PERK介导的内质网应激参与血管紧张素Ⅱ诱导心肌肥大的机制

目的: 研究蛋白激酶R样内质网激酶(PERK)介导的内质网应激(ERS)反应在血管紧张素Ⅱ(AngⅡ)诱导心肌细胞肥大中的作用。方法: 在AngⅡ诱导原代培养的乳大鼠心肌细胞肥大模型上,采用[³H]-亮氨酸掺入、心肌细胞表面积测定等评估心肌细胞肥大程度;以实时定量PCR、RT-PCR和Western blotting检测ERS标志性分子葡萄糖调节蛋白78(GRP78)、钙网蛋白(CRT)、蛋白激酶R样内质网激酶(PERK)、真核细胞翻译起始因子2α(eIF2α)和C/EBP同源蛋白(CHOP)mRNA和蛋白表达变化。结果: 与正常对照组比较,AngⅡ组CRT mRNA和蛋白表达分别高146.4%和125.3%(P<0.05),GRP78 mRNA和蛋白的表达分别高84.0%和77.6% (P<0.05),PERK mRNA和蛋白表达分别高165.4%和132.1%(P<0.05),eIF2α mRNA和蛋白表达分别高110.9%和46.5%(P<0.05),CHOP mRNA和蛋白表达分别高117.7%和63.3%(P<0.05)。结论: PERK介导的内质网应激反应参与了AngⅡ诱导的心肌细胞肥大。     

沉默葡萄糖调节蛋白78基因增强高氧诱导的肺泡上皮细胞凋亡北大核心CSCD

目的探讨小干扰RNA(siRNA)干扰内质网应激相关因子葡萄糖调节蛋白78(GRP78)基因表达对高氧诱导肺泡上皮细胞凋亡的影响及作用机制。方法培养人肺腺癌A549细胞,用构建的siRNA片段通过脂质体LipofectamineTM2000转染A549细胞,将细胞分为高氧空白组、阴性对照(阴性siRNA)组和实验(GRP78-siRNA)组3组,利用950 mL/L O2建立高氧细胞损伤模型。48 h后通过实时定量PCR检测C/EBP同源蛋白(CHOP)mRNA的表达,Western blot技术检测CHOP蛋白表达,流式细胞仪检测细胞凋亡。结果应用siRNA抑制GRP78表达后,GRP78 mRNA和蛋白表达下调、CHOP mRNA和蛋白表达上调、A549细胞凋亡增加。结论下调GRP78基因可增强高氧活化的CHOP凋亡途径,诱导肺泡上皮细胞凋亡。     

沉默葡萄糖调节蛋白78基因增强高氧诱导的肺泡上皮细胞凋亡

目的探讨小干扰RNA(siRNA)干扰内质网应激相关因子葡萄糖调节蛋白78(GRP78)基因表达对高氧诱导肺泡上皮细胞凋亡的影响及作用机制。方法培养人肺腺癌A549细胞,用构建的siRNA片段通过脂质体LipofectamineTM2000转染A549细胞,将细胞分为高氧空白组、阴性对照(阴性siRNA)组和实验(GRP78-siRNA)组3组,利用950 mL/L O2建立高氧细胞损伤模型。48 h后通过实时定量PCR检测C/EBP同源蛋白(CHOP)mRNA的表达,Western blot技术检测CHOP蛋白表达,流式细胞仪检测细胞凋亡。结果应用siRNA抑制GRP78表达后,GRP78 mRNA和蛋白表达下调、CHOP mRNA和蛋白表达上调、A549细胞凋亡增加。结论下调GRP78基因可增强高氧活化的CHOP凋亡途径,诱导肺泡上皮细胞凋亡。     

