs requires a PTX-sensitive G protein, stimulation of the PLC pathway, and subsequent activation of the PKC and PTK pathways.     

ERK1/2通路参与ET_A和ET_B受体介导的离体肠系膜上动脉收缩研究

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号转导通路在内皮素1(ET-1)的两个G蛋白偶联受体ET_A和ET_B介导的收缩机制。方法用大鼠肠系膜上动脉器官培养模型,以敏感的离体药理学实验方法记录培养前后血管平滑肌张力,实时定量的PCR测定培养前后受体mRNA表达水平的变化。结果S6c不引起新鲜的肠系膜上动脉收缩,培养后ETB受体mRNA表达水平上调,介导的收缩明显增强(P<005);而ETA受体介导的收缩功能和mRNA均变化不大。低浓度的SB386023(10-5mol·L-1)降低S6c引起的最大收缩(Emax从239%±26%降至89%±13%,P<001),而对ET1引起的最大收缩并无影响(Emax271%±19%vs251%±16%,P>005);高浓度的SB386023(10-4mol·L-1)明显抑制ETA受体介导的收缩。结论ET-1通过ET_A受体介导新鲜动脉的收缩;动脉培养后表达ET_B受体;ERK1/2信号转导通路对ET_B受体的作用强于ET_A受体。     

ERK1/2 activation plays important roles in the opposite effects of Trichostatin A in non-cancer and cancer cells

Histone deacetylase (HDAC) inhibitors are candidates of anti-cancer drugs as they can effectively kill cancer cells while have little or no toxicity to non-cancer cells, but the molecular mechanism underlying this process remains unclear. We previously reported that HDAC inhibitors could protect normal mouse hepatocytes from apoptosis induced by transforming growth factor-β1 (TGF-β1) with the requirement of extracellular signal-regulated kinase 1/2 (ERK1/2). In this study, we investigate the roles of trichostatin A (TSA), a typical HDAC inhibitor, on three non-cancer cell lines AML-12, MDCK and 3T3-L1, and four cancer cell lines Hep-3B, HeLa, A549 and MCF-7. TSA is a fermentation product of Streptomyces originally used as an antifungal agent. Our results showed that TSA blocked not only the TGF-β1-induced apoptosis but also serum starvation-induced apoptosis in all the non-cancer cells, whereas it could induce strong apoptosis in all the cancer cells. Further investigation revealed that TSA can induce the activation of ERK1/2 in the three non-cancer cells but not in the cancer cells. In summary, these findings indicated that TSA protect non-cancer cells from apoptosis via activating ERK1/2, providing a useful insight into the better application of HDAC inhibitors in cancer therapy.     

Splenectomy enhances the Ly6Clow phenotype in hepatic macrophages by activating the ERK1/2 pathway during liver fibrosis

Background and aim: Splenectomy has been reported to attenuate liver fibrosis. In addition, phenotype transitions of infiltrating macrophages, including Ly6C^high and Ly6C^low, play an essential role in the liver fibrosis. However, whether the spleen can regulate the phenotype switch of macrophages and the underlying mechanism still remain unclear. Methods: Chronic liver fibrosis in mice was induced by intraperitoneal injection with carbon tetrachloride. Splenectomy or sham operation was performed with or without depletion of macrophages during liver fibrosis. Liver fibrosis and the proportion of Ly6C^high and Ly6C^low macrophages were analyzed. Western blotting of ERK1/2 signals was performed in isolated macrophages to investigate the underlying mechanism of phenotype transition. RAW264.7 cells were stimulated by liver total cells conditioned medium with or without preincubation of SCH772984, the ERK1/2 inhibitor, and the phenotype switch of RAW264.7 cells was examined. In vivo, intraperitoneal injection of SCH772984 was performed on the splenectomy mice and the phenotype switch of liver infiltrating macrophages was tested. Results: Splenectomy alleviated the liver inflammation and fibrosis and also promoted the phenotypic switch of infiltrating macrophages to a Ly6C^low phenotype in fibrotic liver. The p-ERK1/2 expression was upregulated in macrophages at the same time. Furthermore, splenectomy increased the percentage of Ly6C^low macrophages and decreased the percentage of Ly6C^high macrophages both in vivo and in vitro, which was reversed by SCH772984. Conclusions: Splenectomy attenuates both the liver fibrosis and inflammation, and promotes the phenotype switch of infiltrating macrophages to an anti-inflammatory Ly6C^low phenotype by activating the ERK1/2 pathway during liver fibrosis.     

