Pterostilbene from Vitis coignetiae protect H2O2-induced inhibition of gap junctional intercellular communication in rat liver cell line

In this study, we identified various stilbenoids derived from Vitis coignetiae and investigated the protective effect of the main component, pterostilbene, against the inhibition of gap junctional intercellular communication (GJIC) induced by H₂O₂ in WB-F344 rat liver epithelial cells. We analyzed seven kinds of stilbenoids, pterostilbene, astringin, piceid, vitisin, rhaponticin, resveratrol, and rhapontigenin, using DAD/UV HPLC. Total stilbenoid content was 127.37 ± 19.29 mg/100 g dry weight. Pretreatment with 0.5, 1.0, and 5.0 μM pterostilbene for 24 h was shown to recover GJIC blocked by 500 μM H₂O₂. Pretreatment with pterostilbene prevented the inhibition of GJIC via the down-regulation of connexin43 phosphorylation by the inactivation of ERK1/2 and p38 MAP kinase. Our results suggest that pterostilbene may be a functional chemopreventative agent and that dietary exposure of pterostilbene would be helpful for improving health.     

EXPRESSION OF TIGHT AND GAP JUNCTIONAL PROTEINS IN THE PERINEURIAL WINDOW MODEL OF THE RAT SCIATIC NERVE

Limited perineurial injury, known as a perineurial window, can lead to neuropathic pain. This article hypothesizes that the recovery of the perineurium is associated with the intercellular junctional proteins. It analyzes the expressions of occludin, ZO-1, and connexin 43 by immunoconfocal microscopy. Seven days after injury, immunoreactivities for occludin and ZO-1 were observed, although there was no connexin 43 detected. Then, 21 days after injury, immunoreactivity for connexin 43 were observed. These results indicate that recovery of the perineurium is associated with the intercellular junctional proteins and that the recovery of gap junctions is delayed compared with that of tight junctions.     

Disruption of oligodendrocyte gap junctions in experimental autoimmune encephalomyelitis

Gap junctions (GJs) are vital for oligodendrocyte survival and myelination. In order to examine how different stages of inflammatory demyelination affect oligodendrocyte GJs, we studied the expression of oligodendrocytic connexin32 (Cx32) and Cx47 and astrocytic Cx43 in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS) induced by recombinant myelin oligodendrocyte glycoprotein. EAE was characterized by remissions and relapses with demyelination and axonal loss. Formation of GJ plaques was quantified in relation to the lesions and in normal appearing white matter (NAWM). During acute EAE at 14 days postimmunization (dpi) both Cx47 and Cx32 GJs were severely reduced within and around lesions but also in the NAWM. Cx47 was localized intracellularly in oligodendrocytes while protein levels remained unchanged, and this redistribution coincided with the loss of Cx43 GJs in astrocytes. Cx47 and Cx32 expression increased during remyelination at 28 dpi but decreased again at 50 dpi in the relapsing phase. Oligodendrocyte GJs remained reduced even in NAWM, despite increased formation of Cx43 GJs toward lesions indicating astrogliosis. EAE induced in Cx32 knockout mice resulted in an exacerbated clinical course with more demyelination and axonal loss compared with wild-type EAE mice of the same backcross, despite similar degree of inflammation, and an overall milder loss of Cx47 and Cx43 GJs. Thus, EAE causes persistent impairment of both intra- and intercellular oligodendrocyte GJs even in the NAWM, which may be an important mechanism of MS progression. Furthermore, GJ deficient myelinated fibers appear more vulnerable to CNS inflammatory demyelination.     

Myocardial Morphological Characteristics and Proarrhythmic Substrate in the Rat Model of Heart Failure Due to Chronic Volume Overload

