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1.
目的探讨人参皂甙Rbl(Gs—Rbl)改善阿霉素所致心力衰竭(HF)效应是否与调整蛋白激酶R样内质网激酶(PERK)通路有关。方法阿霉素(Adr)诱导的HF大鼠随机分为HF组(n=15)和Gs.Rbl组(70rng/kg/d,n=17),另随机选取同龄大鼠作为对照组(n=10)。干预结束并进行心脏超声检查后,TUNNEL检测心肌细胞凋亡率(AR),Western blot和Rt-PCR检测葡萄糖调节蛋白78(GRP78)、PERK、p-PERK、真核细胞起始因子2-(elF2a)、p-elF2a、C/EBP同源蛋白(CHOP)和cleavedcaspase-12。结果1.Adr干预成功构建HF模型,Gs—Rbl显著提高左室射血分数(LVEF)和降低心肌细胞AR(P〈O.01);2.HF组GRP78mRNA和蛋白均显著高于对照组,Gs—Rbl显著低于HF组和对照组二组的表达(P〈0.01);3.Gs—Rbl显著降低HF大鼠PERK和p-PERK表达(P〈0.01);4.HF导致elF2amRNA和蛋白、p-elF2a显著升高,Gs—Rbl显著下调三者的表达(P〈O.01);5.HF组CHOPmRNA和蛋白显著高于对照组,Gs—Rbl组显著抑制其表达(P〈0.01);6.Gs—Rbl显著抑制阿霉素所致的caspase-121TIRNA和cleaved caspase-12蛋白表达(P〈0.01)。结论Gs—Rbl通过调节PERK内质网通路介导其改善HF效应。  相似文献   

2.
目的探讨在非小细胞肺癌(non small cell lung cancer,NSCLC)中的Livin、JNKI蛋白的表达及相关性。方法采用免疫组织化学(二步法)检测187例NSCLC肺癌组织Livin、JNK1[包括总JN1蛋白(t-JNK1),磷酸化JNK1(p-JNK1)]蛋白的表达情况,采用Spearman等级相关分析探讨其相关性。结果Livin主要在胞浆中定位表达,在肺癌中的表达阳性率明显高于肺良性病变对照组;在鳞癌中表达阳性率明显高于腺癌和其他癌类(P〈0.05)。其在肺癌表达的积分明显高于良性对照组(P〈0.05),其在鳞癌中表达的积分明显高于腺癌及其他癌类组织。t-JNK1、p-JNK1在肺癌细胞上及在假复层纤毛柱状上皮均可见表达,主要在胞浆中定位表达,t—JNKl未见核内定位表达,而p-JNKl偶见核内定位表达。t—JNK1在肺癌组表达阳性率明显低于对照组(P〈0.05),t-JNKl在肺癌中表达的积分低于对照组;而p-JNK1在肺癌组表达阳性率明显高于对照组(P〈0.05)。p-JNK1在肺癌中表达的积分高于对照组,但两者无统计学意义(P〉0.05)。Livin与t-JNK1呈现显著的负相关,Livin与p-JNK1呈现显著的正相关。结论Livin在肺癌中高表达,其在鳞癌中表达高于腺癌和其他类型癌。t-JNK1在肺癌中低表达,而p-JNK1在肺癌中高表达。Livin与p-JNK1表达正相关,推测Livin可促JNK1蛋白磷酸化。  相似文献   

