淫羊藿苷对人肝癌HepG2细胞的作用及其机制研究

目的:探讨淫羊藿苷(ICA)对HepG2肝癌细胞的作用及其可能机制。方法:给人肝癌HepG2细胞施加不同浓度的ICA(0～40μM)培养,用CCK-8法和集落形成试验测定细胞增殖能力,通过流式细胞术分析检测细胞周期进程和细胞凋亡情况,蛋白免疫印迹法分析细胞周期相关蛋白CyclinD1、CDK4、凋亡相关蛋白Bcl-2、Bax、以及芳烃受体(AHR)蛋白的表达水平。结果:ICA可抑制HepG2细胞的集落形成和细胞增殖,诱导细胞周期阻滞于G0～G1期,并诱导肝癌细胞凋亡。此外,ICA上调AHR蛋白表达,且其对HepG2细胞增殖的影响被AHR拮抗剂CH223191抑制。结论:ICA可能通过激活AHR信号抑制人肝癌HepG2细胞的增殖和诱导细胞周期阻滞于G0～G1期,并诱导细胞凋亡。     

P38MAPK磷酸化对HepG2细胞耐药的影响及其意义

目的 探讨p38MAPK磷酸化对HepG2细胞耐药的影响及其意义.方法 采用浓度递增法,建立动态HepG2/顺铂(CDDP)耐药模型,Western blot检测磷酸化p38的表达;予以p38MAPK特异性抑制剂SB203580分别处理24、48、72 h后,流式细胞仪动态分析HepG2/CDDP耐药细胞周期、四甲基偶氮唑盐法检测顺铂对HepG2/CDDP耐药细胞的半数药物抑制浓度(IC50)、Western blot检测HepG2/CDDP耐药细胞凋亡分子Bcl-2、Bax及P-gp蛋白的表达.动态耐药模型磷酸化P38的检测行单因素方差分析; SB203580处理后计量资料行3×3析因设计方差分析,率的比较采用x2检验.结果 成功建立HepG2/CDDP耐药细胞株,其对CDDP的IG50值与对照HepG2细胞差异具有统计学意义(t=99.30,P<0.01);随着HepG2/CDDP耐药细胞株耐药表型的增强,P38MAPK活化水平逐渐增强(F=69.39,P<0.01).P38MAPK特异性抑制剂SB203580可逐渐降低HepG2/CDDP对顺铂的IC50值(F=2350,P<0.01)、G0/Gl期细胞比例(x2=520,P<0.01)、Bcl-2/Bax表达比值(F=83.8,P<0.01)及细胞膜药物转运相关蛋白P-gP的表达(F=107,P<0.01).结论 P38活化水平随着肝癌耐药细胞株耐药表型的增强而逐渐升高;抑制P38的活化可有效逆转肝癌HepG2/CDDP耐药细胞株的耐药性.     

P38MAPK信号通路影响血管内皮细胞生长因子诱导肝癌细胞超微结构变化

目的研究p38信号传导通路在血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)诱导肝癌细胞超微结构变化中的作用.方法采用扫描电子显微镜观察VEGF以及预先用SB203580特异性阻断p38MAPK信号传导通路后VEGF诱导肝癌细胞HepG2形态学改变. 结果扫描电镜显示肝癌细胞呈梭形,表面有细而稀疏的突起,VEGF诱导后肝癌细胞成椭圆形,细胞表面伪足增多、变粗.阻断p38MAPK信号通路后,可抑制VEGF诱导的肝癌细胞表面伪足增多、变粗,促进肝癌细胞融合.结论VEGF通过p38MAPK信号通路诱导肝癌细胞伪足增多、变粗,降低细胞间粘附作用.     

