P38/MAPK介导的蓖麻毒素诱导小鼠淋巴细胞凋亡信号传导通路的研究

蓖麻毒素属于Ⅱ型核糖体失活蛋白,由具有催化活性的A链和凝集活性的B链组成,二者之间经二硫键连接。本研究旨在探讨蓖麻毒素在诱导小鼠淋巴细胞凋亡过程中信号转导通路的激活反应。通过MTS法检测蓖麻毒素的细胞毒性后,用流式细胞术分析胞内活性氧(ROS)水平,进而通过westernblot研究信号分子的表达。结果表明:在蓖麻毒素诱导小鼠淋巴细胞凋亡的过程中,胞内活性氧水平显著增高(P<0.05),伴随信号转导分子——促分裂原活化蛋白激酶(MAPKs)中磷酸化c-jun氨基末端激酶(JNK1/2)和应激激活蛋白激酶(p38)的表达,另一信号分子胞外调节激酶(ERK1/2)无明显反应;加入信号分子的相应特异性抑制剂后,只有磷酸化p38的表达受到明显抑制。由此可得出:蓖麻毒素诱导淋巴细胞的凋亡极有可能通过激活p38/MAPKs通路来实现。     

表皮生长因子调控猪肠上皮细胞中钠依赖Ⅱb型磷转运蛋白表达的细胞信号通路研究

本研究旨在探讨表皮生长因子(EGF)调控猪小肠上皮细胞IPEC-J2中钠依赖Ⅱb型磷转运蛋白(NaPi-Ⅱb)表达的分子机制。试验分别用EGF受体酪氨酸激酶抑制剂(tyrphostin AG1478)、蛋白激酶A(PKA)抑制剂(H89)、蛋白激酶C(PKC)抑制剂(k4393)、p38抑制剂(SB203580)、细胞外信号调节激酶(ERK)抑制剂(PD98059)、c-Jun氨基末端激酶(JNK)抑制剂(anisomycin)与EGF共同处理IPEC-J2细胞,利用Western blot检测相关通路蛋白及目的蛋白(NaPi-Ⅱb)的表达水平。结果显示:相较于对照组,EGF处理后NaPi-Ⅱb表达水平显著降低(P0.05);相较于无抑制剂组,EGF受体、PKA、PKC、丝裂原活化蛋白激酶(MAPK)/p38、MAPK/ERK1/2、MAPK/JNK的特异性抑制剂处理IPEC-J2后,NaPi-Ⅱb表达水平显著提高(P0.05),其中添加MAPK/ERK1/2特异性抑制剂显著降低了MAPK/ERK1/2在Tyr204位点的磷酸化水平(P0.05),添加MAPK/JNK的特异性抑制剂显著降低了MAPK/JNK1/2/3在Thr183和Tyr185位点的磷酸化水平(P0.05),说明该2组抑制剂对该通路的抑制作用是通过降低上述位点的磷酸化水平实现的。本研究结果表明EGF受体、PKA、PKC、p38、ERK和JNK均介导了EGF调控IPEC-J2细胞中NaPi-Ⅱb的表达。     

c-Jun氨基末端激酶的激活在猪繁殖与呼吸综合征病毒诱导的细胞凋亡和病毒复制中的作用研究

宿主细胞凋亡在病毒感染发病过程中起着至关重要的作用。MAPK激酶,尤其是应激活化蛋白激酶c-Jun氨基末端激酶(SAPK/JNK)和p38往往参与病毒介导的细胞凋亡。研究证实,猪繁殖与呼吸综合征病毒(PRRSV)感染在体内和体外都会导致宿主细胞的凋亡。本研究旨在确定应激活化蛋白激酶JNK和p38在PRRSV感染诱导的细胞凋亡中是否发挥作用。对JNK和p38磷酸化的检测发现,在PRRSV感染应答中,JNK被激活,而p38没有被激活。应用特异性抑制剂研究这个激酶对细胞凋亡诱导和病毒复制的影响,研究结果发现,JNK抑制剂SP600125引起的JNK抑制能阻断PRRSV介导的细胞凋亡,但并不抑制病毒的复制。进一步研究结果表明,ROS的产生参与了JNK的活化,Bcl-2家族抗凋亡蛋白Mcl-1和Bcl-xl是JNK介导细胞凋亡的下游靶标。因此,JNK信号通路的激活是PRRSV介导的细胞凋亡所必需的,但并非病毒复制所必需。     

磷脂酰激醇3-激酶（PI 3-K）研究新进展

磷脂酰激醇3-激酶(PI3-K)是细胞内一种重要的激酶,通过活化Akt/pkb、MAPK、PKC等信号途径参与细胞内信号转导,引起细胞发生许多生化反应.PI3-K还具有丝/苏氨酸蛋白激酶活性,可以调节自身磷酸肌醇激酶活性;同时PI3-K的活化与细胞的生长、分化、游动性、胰岛素的活性和细胞的生存有很大的联系.     