3.
目的研究内质网跨膜蛋白肌醇酶1α(inositol-requiring enzyme 1α,IRE1α)对其下游转录因子XBP1启动子转录活性的影响。方法在成功构建人IREla基因全长重组质粒的基础上,设计合成并构建靶向IRE1α基因的siRNA干扰载体(pS1;pS2);采用巢式PCR扩增XBP1启动子,插入到pGL3.Basic载体,构建携带XBP1启动子的报告基因重组质粒pGL3-XBP1。分别将siIRE1α干扰质粒与pGL3-XBPl报告基因重组质粒共转染人SW1353细胞和Saos-2细胞中,48h后采用双荧光报告系统检测IRE1α对XBP1启动子转录活性的调控作用。结果酶切及测序结果证实siRNA1干扰质粒和XBPl启动子真核表达载体均构建成功,双荧光报告系统检测显示SW1353细胞和Saos-2细胞中,直接载体pcDNA3.15(-)IRE1α与pGL3-XBP1共转实验组的荧光素酶活性明显高于其他实验组(P〈0.5),并随着IRE1α转染剂量的增加逐渐达到饱和;而silRE1α与pGL3-XBP1共转实验组的荧光素酶活性明显降低。免疫印迹结果显示,过表达IRE1α明显上调剪接型XBPlS蛋白的表达。结论人恶性骨肿瘤细胞中,过表达IRE1α明显上调XBP1启动子的转录与表达,并能有效促进XBPIU剪切生成XBPIU;通过siRNA方法抑制IRE1α后,XBP1启动子的转录与表达受到抑制。本实验为进一步研究骨肿瘤细胞中IRE1α调控XBP1启动子转录与剪接的分子机制奠定基础,为临床骨肿瘤的诊断与治疗提供一定的实验依据。  相似文献   

4.
缺血后处理对肺缺血/再灌注损伤的保护作用及其机制   总被引:1,自引:0,他引:1  
目的:探讨缺血后处理(聃)是否通过抑制P38丝裂原活化蛋白激酶(P38MAPK)活化来减轻再灌注损伤肺细胞的凋亡。方法:雄性SD大鼠40只,随机分成5组(n=8),即对照组(C组)、肺缺血/再灌注组(I/R组)、肺缺血/再灌注+缺血后处理组(IPO组)、缺血后处理+溶剂对照组(D组)、缺血后处理+SB203580组(SB组)。各组分别于再灌注2h留取左肺组织,检测肺组织湿/干重比(W/D)和总肺含水量(TLW);光镜观察肺组织形态学结构改变并进行肺组织损伤定量评估(IQA);原住末端标记法(TUNEL)检测肺细胞凋亡情况并计算凋亡指数(AI);RT-PCR和免疫组化法测定Bax、Bcl-2基因和蛋白的表达。结果:与C组相比,I/R组W/D、TLW、IQA和AI均显著升高(P〈0.05,P〈0.01),肺组织结构发生明显损伤;Bcl-2、Bcl-2/Bax基因及蛋白表达明显降低,Bax基因及蛋白表达明显升高(P〈0.05,P〈0.01);IPO组、D组、SB组与I/R组相比,w/D、TLW、IQA和AI均显著降低(P〈0.05,P〈0.01),肺组织结构损伤情况有所改善;Bcl-2、Bcl-2/Bax基因及蛋白表达明显升高,Bax基因及蛋白表达明显降低(P〈0.05,P〈0.01);D组与IPO组比较各项指标均无明显差异(均P〉0.05);SB组与IPO组相比,肺组织W/D、TLW、IQA和AI均显著降低(P〈0.05,P〈0.01),肺组织结构未见明显损伤;Bcl-2、Bcl-2/Bax基因及蛋白表达明显升高,Bax基因及蛋白表达明显降低(P〈0.05,P〈0.01)。结论:I/R通过激活P38MAPK导致大鼠肺泡结构严重破坏,肺内细胞大量凋亡;IPO可能是通过抑制P38MAPK通路的激活而减轻L/R损伤。  相似文献   

5.
目的:研究细胞自噬对酒精诱导的人肝细胞系(CL-1)的保护作用。方法:培养正常肝细胞系CL-1细胞,80mmol/L酒精常规处理24小时,采用CCK-8法观察酒精对细胞活力的影响;流式细胞技术观察酒精对细胞凋亡的影响;免疫蛋白印迹及转染GFP-LC3法检测细胞自噬水平;选用rapamycin和3-MA调节细胞自噬,观察酒精处理后细胞活力及凋亡的变化。结果:酒精处理体外培养的CL-1细胞,实验组较对照组细胞活力下降(P〈0.05);实验组细胞46.2%发生凋亡,显著高于对照组8.4%;LC3II及Beclinl水平显著高于对照组;GFP-LC3荧光数显著高于对照组(P〈0.05);调节细胞自噬水平,rapamycin组细胞活性增加(P〈0.01),31.1%(46.2%)细胞发生凋亡;3-MA组细胞活性降低(P〈0.05),54.1%(46.2%)细胞发生凋亡。结论:酒精处理降低CL-1细胞活性,促进凋亡,提高自噬水平;提高或降低细胞自噬水平,细胞凋亡及活力随之降低和增加;细胞自噬能够对抗酒精诱导的肝细胞凋亡。  相似文献   

