雌激素提高去卵巢大鼠海马CA4区ERK2的磷酸化水平

目的研究雌激素对去卵巢大鼠海马CA4区神经元细胞外信号调节激酶1/2(extrauancellular sig-nal-regulated kinase-1/2,ERK1/2)磷酸化水平的影响。方法成年Wistar雌性大鼠随机分为正常对照组(INT)、去卵巢组(OVX)和去卵巢加雌激素组(OVX+estrogen)。用放射免疫分析方法测定血中雌二醇含量,用免疫印迹方法检测ERK1/2的磷酸化水平。结果去卵巢组大鼠体内雌激素水平明显降低,与对照组和加雌激素组比较均有显著性差异;免疫印迹法检测结果表明去卵巢大鼠海马组p-ERK2表达减少,雌激素治疗组p-ERK2表达比去卵巢组明显增加;各组间p-ERK1的表达差异不显著。结论雌激素提高衰老雌性大鼠脑内ERK2磷酸化水平,提示雌激素可通过对衰老状态下ERK信号通路的调节作用达到改善学习和记忆的目的。     

糖尿病大鼠脑缺血再灌注海马CA4区神经元中磷酸化ERK1/2的表达上调

目的 探讨细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)在糖尿病脑缺血再灌注大鼠海马CA4区神经元表达的意义.方法 在链脲佐菌素性糖尿病大鼠脑缺血再灌注模型基础中,应用TUNEL、免疫组化方法 观察糖尿病脑缺血组与正常血糖脑缺血组在全脑缺血15 min、再灌注1 h海马CA4区神经元凋亡和磷酸化ERK1/2(P-ERK1/2)的表达变化.结果 糖尿病脑缺血组在缺血15 min、再灌注1 h海马CA4区神经元凋亡发生率均明显高于正常血糖脑缺血组(P<0.05);糖尿病脑缺血组各时间点磷酸化ERK1/2明显增高,于再灌注1 h明显高于正常血糖组(P<0.01).结论 糖尿病加重脑缺血再灌注神经元的损伤,其机制可能与ERK1/2的激活有关.     

丙泊酚对大鼠脑缺血再灌注后脑水肿及EGFR与ERK1/2磷酸化的影响

目的观察丙泊酚对大鼠脑缺血再灌注后脑水肿及表皮生长因子受体(EGFR)和细胞外信号调节蛋白激酶(ERK1/2)磷酸化的影响。方法将60只SD大鼠按随机数字表法分为6组:假手术组(Sham组)、大脑中动脉栓塞(MCAO)2h模型组(I组)、MCAO 2h再灌注24h模型组(I/R组)、丙泊酚预处理假手术组(Pro组)、丙泊酚预处理MCAO 2h模型组(Pro+I组)及丙泊酚预处理MCAO 2h再灌注24h模型组(Pro+I/R组),每组10只。将各组制模成功后大鼠断头取脑组织,采用干重法测定缺血区脑组织含水量,采用Western Blot检测EGFR和ERK1/2磷酸化水平。结果 I/R组缺血区脑组织含水量较I组和Sham组显著增加(P<0.05),EGFR和ERK1/2磷酸化水平较I组和Sham组显著升高(P<0.05);Pro+I/R组脑组织含水量较I/R组显著降低(P<0.05),EGFR和ERK1/2磷酸化水平较I/R组显著下降(P<0.05)。Sham组、I组、Pro组、Pro+I组及Pro+I/R组的缺血区脑组织含水量、EGFR和ERK1/2磷酸化水平比较差异均无统计学意义(P>0.05)。结论丙泊酚能有效通过抑制EGFR和ERK1/2磷酸化降低脑缺血再灌注大鼠脑水肿程度。     