寄生虫丝裂原活化蛋白激酶的研究进展

寄生虫丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)由丝/苏氨酸蛋白激酶组成,相关构象研究表明,MAPKs的结构与功能间存在密切的关系。论文详细阐述了寄生虫p38MAPK、c-Jun氨基末端激酶(JNK)、细胞外信号调节激酶(ERK)等MAPK亚族成员,并对寄生虫MAPK信号转导通路进行了简要介绍。同时,概述了寄生虫对宿主MAPK信号通路的影响,并对未来寄生虫丝裂原活化蛋白激酶的相关研究做了展望。     

磷脂酰肌醇3-激酶/丝苏氨酸蛋白激酶信号途径及其在肌肉生长发育中的调控机制

磷脂酰肌醇3-激酶/丝苏氨酸蛋白激酶(PI3K/Akt)是一类特异性催化磷脂酰肌醇(PI)的激酶,其信号途径是细胞内重要的信号转导通路之一,广泛参与动物机体的新陈代谢过程。本文就PI3K/Akt信号途径的结构、活化机制及其在细胞凋亡和肌肉生长发育中的调控机制进行综述。     

酿酒酵母菌诱导绵羊瘤胃上皮细胞SBD-1表达的信号通路初步研究

文章旨在探索核因子-κB(NF-κB)和丝裂原活化蛋白激酶(MAPKs)通路是否介导益生性酿酒酵母菌(Saccharomyces cerevisiae)诱导绵羊瘤胃上皮细胞(RECs)β-防御素-1(SBD-1)基因的转录。首先建立绵羊RECs培养体系作为体外试验模型,选用诱导SBD-1转录最高的菌液浓度和诱导培养时间进行信号通路初步研究,采用实时荧光定量逆转录PCR(RT-qPCR)对已建立的诱导SBD-1转录模型中的细胞膜受体——Toll样受体2(TLR2)、信号衔接蛋白——髓样分化因子(MyD88)以及NF-κB和MAPKs通路中的相关因子基因转录变化进行检测;然后选用NF-κB和MAPKs通路中的4种特异性抑制剂(即NF-κB通路特异性抑制剂PDTC、P38通路特异性抑制剂SB202190、ERK 1/2通路特异性抑制剂PD98059、JNK通路特异性抑制剂SP600125)通过单独或相互组合处理细胞后再进行诱导培养,同时采用RT-qPCR的方法检测用抑制剂处理绵羊RECs后SBD-1mRNA的转录水平。结果表明:酿酒酵母菌刺激RECs后,NF-κB和MAPKs通路中各因子NF-κB、P38、JNK、ERK1/2、细胞膜受体TLR2与信号衔接蛋白MyD88的mRNA水平与未刺激组相比均有所升高,且呈显著性差异(P0.01或P0.05);通过单独或组合添加抑制剂后再诱导,均发现特异性抑制剂PDTC、SB202190、SP600125、PD98059可极显著抑制酿酒酵母菌对RECs SBD-1的上调作用(P0.01),且P38通路特异性抑制剂SB202190的抑制效果最明显。结果提示,酿酒酵母菌诱导绵羊RECs SBD-1的转录可能与TLR2-MyD88-NF-κB/MAPKs通路有关,但以TLR2-MyD88-MAPKs中的TLR2-MyD88-P38通路为主要的信号通路。     