6.
双歧杆菌DNA对巨噬细胞MAPK的影响   总被引:5,自引:0,他引:5  
目的 探索青春型双歧杆菌的DNA对巨噬细胞丝裂素活化的蛋白激酶(MAPK)活性的影响。方法 以激光共聚焦显微镜定量测定小鼠腹腔巨噬细胞MAPK家系中ERK1/2、JNK和p38的含量。结果 双歧杆菌DNA注射组小鼠腹腔巨噬细胞ERK1/2的平均荧光强度明显高于对照组(P〈0.01),而JNK和p38的平均荧光强度在2组间则差异无显著性(P〉0.05)。结论 青春型双歧杆菌的DNA能提高巨噬细胞ERK1/2的活性,这可能是其激活巨噬细胞的途径之一。  相似文献   

7.
目的探讨电针防治帕金森病的作用机制。方法采用1-甲基-4-苯基1,2,3,6四氢吡啶腹腔注射建立帕金森病小鼠模型,利用组织化学技术以及免疫组织化学技术观察电针对帕金森病小鼠脑黑质铁染色细胞和铁蛋白表达的影响。结果模型组1周、2周、4周小鼠黑质铁染色阳性细胞的数量较正常组明显增多,染色强度也明显增强(P〈0.001;P〈0.01;P〈0.001),电针在3个存活期内均可明显减少帕金森病小鼠黑质铁阳性细胞的数量和染色强度(P〈0.001;P〈0.05;P〈0.001);模型组1周、2周黑质部位铁蛋白的表达与正常组相比都有不同程度下降(P〈0.01;P〈0.001),而电针组2周帕金森病小鼠黑质铁蛋白的表达与模型组相比显著增加(P〈0.01)。结论电针可以通过降低脑内铁的含量,同时增加铁蛋白的表达,从而提高帕金森病模型小鼠脑抗氧化能力,达到保护黑质多巴胺能神经元的目的。  相似文献   

8.
庞晓斌  谢欣梅  李晓婷  赵艳 《生物磁学》2013,(34):6638-6641
目的:研究脉络宁注射液对SH—SY5Y细胞氧糖剥夺/再复氧糖(OGI)/R)损伤的保护作用,并探讨其可能的作用机制。方法:体外培养SH-SY5Y细胞,将细胞随机分为正常组、氧糖剥夺模型组和脉络宁组(1.0mL·L^-1),建立体外OGD/R细胞模型。倒置显微镜观察细胞形态;MTT法测定细胞存活率;测定乳酸脱氢酶(LDH)漏出量;Western Blot检测凋亡相关蛋白Bcl-2、Bax蛋白表达的变化。结果:与模型组相比,脉络宁能减轻OGD/R引起的SH-SY5Y细胞的损伤,明显提高细胞存活率(P〈0.05),减少LDH的释放量(P〈0.05),有效抑制Bax蛋白的表达(P〈0.05),上调Bcl-2的表达(P〈0.05)。结论:脉络宁注射液对OGD/R引起的SH-sY5Y细胞损伤有保护作用,其机制可能与影响凋亡相关基因Bcl-2、Bax的表达有关。  相似文献   