丝裂原活化蛋白激酶信号通路对软脂酸培养的肌细胞过氧化物酶体增殖物激活受体γ辅激活子表达的调控作用

目的研究软脂酸对C2C12细胞过氧化物酶体增殖物激活受体γ辅激活子(PGC-1α)表达的影响,揭示丝裂原活化蛋白激酶(MAPKs)信号通路与PGC-1α表达的关系,寻找软脂酸诱导C2C12细胞PGC-1α表达变化的上游调节通路。方法检测软脂酸培养的C2C12细胞PGC-1α、细胞外信号调节激酶(ERK)、jnk氨基末端激酶(JNK)、p38丝裂原活化蛋白激酶(p38MAPK)及其磷酸化蛋白phosp-38MAPK(P-p38MAPK)的表达变化。寻找发生变化的MAPKs信号通路,用筛选到的p38MAPK抑制剂进行干预,分为对照(Con)组、软脂酸(Palmitate)组、p38MAPK抑制剂组和Palmitate+p38MAPK抑制剂组,测定PGC-1α、p38MAPK总蛋白及其P-p38MAPK的表达。结果软脂酸培养的C2C12细胞PGC-1α蛋白表达下降,呈时间依赖性;ERK、phospho-ERK、JNK、phospho-JNK及p38MAPK表达无变化,P-p38MAPK表达升高。用p38MAPK抑制剂干预C2C12细胞,PGC-1α表达在Palmitate组最低,p38MAPK抑制剂组和Palmitate+p38MAPK抑制剂组较Palmitate组升高,p38MAPK抑制剂组最高。结论软脂酸诱导的PGC-1α表达下降可能由p38MAPK信号通路调控。     

大黄提取物激活p38MAPK促进HepG2肝癌细胞凋亡

目的 探讨中药大黄提取物对HepG2肝癌细胞增殖与凋亡的影响和作用机制。方法 采用MTS、平板克隆形成实验、钙黄绿素/PI染色观察大黄提取物对HepG2肝癌细胞活性的影响,然后通过Hoechst33342/PI染色观察大黄提取物对肝癌细胞凋亡的影响,最后采用蛋白印迹检测大黄提取物对肝癌细胞内p38MAPK以及凋亡相关蛋白Bcl-2、Bax表达水平的影响。结果 MTS、平板克隆形成实验、钙黄绿素/PI染色提示大黄提取物显著抑制肝癌细胞的活性。Hoechst33342/PI染色提示大黄提取物能够浓度依赖性地促进肝癌细胞凋亡。此外,大黄提取物处理后显著增加肝癌细胞内p38MAPK的磷酸化水平,上调凋亡相关蛋白Bax的表达水平并下调Bcl-2表达。使用p38MAPK抑制剂能部分减弱大黄提取物的抗肝癌作用。结论 大黄提取物具有抑制肝癌细胞活性的作用,其作用机制可能是通过激活p38MAPK信号通路诱导肝癌细胞凋亡发挥的。     

抑癌基因Smad4对HepG2生长的影响及其机制探讨

目的研究抑癌基因Smad4对人肝癌细胞株HepG2．生长的影响，并探讨其作用机制。方法采用脂质体瞬时转染法转染PFTX-5Smad4质粒至人肝癌细胞株HepG2（实验组），以转染空质粒PFTX-5的HepG2细胞为对照组，野生型HepG2细胞为空白组，利用免疫印迹（Westernblot）和逆转录聚合酶链反应（RT—PCR）检测Smad4及血管内皮生长因子（VEGF）表达的变化；用MTT法检测细胞增殖及用FITC—AnnexinV、碘化吡啶（propidiumiodide，PI）双染后流式细胞仪（FCM）检测细胞周期和凋亡情况，Westernblot检测细胞周期素D1（CyclinD1）表达差异。结果PFTX-5Smad4瞬时转染到肝癌细胞后，实验组与对照组和空白组相比，Smad4mRNA和蛋白表达明显上升（P〈0．05），而VEGFmRNA和蛋白的表达显著降低（P〈0．05）。实验组细胞CyclinD1表达明显下降（P〈0．05），细胞周期时相分布出现合成前期（G。／G．期）阻滞。实验组细胞凋亡率明显升高（P〈0．01），增殖受到明显抑制。结论Smad4高表达可能通过降低肝癌细胞中VEGF的表达，抑制eyclinD1转录合成，并诱导强烈的G1期阻滞和细胞凋亡，对细胞的增殖有明显的抑制作用，为进-步研究肝癌生长增殖机制提供了实验基础。     