基于HIF-1α/ERK通路探讨异丙酚对缺氧大鼠海马神经元线粒体损伤的影响

目的 观察异丙酚通过缺氧诱导因子-1α(HIF-1α)/细胞外调节激酶(ERK)通路对缺氧大鼠海马神经元线粒体损伤的影响。方法 新生SD大鼠离体培养原代海马神经元细胞,随机分为6组。对照组(高糖DMEM培养液)、缺氧组(低糖DMEM培养液)、LP组(3 mg/L异丙酚的低糖DMEM培养液)、MP组(6 mg/L异丙酚的低糖DMEM培养液)、HP组(12 mg/L异丙酚的低糖DMEM培养液)及U0126组(终浓度12 mg/L异丙酚+50μmol/L U0126的低糖DMEM培养液)。缺氧组、LP组、MP组、HP组及U0126组于缺氧条件培养24 h;对照组于有氧条件培养24 h。透射电镜观察各组海马神经元细胞形态;MTT法检测细胞存活率;激光共聚焦显微镜检测Ca²⁺、活性氧(ROS)的荧光强度;酶联免疫吸附试验检测细胞钙调神经磷酸酶(GaN)活性;Western blotting检测HIF-1α、ERK1/2、p-ERK1/2、天冬氨酸特异性半胱氨酸蛋白酶3(Caspase-3)蛋白表达。结果 缺氧组线粒体损伤严重,LP、MP和HP组线粒体损伤均有所减轻,HP组减...     

丙戊酸钠对大鼠海马神经元癫痫样放电后细胞外信号调节激酶磷酸化水平的影响

目的细胞外信号调节激酶(extracellular signal-regulated kinase 1/2,ERK1/2)参与癫痫的发生,但其与抗癫痫药物之间的关系不明确,文中旨在观察丙戊酸钠对大鼠海马神经元癫痫样放电后磷酸化ERK1/2(p-ERK1/2)的影响。方法取24 h内新生Wistar大鼠,雌雄不拘,迅速断头取脑。建立神经元癫痫样放电模型,将神经元分为空白对照组和丙戊酸钠组,量效实验中,于神经元癫痫样放电前30 min时加入不同浓度的丙戊酸钠(50 mg/L、75 mg/L、100 mg/L),运用免疫荧光技术测定p-ERK1/2在不同浓度时的表达;时效实验中,分别于癫痫样放电前30 min,放电后0 min、30 min、2 h和6 h加入50mg/L丙戊酸钠,采用Wester blot观察p-ERK1/2的变化。结果量效实验中,不同浓度的丙戊酸钠均能降低ERK1/2的磷酸化水平,且无显著性差异。时效实验中,于放电前30 min时加入丙戊酸钠对ERK1/2的磷酸化水平抑制最明显,与以后各时间点间都有显著性差异。结论海马神经元癫痫样放电后ERK1/2被过度持久的激活,在早期小剂量有效浓度的丙戊酸钠能显著抑制此反应中ERK1/2的磷酸化水平。     

应激对大鼠海马CREB、ERK活性的影响

目的 探讨不同时段的应激对大鼠海马环磷酸腺苷反应元件结合蛋白(CREB)、细胞外信号调节激酶(ERK)活性的影响.方法 将成年雄性Sprague Dawley(SD)大鼠随机分为5组:对照组(A)、应激1次组(B)、应激1周组(C)、应激2周组(D)和应激4周组(E),每组动物6只.应激模型为强迫大鼠游泳,每天15min.采用Western blot检测各组大鼠海马CREB、ERK的磷酸化及总蛋白(p-CREB和t-CREB、p-ERK/MAPK和ERK/MAPK)水平.结果 B和C组大鼠海马组织的p-CREB水平与对照组比较差异无显著性(P＞0.05);D和E组大鼠海马组织的p-CREB明显低于对照组(P＜0.05).各应激组t-CREB水平与对照组比较均差异无显著性(P＞0.05).C、D和E组大鼠海马p-ERK44和p-ERK42的水平显著低于对照组(P＜0.05或P＜0.01);B组大鼠海马p-ERK44和p-ERK42的水平与对照组比较差异无显著性(P＞0.05).各应激组和对照组大鼠海马ERK44和ERK42的总蛋白水平比较均差异无显著性(P＞0.05).结论 慢性应激降低大鼠海马CREB的活性,短时间和慢性应激均能降低大鼠海马ERK的活性.     