丝状支原体丝状亚种的脂质相关蛋白通过MAPK信号通路诱导EBL细胞分泌IL-1β的研究

丝裂原活化蛋白激酶(MAPK)信号通路是经典的细胞信号通路之一,主要包括JNK、P38、ERK 3条信号途径。为明确丝状支原体丝状亚种(Mmm)的脂质相关蛋白(LAMPs)是通过MAPK信号通路诱导胎牛肺细胞(EBL)IL-1β的分泌,本研究通过荧光定量PCR检测IL-1β的m RNA表达水平,结果显示LAMPs可以诱导EBL细胞分泌IL-1β,并确定Mmm的LAMPs刺激EBL细胞的最佳时间为6 h,最佳剂量为1μg/m L;将JNK、P38及ERK抑制剂分别加入培养的EBL细胞后,IL-1β的m RNA表达水平均受到显著抑制(p0.05);将p CMV-HA-TLR2转染EBL细胞使其过表达和抗体封闭TLR2两种方式处理,并采用JNK、P38及ERK抑制剂分别处理EBL细胞,经检测过表达TLR2实验组的IL-1βm RNA表达水平上调,然而抗体封闭TLR2实验组的IL-1βm RNA表达水平下调,表明TLR2通过MAPK信号通路对IL-1β的分泌进行调控;采用牛IL-1β的ELISA试剂盒对细胞上清液中IL-1β浓度的检测结果显示,其与IL-1β的m RNA表达水平变化趋势一致。上述结果表明Mmm的LAMPs可以诱导EBL细胞,激活TLR2依赖的MAPK信号通路,从而引起IL-1β的分泌。     

MAPK与寄生虫的关系

丝裂酶原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是一类丝氨酸/苏氨酸蛋白激酶,普遍存在于多种生物,包括酵母和哺乳动物细胞,是细胞外信号传递至细胞核内的重要信号通路。目前,在真核生物细胞中,已确定的至少有4个MAPK亚家族,即细胞外调节蛋白激酶ERK(extracellular signal regulated kinase,ERK)、p38 MAPK、c-Jun氨基末端激酶(c-Jun Nterminal kinases,JNK)及ERK5。     

植物乳杆菌诱导绵羊瘤胃上皮细胞SBD-1表达的信号通路途径初探

本试验旨在探索乳酸杆菌诱导绵羊瘤胃上皮细胞SBD-1表达的可能途径。采用实时荧光定量PCR(RTqPCR)的方法,对已建立的诱导SBD-1表达模型中Toll样受体2(Toll like receptor 2,TLR2)及其相关因子的基因表达变化进行检测;然后选用3种信号通路抑制剂,即NF-κB信号通路抑制剂PDTC、ERK 1/2信号通路抑制剂PD98059和JNK信号通路抑制剂SP600125,将细胞分为8组:细胞组:不作处理;阳性对照组:只添加植物乳杆菌P-8(L.plantarum P-8)诱导;PDTC组:只添加PDTC预处理细胞(PDTC);PDTC+L.plantarum P-8组:PDTC+L.plantarum P-8诱导;PD98059组:PD98059;SP600125组:SP600125;PD98059+L.plantarum P-8组:PD98059+L.plantarum P-8;SP600125+L.plantarum P-8组:SP600125+L.plantarum P-8。采用RT-qPCR的方法检测各组SBD-1mRNA表达水平。结果表明,绵羊瘤胃上皮细胞被L.plantarum P-8诱导后,TLR2及其转接蛋白MyD88、NF-κB和ERK1/2、JNK各基因的mRNA水平都较空白组细胞内的表达极显著增加(P0.01);添加抑制剂后再诱导,发现抑制剂PD98059和SP600125均能极显著(P0.01)抑制细胞内SBD-1 mRNA的表达,而PDTC仅能显著抑制(P0.05)SBD-1的表达。结果表明,L.plantarum P-8可促进绵羊瘤胃上皮细胞TLR2、MyD88、NF-κB、JNK和ERK1/2基因的mRNA表达;添加NF-κB信号通路抑制剂PDTC、ERK 1/2信号通路抑制剂PD98059和JNK信号通路抑制剂SP600125,可抑制L.plantarum P-8对SBD-1mRNA的诱导作用。综上表明,L.plantarum P-8有可能通过激活绵羊瘤胃上皮细胞内NF-κB、JNK、ERK1/2等信号通路促进SBD-1的表达。     

MAPK信号通路介导的自噬调控破骨细胞分化的研究进展

破骨细胞具有骨吸收活性,与骨组织稳态密切相关。丝裂原活化蛋白激酶(MAPK)通路是细胞介导胞内外刺激传导的信号通路,参与细胞的增殖、分化、自噬等多种生理过程。MAPK介导的自噬在调控破骨细胞分化中具有重要作用。探究MAPK的三条经典通路(ERK1/2、JNK及p38 MAPK信号通路)介导的自噬与破骨细胞分化之间的关系,对于寻找与破骨细胞相关的骨代谢疾病的新疗法具有重要意义。     