针对STAT3的siRNA抑制HepG2细胞增殖和p44/42MAPK蛋白的表达

目的研究STAT3信号传导通路对HepG2细胞p44/42MAPK蛋白表达和细胞生长的影响。方法将针对STAT3的siRNA转染入HepG2细胞以沉默STAT3基因的表达,采用MTT法检测细胞生长,采用Western blot法检测STAT3和p44/42MAPK蛋白的表达。结果对照组和lipofectamineTM2000处理组细胞生长无明显差异(q=0.97,P0.05),而siRNA处理组细胞生长被明显抑制(q=9.36,P0.05);SiRNA转染后24h、48h、72h和96h,细胞抑制率分别为33.2%、39.6%4、3.1%和33.9%s,iRNA在96h后抑制作用减弱,细胞开始重新繁殖;转染细胞72h和96h后,可见STAT3蛋白表达均被抑制(t=14.12,P0.05),p44/42MAPK蛋白表达未被抑制(F=3.99,P0.05),而p-p44/42MAPK蛋白表达增加(t=16.30,P0.05)。结论 STAT3信号传导通路可以影响细胞生长及p44/42MAPK蛋白质的磷酸化,而p-p44/42MAPK的表达增加可能代偿了沉默STAT3引起的细胞生长抑制,使细胞重新繁殖。     

阿魏酸钠对MCAO大鼠缺血／再灌注损伤p38MAPK信号通路的影响

目的 探讨阿魏酸钠(SF)在抗大鼠脑缺血/再灌注损伤过程中对p38MAPK信号通路的影响.方法 采用线栓法阻塞大鼠大脑中动脉(MCAO)建立大鼠局灶性脑缺血/再灌注损伤模型,大鼠于MCAO前1 h股静脉注射不同剂量SF100 mg/kg、50 mg/kg、20 mg/kg及p38MAPK抑制剂SB203580,观察各组脑缺血再灌注损伤后大鼠神经学评分,TUNEL法检测神经细胞的凋亡及Westernblot检测p38MAPK、p-p38MAPK蛋白表达.结果 假手术组无神经学改变,SF100 mg/kg、50 mg/kg及p38MAPK抑制剂SB203580组神经学评分明显低于缺血再灌注组(P<0.05),各用药组间无明显差异.SF100 mg/kg、50 mg/kg、20 mg/kg及p38MAPK抑制剂SB203580TUNEL阳性细胞率(%)均较缺血再灌注组明显下降.假手术组未见p-p38MAPK表达,缺血再灌注组p-p38MAPK显著增高,SF100 mg/kg、50 mg/kg、20 mg/kg对脑组织总p38MAPK表达影响不明显(P>0.05),主要下调p-p38MAPK表达.结论 阿魏酸钠对大鼠缺血再灌注损伤的保护作用机制可能与抑制p38MAPK信号传导通路有关.     

基于p38MAPK/Nrf2/HO-1通路探讨清达颗粒对脂多糖诱导活化的小胶质细胞抗氧化作用研究

目的观察清达颗粒(QDG)对脂多糖(LPS)诱导活化的小胶质细胞抗氧化作用,探讨其与p38MAPK/Nrf2/HO-1抗氧化信号通路的可能联系。方法体外培育BV-2小胶质细胞,采用MTT法筛选合适的药物浓度,之后采用LPS(1μg/mL)诱导炎症模型,将其分为对照组、LPS组、LPS+QDG组,并LPS+QDG组设置31.25μg/mL、62.50μg/mL、125.00μg/mL 3个质量浓度亚组,检测各组细胞活性氧(ROS)、肿瘤坏死因子-α(TNF-α)、丙二醛(MDA)以及谷胱甘肽过氧化物酶(GSH-Px)的表达情况,Western Blot法检测磷酸化p38MAPK(p-p38)、p38MAPK(p38)、转录因子Nrf2、细胞核内Nrf2、血红素加氧酶-1(HO-1)的蛋白表达情况以及p38抑制剂干预LPS诱导的炎症细胞HO-1蛋白的表达情况。结果 LPS+QDG组ROS、TNF-α、MDA的释放抑制受到一定程度上促进GSH-Px的释放,并升高p-p38、细胞核内Nrf2以及HO-1蛋白的表达。p38抑制剂干预后下调了LPS+QDG组HO-1蛋白的表达。结论清达颗粒可有效减轻LPS诱导的BV-2小胶质细胞的氧化应激损伤,这可能与激活p38MAPK/Nrf2/HO-1信号转导通路有关。     