应激对大鼠海马CREB、ERK活性的影响

目的探讨不同时段的应激对大鼠海马环磷酸腺苷反应元件结合蛋白(CREB)、细胞外信号调节激酶(ERK)活性的影响。方法将成年雄性Sprague Dawley(SD)大鼠随机分为5组:对照组(A)、应激1次组(B)、应激1周组(C)、应激2周组(D)和应激4周组(E),每组动物6只。应激模型为强迫大鼠游泳,每天15min。采用Westernblot检测各组大鼠海马CREB、ERK的磷酸化及总蛋白(p-CREB和t-CREB、p-ERK/MAPK和ERK/MAPK)水平。结果B和C组大鼠海马组织的p-CREB水平与对照组比较差异无显著性(P>0.05);D和E组大鼠海马组织的p-CREB明显低于对照组(P<0.05)。各应激组t-CREB水平与对照组比较均差异无显著性(P>0.05)。C、D和E组大鼠海马p-ERK44和p-ERK42的水平显著低于对照组(P<0.05或P<0.01);B组大鼠海马p-ERK44和p-ERK42的水平与对照组比较差异无显著性(P>0.05)。各应激组和对照组大鼠海马ERK44和ERK42的总蛋白水平比较均差异无显著性(P>0.05)。结论慢性应激降低大鼠海马CREB的活性,短时间和慢性应激均能降低大鼠海马ERK的活性。     

灵芝酸对痫样放电海马神经元ERK1/2磷酸化水平的影响

目的：探讨灵芝酸对癫痫样放电海马神经元ERK1/2磷酸化水平的影响。方法：以24h内新生Wistar大鼠为细胞源,用Neurobasal培养液培养海马神经元。①应用激光共聚焦显微镜检测培养神经元的纯度及其鉴定。②应用MTT法测灵芝酸的最适无毒浓度。③在研究最大无毒浓度的灵芝酸对痫样放电海马神经元ERK1/2磷酸化水平影响的实验中,将细胞随机分为：①正常对照组：将培养至第9天的海马神经元全液换成正常细胞外液处理3h,然后恢复正常培养3h后取材;②模型组：将培养至第9天的海马神经元全液换成无镁细胞外液处理3h,然后恢复正常培养3h后取材;③治疗组：将培养至第9天的海马神经元根据灵芝酸浓度不同,分为低、中、高三个浓度组（浓度分别为20μg/mL、80μg/mL、320μg/mL）,将各组的维持培养液全液换成无镁细胞外液处理3h,然后全液换成维持培养液配制不同浓度灵芝酸混悬液处理3h后取材。ELISA方法检测ERK1/2磷酸化水平的表达。结果：成功离体培养海马神经元,并用酶联免疫分析ERK1/2磷酸化水平的表达。各实验组中均有ERK1/2的表达并且各治疗组均能降低ERK1/2磷酸化水平,浓度为80μg/mL时结果最明显。结论：海马神经元癫痫样放电后ERK1/2被激活,在有效浓度的灵芝酸能显著抑制此反应中ERK1/2的磷酸化水平。     

细胞外信号调节激酶ERK1/ERK2对胰腺癌生长的影响

目的研究胰腺癌患者组织中及细胞系Pane-1及Jf-305中的ERK1／ERK2蛋白表达情况,以探讨其在胰腺癌发生、发展过程中的意义。方法应用免疫组织化学技术检测临床胰腺癌患者组织中ERK1／ERK2蛋白表达;Western blot方法观察胰腺癌细胞系Pane-1及Jf-305中的ERK1／ERK2蛋白表达。结果胰腺癌患者组织ERK1和ERK2蛋白均呈显著性高表达,阳性率分别为86．7％(39／45)和84．44％(38／45),而对照组织的阳性率分别为30％(6／20)和20％(4／20),胰腺癌组织和对照组织阳性率有显著差异;胰腺癌细胞系Panc-1及Jf-305中均有ERK1／ERK2蛋白的高表达。结论胰腺癌患者组织及细胞系中均有ERK1／ERK2蛋白强表达,ERK1和ERK2蛋白的过表达可能在人胰腺癌的发生、发展过程中起重要作用。     

Role of extracellular signal-regulated kinase 1/2 in cigarette smoke-induced mucus hypersecretion in a rat model

Background  Airway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease, which is closely associated with cigarette smoking. However, the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known. This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 (ERK 1/2) in airway mucus hypersecretion induced by cigarette smoke in rats.