巴戟天寡糖单体HexB减轻HUVECs缺氧复氧损伤

目的:研究巴戟天寡糖单体HexB减轻缺氧复氧(H/R)损伤诱导人脐静脉内皮细胞(HUVECs)凋亡的分子机制。方法:HUVECs分别用HexB、内质网应激(ERS)抑制剂4-苯基丁酸(4-PBA)和ERS诱导剂毒胡萝卜素(TG)干预,并将培养的HUVECs分为:对照组、HexB组、H/R组、HexB+H/R组、4-PBA+H/R组、TG组和HexB+TG组。CCK-8法和流式细胞术检测细胞活性及凋亡率,Western blot法检测ERS标志分子葡萄糖调节蛋白78(GRP78)和C/EBP同源蛋白(CHOP)、凋亡相关蛋白caspase-12及磷酸化c-Jun氨基末端激酶(p-JNK)的蛋白水平。结果:与对照组比较,H/R组和TG组的细胞凋亡率明显增加,细胞活力明显降低,GRP78、CHOP、caspase-12及p-JNK的蛋白水平上调(P0.05);与H/R组比较,HexB+H/R组及4-PBA+H/R组的细胞凋亡率显著降低,细胞活力升高,GRP78、CHOP、caspase-12及p-JNK的蛋白水平降低(P0.05);与TG组比较,HexB+TG组的细胞凋亡率显著降低,细胞活力升高,GRP78、CHOP、caspase-12及p-JNK的蛋白水平降低(P0.05);HexB+H/R组与4-PBA+H/R组比较,细胞凋亡率、细胞活力及GRP78、CHOP、caspase-12、p-JNK蛋白水平的差异无统计学显著性。结论:通过抑制内质网应激,HexB可以减轻H/R诱导的HUVECs凋亡,其机制可能与下调GRP78、CHOP、p-JNK及caspase-12的水平有关。     

西洋参茎叶总皂苷通过抑制内质网应激减轻毒胡萝卜素诱导的心肌细胞凋亡

 目的:研究西洋参茎叶总皂苷 (PQS) 减轻毒胡萝卜素(TG) 诱导的心肌细胞凋亡的分子机制。方法:原代培养的心肌细胞分为：control组、TG组、PQS (40 mg/L、80 mg/L及160 mg/L)+TG组、牛磺酸 (40 mmol/L)+TG组、蛋白激酶R样内质网激酶 (PERK)敲低+TG组及随机双链RNA转染对照 (mock)+TG组。通过向培养液中加入1 μmol/L TG作用24 h诱导离体培养的乳大鼠心肌细胞凋亡。以RNAi方法敲低心肌细胞PERK基因。CCK-8法和流式细胞术检测细胞活性及凋亡率,Western blotting方法检测内质网应激(ERS)标志分子葡萄糖调节蛋白78 (GRP 78)、钙网蛋白 (CRT)、PERK、p-PERK、真核起始因子2α (eIF2α)、p- eIF2α、活化转录因子4 (ATF4)、C/EBP同源蛋白 (CHOP) 及凋亡相关蛋白Bcl-2、Bax的表达。结果:与对照组比较,TG孵育明显诱导细胞凋亡,降低细胞活力,上调PERK和eIF2α磷酸化以及GRP78、CRT、ATF4、CHOP及促凋亡蛋白Bax表达,降低抗凋亡蛋白Bcl-2表达 (P<0.05);与TG组比较,PQS 160 mg/L及敲低PERK均可显著降低细胞凋亡率,升高细胞活力 (P<0.05);3种不同浓度的PQS可呈剂量依赖性降低Bax蛋白表达,升高Bcl-2蛋白表达 (P<0.05),敲低PERK基因及PQS (160 mg/L) 预处理均可降低ERS分子GRP78、CRT、ATF4及CHOP表达,降低PERK及eIF2α的磷酸化水平 (P<0.05)。结论: PQS 160 mg/L 减轻ERS诱导剂TG诱导的心肌细胞凋亡,PQS预处理可以模拟PERK敲低减轻TG致心肌细胞凋亡的效应,提示PERK-eIF2α-ATF4-CHOP途径参与PQS减轻ERS相关凋亡的作用。     

