不同缺氧时间对神经细胞中外源性HSP70表达的影响

目的:探讨不同缺氧时间刺激对神经细胞中外源性HSP70基因表达的影响.方法:采用重组腺病毒vAd-HSP70感染体外原代培养的神经细胞,48h后给予感染细胞不同缺氧时间(缺氧0h,0.5h,1h,2h,3h,4h)刺激,再复氧处理后,用RT-PCR和Westernblotting检测重组腺病毒介导的HSP70基因在神经元和胶质细胞中的转录表达.结果:重组腺病毒vAd-HSP70感染的神经细胞可检测到外源性HSP70基因的转录表达.经缺氧再复氧处理后,随着缺氧时间延长,HSP70转录水平和蛋白表达都增加,缺氧1h时最高(1.1539±0.0315,0.9699±0.0023),其次是缺氧2h时(1.0398±0.0723,0.9622±0.0026),随后依次降低,缺氧4h组(0.7477±0.0328,0.9335±0.0034)HSP70表达最低,与其他各组比较有统计学意义(P<0.05).结论:缺氧2h内给予神经细胞再复氧处理外源性HSP70表达水平较高,有利于神经细胞功能的恢复.     

藏药镰形棘豆对缺氧/复氧H_9C_2心肌细胞损伤的保护作用

探讨藏药镰形棘豆(OFB)提取物对缺氧/复氧(H/R)H_9C_2心肌细胞损伤的保护作用。方法:采用三气培养箱培养法建立H_9C_2心肌细胞缺氧/复氧损伤模型。实验分为3组:正常细胞对照组;缺氧/复氧损伤组(H/R);7个药物加缺氧/复氧损伤组(H/R+OFB);于缺氧/复氧开始时分别加入终浓度为0.1 mg/L、1 mg/L、10 mg/L、100 mg/L、300 mg/L、600 mg/L、1 000 mg/L的镰形棘豆提取物,以细胞活力为指标,利用MTS法测定药物对细胞活力的影响。检测镰形棘豆乙酸乙酯水甲醇压柱组分与氯仿萃取组分不同给药剂量对H_9C_2心肌细胞缺氧/复氧损伤的影响。结果:与模型组相比药物处理组显著升高缺氧/复氧H_9C_2心肌细胞活力(p0.05)。结论:藏药镰形棘豆对H_9C_2心肌细胞的缺氧/复氧损伤有保护作用。     

番茄红素对大鼠乳鼠心肌细胞缺氧复氧的保护作用以及其机制

目的:探讨番茄红素对心肌细胞缺氧复氧的保护作用以及其分子机制。方法:采用原代培养心肌细胞建立缺氧/复氧损伤模型,实验分8组:正常对照组,H/R组,H/R+番茄红素(1,2,4,8,16,32μmol/L)剂量组。观察各组细胞经H/R损伤后,细胞内天冬氨酸氨基转移酶(AST)、肌酸激酶(CK)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量的变化情况,选择正常对照组,H/R组,最佳番茄红素剂量组做MTT分析细胞凋亡,Western检测TRL 4以及NF-κB的表达。结果:番茄红素(16,8,4,2μmol/L)剂量组可显著降低缺氧/复氧损伤心肌细胞内AST、CK、LDH释放量及MDA的生成,并能提高SOD活性。此外番茄红素可减少心肌细胞缺氧/复氧损伤后的心肌凋亡,减少TRL 4受体以及NF-κB的表达。结论:番茄红素具有抗缺氧/复氧损伤,保护心肌细胞的作用,其机制可能是通过抑制TRL 4通路来实现的。     