Methods  A rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks．Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126, a specific MEK1 kinase inhibitor, at doses of 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg for 14 days. Expression of MUC5AC mRNA and protein, ERK 1/2 and phosphorylated-ERK 1/2 (p-ERK 1/2) were detected by RT-PCR, immunohistochemistry and Western blotting.

Results  Cigarette smoke significantly increased airway goblet cells metaplasia, induced the overexpression of MUC5AC mRNA and protein in bronchial epithelia, and increased the ratio of p-ERK 1/2 and ERK 1/2. U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke (P <0.05). Moreover, there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein (P <0.05).

Conclusions  Inhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein. ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.

     

细胞外信号调节激酶在应激性溃疡发病中的作用

目的:探讨细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)在实验性应激性溃疡发病中的作用.方法:采用浸水束缚(water-immersion restraint,WIR)应激实验复制大鼠应激性溃疡模型.检测WIR应激5 min、15 min、30 min、1 h、2 h和3.5 h各时间点的胃黏膜ERK1/2活化情况,并观察特异性ERK1/2抑制剂PD98059预处理(1 mg/kg,iv.)对胃黏膜损伤的影响.采用Western印迹检测ERK1/2和caspase-3表达,激酶活性分析方法检测ERK1/2活性,电泳迁移率变动分析(EMSA)检测转录因子活化蛋白1(AP-1)和核因子κB(NF-κB)DNA结合活性,Northern印迹检测TNF-α和IL-1β mRNA表达,采用溃疡指数(UI)计分和病理学检查评价胃黏膜病变程度, TUNEL技术检测和定量细胞凋亡.结果:正常大鼠胃黏膜仅检测到极微弱的磷酸化ERK1/2表达,WIR应激15 min后胃黏膜ERK1/2即发生活化并于3.5 h后达到峰值.PD98059抑制ERK活化后,胃黏膜AP-1和NF-κB活性及TNF-α和IL-1β mRNA表达显著下调,胃黏膜损伤程度也明显减轻,同时伴有胃黏膜caspase-3活化和凋亡轻度增加.结论:ERK1/2活化在浸水束缚应激诱导的胃黏膜损伤中发挥了重要作用.     

磷酸化胞外信号调节激酶1/2参与雌激素对切口痛大鼠的伤害性感受调节

目的通过大鼠切口痛模型来探讨磷酸化胞外信号调节激酶1/2(pERK1/2)参与雌激素对伤害性感受调节的作用机制。方法成年Sprague-Dawley雌鼠32只,在卵巢切除术(OVX)后第15天建立切口痛模型,随机分为4组,每组8只:雌激素替代(50μg雌二醇溶于100μL橄榄油)+切口痛假手术组(E+S组),溶剂替代(100μL橄榄油)+切口痛假手术组(V+S组),雌激素替代(50μg雌二醇溶于100μL橄榄油)+切口痛组(E+I组),溶剂替代(100μL橄榄油)+切口痛组(V+I组)。雌激素替代组于OVX术后第14天起每2 d腹腔注射雌激素1次至完成行为学实验,溶剂替代组注射等体积橄榄油。于OVX术前,切口痛术前当天(即OVX术后第15天),切口痛术后第1、3、5、7天(即OVX术后第16、18、20、22天)进行热痛实验,记录热缩足潜伏期(PWTL)。完成行为学实验后,采用免疫印迹法检测大鼠脊髓背角pERK1/2水平。结果 V+S组OVX术后第16、18、20、22天大鼠PWTL值较E+S组显著延长(P值均<0.05)。E+I组、V+I组大鼠切口痛术后(OVX术后第16、18、20、22天)手术侧PWTL...     