分裂原激活蛋白激酶p38信号传导通路在抑制血管内皮细胞生长因子诱导肝癌转移的实验研究

目的　研究血管内皮细胞生长因子 (vascularendothelialgrowthfactor ,VEGF)诱导肝癌转移时 p38分裂原激活蛋白激酶 (mitogen activatedproteinkinase,MAPK)信号传导通路的作用。 方法　运用Bodyenchamber膜侵袭培养系统 ,应用 p38MAPK信号传导通路特异性阻断剂SB 2 0 35 80 ,观察其对VEGF诱导肝癌细胞转移抑制作用。结果　 5ng/mlVEGF培养 5h ,向下室浸润肝癌细胞数为 16×10 4 /ml,高于对照组 1.2 5× 10 4 /ml,P <0 .0 1。预先用 5 μMSB 2 0 35 80 ,可阻断 95 %VEGF诱导肝癌转移能力。结论　VEGF通过 p38MAPK信号传导通路诱导肝癌细胞转移 ,阻断p38MAPK信号传导通路 ,可抑制VEGF诱导的肝癌细胞转移。     

Histone deacetylase inhibitor MS-275 alone or combined with bortezomib or sorafenib exhibits strong antiproliferative action in human cholangiocarcinoma cells

AIM: To investigate the antiproliferative effect of the histone deacetylase (HDAC) inhibitor MS-275 on cholangiocarcinoma cells alone and in combination with conventional cytostatic drugs (gemcitabine or doxorubicin) or the novel anticancer agents sorafenib or bortezomib. METHODS: Two human bile duct adenocarcinoma cell lines (EGI-1 and TFK-1) were studied. Crystal violet staining was used for detection of cell number changes. Cytotoxicity was determined by measuring the release of the cytoplasmic enzyme lactate dehydrogenase (LDH). Apoptosis was determined by measuring the enzyme activity of caspase-3. Cell cycle status reflected by the DNA content was detected by flow cytometry.RESULTS: MS-275 treatment potently inhibited the proliferation of EGI-1 and TFK-1 cholangiocarcinoma cells by inducing apoptosis and cell cycle arrest. MS-275-induced apoptosis was characterized by activation of caspase-3, up-regulation of Bax and down-regulation of Bcl-2. Cell cycle was predominantly arrested at the G1/S checkpoint, which was associated with induction of the cyclin-dependent kinase inhibitor p21Waf/CIP1. Furthermore, additive anti-neoplastic effects were observed when MS-275 treatment was combined with gemcitabine or doxorubicin, while combination with the multi-kinase inhibitor sorafenib or the proteasome inhibitor bortezomib resulted in overadditive anti-neoplastic effects.CONCLUSION: The growth of human cholangiocarcinoma cells can be potently inhibited by MS-275 alone or in combination with conventional cytostatic drugs or new, targeted anticancer agents.     

Antiproliferative and proapoptotic effects of histone deacetylase inhibitors on gastrointestinal neuroendocrine tumor cells

Treatment options of advanced neuroendocrine tumors (NETs) are unsatisfactory. Hence, innovative therapeutic approaches are urgently needed. Inhibition of histone deacetylases (HDACs) is a promising new approach in cancer therapy. While several HDAC inhibitors have already entered clinical trials, the effect of HDAC inhibition on NET has not been investigated. Therefore, we evaluated the antineoplastic effects of three different HDAC inhibitors, trichostatin A (TSA), sodium butyrate (NaB), and MS-275, on growth and apoptosis of the gastrointestinal NET cell lines CM and BON. We could demonstrate that HDAC inhibition dose-dependently inhibited proliferation of both cell lines with IC50 values varying from the millimolar (NaB) to the micromolar (MS-275) and the nanomolar range (TSA). Moreover, HDAC inhibition potently induced apoptosis, which was accompanied by DNA-fragmentation, an up to 12-fold caspase-3 activation and downregulated Bcl-2 expression. Furthermore, HDAC inhibition resulted in cell cycle arrest at the G1-S-transition, which was associated with the suppression of cyclin D1 expression and induction of p21 and p27 expression. For BON cells, we observed an additional block in the G2/M phase, which was aligned with a downregulation of cyclin B1. In addition, combined treatment with MS-275 and somatostatin or the synthetic somatostatin analog octreotide was evaluated. Neither somatostatin nor its stable analog octreotide augmented the antiproliferative effect of MS-275 in NET cells. To conclude, our data show that HDAC inhibition is a promising new approach in the treatment of NET disease, which should be evaluated in clinical studies.     