栀子苷元通过抑制内质网应激改善脾细胞凋亡治疗脓毒症的机制研究

目的 评估栀子苷元 (Genipin) 对盲肠结扎穿孔术 (Cecal Ligation and Puncture,CLP) 诱导的脓毒症小鼠模型的治疗效果,并探讨其潜在的作用机制。方法 将雄性 C57BL/6 小鼠分为假手术组 (Sham组)、脓毒症模型组 (CLP组)、栀子苷元治疗脓毒症组 (Genipin+CLP组)。栀子苷元治疗组在建模后0 h和20 h经尾静脉注射2.5 mg/kg栀子苷元,而Sham组和CLP组注射等体积生理盐水。实验1,观察栀子苷元对脓毒症小鼠120 h生存率的影响;实验2,基于生存率的结果选取建模后36 h为采样时间点,并行以下方法检测：利用酶联免疫吸附法 (Enzym-linked Immunosorbent Assay,ELISA) 检测肿瘤坏死因子α (Tumor Necrosis Factor α,TNF-α) 和白细胞介素6(Interleukin 6,IL-6) 含量;常规苏木精-伊红 (Hematoxylineosin,HE) 染色法检测肺和肝组织病理损害;Western Blot法检测脾脏内质网应激相关蛋白的表达变化,包括葡萄糖调节蛋白 78 (Glucose Regulated Protein 78,GRP78)、蛋白激酶 R 样内质网激酶 (Protein Kinase R-like Endoplasmic Reticulum Ki‐ nase,PERK)、磷酸化真核翻译起始因子2α (p-eukaryotic Initation Factor 2α,p-eIF2α) 和C/EBP同源蛋白 (C/EBP Homolo‐ gous Protein,CHOP);脱氧核糖核苷酸末端转移酶介导的缺口末端标记法 (Terminal-deoxynucleotidyl Transferase-mediated dUTP Nick End-labeling,TUNEL) 检测脾细胞凋亡变化。结果 与 CLP组相比,栀子苷元治疗提高了脓毒症小鼠 120 h生存率 (P＜0.05),降低了血清 TNF-α和 IL-6含量 (P＜0.05),并减轻肺和肝脏组织病理学损伤。另外,栀子苷元显著下调了脾脏GRP78、PERK、p-eIF2α和CHOP蛋白的表达水平 (P＜0.05),并进而减少内质网应激诱导的脾细胞凋亡数量 (P＜0.05)。 结论 栀子苷元能够明显减轻脓毒症所导致的脏器损害和过度炎症反应,改善脓毒症预后。该效应的作用机制可能与栀子苷元减轻脾脏内质网应激导致其凋亡机制的启动被阻断,进而减轻脾细胞凋亡有关。     

ox-LDL对巨噬细胞内皮脂肪酶表达的影响

目的: 探讨氧化低密度脂蛋白(ox-LDL)对鼠源RAW264.7巨噬细胞内皮脂肪酶(EL)表达的影响。方法: 用ox-LDL(0~100 mg/L)孵育RAW264.7细胞24 h使其活化,或者用PDTC (NF-κB抑制剂)预孵育RAW264.7细胞30 min再与ox-LDL(50 mg/L)共同孵育24 h;应用Western blotting 检测EL和NF-κB p65的表达。结果: ox-LDL孵育RAW264.7细胞明显增加EL表达(P<0.05);ox-LDL(50 mg/L)孵育RAW264.7细胞15~30 min可激活NF-κB,用NF-κB 抑制剂PDTC处理后则明显抑制ox-LDL诱导的EL表达增加(P<0.05)。结论: ox-LDL可明显增加RAW264.7细胞EL的表达,可能与NF-κB活化有关。     