MAPK信号通路对脂肪细胞分化的调控

促分裂原活化的蛋白激酶(MAPK)通路主要包括胞外信号调控激酶(ERK)、p38MAPK和氨基末端蛋白激酶(JNK)三条途径,参与调节细胞增殖、分化、凋亡及细胞间的功能同步等过程,是细胞信号转导方面最为活跃的研究领域之一。研究显示MAPK也参与脂肪细胞的分化调节并发挥重要作用。ERK和p38MAPK信号通路对脂肪细胞分化的调节在不同的实验模型中表现为正调控和负调控两种不同形式;而另一成员JNK能使胰岛素受体底物1的丝氨酸发生磷酸化,进而干扰胰岛素信号,从而抑制骨髓间充质干细胞(BMSCs)的成脂分化,即对脂肪细胞分化发挥负调控作用。论文就MAPK信号通路在脂肪细胞分化中的功能进行综述,为脂类代谢性疾病的诊断和治疗提供参考。     

Involvement of the ERK1/2 MAPK pathway in insulin-induced S6K1 activation in avian cells

In mammals, insulin regulates S6K1, a key enzyme involved in the control of protein synthesis, via the well-documented phosphoinositide-3'kinase (PI3K) pathway. Conversely, S6K1 is activated by insulin in avian muscle despite the relative insulin insensitivity of the PI3K pathway in this tissue. Mitogen-activated protein kinase (MAPK) cascade is another insulin sensitive pathway. The aim of this study was to explore the potential involvement of the ERK1/2 MAPK pathway in the control of p70 S6 kinase (S6K1) in avian species. Firstly, we characterized ERK1/2 MAPK in various chicken tissues. ERK2 was the only isoform detected in avian species whatever the tissue studied. We also showed that ERK2 is activated in vivo by insulin in chicken muscle. The regulation and the role of ERK2 in insulin signaling were next investigated in chicken hepatoma cells (LMH) and primary myoblasts. Insulin stimulation led to ERK2 and S6K1 phosphorylation, and concomitantly increased kinase activity. U0126, an inhibitor of the ERK MAPK pathway, completely abolished insulin-induced S6K1 phosphorylation and activity in chicken myoblasts, whereas its effect was only partial in LMH cells. In conclusion, these results show that ERK1/2 MAPK is involved in the control of S6K1 by insulin in chicken cells, particularly myoblasts.     

牛多形核白细胞(PMN)中P38 MAPK,PKC和PI3-K介导的NADPH氧化酶激活和吞噬作用(英文)

用血清调理的酵母聚糖（ｓｅｒｕｍ－ｏｐｓｏｎｉｚｅｄ　ｚｙｍｏｓａｎ　,ｓＯＺ）刺激多性核白细胞（ＰＭＮ）引起的Ｏ２－产生受到ｐ３８ＭＡＰＫ抑制物（ＳＢ２０３５８０）,ＰＩ３－Ｋ抑制物（ｗｏｒｔｍａｎｎｉｎ）和ＰＫＣ抑制物（ＧＦ１０９２０３Ｘ）的明显抑制,这些抑制物也明显引起ｓＯＺ诱导的一种ＮＡＤＰＨ氧化酶的胞浆成分 ｐ４７ｐｈｏｘ的磷酸化。可是流式细胞分析表明,ＳＢ２０３５８０和 ｗｏｒｔｍａｎｎｉｎ使吞噬作用减弱,而 ＧＦＩＯ９２０３Ｘ使吞噬作用增强。这些结果表明,虽然ＰＩ３－Ｋ和ｐ３８ ＭＡＰＫ都参与ＮＡＤＰＨ氧化酶激活和吞噬作用的信号传导,但ＮＡＤＰＨ氧化酶激活的信号传导途径与吞噬作用的信号传导途径不同。     

Involvement of TNF-α and MAPK pathway in the intramammary MMP-9 release via degranulation of cow neutrophils during acute mammary gland involution