内质网应激预处理对人正常肝细胞缺氧再复氧损伤的保护作用

目的:研究内质网应激预处理对人肝细胞缺氧复氧损伤的保护作用。方法:将培养的人肝细胞分为4组:正常对照(C)组、细胞缺氧复氧损伤(H/R)组、内质网应激(ER)组、内质网应激预处理(ERP+H/R)组。收集各组细胞,以流式细胞仪检测细胞凋亡,Western-bloting及RT-PCR检测内质网应激特异蛋白GRP78表达水平,并通过透射电镜观察各组细胞超微结构改变。结果:ERP+H/R组细胞凋亡率明显低于H/R组(P<0.05),ER及ERP+H/R组GRP78蛋白表达明显高于H/R组(P<0.05)。结论:内质网应激预处理对肝细胞缺氧复氧损伤具有明显的保护作用,内质网应激特异性蛋白GRP78可能在肝细胞缺氧复氧损伤中作为一种关键性的保护蛋白出现。     

SIRT3抑制线粒体自噬并减轻高糖加重的神经元缺氧再灌注损伤*

目的:探讨SIRT3调控的线粒体自噬对高糖加重神经元缺氧再灌注损伤的影响及机制。方法:高糖(50 mmol/L)干预HT22细胞后,构建细胞缺氧/复氧模型,利用SIRT3抑制剂3-TYP抑制SIRT3表达。倒置显微镜观察细胞形态改变,CCK8法检测细胞存活率,流式细胞术检测细胞凋亡率,TMRE荧光试剂盒检测细胞线粒体膜电位,RT-qPCR、Western blot检测相关分子的基因和蛋白质表达。结果:高糖使神经元缺氧再灌注后的细胞碎片进一步增加,细胞存活率降低,细胞凋亡率升高(P<0.05)。此外,高糖降低了神经元缺氧再灌注后的线粒体膜电位(P<0.05)。进一步研究发现,高糖上调神经元缺氧再灌注后线粒体分裂相关蛋白DRP1的表达水平,降低了线粒体融合相关蛋白OPA1和线粒体外膜蛋白TOM20的表达;并且增加了自噬相关蛋白LC3Ⅱ、Beclin-1和线粒体自噬相关蛋白PINK1、Parkin的表达;同时,高糖升高了SIRT3的基因和蛋白质表达(P<0.05)。而SIRT3抑制剂3-TYP使神经元高糖缺氧再灌注损伤加重,同时进一步上调DRP1、LC3Ⅱ和PINK1的蛋白质表达(P<0.05)。结论:高糖可显著加重神经元缺氧再灌注损伤,破坏细胞线粒体功能,激活细胞线粒体自噬;SIRT3可抑制PINK1-Parkin通路介导的线粒体自噬并减轻神经元高糖缺氧再灌注损伤。     

13-甲基十四烷酸对大鼠脑皮层星形胶质细胞氧反常的影响

目的研究13-甲基十四烷酸(13-methyltetradecanoic Acid,13-MTD)对大鼠脑皮质星形胶质细胞氧反常的保护作用。方法传代培养新生SD乳鼠大脑皮质星形胶质原代细胞,以氧糖剥夺/再复氧糖(OGD/R)方法复制氧反常模型,OGD 10 h/R 24 h,于再复氧糖即刻分别给予13-MTD 20,40,80μg/m L(M20,M40,M80)干预,倒置显微镜动态观察星形胶质细胞形态,细胞免疫化学鉴定角质纤维酸性蛋白(glial fibrillary acidic protein,GFAP),MTT法检测线粒体活性,免疫组化法检测星形胶质细胞的水通道蛋白4(aquaporin 4,AQP4)蛋白表达。结果OGD 10 h/R 24 h损伤后,体外培养的SD乳鼠脑皮质星形胶质细胞出现明显损伤,线粒体活性显著下降(P0.01),星形胶质细胞膜AQP4蛋白表达量明显增加(P0.01);与模型组比较,13-MTD 20,40,80μg/m L可减少损伤,使线粒体活性上升、AQP4蛋白表达减少,以80μg/m L效果最好(P0.01)。结论 13-MTD可通过降低AQP4的表达,提高线粒体活性,减轻细胞水肿,进而保护氧反常诱导的星形胶质细胞损伤。     