肝纤维化大鼠肝细胞内质网形态及GRP78表达的研究

目的: 观察四氯化碳(CCl₄)致大鼠肝纤维化过程中内质网形态及内质网应激标志性蛋白——葡萄糖调节蛋白78(GRP78)的表达变化, 探讨内质网应激在肝纤维化发病机制中可能的作用。方法: 雄性Wistar大鼠皮下注射CCl₄制备肝纤维化模型,分别在4周及8周处死大鼠测定肝脏指数、血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活性, 观察肝组织病理改变和肝细胞内质网形态, 免疫组化和real-time PCR分别检测肝组织GRP78蛋白及mRNA表达变化。结果: 肝纤维化组大鼠肝脏指数、血清ALT和AST活性显著高于正常对照组(P<0.01),肝纤维化明显, 电镜下见肝细胞内质网扩张,数量明显减少; 肝细胞胞浆中GRP78蛋白表达量及mRNA表达量较正常组显著增加(P<0.01)。结论: 在CCl₄诱导的肝纤维化发生过程中肝细胞内质网形态有明显损伤性变化, 内质网应激蛋白GRP78蛋白及基因表达水平明显增加, 提示内质网应激参与肝纤维化发生发展。     

自噬通过抑制C/EBP同源蛋白表达减轻氧化低密度脂蛋白诱导的巨噬细胞凋亡

目的:研究自噬对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)所致巨噬细胞凋亡的影响,并探讨可能的分子机制。方法:体外培养RAW264.7巨噬细胞,分别给予3-甲基腺嘌呤(3-methyladenine,3-MA;3 mmol/L)、雷帕霉素(rapamycin,Rap;1μmol/L)或4-苯丁酸(4-phenylbutyric acid,PBA;4 mmol/L)预处理1 h,再加入ox-LDL(100 mg/L)继续培养12 h。分别采用MTT法和Annexin V-FITC双染法检测细胞活力和凋亡情况;相应试剂盒测定培养液乳酸脱氢酶(lactic dehydrogenase,LDH)和细胞内caspase-3活性;采用Western blot法检测自噬标志分子beclin-1和内质网应激标志分子葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)及促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein,CHOP)表达的变化;采用激光共聚焦显微镜观测细胞内微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的变化。结果:ox-LDL处理可显著降低巨噬细胞活力,并增加LDH漏出、凋亡率及caspase-3活性;ox-LDL对细胞的上述损伤作用可被自噬抑制剂3-MA促进而被自噬诱导剂雷帕霉素拮抗。ox-LDL诱导巨噬细胞自噬反应,表现为beclin-1表达上调,LC3颗粒化显著;ox-LDL对自噬的诱导作用可被3-MA抑制而被Rap增强。另外,3-MA可促进ox-LDL所诱导的CHOP进一步上调,而Rap可明显拮抗ox-LDL对CHOP的诱导作用。PBA可显著抑制ox-LDL所诱导的GRP78上调,且明显减轻ox-LDL所诱导的自噬反应,表现为beclin-1表达下调,LC3颗粒化程度减弱。结论:内质网应激介导ox-LDL对巨噬细胞自噬的诱导作用,而一定程度的自噬可通过抑制CHOP表达从而减轻ox-LDL所诱导的巨噬细胞凋亡。     