Neutrophil-derived MMP-9 activity is regulated more promptly and efficiently at the level of degranulation than at other levels of regulation. In human neutrophils, degranulation is one of the earliest responses to TNF-α stimulation, which involves protein kinase C and mitogen-activating protein kinase (MAPK) pathways. The level of MMP-9 in mammary secretion of cows increases drastically following milk-stasis, which is partially explained by increases of both neutrophil infiltration and neutrophil degranulation per se. Since MMP-9 represents one of the major remodeling capacities in the mammary gland of cows during early dry period, the current study attempted to explore the involved intracellular mechanisms in the up-regulated MMP-9 secretion. We repeatedly measured on the somatic cells of mammary secretion along the early dry period of cows the expression of TNF-α protein and the phosphorylation of p38 MAPK, ERK, and JNK. Also, cultures of bovine peripheral neutrophils were conducted to examine the mode of short-term MMP-9 secretion in response to TNF-α stimulation and the blocking effects of TNF-α antibody and inhibitors of MAPK pathways. Ex vivo measurements show that conventional cow milk has fully transformed into a neutrophil-abundant, lactoferrin-rich, and high-MMP-9 mammary secretion by d 7 in milk-stasis. No significant (P>0.05) change, however, was found in the expression of TNF-α or the phosphorylation extent of MAPK pathway intermediates on the somatic cells of mammary secretion during the first 3 weeks in milk-stasis. In vitro studies indicate linear increase of short-term MMP-9 release in response to TNF-α stimulation in dosages between 0.1 and 10 ng/ml. In the presence of preparations of d 7-dry secretion of cows, the short-term release of MMP-9 from bovine peripheral neutrophils was significantly (P<0.05) blocked by inhibitor of p38 MAPK but was significantly (P<0.05) promoted by ERK inhibitor while TNF-α antibody or JNK inhibitor exerted no effect. In conclusion, the current ex vivo measurements suggest no apparent association of TNF-α and MAPK pathway with long term intramammary accumulation of MMP-9 during the early dry period of cows, whereas cultures of bovine peripheral neutrophils under a simulated acute involution intramammary environment of cows suggest a role played by TNF-α and MAPK pathways in the short-term MMP-9 release via degranulation.     

Tamoxifen induces vasorelaxation via inhibition of mitogen-activated protein kinase in rat aortic smooth muscle

The contribution of the mitogen-activated protein kinase (MAPK) pathway to the relaxation induced by tamoxifen, a synthetic non-steroidal anti-estrogen, was examined in rat vascular smooth muscle. Tamoxifen (0.1-300 microM) inhibited the contraction induced by endothelin-1 (ET-1, 3 nM) in aortic smooth muscle in a concentration-dependent manner. The inhibitory effect of tamoxifen was not attenuated by 10 microM ICI 182,780, a selective antagonist of estrogen receptors. In the Ca(2+) channel inhibitor verapamil (1 microM)-pretreated strips, tamoxifen also inhibited the contraction induced by ET-1. Both PD098059 and SB203580, inhibitors of MAPK/extracellular signal-regulated kinase (ERK) kinase and p38 MAPK, respectively, inhibited ET-1-induced contraction in aortic smooth muscle. In Western blot analysis with anti-phosphorylated MAPK antibodies, ET-1 (3 nM) enhanced activities of both ERK1/2 and p38 MAPK in aortic muscle strips, which were not attenuated by the treatment with 4 mM EGTA. Tamoxifen (100 microM) inhibited the activities of ERK1/2 and p38 MAPK induced by ET-1 without significant changes in the expression of these kinases. These results suggest that tamoxifen induces relaxation of rat vascular smooth muscle, and that this is, at least in part, mediated by the inhibition of the Ca(2+)-independent MAPK pathway.     

Apoptosis of Granulosa Cells: A Review on the Role of MAPK-signalling modules

Recent studies suggest that ovarian follicular atresia is associated with DNA fragmentation and degeneration of granulosa cells, the hallmark of programmed cell death or apoptosis. Apoptosis of granulosa cells play a major role in follicular atresia. These studies have also demonstrated the involvement of tumour suppressors, apoptotic proteins and survival factors. These factors contribute to the developmental decision as to whether the ovarian follicles mature or undergo atresia. However, the precise temporal and molecular events involved in the apoptotic pathways in this process need to be elucidated. The present report summarizes the role of Jun N‐terminal kinase (JNK), p38 mitogen activated protein kinase (p38 MAPK), and extracellular‐signal regulated kinase (ERK)‐signalling module in the regulation of pro‐ and anti‐apoptotic factors of the granulosa cells in regulating follicular atresia. The findings presented here suggest that the loss of tropic hormone support is translated into the attenuation of Raf‐1‐MAPK/ERK kinase (MEK)‐ERK‐signalling pathway of the granulosa cells and this results in the decreased phosphorylation of the pro‐apoptotic BAD.