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     

汉黄芩苷对缺氧复氧诱导H9c2心肌细胞损伤及P38和ERK1/2表达的影响

目的 探讨汉黄芩苷（Wog）对缺氧复氧诱导H9c2心肌细胞损伤及P38和ERK1/2表达的影响。方法 培养心肌细胞H9c2构建缺氧复氧模型（A/R）,给予Wog低、中、高剂量（12.5、25、50 μM）处理24h,采用CCK8检测48 h后细胞活力,Hochest染色检测细胞凋亡,ELISA检测细胞上清液中心肌损伤标记物［肌酸激酶（CK）,肌酸激酶同工酶（CK MB）、肌红蛋白（Mb）］和炎性细胞因子［白介素6（IL-6）、白介素1β（IL-1β）和诱导型一氧化氮合酶（iNOS）］的含量,生化试剂盒检测氧化应激指标［超氧化物歧化酶（SOD）、丙二醛（MDA）及乳酸脱氢酶（LDH）］的含量,Western blot检测相关蛋白［B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关x蛋白（Bax）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）、Caspase-9］、丝裂原活化蛋白激酶P38及细胞外调节激酶（ERK1/2）的表达。 结果 低、中、高剂量Wog处理缺氧复氧模型细胞H9c2,细胞存活率、Bcl-2及SOD的表达显著升高,凋亡率、Bax、Caspase-3、Caspase-9、CK、CK-MB、Mb、IL-6、IL-1β、iNOS、MDA、LDH、P38及ERK1/2的表达显著降低（均P＜0.05）。 结论 汉黄芩苷可通过改善炎症反应和氧化应激损伤,减少心肌细胞的凋亡,对缺氧复氧诱导H9c2心肌细胞损伤具有一定的保护作用,可能与下调P38、ERK1/2磷酸化有关。     

General anesthetics selectively modulate glutamatergic and dopaminergic signaling via site-specific phosphorylation in vivo

Isoflurane, propofol and ketamine are representative general anesthetics with distinct molecular mechanisms of action that have neuroprotective properties in models of excitotoxic ischemic damage. We characterized the effects of these agents on neuronal glutamate and dopamine signaling by profiling drug-induced changes in brain intracellular protein phosphorylation in vivo to test the hypothesis that they affect common downstream effectors. Anesthetic-treated and control mice were killed instantly by focused microwave irradiation, frontal cortex and striatum were removed, and the phosphorylation profile of specific neuronal signaling proteins was analyzed by immunoblotting with a panel of phospho-specific antibodies. At anesthetic doses that produced loss of righting reflex, isoflurane, propofol, and ketamine all reduced phosphorylation of the activating residue T183 of ERK2 (but not of ERK1); S897 of the NR1 NMDA receptor subunit; and S831 (but not S845) of the GluR1 AMPA receptor subunit in cerebral cortex. At sub-anesthetic doses, these drugs only reduced phosphorylation of ERK2. Isoflurane and ketamine also reduced phosphorylation of spinophilin at S94, but oppositely regulated phosphorylation of presynaptic (tyrosine hydroxylase) and postsynaptic (DARPP-32) markers of dopaminergic neurotransmission in striatum. These data reveal both shared and agent-specific actions of CNS depressant drugs on critical intracellular protein phosphorylation signaling pathways that integrate multiple second messenger systems. Reduced phosphorylation of ionotropic glutamate receptors by all three anesthetics indicates depression of normal glutamatergic synaptic transmission and reduced potential excitotoxicity. This novel approach indicates a role for phosphorylation-mediated down-regulation of glutamatergic synaptic transmission by general anesthetics and identifies specific in vivo targets for focused evaluation of anesthetic mechanisms.     