西洋参茎叶总皂苷通过抑制过度内质网应激减轻大鼠心肌细胞缺氧/复氧损伤

目的: 观察西洋参茎叶总皂苷 (PQS) 对缺氧/复氧 (H/R) 心肌细胞的保护作用,并从内质网应激 (ERS)的角度探讨其分子机制。方法: 建立心肌细胞缺氧/复氧 (H/R) 损伤模型,以台盼蓝染色法、乳酸脱氢酶活性以及流式细胞术检测细胞损伤及凋亡情况;以RT-PCR和Western blotting方法,检测ERS标志分子葡萄糖调节蛋白78 (GRP78)、钙网蛋白 (CRT)、C/EBP同源蛋白 (CHOP)、caspase-12及凋亡相关蛋白Bcl-2、Bax的表达。结果: 和H/R组心肌细胞相比,PQS+H/R组:(1) 细胞凋亡率降低4.19% (P<0.05),存活率升高21.2% (P<0.05),LDH活性降低66.58% (P<0.05);(2) Bcl-2 mRNA和蛋白表达分别升高30.9%和48.0% (P<0.05),Bax mRNA和蛋白表达分别降低39.7%和48.4% (P<0.05);(3) GRP78 mRNA和蛋白表达分别降低61.6%和37.7% (P<0.05),CRT mRNA和蛋白表达分别降低35.7%和52.2% (P<0.05); CHOP mRNA和蛋白表达分别降低57.0%和51.7% (P<0.05);剪切后的caspase-12蛋白表达降低34.9% (P<0.05)。结论: PQS可减轻H/R诱导的心肌细胞损伤,其机制是降低H/R诱导的GRP78、CRT mRNA和蛋白表达,抑制CHOP、caspase-12等内质网凋亡通路激活,从而抑制过度ERS介导的细胞凋亡。     

β_1-肾上腺素受体自身抗体通过激活内质网应激诱导心肌细胞凋亡

目的:探讨内质网应激在β_1-肾上腺素受体自身抗体(β_1-AA)引起心肌细胞凋亡中的作用。方法:采用β_1-肾上腺素受体细胞外第二环抗原肽段主动免疫大鼠,应用SA-ELISA法检测大鼠血清中β_1-AA的水平,TUNEL染色检测心肌组织的凋亡水平,Western blot法和免疫组化法检测心肌组织中葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)及caspase-12的蛋白表达。利用亲和层析法纯化的β_1-AA处理H9c2心肌细胞,CCK-8法检测细胞活力,Annexin V-FITC/PI双染流式细胞术检测心肌细胞凋亡;采用内质网应激抑制剂4-苯基丁酸(4-PBA)预处理H9c2心肌细胞,再给予β_1-AA干预,CCK-8法和流式细胞术分别检测细胞活力及凋亡的变化情况。结果:与对照组相比,主动免疫大鼠血清中β_1-AA水平在免疫2周时显著增加,进一步增加至8周,并且主动免疫2周大鼠心肌组织凋亡率明显升高,持续升高至8周。与对照组相比,主动免疫大鼠心肌组织中GRP78、CHOP及caspase-12的蛋白表达在免疫4周和8周时均明显增加。β_1-AA引起H9c2心肌细胞活力持续降低,凋亡明显增加。与β_1-AA单独处理组相比,内质网应激抑制剂4-PBA预处理H9c2心肌细胞可以有效逆转β_1-AA诱导的细胞凋亡增加和活力下降。结论:β_1-AA可以通过激活内质网应激诱导心肌细胞凋亡。     

肿瘤坏死因子α通过PI3K-IP3R-Ca2+途径诱导乳鼠心肌肥大

 目的: 探讨肿瘤坏死因子α(TNF-α)是否通过PI3K-IP₃R-Ca²⁺信号途径诱导心肌肥大。方法: 以培养的乳鼠心肌细胞为模型,采用Lowry法测心肌细胞蛋白含量;[³H]-亮氨酸掺入法测定心肌细胞蛋白合成;计算机图像分析系统测心肌细胞体积;Till阳离子测定系统测定心肌细胞内Ca²⁺浓度。结果: PI3K阻断剂LY294002(50 μmol/L)明显抑制TNF-α(100 μg/L)诱导的心肌[Ca²⁺]_i增高(P<0.01),对正常心肌[Ca²⁺]_i无明显影响;其抑制程度与合用2-氨基乙基二苯硼酸盐(2-APB)组相近,但小于与兰尼碱(RYA)合用组(P<0.05)。PI3K阻断剂LY294002(50 μmol/L)明显抑制TNF-α(100 μg/L)诱导的心肌细胞蛋白含量、蛋白合成及细胞体积的增加;其抑制程度与合用2-APB组无差异,但明显大于单用2-APB组,小于合用RYA组(P<0.05)。PI3K阻断剂LY294002(50 μmol/L)对正常心肌细胞蛋白含量、蛋白合成及细胞体积无明显影响。结论: TNF-α通过PI3K-IP₃R-Ca²⁺途径诱导心肌肥大。     

