ERK1/2信号途径与弓形虫侵入细胞及胞内增殖关系的研究

目的探讨阻断细胞外蛋白调节激酶(ERK1/2)信号途径对弓形虫速殖子侵入宿主细胞及在胞内增殖的影响。方法姬氏染色法检测ERK1/2途径不同时间及不同剂量的抑制剂U0126或PD98059作用下宿主细胞感染弓形虫速殖子的百分率,根据细胞感染率和感染细胞内虫荷量分析ERK1/2途径抑制剂对速殖子在细胞中增殖的影响。结果速殖子在细胞培养系统中以1、10和100μmol/L U0126或PD98059作用3 h、6 h和9 h后,前者细胞培养孔中的细胞感染率分别较对照组平均下降34.62%(P<0.01)、53.55%(P<0.01)和67.76%(P<0.01),后者分别较对照组平均下降22.67%(P<0.01)、52.21%(P<0.01)和58.99%(P<0.01);感染细胞感染速殖子率与对照组比较差异无统计学意义(P>0.05)。结论阻断ERK1/2途径的不同信号位点对弓形虫速殖子侵入宿主细胞影响不同,ERK1/2途径在速殖子侵入宿主细胞起主要作用,但对侵入后的增殖无明显影响。     

桂皮醛及其代谢产物肉桂酸体外抗柯萨奇病毒B_3的作用机制研究

目的探讨桂皮醛(cinnamaldehyde,CA)及其代谢产物肉桂酸(cinnamic acid,CD)体外抗柯萨奇病毒B3(CVB3)的作用机制。方法用CVB3感染SD乳鼠心肌细胞,建立病毒性心肌炎(VMC)细胞模型。1)MTT法测定CA和CD对正常心肌细胞的IC50;2)MTT法测定不同剂量CA、CD对CVB3感染心肌细胞72h存活率;3)分别于接种CVB3的同时(0h)、接种前1h(-1h)和接种后1h(1h)给予100μmol/LCA、CD各100μl,观察CPE,MTT测定72h细胞存活率,并进行病毒中和抗体、免疫荧光和电镜观察。结果 1)CA对正常心肌细胞的LogIC50为-4.125,0.1～1000μmoL/L剂量的CD组心肌细胞存活率显著高于CA组(P0.01);2)10～1000μmol/LCD组细胞存活率高于CA各组和CVB3组,心肌细胞病毒滴度差异有统计学意义(P0.01)。100～1000μmol/LCA组病毒滴度低于CVB3组(P0.01),细胞存活率与CVB3组比较差异无统计学意义(P0.05);3)接种CVB3-1h、0h和1h,分别给予100μmol/LCD100μl,其病毒滴度均显著低于CVB3组(P0.01),细胞存活率高于CA组和CVB3组(P0.01),不同时间接种CVB3组与CD组比较差异无统计学意义(P0.05),电镜和免疫荧光试验显示,CD对CVB3有直接灭活和抑制细胞内复制作用。接种CVB30和1h给予CA,其病毒滴度低于CVB3组(P0.01),但细胞存活率与CVB3组比较差异无统计学意义(P0.05)。结论 CA在体外可直接灭活CVB3,但对心肌细胞具有明显毒性,可能与其醛基结构相关。CD可直接杀灭CVB3,抑制CVB3在细胞内的复制,但对CVB3感染无预防作用。     

Akt和胞外信号调节激酶通路间的信号交流对内质网应激条件下肝癌细胞周期的调控

目的 研究内质网应激介导的磷脂酰肌醇3激酶(PI3K)/Akt和丝裂原活化蛋白激酶(MEK)/胞外信号调节激酶(ERK)途径间的信号交流及其对内质网应激条件下肝癌细胞周期的调控作用.方法 采用PI3K抑制剂LY294002、Akt激活型突变载体myr-Akt和MEK抑制剂U0126分别阻断或激活内质网应激介导的Akt和ERK活化,并利用Western blot和流式细胞技术分析内质网应激条件下PI3K/Akt和MEK/ERK途径间的信号交流及其对肝癌细胞株SMMC-7721、Hep3B和HepG2细胞周期的调控作用.数据处理采用Sperman等级相关分析,P＜0.05为差异有统计学意义.结果 阻断PI3K/Akt明显促进内质网应激介导的MEK/ERK活化,而过度激活PI3K/Akt则抑制内质网应激介导的MEK/ERK活化.阻断MEK/ERK对内质网应激介导的PI3K/Akt活化无影响.持续活化的Akt突变载体myr-Akt和MEK抑制剂U0126均明显抑制了内质网应激诱导的压力细胞G0/G1期阻滞.结论 PI3K/Akt和MEK/ERK信号途径在内质网应激肝癌细胞中存在信号交流,该信号交流对细胞周期起重要调控作用.     