微囊蛋白-1蛋白参与哮喘气道平滑肌细胞增殖中ERK1／2调控以及罗红霉素的干预

目的：在细胞水平研究微囊蛋白-1（caveo-1in-1）对气道平滑肌细胞（ASMCs）的增殖作用以及对ERK1／2的通路调控,探讨caveolin-1抑制ASMCs增殖的可能机制。方法：复制哮喘大鼠模型,光镜观察肺组织病理变化,透射电镜观察ASMCs上微囊（caveolae）的结构及变化,用组织贴壁法体外培养气道平滑肌细胞,实验设正常对照组（A组）、哮喘组（B组）、ERKl／2信号通路阻断剂PD98059（C组）、罗红霉素组（D组）、caveo—lae结构破坏药物8-甲基环糊精组（E组）;用CCK-8法检测各组细胞的增殖情况,逆转录-聚合酶链测定（RT-PCR）和蛋白质印迹法（WesternBlot）检测ERKmRNA和p-ERK1／2蛋白的表达;WesternBlot法检测各组caveolin-1蛋白细胞表达。结果：CCK-8检测结果显示D组及C组ASMCs增殖反应低于B组,D组（0．59±0．15） vs B组（0．96±0．14）,P〈0．05,C组（0．63±0．11） vs （0．96±0．14）,P〈0．05;E组ASMCs增殖反应较B组进一步活跃（1．26±0．21） vs （0．96±0．14）,P〈0．05。D组及C组ASMCs上caveolin-1的表达较B组明显升高,P〈0．01,pERKl／2蛋白以及ERKInRNA表达较B组降低,E组相反。D组ASMCs的ERK mR-NA和p-ERK1／2蛋白表达水平均显著低于B组及E组（P〈0．01）。结论：caveolin-1可能是ASMCs内信号转导的负调控蛋白,这-作用可能是通过抑制ERK信号通路实现。罗红霉素可能上调caweolin-1蛋白的表达量,抑制ERK mR-NA表达和翻译,从而抑制哮喘大鼠ASMCs的增殖。     

Emodin protected against synaptic impairment and oxidative stress induced by fluoride in SH‐SY5Y cells by modulating ERK1/2/Nrf2/HO‐1 pathway

Excessive fluoride exposure contributes to neurotoxic effects. Emodin exhibits antioxidative functions in the central nervous system (CNS); however, its neuroprotective mechanism against fluoride remains to be elucidated. Our aim was to explore the neuroprotective efficacy and the possible mechanisms of emodin. In our study, synaptic proteins and oxidative stress damage were examined after human neuroblastoma SH‐SY5Y cells were treated with high doses of NaF for 24 hours. Moreover, pretreatment with emodin was used to shed light on the neuroprotective effects in NaF‐induced toxicity in SH‐SY5Y cells. We found that NaF significantly lowered the protein expressions of SNAP 25, synaptophysin and PSD 95 in SH‐SY5Y cells. In addition, NaF exposure increased the protein expression of p‐ERK1/2 and decreased the protein expressions of Nrf2 and HO‐1, as well as facilitated increasing ROS, 4‐hydroxynonenal (4‐HNE), and 8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG). Pretreatment with emodin significantly recovered these alterations caused by NaF. These data implied that the neuroprotective effects of emodin and pointed to the promising utilization for protecting against neurotoxicity induced by fluoride.