9.
刘浩  邢益平  贾一琼  李军  王世霞  卢山  黄祖瑚 《生物磁学》2011,(13):2443-2446,2431
目的:研究N-糖基化移位对乙型肝炎病毒表面抗原中蛋白核酸疫苗体外蛋白表达及小鼠体内体液免疫及细胞免疫应答的影响。方法:通过基因工程中定点突变技术,将乙型肝炎病毒表面抗原中蛋白(MHBs)中第4位氨基酸上连接的糖链去除,或将糖链依次移位至第5、6或7位氨基酸,来构建N-糖基化去除及移位的核酸疫苗,分别命名为Adr—dN4、Adr-N4—5、Adr—N4.6、Adr—N4.7。用上述核酸疫苗与野生型MHBs核酸疫苗(pSW3891/MHBs/Adr,简称Adr)及空载体质粒pSW3891分别用脂质体瞬时转染293T细胞,应用蛋白印迹法检测MHBs的表达。采用肌肉注射法,以各组疫苗分别对BALB/c小鼠于第0、2、4和6周进行免疫.用ELISA法检测小鼠血清中抗.HBs抗体、ELISPOT法检测小鼠表面抗原多肽特异性分泌IFN-T的脾细胞数量。结果:蛋白印迹法结果显示Adr、Adr-dN4、Adr—N4.5、Adr—N4—6、Adr-N4—7体外转染293T细胞后,均可以在293T细胞内表达,且Adr、Adr-N4—5、Adr-N4—7可将表达产物分泌到细胞外。ELISA及EISPOT结果表明:Adr免疫组小鼠抗-HBs终点滴度及表面抗原特异性分泌IFN-γ的脾细胞数量,均略高于其他免疫组小鼠,但与Adr—N4—5、Adr.N4—7相比无统计学差异(P〉0.05),与Adr-dN4和Adr—N4—6组相比有显著的统计学差异(P〈0.05)。结论:在第5或7位氨基酸附加N-连接糖链,能修补或替代Asn4连接糖链引导MHBs分泌的功能。HBs表达蛋白分泌到细胞外对诱导机体产生特异性细胞和体液免疫是至关重要的。  相似文献   

10.
目的:探讨缺血后处理对再灌注损伤肺细胞凋亡的影响。方法:健康雄性sD大鼠24只,随机分为对照组(C组)、缺血/再灌注组(I/n组)和缺血后处理组(IPostC组)(n=8)。对比观察各组血清中丙二醛(MDA)、超氧化物歧化酶(SOD)、髓过氧化物酶(MPO)活力及含量变化,原位缺口末端标记法(TUNEL)检测肺组织细胞凋亡情况,免疫组化及RT-PCR法检测肺组织中Bax、Bcl一2蛋白和基因的表达。结果:I/R组与c组相比,MDA含量、MPO活力明显升高,SOD活力明显下降(均P〈0.01),肺组织原位细胞凋亡检测示I/R组凋亡指数(AI)(39.03±3.46)显著高于C组(2.88±0.34),Bcl-2/Bax比值在蛋白和基因水平明显降低(均P〈0.01);IPostC组与I/R组相比MDA含量显著降低(P〈0.05),MPO活力显著降低(P〈0.01),SOD活性升高(P〈0.01),AI为8.03±0.88显著低于L/R组,并能明显升高Bcl-2/Bax比值(均P〈0.01)。结论:缺血后处理通过减轻脂质过氧化反应及中性粒细胞聚集,降低Bax/Bel.2比值,使肺组织细胞凋亡减少,从而有效地减轻肺缺血/再灌注损伤。  相似文献   

11.
目的观察GPR30受体激动剂G1对高糖诱导的EA.hy926内皮细胞内质网应激(endoplasmic reticulum stress,ERS)的影响。方法选用EA.hy926内皮细胞为研究对象,分为3组:正常对照组(Con,17.51mmol/L葡萄糖)、高糖组(HG,33.3mmol/L葡萄糖)、高糖+G1组(HG+G1,HG+1umol/L G1),利用流式细胞术检测3组细胞凋亡率,Western blot法检测ERS相关分子Bip、IRE1、PERK及凋亡分子Bax、Bcl-2的表达变化,RT-PCR法检测Bip和CHOP的mRNA表达变化。结果 HG组与Con组比较,细胞凋亡率明显升高(P0.01),Bip、IRE1、PERK及凋亡分子Bax表达上调(P0.01,P0.05或P0.001),Bcl-2的表达下调(P0.01),Bip mRNA、CHOP mRNA表达上调(P0.001及P0.01);HG+G1组与HG组比较,细胞凋亡率明显降低(P0.05),Bip、IRE1、PERK及凋亡分子Bax表达下调(P0.05或P0.01),Bcl-2的表达上调(P0.05),Bip mRNA、CHOP mRNA表达下调(P0.001及P0.01)。结论 GPR30受体激动剂G1可抑制EA.hy926内皮细胞内质网应激。  相似文献   