褪黑素对创伤性脑损伤大鼠ERK-MAPK信号传导机制的影响

目的探讨褪黑素(MT)对创伤性脑损伤(TBI)急性期ERK-丝裂原活化蛋白酶(ERK-MAPK)信号通路的影响。方法制作大鼠创伤性脑损伤模型,打击损伤前30 min自尾静脉注射MT。细胞凋亡原位检测(TUNEL)荧光法检测细胞凋亡,Western印迹法检测损伤脑皮质p ERK表达水平。结果 TUNEL荧光法检测显示TBI+MT组损伤灶周围皮层TUNEL阳性细胞少于TBI组,凋亡指数降低(P0.05)。Western印迹法检测脑损伤后各个时间点p ERK表达,在损伤后1 h起迅速升高,3 d为表达高峰,其后逐渐下降,但直到损伤后5 d仍未恢复至基础水平。TBI+MT组p ERK水平较TBI组明显降低(P0.01),而且MT 20 mg/kg组的p ERK1/2水平低于10 mg/kg组(P0.05)。结论创伤性脑损伤诱导了强烈的ERK-MAPK信号通路激活,MT能够剂量依赖性抑制ERK1/2-MAPK信号通路激活,并抑制急性期神经细胞凋亡。     

细胞外信号调节激酶1/2途径参与甲状旁腺素1-34诱导的心肌细胞肥大

目的 观察丝裂素活化蛋白激酶的上游激酶1(MAPKK,MEK1)抑制剂PD98059对大鼠甲状旁腺素1-34(rPTH1-34)诱导的心室肌细胞肥大的影响及细胞外信号调节激酶1/2(ERK1/2)表达的变化,分析MEK/ERK途径的可能作用. 方法 体外培养新生大鼠心室肌细胞,10-7mol/LrPTH1-34诱导建立心肌细胞肥大模型,在模型中加入2×10-5mol/L PD98059,Motic Images Advanced 3.0软件测定细胞直径、3H-亮氨酸掺入实验检测细胞蛋白合成速率、RT-PCR半定量测定心房利钠肽mRNA、Western blot方法观察ERK1/2、磷酸化ERK1/2蛋白表达的变化. 结果 与正常组相比,10-7mol/L rPTH1-34孵育24 h可使体外培养的心肌细胞直径增加13.6 μm、细胞蛋白合成速率增加898 cpm/well,使心房利钠肽mRNA表达增加73.9%,p-ERK1/2蛋白表达增加15%(P＜0.05).与PTH组相比,预先给予PD98059可使细胞直径减少7.1 μm,细胞蛋白合成速率减少644 cpm/well,心房利钠肽mRNA表达下降52.2%,磷酸化ERK1/2蛋白表达减少18%(P＜0.05).单独使用PD98059对正常心肌细胞没有影响(P＞0.05). 结论 PD98059通过抑制ERK1/2、磷酸化ERK1/2的表达阻断了心肌细胞肥大反应,MEK/ERK1/2途径的活化参与了rPTH1-34的致肥大作用.     