Chronic volume overload leads to cardiac hypertrophy and later to heart failure (HF), which are both associated with increased risk of cardiac arrhythmias. The goal of this study was to describe changes in myocardial morphology and to characterize arrhythmogenic substrate in rat model of developing HF due to volume overload. An arteriovenous fistula (AVF) was created in male Wistar rats between the inferior vena cava and abdominal aorta using needle technique. Myocardial morphology, tissue fibrosis, and connexin43 distribution, localization and phosphorylation were examined using confocal microscopy and Western blotting in the stage of compensated hypertrophy (11 weeks), and decompensated HF (21 weeks). Heart to body weight (BW) ratio was 89% and 133% higher in AVF rats at 11 and 21 weeks, respectively. At 21 weeks but not 11 weeks, AVF rats had pulmonary congestion (increased lung to BW ratio) indicating presence of decompensated HF. The myocytes in left ventricular midmyocardium were significantly thicker (+8% and +45%) and longer (+88% and +97%). Despite extensive hypertrophy, there was no excessive fibrosis in the AVF ventricles. Distribution and localization of connexin43 were similar between groups, but its phosphorylation was significantly lower in AVF hearts at 21st week, but not 11th week, suggesting that HF, rather than hypertrophy contributes to the connexin43 hypophosphorylation. In conclusion, volume overload leads to extensive eccentric hypertrophy, but not to myocardial fibrosis. Increased vulnerability to arrhythmia in this HF model is possibly related to gap junction remodeling with hypophosphorylation of connexin43. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

缝隙连接蛋白43在七氟醚后处理保护大鼠离体心肌缺血再灌注中的作用

目的探讨缝隙连接蛋白(connexin,Cx)43在七氟醚后处理保护大鼠离体心肌缺血再灌注中的作用。方法健康成年雄性SD大鼠40只,体质量200～250 g,采用随机数字表法将其分为4组(n=10):缺血再灌注组(IR组)、七氟醚后处理组(S组)、七氟醚混合5-羟基葵酸钠(5-HD)组(SH组)及5-HD组(H组)。采用Langendorff装置进行离体心脏灌注。各组均在阻断左冠状动脉(LAD)前维持灌注10 min,然后阻断LAD血流30 min后开放,再灌注120 min,其中S、SH及H组分别于LAD开放后使用3%七氟醚预饱和的K-H液、3%预饱和的七氟醚预充饱和的K-H加5-HD(100μmol/L)及加入5-HD(100μmol/L)的K-H液灌注15 min,然后使用普通K-H液继续灌注105 min。分别于阻断LAD前即刻(T0),开放LAD即刻(T1),开放LAD后15 min(T2),开放LAD后135 min(T3)时记录HR、左心室收缩压(LVSP)、左心室舒张压(LVDP)、左心室最大上升及下降速率(±dp/dtmax)。再灌注结束后,取左心室TTC染色测定心肌梗死面积,采用免疫组化染色观测Cx43蛋白表达情况,采用Western blot法检测Cx43蛋白及磷酸化Cx43(p-Cx43)蛋白含量。结果与IR组比较,S组的HR、LVSP、±dp/dtmax较高,LVDP降低,心肌梗死面积小,Cx43蛋白及p-Cx43蛋白含量增加,有统计学意义(P<0.05)。IR组与SH组及H组之间相比HR、LVSP、±dp/dtmax、LVDP、心肌梗死面积、Cx43蛋白和p-Cx43蛋白差异无显著性(P>0.05)。结论七氟醚后处理减轻离体大鼠心肌IR损伤的作用可能与其开放线粒体敏感钾通道从而促进Cx43蛋白表达及其磷酸化有关。     