Phase 1 and pharmacologic study of MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias

MS-275 is a benzamide derivative with potent histone deacetylase (HDAC) inhibitory and antitumor activity in preclinical models. We conducted a phase 1 trial of orally administered MS-275 in 38 adults with advanced acute leukemias. Cohorts of patients were treated with MS-275 initially once weekly x 2, repeated every 4 weeks from 4 to 8 mg/m2, and after 13 patients were treated, once weekly x 4, repeated every 6 weeks from 8 to 10 mg/m2. The maximum-tolerated dose was 8 mg/m2 weekly for 4 weeks every 6 weeks. Dose-limiting toxicities (DLTs) included infections and neurologic toxicity manifesting as unsteady gait and somnolence. Other frequent non-DLTs were fatigue, anorexia, nausea, vomiting, hypoalbuminemia, and hypocalcemia. Treatment with MS-275 induced increase in protein and histone H3/H4 acetylation, p21 expression, and caspase-3 activation in bone marrow mononuclear cells. No responses by classical criteria were seen. Our results show that MS-275 effectively inhibits HDAC in vivo in patients with advanced myeloid leukemias and should be further tested, preferably in patients with less-advanced disease.     

PEG10基因siRNA真核表达载体对HepG2细胞周期的影响

目的观察针对遗传印记基因PEG10的siRNA真核表达载体对人肝癌HepG2细胞周期及细胞周期调控蛋白表达水平的影响。方法以脂质体法将psiRNA-PEG10真核表达载体转染HepG2细胞，RT-PCR检测PEG10mRNA的表达，流式细胞仪观察细胞周期分布的变化；RT-PCR和Western Blotting法分别检测细胞周期调节蛋白D1（Cyclin D1）和P27mRNA和蛋白水平的表达变化。结果RT-PCR结果显示，转染psiRNA-PEG10的HepG2细胞中PEG10mRNA的表达明显降低；流式细胞仪检测发现，与未转染组和空载体组相比，转染psiRNA-PEG10后处于G0／G1期的HepG2细胞百分比明显升高（P〈0．01），而G2／M期的细胞百分比明显降低（P〈0．01）；在mRNA和蛋白水平上，转染psiRNA-PEG10的细胞Cyclin D1表达明显降低；而P27表达明显升高（P〈0．01）。结论PEG10可通过影响细胞周期调控蛋白CyclinD1和P27表达，干扰肿瘤细胞的细胞周期发挥抗瘤作用，针对PEG10的siRNA真核表达载体有望成为研究肝癌治疗的有力工具。     

辛伐他汀抑制HepG2细胞增殖作用的机制

目的 体外观察辛伐他汀对人肝癌细胞HepG2增殖、细胞周期及细胞周期蛋白依赖激酶抑制因子p21蛋白表达的影响.方法 采用四甲基偶氮唑盐(MTT)法观察辛伐他汀对HepG2细胞增殖的影响,用流式细胞仪检测辛伐他汀对细胞周期的作用,用免疫细胞化学法观察辛伐他汀对细胞周期蛋白依赖激酶抑制因子p2l蛋白表达的影响.对数据进行析因设计与单因素方差分析.结果 体外辛伐他汀可抑制HepG2细胞的增殖(F浓度=1264,P＜0.001 ;F时间=17.466,P＜0.001;F浓度*时间=35.053,P＜0.001).辛伐他汀处理组G0/G1期细胞增多,但细胞凋亡不明显;体外辛伐他汀可增强HepG2细胞周期蛋白依赖激酶抑制因子p21蛋白的表达(F=512.133,P＜0.001).结论 体外辛伐他汀对HepG2细胞增殖有抑制作用,该作用可能与其使细胞生长阻滞于G0/G1期及增强细胞周期蛋白依赖激酶抑制因子p21蛋白表达有关.     