MAPK信号通路在力学刺激对MG-63成骨样细胞护骨素表达中的作用

目的观察力学刺激对MG-63成骨样细胞护骨素(osteoprotegrin,OPG)表达及丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的影响,以及探讨MAPK信号通路在OPG表达中的作用。方法将不同作用时间的机械应变分别作用于MG-63成骨样细胞,采用免疫印迹(Western blot)法检测OPG蛋白、磷酸化的细胞外信号调节激酶1/2 (p-ERK1/2)、p38MAPK (p-p38MAPK)、JNK (p-JNK)表达,采用RT-PCR方法检测OPG mRNA的表达,同时应用信号通路阻断剂观察MAPK各信号通路在OPG表达中的作用。结果机械力学刺激明显促进了MG-63成骨样细胞OPG蛋白及mRNA的表达,加力1、3、6、12 h后OPG蛋白表达分别是对照组的2. 3、4. 2、5. 9、6. 6倍(P<0. 05),加力3、6、12 h后OPG mRNA表达分别是对照组的4. 8、6. 8、7. 5倍(P<0. 05),加力12 h组作用最明显。力学刺激能够激活ERK1/2信号通路,加力60 min后激活最显著,p-ERK1/2表达是对照组的3. 2倍(P <0. 05)。力学刺激也能激活JNK信号通路,加力10 min后开始活化,加力30 min后最显著,加力10、15、30 min后p-JNK表达分别是对照组的2. 9、4. 3、5. 1倍(P<0. 05)。但力学刺激不能激活p38MAPK信号通路。分别应用ERK1/2、JNK的信号通路阻断剂后,发现当抑制ERK1/2信号通路时,OPG蛋白表达也受到了抑制,差异有统计学意义(P<0. 05)。结论机械力学刺激上调了MG-63成骨样细胞OPG蛋白及mRNA的表达,ERK1/2信号通路可能参与了此过程。     

重组人硫氧还蛋白抗柯萨奇B3m病毒损伤的研究

目的 观察重组人硫氧还蛋白(TRX)对柯萨奇B3m病毒(CVB3m)致HeLa细胞损伤的保护作用,并检测TRX对病毒复制的抑制作用.方法 采用IOTCID50的CVB3m病毒感染HeLa细胞,给予不同剂量的TRX(2、5、10mg/L)加以保护,实验设TRX保护组、病毒感染组、TRX对照组和对照组,每组设6个复孔.采用四甲基偶氮唑盐(MTT)和相差显微镜观察细胞生长情况.结果 相差显微镜下对照组HeLa细胞排列紧密、呈多角形;病毒感染组HeLa细胞排列不紧密,细胞明显变网、脱落;TRX保护组(2、5、10 mg/L)随TRX剂量增加HeLa细胞形态明显改善.MTT结果显示,TRX对照组(2、5、10 mg/L)生长抑制率(1.2%、2.9%、6.3%)与对照组(0)比较,差异无统计学意义(P均>0.05);TRX保护组(2、5、10 mg/L)生长抑制率(32.0%、28.0%、27.0%)与病毒感染组(51.7%)比较,均有降低(P均<0.05).病毒复制抑制作用结果显示,TRX保护组(2、5、10 mg/L)生长抑制率(26.0%、27.0%、10.9%)与病毒感染组(60.0%)比较,均有降低(P均<0.05);TRX各保护组之间比较,低剂量组(2、5 mg/L)与高剂量组(10 mg/L)间差异有统计学意义(P均<0.05).结论 重组人TRX(2、5、10mg/L)可以减轻CVB3m病毒导致的HeLa细胞损伤,对细胞无明显毒性作用,并具有抑制病毒复制的作用.     