调肝与健脾对节育器致人子宫异常出血症子宫内膜组织ERK1/2、PI_3K信号通路的影响差异

目的:探讨柴胡止血液对置器后子宫异常出血患者的影响机制。方法:原代培养置器后子宫异常出血患者的宫内膜组织,给含药血清后,分别采用western blot和ELISA的方法测定ERK1/2和PI3K磷酸化的水平。结果:4号组织的归脾汤组升高了PI3K磷酸化水平,P<0.05;1号组织的柴胡止血液组有升高ERK1/2磷酸化水平的趋势;3号组织的柴胡止血液组有升高ERK1/2磷酸化水平的趋势;5号组织的柴胡止血液组有升高PI3K磷酸化水平的趋势;2号组织的归脾汤组有升高ERK1/2磷酸化水平的趋势;4号组织的归脾汤组有升高ERK1/2和PI3K磷酸化水平的趋势。结论:对于出血量大的肝旺患者,柴胡止血液可能通过激活ERK1/2通路来增强血管的收缩从而达到止血的目的;对于出血量较小的肝旺患者证,柴止血液可能通过激活PI3K通路来调节组织抗炎作用,从而达到治疗效果。     

隐丹参酮对IGF-1促PC12细胞存活的作用及机制探讨

目的:研究隐丹参酮对类胰岛素样生长因子-1(IGF-1)促进PC12细胞存活的影响及可能机制.方法:首先采用剥夺血清造成细胞损伤模型确定IGF-1发挥促存活作用的剂量,然后不同剂量的隐丹参酮预先作用PC12细胞,再加入IGF-1处理,MTT检测隐丹参酮对IGF-1促PC12细胞存活的影响;Western blot检测隐丹参酮对IGF-t诱导其受体(1GF-IR)磷酸化及下游信号通路磷酯酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)及细胞外信号调节激酶(ERK) 1/2的影响.结果:MTT显示隐丹参酮能剂量依赖性地阻断IGF-1的促存活作用,抑制PC12细胞的增殖;Western blot结果显示IGF-1能明显诱导其受体(IGF-1R)磷酸化,而隐丹参酮则起抑制作用,并使下游Akt及ERK1/2的磷酸化水平下调,呈明显的剂量依赖性.结论:隐丹参酮可通过抑制IGF-1R及其下游信号分子Akt和ERK1/2的磷酸化,从而抑制IGF-1的促存活功能,发挥其自身抑制细胞存活,促使细胞凋亡的作用.     

Astrocytes in the damaged brain: Molecular and cellular insights into their reactive response and healing potential

Long considered merely a trophic and mechanical support to neurons, astrocytes have progressively taken the center stage as their ability to react to acute and chronic neurodegenerative situations became increasingly clear. Reactive astrogliosis starts when trigger molecules produced at the injury site drive astrocytes to leave their quiescent state and become activated. Distinctive morphological and biochemical features characterize this process (cell hypertrophy, upregulation of intermediate filaments, and increased cell proliferation). Moreover, reactive astrocytes migrate towards the injured area to constitute the glial scar, and release factors mediating the tissue inflammatory response and remodeling after lesion. A novel view of astrogliosis derives from the finding that subsets of reactive astrocytes can recapitulate stem cell/progenitor features after damage, fostering the concept of astroglia as a promising target for reparative therapies. But which biochemical/signaling pathways modulate astrogliosis with respect to both the time after injury and the type of damage? Are reactive astrocytes overall beneficial or detrimental for neuroprotection and tissue regeneration? This debate has been animating this research field for several years now, and an integrated view on the results obtained and the possible future perspectives is needed. With this Commentary article we have attempted to answer the above-mentioned questions by reviewing the current knowledge on the molecular mechanisms controlling and sustaining the reaction of astroglia to injury and its stem cell-like properties. Moreover, the cellular/molecular mechanisms supporting the detrimental or beneficial features of astrogliosis have been scrutinized to gain insights on possible pharmacological approaches to enhance astrocyte neuroprotective activities.     

促分裂原活化的蛋白激酶信号通路与能量代谢的关系

能量代谢每时每刻都伴随着物质代谢在生命体内发生。生物通过利用物质代谢产生的高能磷酸化合物以维持各项生命功能的运转,不同状态下的细胞通过利用各种物质通过不同的通路进行不同的代谢方式。促分裂原活化的蛋白激酶(MAPK)通路作为生命体里广泛存在的一类信号蛋白,影响着细胞、生物体的生命活动。MAPK通路在能量代谢的各个环节起着非常重要的作用。通过研究MAPK通路中的胞外信号调节激酶(ERK)通路、Jun激酶(JNK)通路、P38通路均与能量代谢有关的蛋白、靶点、关键酶、转运体,与三大营养物质能量代谢相结合,从而为能量代谢相关的细胞功能进一步研究铺垫,并为今后治疗肿瘤性疾病、感染性疾病、代谢性疾病等提供新思路。     