Basic fibroblast growth factor inhibits p38-mediated cell differentiation and growth inhibition by activin A but not by histone deacetylase inhibitors in CML cells

The p38 mitogen-activated protein kinase (p38) is involved in multiple cellular functions such as cell proliferation and differentiation. Previously, we found that activin A mediated hemoglobin synthesis and cell growth inhibition through p38, whereas, basic fibroblast growth factor (bFGF) inactivated p38 to antagonize the activin A effects. In this study, we selected three structurally different histone deacetylase (HDAC) inhibitors, apicidin, MS275, and sodium butyrate that activate p38, to probe the signal pathway from activin A to p38 in chronic myeloid leukemia (CML)-derived K562 cells. HDAC inhibitors and activin A showed additive p38 phosphorylation. The enhanced phosphorylation of p38 was correlated with increased cell differentiation and decreased cell proliferation. The use of p38 inhibitor SB203580 in conjunction with activin A or with the HDAC inhibitors inhibited cell differentiation and restored cell proliferation, indicating that activin A and the HDAC inhibitors exert their effects through p38 activation. However, bFGF did not affect HDAC inhibitors-induced cell differentiation or growth inhibition. Western blots showed that p38 phosphorylation remained at similar levels with or without bFGF in the presence of HDAC inhibitors. Thus, the HDAC inhibitors activate p38 in a manner different from the activin A pathway. Furthermore, mRNA expressions for activin type I, IB, II, and IIB receptors remained constant in the presence of activin A, bFGF, or both activin A and bFGF. These results indicate that bFGF does not directly act on p38 nor on the mRNA expression levels of activin receptors but inhibit activin A activation of p38 upstream of p38 in K562 cells.     

Helicobacter pylori and mitogen-activated protein kinases regulate the cell cycle, proliferation and apoptosis in gastric epithelial cells

Background and Aims:  Helicobacter pylori infection activates mitogen-activated protein kinases (MAPK) and modulates cell proliferation and apoptosis. However, the relationship between H. pylori infection and MAPK signaling in controlling cell proliferation and apoptosis is not clear, nor has the role of MAPK on the gastric epithelial cell cycle and proliferation been established. Therefore, we investigated the effects of H. pylori infection and MAPK inhibition on these processes.
Methods:  Gastric epithelial cell lines (AGS and MKN45) were infected with H. pylori and/or treated with MAPK inhibitors. Cell cycle and apoptosis were measured by flow cytometry. Cell cycle proteins and proliferation were monitored by western blot and cell count, respectively.
Results:  Infection with H. pylori resulted in dose-dependent MAPK activation, cell cycle arrest, reduced proliferation and increased apoptosis. The effect of H. pylori and MAPK at various cell cycle checkpoints was noted: MEK1/2 and p38 inhibition increased H. pylori -induced cell cycle G₁ arrest, while JNK inhibition reduced G₁ arrest. MEK1/2 inhibition increased p21, p27 and cyclin E and JNK inhibition additionally increased cyclin D1 expression. Both inhibitors decreased cell proliferation. All inhibitors enhanced apoptosis after H. pylori infection. We also detected MAPK cross-talk in AGS cells: p38 and JNK inhibitors increased ERK activation. The p38 inhibitor increased JNK and the MEK1/2 inhibitor decreased JNK activation only during H. pylori infection.
Conclusions:  These results suggest H. pylori and MAPK differentially regulate the cell cycle, proliferation and apoptosis in gastric epithelial cells. The imbalance between H. pylori infection and MAPK activation likely contributes to the H. pylori -induced pathogenesis.     

慢病毒介导p38MAPK基因沉默对高糖诱导成骨细胞凋亡的影响

目的 观察p38丝裂原活化的蛋白激酶(MAPK)在高糖诱导的MC3T3-E1成骨细胞凋亡中的作用,并探讨其对凋亡相关信号分子caspase-3、bax及bcl-2表达的影响.方法 构建靶向p38MAPK的shRNA慢病毒载体,将体外培养的MC3T3-E1成骨细胞分为正常对照组(A组)、高糖组(B组)、p38MAPK-shRNA慢病毒转染组(C组)、信号转导阻断剂组(D组)和无关shRNA转染组(E组).RT-PCR检测细胞p38MAPK mRNA的表达,流式细胞术检测细胞凋亡,Western印迹检测p38MAPK、p-p38 MAPK、caspase-3、bax、bcl-2蛋白水平,透射电镜观察细胞超微结构.结果 构建靶向p38MAPK的shRNA慢病毒载体,并成功导入MC3T3-E1成骨细胞.与高糖组和无关转染组相比,p38MAPK-shRNA转染能显著抑制高糖诱导的MC3T3-E1细胞p38MAPK过度活化,明显减少细胞的凋亡(P＜0.01);同时,p38MAPK-shRNA转染及p38MAPK阻断剂明显降低MC3T3-E1细胞中p38MAPK、p-p38MAPK、caspase-3及促凋亡基因bax蛋白表达,上调凋亡抑制基因bcl-2,与高糖组和无关转染组相比,差异有统计学意义(P＜0.01,P＜0.05).结论 慢病毒介导p38MAPK靶向RNA干扰可通过抑制p38MAPK 信号通路的活化,降低p-p38MAPK、caspase-3、bax表达,上调bcl-2表达,最终抑制高糖所诱导的MC3T3-E1成骨细胞的凋亡.