缺氧复氧诱导自噬基因表达对滋养细胞生长的影响与机制

为探索子痫前期孕妇胎盘病变的发病机制,我们模拟体内缺血再灌注微环境,在体外建立胎盘滋养细胞HTR8/SVneo缺氧复氧模型,以探究缺氧复氧对细胞自噬的诱导作用及对细胞生长的影响。将实验分为对照组、缺氧复氧组及自噬抑制剂3-MA+缺氧复氧组,应用吖啶橙染色及LC3-Ⅱ免疫荧光染色检测经处理24 h后细胞自噬水平,MTT法检测细胞增殖能力,Real time PCR检测自噬基因Beclin-1、LC3-Ⅱ的表达,Western blot分析相应自噬蛋白的表达。结果显示缺氧复氧组HTR8/SVneo细胞自噬水平明显升高(p0.01),伴随Beclin-1、LC3-Ⅱ基因及蛋白表达显著增高(p0.01),细胞增殖同时显著受抑(p0.01),而加入3-MA后缺氧复氧组细胞自噬水平明显受抑(p0.01),Beclin-1、LC3-Ⅱ基因及蛋白表达显著下降(p0.01),细胞增殖能力显著提高(p0.01)。这表明滋养细胞HTR8/SVneo在缺氧复氧环境中启动细胞自噬,过度自噬时可能通过诱导Ⅱ型程序性死亡影响细胞增殖,当自噬被抑制后,细胞增殖能力明显恢复。     

线粒体大麻素受体1在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响

目的:探讨线粒体CB1受体(mitochondrial cannabinoid receptor1,mtCB1)在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响。方法:原代培养新生的Wistar大鼠海马神经元,将培养至第8天的海马神经元采用随机数字表分为5组(n=60):正常组(N组):正常培养,不做任何处理;缺氧复氧组(H/R组):采用氧糖剥夺法构建海马神经元缺氧复氧损伤模型,缺氧6h,复氧20 h;缺氧复氧组+ACEA+AM251组(H/R+ACEA+AM251组):缺氧6 h结束后立即加入ACEA和AM251,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+ACEA+Hemopressin(H/R+ACEA+Hemo组):缺氧6h结束后立即加入ACEA和Hemopressin,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+赋形剂组(H/R+V组):同样于缺氧6h结束后立即加入二甲基亚砜(DMSO),终浓度0.1%,复氧20 h。使用激光共聚焦显微镜检测细胞内Ca~(2+)的浓度,流式细胞仪检测细胞凋亡率,Western blot检测凋亡诱导因子(AIF)、线粒体分裂相关蛋白Drp1、Fis1,细胞凋亡相关蛋白细胞色素C(Cytc)和Rho相关的卷曲蛋白激酶1(ROCK1)的表达。结果:与N组相比,H/R组、H/R+ACEA+AM251组、H/R+ACEA+Hemo组和H/R+V组的细胞内Ca~(2+)浓度、细胞凋亡率、以及AIF、Drp1、Fis1、Cytc、ROCK1蛋白的表达水平均明显增加(P0.05);与H/R组相比,H/R+ACEA+Hem组上述各检测指标明显降低(P0.05),H/R+ACEA+AM251组和H/R+V组各指标比较差异无统计学意义(P0.05)。结论:线粒体CB1受体(mtCB1受体)可能通过降低细胞内ROS的含量来减少细胞内Ca~(2+)浓度和ROCK1的表达,进而抑制线粒体分裂,并最终减轻海马神经元缺氧复氧损伤。     