MAPK信号通路在力学刺激对MG-63成骨样细胞护骨素表达中的作用

目的观察力学刺激对MG-63成骨样细胞护骨素(osteoprotegrin,OPG)表达及丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的影响,以及探讨MAPK信号通路在OPG表达中的作用。方法将不同作用时间的机械应变分别作用于MG-63成骨样细胞,采用免疫印迹(Western blot)法检测OPG蛋白、磷酸化的细胞外信号调节激酶1/2 (p-ERK1/2)、p38MAPK (p-p38MAPK)、JNK (p-JNK)表达,采用RT-PCR方法检测OPG mRNA的表达,同时应用信号通路阻断剂观察MAPK各信号通路在OPG表达中的作用。结果机械力学刺激明显促进了MG-63成骨样细胞OPG蛋白及mRNA的表达,加力1、3、6、12 h后OPG蛋白表达分别是对照组的2. 3、4. 2、5. 9、6. 6倍(P0. 05),加力3、6、12 h后OPG mRNA表达分别是对照组的4. 8、6. 8、7. 5倍(P0. 05),加力12 h组作用最明显。力学刺激能够激活ERK1/2信号通路,加力60 min后激活最显著,p-ERK1/2表达是对照组的3. 2倍(P 0. 05)。力学刺激也能激活JNK信号通路,加力10 min后开始活化,加力30 min后最显著,加力10、15、30 min后p-JNK表达分别是对照组的2. 9、4. 3、5. 1倍(P0. 05)。但力学刺激不能激活p38MAPK信号通路。分别应用ERK1/2、JNK的信号通路阻断剂后,发现当抑制ERK1/2信号通路时,OPG蛋白表达也受到了抑制,差异有统计学意义(P0. 05)。结论机械力学刺激上调了MG-63成骨样细胞OPG蛋白及mRNA的表达,ERK1/2信号通路可能参与了此过程。     

洛伐他汀经PI3K/Akt和ERK1/2信号通路抑制大鼠骨髓间充质干细胞凋亡

目的 体外以缺氧无血清条件模拟心肌梗死后的心脏缺血微环境,研究洛伐他汀能否抑制缺氧无血清引起的骨髓间充质干细胞(MSC)凋亡并探讨其机制.方法 以Hocchst33342染色荧光显微镜观察法及Annexin V/PI流式细胞术检测洛伐他汀的抗凋亡作用,并进一步采用Westernblot方法 检测洛伐他汀对线粒体凋亡途径的抑制作用以及对磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸激酶(Akt)途径和丝裂原活化的蛋白激酶(MAPK)的激酶(MEK)/细胞内信号调节蛋白激酶(ERK1/2)途径的激活作用.结果 0.01～1 μmol/L浓度范围的洛伐他汀能够有效地抑制缺氧无血清引起的MSC凋亡.洛伐他汀抑制线粒体凋亡途径,洛伐他汀抑制细胞色素C释放,降低天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)活化,从而保护线粒体功能.洛伐他汀的抗凋亡效应以及其抑制细胞色素C释放的作用均可被PI3K抑制剂LY294002和MEK抑制剂U0126阻断.洛伐他汀能够激活PI3K/Akt和MEK/ERK1/2两条细胞存活信号通路,分别导致Akt和GSK-3β及ERK1/2磷酸化.结论 洛伐他汀能够抑制线粒体凋亡途径,并激活PI3K/Akt和MEK/ERK1/2细胞存活通路,最终发挥抗缺氧无血清引起的MSC凋亡.该研究为提高移植干细胞的存活率提供了一种可能有效的干预措施.     

穿孔素在慢性心肌损伤中的表达及其意义

目的：探讨穿孔素在阿萨奇病毒B组3型（CVB3m)至T－2毒素染毒小鼠心肌损伤中的表达及其意义,方法：实验组BALB／C小鼠1．0mg/kg体重T－2毒素隔日灌胃,3周后腹腔接种0．1ml内含1000TCID50的CVB3m病毒液,对照组生理盐水灌胃,腹腔接种0．1ml 1640营养液,观察心肌病理改变,同时用RTPCR技术对心肌组织中CVB3m RNA和穿孔素mRNA表达进行检测,结果：CVB3m感染T－2毒素染毒小鼠心肌出现慢性损伤,可见纤维结缔组织增生和坏死灶癜痕修复。心肌组织中持续表达CVB34m RNA和穿孔素mRNA。结论CVB3m RNA和穿孔素mRNA的持续表达可能是CVB3m致T－2毒素染毒小鼠心肌慢性损伤的机制之一。     

Melanocortin 5 receptor signaling and internalization: Role of MAPK/ERK pathway and β-arrestins 1/2

The Melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) that exhibits high affinity for α-MSH. Here we present evidence for MC5R-GFP internalization and subsequent recycling to cell surface, in α-MSH-stimulated HeLa cells. This melanocortin induces a biphasic activation of ERK1/2 with an early peak at 15min, a G(i)-protein driven, β-arrestins 1/2 independent process, and a late sustained activation that is regulated by β-arrestins 1/2. ERK1/2 lead to downstream phosphorylation of 90-kDa ribosomal S6 kinases (p90RSK) and mitogen- and stress-activated protein kinase 1 (MSK1). Only a small fraction (10%) of phosphorylated p90RSK and ERK1/2 translocates to the nucleus inducing c-Fos expression. α-MSH also activates CREB through cAMP/PKA pathway. In 3T3-L1 adipocytes, where MC5R is endogenously expressed, α-MSH also induces phosphorylation and cytosolic retention of the same signaling molecules. These findings provide new evidence on the signaling mechanisms underlying MC5R biological response to α-MSH.     