抑制ERK1/2通路激活改善大鼠心房纤维化和缝隙连接蛋白40重构

目的 探讨ERK1/2通路抑制剂U0126对盐酸异丙基肾上腺素(ISO)诱导的大鼠心房纤维化和缝隙连接蛋白40(Cx40)重构的影响。 方法 将32只雄性SD大鼠随机等分为空白对照组、DMSO组、ISO [5 mg/(kg·d) ]+DMSO组(模型组)和ISO [5 mg/(kg·d) ]+U0126 [0.5 mg/(kg·d) ]+DMSO组(干预组)。每组1次/d给予相关试剂,连续7 d后处死大鼠并取心肌组织。用放射免疫法测血管紧张素Ⅱ(AngⅡ)含量;H-E和Masson染色法观察心肌纤维化程度;免疫组化法测定磷酸化丝裂原活化蛋白激酶激酶1/2(p-MEK1/2)、磷酸化细胞外信号调节激酶1/2(p-ERK1/2)以及Cx40的表达。 结果 (1)空白对照组 [(242.133±4.870) ng/L ]与DMSO组 [(239.412±1.795) ng/L ]的AngⅡ含量差异无统计学意义,而模型组 [(500.250±8.869) ng/L ]和干预组 [(498.695±9.340) ng/L ]的AngⅡ含量较空白对照组与DMSO组均升高,差异有统计学意义(P均<0.01)。(2)空白对照组与DMSO组无心房纤维化,而干预组心房纤维化程度较模型组减弱(P<0.01)。(3)空白对照组与DMSO组中p-MEK1/2和p-ERK1/2的含量差异无统计学意义,模型组中两者含量较空白对照组与DMSO组均增加(P均<0.01),干预组中两者含量与空白对照组和DMSO组比较差异均无统计学意义,而与模型组比较差异有统计学意义(P<0.01)。(4)空白对照组与DMSO组中Cx40含量差异无统计学意义且呈线性分布于心肌细胞闰盘处,模型组中Cx40含量较空白对照组和DMSO组均减少(P均<0.01)且分布无规律性,而干预组中Cx40含量与空白对照组和DMSO组比较差异均无统计学意义且部分呈线性分布于心肌细胞闰盘处;干预组中Cx40含量减少程度较模型组减弱(P<0.01),且部分呈线性分布于心肌细胞闰盘处。 结论 心肌组织中AngⅡ含量长期升高可能参与了心房纤维化的形成和Cx40重构,U0126通过抑制ERK1/2通路激活可有效改善心房纤维化程度和Cx40重构。     

Two Iranian families with a novel mutation in GJB2 causing autosomal dominant nonsyndromic hearing loss

Mutations in GJB2, encoding connexin 26 (Cx26), cause both autosomal dominant and autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNA3 and DFNB1 loci, respectively. Most of the over 100 described GJB2 mutations cause ARNSHL. Only a minority has been associated with autosomal dominant hearing loss. In this study, we present two families with autosomal dominant nonsyndromic hearing loss caused by a novel mutation in GJB2 (p.Asp46Asn). Both families were ascertained from the same village in northern Iran consistent with a founder effect. This finding implicates the D46N missense mutation in Cx26 as a common cause of deafness in this part of Iran mandating mutation screening of GJB2 for D46N in all persons with hearing loss who originate from this geographic region.     

压力超负荷心力衰竭大鼠心室肌电生理失稳态及缝隙连接蛋白Cx43 的变化

目的观察压力超负荷所致心力衰竭（HF）大鼠心室肌电生理失稳态和缝隙连接蛋白Cx43表达的变化。方法采用腹主动脉缩窄法建立压力超负荷大鼠HF模型,取左室舒张末压≥15mmHg的存活大鼠入HF组,另设假结扎对照组。术后32周两组大鼠以颈总动脉插管法和心脏B超测定心功能,并检测电生理指标,以免疫印迹方法检测心肌细胞Cx43蛋白表达的变化,通过透射电镜观察心室肌缝隙连接分布的变化。结果HF大鼠出现明显电生理失稳态和心功能不全,左室舒张末压明显增高而心室有效不应期明显延长,左室射血分数明显下降,同时大鼠心室肌中缝隙连接蛋白Cx43表达明显下调（0．929±0．095VS1．250±0．083,P〈0．05）并出现空间重构。结论压力超负荷所致HF大鼠心室肌存在明显缝隙连接重构,这可能是导致HF时心肌电生理重构的重要机制。     

缝隙连接蛋白43 (Cx43)与神经炎症的研究进展

 缝隙连接蛋白43(connexin43, Cx43)是中枢神经系统(central nervous system, CNS)中含量最丰富的连接蛋白,主要分布于星形胶质细胞,以缝隙连接(gap junction, GJ)和半通道(hemichannel, HC)的形式存在,分别介导细胞间和细胞与外环境间的信息交流。随着人口的老龄化,阿尔茨海默病(Alzheimer's disease, AD)、脑卒中及术后认知功能障碍等疾病的发生率越来越高,神经炎症是这些CNS疾病的一个共同特征,涉及到白细胞聚集、胶质细胞激活、炎性因子释放等,该过程需要高效的信息交流,研究表明Cx43在该过程中发挥着不可忽视的作用,主要表现为GJ抑制和HC活性增加并影响神经功能。本文重点综述Cx43与神经炎症的关系,为多种CNS疾病的治疗提供新的靶点。