Abstract：

Objective To examine the role of p38MAPK in high glucose-induced apoptosis of osteoblast MC3T3-E1 cell line, and to investigate its effect on the expressions of apoptosis-related molecules including caspase3, bax, and bcl-2. Methods The lentiviral vector containing short hairpin RNA targeting p38MAPK was constructed. The cultured osteoblast MC3T3-E1 cell were divided into 5 groups:normal control group(A group), high glucose group(B group), p38MAPK-shRNA transfection group(C group), signal transduction inhibitor group(D group), and transfection with negative control siRNA group(E group). RT-PCR was used to determine the p38MAPK mRNA expression levels in MC3T3-E1 cells. Flow cytometry(FCM)was employed to detect the cell apoptotic percentage. The protein levels of apoptosis-related molecules p38MAPK, p-p38MAPK, caspase-3, bax, and bcl-2 were assayed by Western blot. Ultrastructural alternation of MC3T3-E1 cell was observed under transmission electron microscopy(TEM). Results The lentiviral vector containing short hairp in RNA targeting p38MAPK was successfully constructed and transfected into MC3T3-E1 cells. RT-PCR result suggested that the siRNA targeting p38MAPK could effectively reduce the p38MAPK mRNA expression level induced by high glucose in MC3T3-E1 cell line. FCM showed siRNA significantly decreased high glucose-induced apoptosis percentage of MC3T3-E1 cells(P＜0.01). Meanwhile, we also found the siRNA significantly attenuated the proteins levels of p38MAPK, p-p38MAPK, caspase-3, and gene bax induced by high glucose in MC3T3-E1 cells, whereas the protein level of gene bcl-2 was enhanced remarkably when compared with high glucose group and negative control siRNA group(P＜0.01, P＜0.05).Conclusion The iRNA targeting p38MAPK suppressed high glucose-induced MC3T3-E1 cell apoptosis via inhibiting the activation of p38MAPK signaling pathway, thereby reducing the expressions levels of p-p38MAPK, caspase-3 and gene bax, and up-regulating the level of gene bcl-2.     

Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin

Pharmacologic stimulation of fetal hemoglobin (HbF) expression may be a promising approach for the treatment of beta-thalassemia. In this study, we have investigated the HbF-inducing activity and molecular mechanisms of specific histone deacetylase (HDAC) inhibitors in human K562 erythroleukemia cells. Apicidin was the most potent agent compared with other HDAC inhibitors (trichostatin A, MS-275, HC-toxin, suberoylanilide hydroxamic acid [SAHA]) and previously tested compounds (butyrate, phenylbutyrate, isobutyramide, hydroxyurea, 5-aza-cytidine), leading to a 10-fold stimulation of HbF expression at nanomolar to micromolar concentrations. Hyperacetylation of histones correlated with the ability of HDAC inhibitors to stimulate HbF synthesis. Furthermore, analysis of different mitogen-activated protein (MAP) kinase signaling pathways revealed that p38 signaling was activated following apicidin treatment of cells and that inhibition of this pathway abolished the HbF-inducing effect of apicidin. Additionally, activation of the Agamma-globin promoter by apicidin could be inhibited by p38 inhibitor SB203580. In summary, the novel HDAC inhibitor apicidin was found to be a potent inducer of HbF synthesis in K562 cells. The present data outline the role of histone hyperacetylation and p38 MAP kinase signaling as molecular targets for pharmacologic stimulation of HbF production in erythroid cells.