Nip21 gene expression reduces coxsackievirus B3 replication by promoting apoptotic cell death via a mitochondria-dependent pathway

Our previous studies, using differential mRNA display, suggested that the mouse Nip21 gene may be involved in myocarditis development in the coxsackievirus B3 (CVB3)-infected mouse heart. Sequence comparison indicated that the mouse Nip21 gene shares high sequence homology to human Nip2. This human protein is known to interact with both the apoptosis inhibitor Bcl-2 and a homologous protein, the adenovirus E1B 19-kDa protein. Such interactions implicate Nip21 gene in cell death pathways. To study the function of this gene, we have cloned Nip21 from mouse hearts and established a Tet-On doxycycline-inducible HeLa cell line and a cardiomyocyte H9c2 cell line expressing Nip21 to characterize gene function in relation to apoptosis. We demonstrated that Nip21 expression could induce apoptosis via caspase-depended mitochondria activation. To further determine the function of Nip21 in CVB3-induced apoptosis, the Tet-On/Nip21 HeLa cell line was induced by doxycycline followed by CVB3 infection. We found that activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase occurred 2 hours earlier than in vector-transfected control cells, suggesting that Nip21 expression enhances CVB3-induced apoptosis. We also demonstrated a significant decrease in HeLa cell and H9c2 cell viability. Particularly, as illustrated by viral plaque assay, CVB3 replication was dramatically reduced in Tet-On HeLa cells, due at least in part to the earlier killing of the host cells by Nip21 overexpression.     

Long-term cardiac gene expression using a coxsackieviral vector

Efficient myocardial gene transfer in the intact adult heart is difficult using conventional transfer vectors. Since coxsackievirus B3 (CVB3) is cardiotropic, it may be possible to exploit its cardiotropic characteristics to design a vector for gene transfer to the intact heart. We generated a recombinant CVB3 cDNA by inserting a green fluorescent protein (GFP) gene immediately upstream from the VP0 capsid protein of CVB3. The infectious virus (rCVB3-GFP) was recovered from the supernatants of the transfected Cos-7 cells, and was grown in HeLa cells to titers of 10(11) pfu/ml. In the rCVB3-GFP infected HeLa cells and neonatal rat cardiac myocytes, GFP protein expression was documented by immunoblot and by fluorescent microscopy. GFP expression was maintained after five passages in HeLa cells. To test in vivo expression of GFP, we infected 8-week-old inbred female Balb/C mice with 10(6) pfu of rCVB3-GFP, intraperitoneally. GFP was present in up to 30% of cardiac myocytes over the 8 weeks post infection (p.i.) and it was co-localized with CVB3 infection. Surprisingly, in spite of detection of GFP up to at least 8 weeks after infection, there was no mortality in the mice. It is possible to express exogenous proteins in the intact heart after an intraperitoneal (i.p.) injection of recombinant coxsackievirus. The duration of expression persisted for at least 8 weeks with little immune response nor mortality. These results demonstrated that the cardiac tropism of CVB3 could be used to design vectors for efficient gene expression in the intact heart.     