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目的 构建靶向人XBP1S的siRNA真核表达载体(pSUPER-XBP1S)并观察其对人HeLa细胞和HepG2细胞增殖能力的影响.方法 设计并合成针对XBP1S基因的siRNA,退火成互补双链后克隆至真核表达载体pSUPER构建重组质粒,并将其转染入HeLa细胞和HepG2细胞中.采用RT-PCR检测转染前后XBP1S在HeLa细胞和HepG2细胞中的转录,Western印迹检测转染前后XBP1S蛋白的表达;MTT法、细胞计数检测重组质粒对HeLa细胞和HepG2细胞增殖能力的影响.结果 重组质粒能有效地抑制HeLa细胞和HepG2细胞中XBP1S基因的转录和表达;转染HeLa细胞和HepG2细胞后,细胞增殖抑制率及细胞增殖数与对照组比较,差异有统计学意义(P〈0.05).结论 成功构建了靶向人XBP1S的siRNA表达载体pSUPER-XBP1S,并且有效的抑制了HeLa细胞和HepG2细胞中XBP1S的转录和表达,有效抑制了细胞的增殖能力.  相似文献   

14.
Advanced oxidation protein products (AOPPs) are formed during chronic oxidative stress as a result of reactions between plasma proteins and chlorinated oxidants. Their levels are elevated during various cardiovascular diseases. Because elevated AOPPs serve as independent risk factors for ischemic heart disease, and cardiomyocyte death is a hallmark of ischemic heart disease, we hypothesized that AOPPs will induce cardiomyocyte death. AOPP-modified mouse serum albumin (AOPP-MSA) induced significant death of neonatal mouse cardiomyocytes that was attenuated by knockdown of the receptor for advanced glycation end products, but not CD36. Notably, TRAF3-interacting protein 2 (TRAF3IP2; also known as CIKS or Act1) knockdown blunted AOPP-induced apoptosis. AOPP-MSA stimulated Nox2/Rac1-dependent superoxide generation, TRAF3IP2 expression, and TRAF3IP2-dependent JNK activation. The superoxide anion generating xanthine/xanthine oxidase system and hydrogen peroxide both induced TRAF3IP2 expression. Further, AOPP-MSA induced mitochondrial Bax translocation and release of cytochrome c into cytoplasm. Moreover, AOPP-MSA suppressed antiapoptotic Bcl-2 and Bcl-xL expression. These effects were reversed by TRAF3IP2 knockdown or forced expression of mutant JNK. Similar to its effects in neonatal cardiomyocytes, AOPP-MSA induced adult cardiomyocyte death in part via TRAF3IP2. These results demonstrate for the first time that AOPPs induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK activation in vitro and suggest that AOPPs may contribute to myocardial injury in vivo. Thus TRAF3IP2 may represent a potential therapeutic target in ischemic heart disease.  相似文献   

15.
TRAF2 and ASK1 play essential roles in tumor necrosis factor alpha (TNF-alpha)-induced mitogen-activated protein kinase signaling. Stimulation through TNF receptor 2 (TNFR2) leads to TRAF2 ubiquitination and subsequent proteasomal degradation. Here we show that TNFR2 signaling also leads to selective ASK1 ubiquitination and degradation in proteasomes. c-IAP1 was identified as the ubiquitin protein ligase for ASK1 ubiquitination, and studies with primary B cells from c-IAP1 knock-out animals revealed that c-IAP1 is required for TNFR2-induced TRAF2 and ASK1 degradation. Moreover, in the absence of c-IAP1 TNFR2-mediated p38 and JNK activation was prolonged. Thus, the ubiquitin protein ligase activity of c-IAP1 is responsible for regulating the duration of TNF signaling in primary cells expressing TNFR2.  相似文献   