柚皮素对心肌缺血/再灌注损伤大鼠PI3K/AKT信号通路和内质网应激及其相关凋亡通路的影响

目的:观察柚皮素对缺血/再灌注(I/R)大鼠心脏损伤的影响,并探讨柚皮素的作用是否涉及PI3K/AKT信号通路和内质网应激及其相关凋亡通路.方法:48只SD大鼠按随机数字表法分成假手术组(sham组)、模型组(I/R组)、柚皮素处理组(NAR组)和柚皮素处理+LY294002组(NL组).结扎大鼠冠状动脉左前降支30 ...     

Nerve Growth Factor Protects the Ischemic Heart via Attenuation of the Endoplasmic Reticulum Stress Induced Apoptosis by Activation of Phosphatidylinositol 3-Kinase

Background: Increased expression of nerve growth factor (NGF) has been found in the myocardium suffered from ischemia and reperfusion (I/R). The pro-survival activity of NGF on ischemic heart has been supposed to be mediated by phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Endoplasmic reticulum (ER) stress, which is activated initially as a defensive response to eliminate the accumulated unfolded proteins, has shown a critical involvement in the ischemia induced myocardial apoptosis. This study was aimed to investigate whether NGF induced heart protection against I/R injury includes a mechanism of attenuation of ER stress-induced myocardial apoptosis by activation of PI3K/Akt pathway.Methods: Isolated adult rat hearts were perfused with a Langendörff perfusion system. Hearts in the Sham group were subjected to 225 min of continuous Krebs-Henseleit buffer (KHB) perfusion without ischemia. Hearts in I/R group were perfused with KHB for a 75-min of equilibration period followed by 30 min of global ischemia and 120 min of KHB reperfusion. Hearts in the NGF group accepted 45 min of euilibration perfusion and 30 min of NGF pretreatment (with a final concentration of 100 ng/ml in the KHB) before 30 min of global ischemia and 120 min of reperfusion. Hearts in K252a and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 groups were pretreated with either a TrkA inhibitor, K252a or a phosphatidyl inositol 3-kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 for 30 min before NGF (100 ng/ml) administration. Cardiac hemodynamics were measured from the beginning of the perfusion. Cardiac enzymes and cardiac troponin I (cTnI) were assayed before ischemia and at the end of reperfusion. Myocardial apoptosis rate was measured by TUNEL staining, and expression of glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, total- and phospho-(Ser473)-Akt were assessed by Western blot analyses.Results: NGF pretreatment significantly improved the recovery of post-ischemia cardiac hemodynamics. Reduced creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) activity and cTnI levels, as well as decreased myocardial apoptosis ratio were observed in the NGF group. The improvement of NGF on recovery of cardiac function and alleviation of myocardial injury were completely abolished by K252a or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. GRP78, caspase-12 and CHOP were highly expressed in ischemic myocardium, while NGF significantly inhibited the overexpression of these proteins which were involved in ER stress-induced myocardial apoptosis. NGF pretreatment also induced phosphorylation of Akt. When the activation of PI3K/Akt pathway is blocked by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, the NGF induced suppression of the apoptosis-related proteins expression was reversed.Conclusions: NGF pretreatment may protect the ischemic heart via inhibition of the ER stress-induced apoptosis; this pro-survival effect is mediated by PI3K/Akt pathway.     