哺乳类雷帕霉素靶蛋白信号通路对柯萨奇病毒B3诱导的心肌成纤维细胞Smad 3蛋白和Ⅰ型胶原表达调控的实验研究

目的 探讨哺乳类雷帕霉素靶蛋白(mTOR)信号通路对柯萨奇病毒B 3(CVB 3)诱导的心肌成纤维细胞Smad 3蛋白(Smad 3)和Ⅰ型胶原表达的调控作用.方法 CVB 3感染原代培养的SD鼠心肌成纤维细胞,用mTOR的抑制剂-雷帕霉素阻断mTOR信号通路,采用逆转录-聚合酶链反应(RT-PCR)和Western blot方法检测细胞Smad 3和Ⅰ型胶原表达.结果 (1)雷帕霉素干预心肌成纤维细胞48 h,RT-PCR检测mTOR/β-肌动蛋白(actin)光密度值分别为1 nmol/L组0.381±0.022、10 nmol/L组0.282±0.014、100 nmol/L组0.263±0.012,均低于对照组1.45±0.04,10 nmol/L组和100 nmol/L组低于1nmol/L组,差异均有统计学意义(P均＜0.05).10 nmol/L雷帕霉素干预心肌成纤维细胞0 h(对照组)、24 h、48 h、72 h,其mTOR/β-actin光密度值分别为0.341±0.022、0.203±0.021、0.163±0.022、0.144±0.013,光密度值随雷帕霉素干预时间的延长而降低(P＜0.05).(2)RT-PCR和Western blot测得对照组、CVB 3组、CVB 3+雷帕霉素组心肌成纤维细胞Smad 3/β-actin光密度值分别为0.63±0.06、1.18±0.03、0.77±0.08,Smad 3/β-actin灰度值分别为0.89±0.07、2.27±0.13、0.131±0.013.RT-PCR测得上述各组Ⅰ型胶原/β-actin光密度值分别为1.13±0.06、1.303±0.012、0.82±0.03.以上结果显示CVB 3组光密度值/灰度值高于对照组,CVB 3+雷帕霉素组低于CVB 3组,差异有统计学意义(P＜0.05).结论 雷帕霉素抑制CVB 3诱导的心肌成纤维细胞Smad 3和Ⅰ型胶原表达,提示mTOR信号通路可能通过调控Smad及Ⅰ型胶原表达,在CVB.3诱导的心肌纤维化过程中起重要作用.     

In vitro anti-coxsackievirus B(3) effect of ethyl acetate extract of Tian-hua-fen

AIM: To investigation the anti-coxsackievirus B(3) (CVB(3m)) effect of the ethyl acetate extract of Tian-hua-fen on HeLa cells infected with CVB(3m). METHODS: HeLa cells were infected with CVB(3m) and the cytopathic effects (CPE) were observed through light microscope and crystal violet staining on 96-well plate and A(600) was detected using spectrophotometer. The protective effect of the extract to HeLa cells and the mechanism of the effect were also evaluated through the change of CPE and value of A(600). RESULTS: The extract had some toxicity to HeLa cells at a higher concentration while had a marked inhibitory effect on cell pathological changes at a lower concentration. Consistent results were got through these two methods. We also investigated the mechanism of its anti-CVB(3m) effect and the results indicated that the extract represented an inhibitory effect through all the processes of CVB(3m) attachment, entry, biosynthesis and assemble in cells. CONCLUSION: The results demonstrate that the ethyl acetate extract of Tian-hua-fen has a significant protective effect on HeLa cells infected with CVB(3m) in a dose-dependent manner and this effect exists through the process of CVB(3m) attachment, entry, biosynthesis and assemble in cells, suggesting that the ethyl acetate extract of Tian-hua-fen can be developed as an anti-virus agent.     

17β-Estradiol Exposure Leads to Activation of ERK and p38 but Not JNK in Vascular Endothelial Cells