16.
Previously, we have shown that ASK1-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP (Ras-GTPase-activating protein) protein family, opens its conformation in response to tumor necrosis factor (TNF), allowing it to form a complex with TRAF2-ASK1 that leads to activation of ASK1-JNK/p38 signaling in endothelial cells (EC). In the present study, we show that a TNF-inducible 14-3-3-binding site on AIP1 is critical for the opening of its conformation and for the AIP1-mediated TNF signaling. Ser-604, located in the C-terminal domain of AIP1, was identified as a 14-3-3-binding site. TNF treatment of EC induces phosphorylation of AIP1 at Ser-604 as detected by a phospho-specific antibody, with a similar kinetics to ASK1-JNK/p38 activation. 14-3-3 associates with an open, active state of AIP1 assessed by an in vitro pulldown assay. Mutation of AIP1 at Ser-604 (AIP1-S604A) blocks TNF-induced complex formation of AIP1 with 14-3-3. TNF treatment normally induces association of AIP1 with TRAF2-ASK1. The interactions with TRAF2 and ASK1 do not occur with AIP1-S604A, suggesting that phosphorylation at this site not only creates a 14-3-3-binding site but also opens up AIP1, allowing binding to TRAF2 and ASK1. Overexpression of AIP1-S604A blocks TNF-induced ASK1-JNK activation. We further show that RIP1 (the Ser/Thr protein kinase receptor-interacting protein) associates with the GAP domain of AIP1 and mediates TNF-induced AIP1 phosphorylation at Ser-604 and JNK/p38 activation as demonstrated by both overexpression and small interfering RNA knockdown of RIP1 in EC. Furthermore, RIP1 synergizes with AIP1 (but not AIP1-S604A) in inducing both JNK/p38 activation and EC apoptosis. Our results demonstrate that RIP1-mediated AIP1 phosphorylation at the 14-3-3-binding site Ser-604 is essential for TNF-induced TRAF2-RIP1-AIP1-ASK1 complex formation and for the activation of ASK1-JNK/p38 apoptotic signaling.  相似文献   

17.
The stress-activated protein kinases (SAPKs, also called c-Jun NH(2)-terminal kinases) and the p38s, two mitogen-activated protein kinase (MAPK) subgroups activated by cytokines of the tumor necrosis factor (TNF) family, are pivotal to the de novo gene expression elicited as part of the inflammatory response. Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAP3K) that activates both the SAPKs and p38s in vivo. Here we show that TNF receptor (TNFR) associated factor 2 (TRAF2), an adapter protein that couples TNFRs to the SAPKs and p38s, can activate ASK1 in vivo and can interact in vivo with the amino- and carboxyl-terminal noncatalytic domains of the ASK1 polypeptide. Expression of the amino-terminal noncatalytic domain of ASK1 can inhibit TNF and TRAF2 activation of SAPK. TNF can stimulate the production of reactive oxygen species (ROS), and the redox-sensing enzyme thioredoxin (Trx) is an endogenous inhibitor of ASK1. We also show that expression of TRAF2 fosters the production of ROS in transfected cells. We demonstrate that Trx significantly inhibits TRAF2 activation of SAPK and blocks the ASK1-TRAF2 interaction in a reaction reversed by oxidants. Finally, the mechanism of ASK1 activation involves, in part, homo-oligomerization. We show that expression of ASK1 with TRAF2 enhances in vivo ASK1 homo-oligomerization in a manner dependent, in part, upon the TRAF2 RING effector domain and the generation of ROS. Thus, activation of ASK1 by TNF requires the ROS-mediated dissociation of Trx possibly followed by the binding of TRAF2 and consequent ASK1 homo-oligomerization.  相似文献   

18.
Here we studied the cellular mechanisms of ursolic acid's anti-bladder cancer ability by focusing on endoplasmic reticulum stress (ER stress) signaling. We show that ursolic acid induces a significant ER stress response in cultured human bladder cancer T24 cells. ER stress inhibitor salubrinal, or PERK silencing, diminishes ursolic acid-induced anti-T24 cell effects. Salubrinal inhibits ursolic acid-induced CHOP expression, Bim ER accumulation and caspase-3 activation in T24 cells. Ursolic acid induces IRE1–TRAF2–ASK1 signaling complex formation to activate pro-apoptotic ASK1–JNK signaling. We suggest that ER stress contributes to ursolic acid's effects against bladder cancer cells.  相似文献   

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