PERK-eIF2α-ATF4信号通路参与调控磷酸三钙磨损颗粒诱导的假体周围骨溶解

目的 探讨蛋白激酶R样内质网激酶（PERK）-真核细胞起始因子2α（eIF2α）-活化转录因子4（ATF4）介导的内质网应激通路在磷酸三钙（TCP）磨损颗粒诱导假体周围骨溶解中的作用。 方法 取雄性ICR小鼠30只,随机分为3组：假手术组（sham,n=10）、TCP磨损颗粒组（模型组,n=10）和salubrinal干预组(SAL,n=10)。采用TCP磨损颗粒诱导小鼠颅骨溶解模型,于术后第2天颅顶局部注射SAL（1 mg/kg）,每隔2 d 1次,持续干预2周。实验结束后处死动物取颅骨和外周血。Western blotting法检测TCP磨损颗粒植入部位周围骨组织中内质网应激分子伴侣葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）表达水平及PERK-eIF2α-ATF4信号通路的活化情况;HE染色和抗酒石酸酸性磷酸酶（TRAP）染色观察SAL对假体周围骨溶解和破骨细胞形成的影响;Real-time PCR检测SAL对破骨细胞活化相关基因抗酒石酸酸性磷酸酶（TRAP）、基质金属蛋白酶-9（MMP-9）和 c-fos的mRNA水平;ELISA法检测SAL对血清中肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）和前列腺素E₂（PGE₂）水平的影响。 结果 TCP磨损颗粒可诱导假体周围骨组织发生内质网应激反应,同时激活PERK-eIF2α-ATF4信号通路,表现为TCP磨损颗粒组小鼠颅骨组织中内质网应激通路蛋白GRP78、CHOP和磷酸化PERK（p-PERK）、磷酸化eIF2α（p-eIF2α）和ATF4蛋白表达均显著上调,而PERK和eIF2α蛋白明显下调（P<0.05）;而颅顶局部注射eIF2α特异性抑制剂SAL可明显阻止TCP磨损颗粒诱导假体周围破骨细胞形成和骨溶解,减少TRAP、MMP-9和cathepsin K 的mRNA水平（P<0.05）;同时抑制TNF-α、PGE2和IL-1β等炎症因子的产生（P<0.05）。 结论 PERK-eIF2α-ATF4介导的内质网应激通路参与调控TCP磨损颗粒诱导的小鼠颅骨溶解;抑制该信号通路可减轻TCP磨损颗粒诱导的假体周围骨溶解和关节松动。     

ABCG1 deficiency promotes endothelial apoptosis by endoplasmic reticulum stress-dependent pathway

The present study was focused on whether ABCG1 deficiency was involved in endothelial apoptosis and its possible mechanism. Human umbilical artery endothelial cells were transfected with ABCG1 siRNA and/or ABCG1 expression plasmid. We observed that silencing of endothelial ABCG1 reduced cholesterol efflux to HDL and increased intracellular lipid content. Moreover, reduction of ABCG1 promoted endothelial apoptosis and expression of endoplasmic reticulum (ER) stress-related molecules GRP78 and CHOP. In contrast, transfection of ABCG1 overexpression plasmid reversed endothelial apoptosis and intracellular lipid accumulation as well as decreased expression of GRP78 and CHOP in ABCG1-deficient endothelial cells. Furthermore, endothelial apoptosis and ER stress-related molecules were induced by repletion of endothelial cells with cholesterol-loaded cyclodextrin, otherwise endothelial apoptotic response and expression of GRP78 and CHOP were suppressed by depletion of cellular cholesterol in ABCG1-deficient endothelial cells. The present results suggest that reduction of ABCG1 induces endothelial apoptosis, which seems associated with intracellular free cholesterol accumulation and subsequent ER stress.     