Estrogen has been shown to increase endothelial cell (EC) production of nitric oxide (NO) by the rapid activation of endothelial nitric oxide synthase (eNOS). To better understand the mechanisms by which estrogen acutely increases endothelial NO production, proteins of the mitogen activated protein kinase (MAPK) family, ERK, JNK, and p38 were examined to determine their potential role in this process. Bovine aortic endothelial cells (BAEC) were grown to confluence in phenol red-free DMEM F-12, supplemented with 10% fetal calf serum, on 35 cm² dishes. After confluence, cells were starved for 12–24 hours and then exposed to 10 nM 17-estradiol (E₂) for 1–60 minutes. The cells were then lysed and proteins resolved by SDS-PAGE followed by transfer to nitrocellulose membranes. Membranes were then probed with anti-phosphospecific antibodies for ERK, JNK, and p38. Antibodies specific for total ERK and JNK were used to assure even protein loading. The level of ERK phosphorylation rapidly increased in biphasic pattern after E2 exposure: 1 min, 4.3 fold; 5 min, 1 fold; and 10 min, 7.3 fold (max). However, the level of JNK phosphorylation did not increase throughout the time course. The level of p38 phosphorylation increased rapidly to 3.6 fold at 10 minutes. We demonstrate that E2 rapidly activates ERK in a biphasic manner and p38 in a monophasic manner, but has no effect on the activity of JNK. Our data suggest that rapid eNOS activation by estrogen in vascular endothelial cells is predominantly mediated through ERK and p38 pathway. Rapid activation of ERK and p38 resulting in NO production may substantially contribute to the atheroprotective effect of estrogen.     

Extracellular signal-regulated kinase 1- and 2-mediated gastric mucosal injury and repair in gastric ischemia–reperfusion of rats

Background The current study was undertaken to investigate the time course of gastric ischemia–reperfusion (GI-R)-induced gastric mucosal injury and repair and whether extracellular signal-regulated kinase 1/2 (ERK1/2) were involved in GI-R-induced gastric mucosal injury and repair. Methods Immunohistochemistry and Western blot analyses were used. Results Gastric mucosal injury induced by ischemia alone was mild. However, the injury worsened after reperfusion, reaching a maximum at 1 h, and was accompanied by increased apoptotic cells and decreased proliferative cells. Then, the gastric mucosal cells began to repair the injury by enhanced proliferation, which peaked at 24 h after reperfusion, and by 72 h the damaged gastric mucosa was mostly repaired. The ERK1/2 (nonactivated ERK1/2) protein expression level and distribution profile showed no significant changes during the entire reperfusion phase, but the p-ERK1/2 (activated ERK1/2) level changed dramatically. The p-ERK1/2 protein level was decreased at 0.5 h after reperfusion began, and then gradually increased, peaking after 3 h of reperfusion; these changes in p-ERK1/2 occurred simultaneously in the cytoplasm and nucleus. On the other hand, inhibition of the activation of ERK1/2, induced by PD98059, a specific ERK1/2 upstream inhibitor, aggravated the gastric mucosal injury, and apoptosis was increased and proliferation was reduced in the gastric mucosal cells after the same duration of reperfusion. Conclusions Serious gastric mucosal damage involving apoptotic cells occurred rapidly at an early stage of reperfusion and was closely related to the suppression of ERK1/2 activation. The activated ERK1/2 signaling transduction pathway played an important role. Activated ERK1/2 participated in the regulation of gastric mucosal injury and repair induced by GI-R, and might be mediated by the inhibition of apoptosis and the promotion of proliferation in gastric mucosal cells.     

The permissive effects of glucose on receptor-operated potentiation of insulin secretion from mouse islets: a role for ERK1/2 activation and cytoskeletal remodelling

Aims/hypothesis

Glucose plays two distinct roles in regulating insulin secretion from beta cells—an initiatory role, and a permissive role enabling receptor-operated secretagogues to potentiate glucose-induced insulin secretion. The molecular mechanisms underlying the permissive effects of glucose on receptor-operated insulin secretion remain uncertain. We have investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and consequent cytoskeletal remodelling in this process.

Methods

Insulin release was measured from groups of isolated mouse islets using static incubation experiments and subsequent radioimmunoassay of samples. ERK1/2 activation was measured by western blotting of islet protein samples for both phosphorylated and total ERK1/2. Rhodamine–phalloidin staining was used to measure filamentous actin in dispersed primary beta cells.

Results

Inhibition of ERK1/2 blocked potentiation of glucose-induced insulin release by the receptor-operated secretagogues kisspeptin, A568, exendin-4 and JWH015, although the agonists alone had minimal effects on ERK1/2 activation, suggesting a permissive rather than causal role for ERK1/2 activation in receptor-operated insulin release. Following pharmacological activation of ERK1/2 all agonists caused a significant increase in insulin release from islets incubated with sub-stimulatory levels of glucose. ERK1/2 inhibition significantly reduced the glucose-dependent decreases in filamentous actin observed in primary beta cells, while pharmacological dissociation of actin filaments enabled all receptor-operated secretagogues tested to significantly stimulate insulin release from islets at a sub-stimulatory glucose concentration.