Glucocorticoid modulation of extracellular signal-regulated protein kinase1/2 and p38 in human ovarian cancer HO-8910 cells

Objective To investigate the signaling pathway through testing the effects of dexamethasone (Dex)on the activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2 and p38 kinase(p38) in HO-8910 cells.Methods Activation of the ERK1/2against the total ERK1/2 and p38 mitogen-activated protein kinases (MAPKs) protein and the phosphorylated forms of them.Results Dex could suppress the activation of ERK1/2, while enhance the activation of p38 rapidly and strongly in a dose- and time- dependent manner. Neither effect could be blocked by RU486, the antagonist of glucocorticoid receptor (GR).Conclusion Dex has rapid effects on the activation of ERK1/2 and p38, and these effects are not mediated by GR.     

Calcitonin gene-related peptide induces proliferation and monocyte chemoattractant protein-1 expression via extracellular signal-regulated kinase activation in rat osteoblasts

Background Calcitonin gene-related peptide （CGRP）, a sensory neuropeptide, affects osteoblast proliferation and bone formation. However, the mechanisms are not fully understood. Monocyte chemoattractant protein-1 （MCP-1） is a chemokine that stimulates the migration of monocytes and plays important roles in regulating bone remolding during fracture repair. In this study, we investigated the effects of CGRP on proliferation and MCP-1 expression in cultured rat osteoblasts. Methods Primary rat osteoblasts were isolated from fetal rats calvariae. Cells were exposed to gradient concentrations （10^-9 to 10^-7 mol/L） of CGRP. Protein and mRNA levels of MCP-1 were quantified by Western blotting and semiquantitative reverse transcdption-polymerase chain reaction, respectively. The protein level of MCP-1 was investigated and compared in cell culture media by enzyme linked immunosorbent assay （ELISA）. Phospho-extracellular signal-regulated kinase （ERK） expression was detected by Western blotting. Cell proliferative activity was measured by 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyl tetrazolium bromide （MTT） and BrdU assay. The effects of MAPK/ERK kinase （MEK）-inhibitor U0126 on CGRP-induced MCP-1 expression in primary rat osteoblasts were examined. Results CGRP effectively enhanced primary rat osteoblast proliferation and led to significant increases in the expression of MCP-1 mRNA and protein in time- and dose-dependent manners. CGRP activated the ERK pathway. Pretreatment of cultured rat osteoblasts with MEK inhibitor U0126 resulted in dose-dependent inhibitions of CGRP-induced MCP-1 mRNA and protein levels. Thus, CGRP promoted cell proliferation and stimulated MCP-1 expression in cultured rat osteoblasts. Conclusion These studies document novel links between CGRP and MCP-1 and illuminate the effects of CGRP in regulating bone remodeling.     

细胞外信号调节激酶在人纤维化肝组织中的表达

目的:研究细胞外信号调节激酶(ERK)在人纤维化肝组织中的表达及其与肝纤维化发生的相关性。方法:收集肝手术标本(纤维化肝组织和远癌的纤维化肝组织,以正常的肝组织做对照),以逆转录聚合酶链反应(RT-PCR)法检测正常肝和不同程度纤维化肝组织中ERK的表达情况,对结果进行统计分析。结果:人肝纤维化的发生发展过程中,ERK的表达增加。结论:ERK参与并介导其中的RAS/RAF/MEK/ERK信号通路,可能与肝纤维化的发生密切相关。     

Effect of Sodium Tanshinone ⅡA Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinasel/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

Objective： To observe the effects of sodium tanshinone ⅡA sulfonate （STS） on angiotensin Ⅱ （Ang Ⅱ）-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase （p-ERK1/2）. Methods： In the primary culture of neonatal rat myocardial cells, the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by [3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells. The expression of p-ERK1/2 was determined using Western blot and immunofluorescence labeling. Results： （1） The total protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ （1 μ mol/L） for 24 h; STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein. （2） After pretreatment of myocardial cells with Ang Ⅱ （1 μmol/L） for 5 min, the p-ERK1/2 protein expression was increased, with the most obvious effect shown at about 10 min; pretreatment of myocardial cells with STS at different doses （2, 10, 50μmol/L） for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner. （3） After the myocardial cells were stimulated by AngⅡ （1 μ mol/L）, the immunofluorescence of ERK1/2 rapidly appeared in the nucleus. The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS. （Conclusion： STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ, and the mechanism may be associated with the inhibition of p-ERK1/2 expression.