4-苯基丁酸钠盐通过抑制内质网应激反应改善大鼠创伤性脑损伤

目的 观察内质网应激相关蛋白葡萄糖调控蛋白78(GRP78)、磷酸化胰腺内质网激酶(p-PERK)和C/EBP同源蛋白(CHOP)在大鼠弥漫性脑创伤后的表达变化,探讨4-苯基丁酸钠盐(4-PBA)通过抑制内质网应激,减轻创伤后脑损伤(TBI)程度的机制.方法 将90只雄性SD大鼠随机分为假手术(sham)组、TBI组和4-PBA组.Marmarou法建立SD大鼠中度弥漫性脑创伤模型;于伤后即刻腹腔注射4-PBA(120 mg/kg)每天1次,共3d.分别于伤后3、6、12、24、48和72 h处死大鼠,观察伤后24、48和72 h大鼠的神经行为表现;HE染色观察病理学改变;免疫组织化学法及Western blotting检测伤后不同时间点皮质区GRP78、p-PERK和CHOP蛋白的表达.结果 4-PBA组大鼠脑创伤后的神经功能缺损明显改善,与TBI组相比差异具有统计学意义(P<0.05).与Sham组相比,TBI组GRP78、p-PERK和CHOP蛋白表达明显升高(P<0.05),GRP78于伤后3 h增加,12 h达高峰,之后逐渐减少,72 h回落至基线水平;p-PERK于12 h达高峰(P<0.05);CHOP于24 h达高峰,48～72h回落,仍高于基线水平(P<0.05);4-PBA组GRP78、p-PERK与CHOP的表达均低于TBI组(P<0.05).结论 脑创伤后启动内质网应激反应,4-PBA对脑创伤大鼠具有脑保护作用,其机制之一是通过阻断内质网应激启动的PERK/CHOP途径而实现的.     

PHOSPHATIDYLINOSITOL 3-KINASE REGULATES PROLIFERATION OF RAW 264.7 MACROPHAGES

Phosphatidylinositol 3-kinase (PI3-kinase) is an enzyme that acts as a direct biochemical link between a novel phosphatidylinositol pathway and a number of proteins containing intrinsic or associated kinase activities. Here we demonstrate that wortmannin, PI3-kinase inhibitor, decreases the proliferation of RAW 264.7 macrophages and that another structurally unrelated inhibitor of PI3-kinase, LY294002, also inhibits the proliferation. These results indicate a possible involvement of PI3-kinase in RAW 264.7 macrophages growth regulation. Wortmannin stimulation of RAW 264.7 macrophages is followed by sustained expression of the mRNA of c-fos and a transient expression of the mRNA of c-jun. We also show that the wortmannin and LY294002 induce a cell cycle arrest in asynchronously growing cells leading to an inhibition of cell proliferation after 12 h of treatment. In addition, wortmannin or LY294002 inhibited the phorbol 12-myristate 13-acetate-induced macrophages proliferation potently. These results suggest that PI3-kinase plays an important role in growth regulation of RAW 264.7 macrophages and that protein kinase C is a down stream effector of PI3-kinase.     

PI3K/Akt调控内质网应激对GRP78的诱导

目的: 研究内质网应激条件下PI3K/Akt信号通路对HEK293细胞中葡萄糖调节蛋白78(GRP78)表达水平的调控作用。方法: 采用PI3K抑制剂LY294002、Akt1失活型突变载体Akt1(K179M)及Akt siRNAs阻断内质网应激介导的Akt活化,采用Akt激活型突变载体Myr-Akt过度激活内质网应激介导的Akt活化,并利用RT-PCR和Western blotting技术分析内质网应激条件下PI3K/Akt信号途径对HEK293细胞中GRP78表达水平的调控作用。结果: LY294002、Akt1(K179M)及Akt1 siRNA均明显抑制了内质网应激对GRP78的诱导。Myr-Akt1明显促进内质网应激对GRP78的诱导。Myr-Akt2/3及Akt2/3 siRNA对GRP78的诱导均无影响。PI3K/Akt信号通路阻断或过度激活对GRP78 mRNA水平的诱导无影响,但是对GRP78的降解有显著影响。结论: HEK293细胞中,PI3K/Akt通过蛋白稳定性调节促进内质网应激对GRP78的诱导。