Cyclopeptide RA-V inhibits angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells

BACKGROUND AND PURPOSE

Anti-angiogenic agents have recently become one of the major adjuvants for cancer therapy. A cyclopeptide, RA-V, has been shown to have anti-tumour activities. Its in vitro anti-angiogenic activities were evaluated in the present study, and the underlying mechanisms were also assessed.

EXPERIMENTAL APPROACH

Two endothelial cell lines, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1), were used. The effects of RA-V on the proliferation, cell cycle phase distribution, migration, tube formation and adhesion were assessed. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant molecules.

KEY RESULTS

RA-V inhibited HUVEC and HMEC-1 proliferation dose-dependently with IC₅₀ values of 1.42 and 4.0 nM respectively. RA-V inhibited migration and tube formation of endothelial cells as well as adhesion to extracellular matrix proteins. RA-V treatment down-regulated the protein and mRNA expression of matrix metalloproteinase-2. Regarding intracellular signal transduction, RA-V interfered with the activation of ERK1/2 in both cell lines. Furthermore, RA-V significantly decreased the phosphorylation of JNK in HUVEC whereas, in HMEC-1, p38 MAPK was decreased.

CONCLUSIONS AND IMPLICATIONS

RA-V exhibited anti-angiogenic activities in HUVEC and HMEC-1 cell lines with changes in function of these endothelial cells. The underlying mechanisms of action involved the ERK1/2 signalling pathway. However, RA-V may regulate different signalling pathways in different endothelial cells. These findings suggest that RA-V has the potential to be further developed as an anti-angiogenic agent.     

Puerarin inhibits angiotensin II-induced cardiac hypertrophy via the redox-sensitive ERK1/2, p38 and NF-κB pathways

Aim： To investigate the effects of puerarin （Pue）, an isoflavone derived from Kudzu roots, on angiotensin II （Ang II）-induced hypertrophy of cardiomyocytes in vivo and in vitro.
Methods： C57BL/6J mice were infused with Ang II and treated with Pue （100 mg·kg-1·d-1, po） for 15 d. After the treatment, systolic blood pressure （SBP） and left ventricular wall thickness were assessed. The ratios of heart weight to body weight （HW/BW） and left ventricular weight to body weight （LVW/BW） were determined, and heart morphometry was assessed. Expression of fetal-type genes （ANP, BNP and β-MHC） in left ventricles was measured using semi-quantitative RT-PCR. Mouse primary cardiomyocytes were treated with Pue （50, 100, 200 μmol/L）, then exposed to Ang II （1 μmol/L）. ROS level was examined with flow cytometry, the binding activity of NF-κB was determined using EMSA. Western blot was used to measure the levels of ERK1/2, p38 and NF-κB pathway proteins. [3H]leucine incorporation was used to measure the rate of protein synthesis.
Results： Oral administration of Pue significantly suppressed Ang II-induced increases in the myocyte surface area, HW/BW, LVW/BW, SBP and left ventricular wall thickness. Furthermore, Pue significantly suppressed Ang II-induced increases in ANP, BNP and β-MHC expression in the left ventricles in vivo. Treatment of cardiomyocytes with Pue （50–500 μmol/L） did not affect the viability of cardiomyocytes in vitro. Pretreatment of cardiomyocytes with Pue dose-dependently inhibited Ang II-induced increases in ROS production, NF-κB binding activity, protein synthesis and cell breadth. Furthermore, pretreatment with Pue significantly suppressed Ang II-induced activation of ERK1/2, p38 and the NF-κB pathway proteins and the expression of ANP and β-MHC in cardiomyocytes. The positive drug valsartan exerted similar effects on Ang II-induced cardiac hypertrophy in vivo and in vitro.
Conclusion： Pue attenuates Ang II-induced cardiac hypertrophy by inhibiting activation of the redox-sensitive ERK1/2, p38 and the NF-κB pathways.