Conclusions/interpretation

Glucose-induced ERK1/2 activation in beta cells mediates the permissive effects of stimulatory glucose concentrations on receptor-operated insulin secretagogues, at least in part through effects on actin depolymerisation and cytoskeletal remodelling.     

Role of Fas/FasL pathway in the activation of infiltrating cells in murine acute myocarditis caused by Coxsackievirus B3

OBJECTIVES: This study was designed to investigate the roles of Fas/FasL pathway in myocardial damage in murine acute myocarditis caused by Coxsackie virus B3 (CVB3). BACKGROUND: Cardiac myocyte apoptosis rarely occurs in murine acute myocarditis caused by CVB3. Fas/FasL belong to the tumor necrosis factor receptor/ligand superfamily of costimulatory molecules and are known to play a critical role in the induction of apoptosis, as well as in the cytotoxicty mediated by T-cells and natural killer cells. METHODS: We first analyzed the expression of Fas on cardiac myocytes in vivo and in vitro. Second, we examined the development of myocardial damage, in C3H/He mice treated with an anti-FasL monoclonal antibody (mAb), and in C3H/He-lpr/lpr mice and C3H/He-gld/gld mice infected with CVB3. Third, to investigate the effects of anti-FasL mAb treatment on the activation of the infiltrating cells, we examined the expression of interferon (IFN)-gamma and interleukin (IL)-2 as activation markers in the heart of mice by semiquantitative polymerase chain reaction. RESULTS: Fas was markedly induced on cardiac myocytes with acute myocarditis. Myocardial inflammation was decreased in mice treated with anti-Fas L mAb, C3H/He-lpr/lpr mice and C3H/He-gld/gld mice. Anti-FasL mAb-treatment also decreased the expression of IFN-gamma, IL-2, inducible nitric oxide synthase and CVB3 genomes in myocardial tissue. CONCLUSIONS: Our findings strongly suggested that the Fas/FasL pathway played a critical role in the development of massive myocardial necrosis through activation of infiltrating cells, and raise the possibility of immunotherapy by blocking the Fas/FasL pathway to prevent myocardial damage and improve the prognosis of patients with viral myocarditis.     

In Vitro Model Systems of Coxsackievirus B3-Induced Myocarditis: Comparison of Commonly Used Cell Lines and Characterization of CVB3-Infected iCell® Cardiomyocytes

Coxsackievirus B3 (CVB3) belongs to the enteroviruses, which are a well-known cause of acute and chronic myocarditis, primarily infecting cardiac myocytes. As primary human cardiomyocytes are difficult to obtain, viral myocarditis is quite frequently studied in vitro in different non-cardiac and cardiac-like cell lines. Recently, cardiomyocytes that have been differentiated from human-induced pluripotent stem cells have been described as a new model system to study CVB3 infection. Here, we compared iCell^® Cardiomyocytes with other cell lines that are commonly used to study CVB3 infection regarding their susceptibility and patterns of infection and the mode of cell death. iCell^® Cardiomyocytes, HeLa cells, HL-1 cells and H9c2 cells were infected with CVB3 (Nancy strain). The viral load, CVB3 RNA genome localization, VP1 expression (including the intracellular localization), cellular morphology and the expression of cell death markers were compared. The various cell lines clearly differed in their permissiveness to CVB3 infection, patterns of infection, viral load, and mode of cell death. When studying the mode of cell death of CVB3-infected iCell^® Cardiomyocytes in more detail, especially regarding the necroptosis key players RIPK1 and RIPK3, we found that RIPK1 is cleaved during CVB3 infection. iCell^® Cardiomyocytes represent well the natural host of CVB3 in the heart and are thus the most appropriate model system to study molecular mechanisms of CVB3-induced myocarditis in vitro. Doubts are raised about the suitability of commonly used cell lines such as HeLa cells, HL-1 cells and H9c2 cells to evaluate molecular pathways and processes occurring in vivo in enteroviral myocarditis.