黄芪甲苷后处理对乳鼠心肌细胞缺氧复氧损伤的作用研究

目的:观察黄芪甲苷(AstragalosideⅣ,AsⅣ)后处理对缺氧复氧损伤(simulated ischemia reperfusion injury,SI/RI)的SD乳鼠心肌细胞是否具有保护作用。方法:将乳鼠原代心肌细胞平均分为五组,即空白对照组(Control)、缺氧复氧处理组(SI/RI)、黄芪甲苷预处理(5,10、20μM)+SI/RI组(AsIV+SI/RI)。各组细胞经处理后,四氮唑溴盐比色法(MTT)检测各组细胞存活率;TUNEL染色法测定各组细胞凋亡率;SOD测试盒检测培养液中超氧化物歧化酶(SOD)含量,总嘌呤氧化酶(XOD)测试盒检测丙二醛(MDA)含量。Western blot法检测各组细胞抗凋亡蛋白Bcl-2和促凋亡蛋白Caspase-3的表达。结果:与空白组相比,缺氧复氧损伤组细胞活力显著下降(P0.05),凋亡率显著上升(P0.05),其培养液中SOD水平显著降低(P0.05),MDA水平显著升高。而不同浓度AsⅣ后处理组的心肌细胞存活率显著上升,凋亡率显著下降,培养液中SOD水平显著上升,MDA水平显著下降(P0.05),且呈浓度呈依赖性。Western blot结果显示AsⅣ后处理组细胞中的Bcl-2表达明显上升,Caspase-3明显下降。结论:黄芪甲苷后处理对缺氧复氧诱导的乳鼠心肌细胞损伤具有显著的保护作用,能够显著上调抗凋亡蛋白Bcl-2的表达,下调促凋亡蛋白Caspase-3的表达。     

缺氧复氧心肌细胞中HSP90对AKT表达的调控作用

研究新生大鼠缺氧复氧心肌细胞中热休克蛋白90(HSP90)的表达对AKT表达的影响,探讨HSP90在PI3K/AKT信号通路中的作用。方法:建立新生大鼠心肌细胞缺氧复氧模型,通过运用HSP90阻断剂Geldanamycin(GA),分另以MTT法检测心肌细胞的活力、透射电镜观察心肌细胞超微结构改变、Western印迹法分析大鼠心肌细胞中HSP90表达变化与总AKT蛋白的相关性。结果:缺氧复氧心肌细胞中HSP90和AKT表达量均有明显升高,阻断HSP90后,AKT表达量明显下降,此时心肌细胞活力明显下降,细胞超微结构受损明显。结论:AKT对缺氧复氧引起的心肌细胞损伤有内源性保护作用,此作用的发挥与HSP90和AKT的结合密切相关,HSP90对缺氧复氧中的心肌细胞AKT表达有显著影响。     

Reoxygenation-induced constriction in murine coronary arteries: the role of endothelial NADPH oxidase (gp91phox) and intracellular superoxide

Previous work suggests that superoxide mediates hypoxia/reoxygenation (H/R)-induced constriction of isolated mouse coronary arteries (CA). To determine the source of superoxide overproduction during H/R we studied CA obtained from transgenic (Tg) mice overexpressing human CuZn-superoxide dismutase (SOD) and mice lacking gp91(phox) using an in vitro vascular ring bioassay. We found that under normoxic conditions CA isolated from wild type (wt) mice, CuZn-SOD Tg mice and gp91(phox) knock-out mice had similar contractile responses to U46619 and hypoxia and similar dilation responses to acetylcholine. In wt CA, 30 min of hypoxia (1% O(2)) followed by reoxygenation (16% O(2)) resulted in further coronary vasoconstriction (internal diameter from 105 +/- 11 to 84.5 +/- 17.9 microm), whereas this response was completely blocked in both CuZn-SOD Tg and gp91(phox) knock-out CA (104.3 +/- 10.5 to 120.7 +/- 14 microm and 143.3 +/- 15.3 to 172.7 +/- 12.5 microm, respectively, p < 0.01). Furthermore, we show that H/R enhances the generation of superoxide radicals in wt CA (25.8 +/- 0.7 relative light units per second (RLU/s)), whereas CuZn-SOD Tg CA (12.2 +/- 0.8 RLU/s, p < 0.01) and gp91(phox) CA (12.5 +/- 0.9 RLU/s, p < 0.01) show reduced levels. These results demonstrate that H/R-induced vasoconstriction is mediated by intracellular superoxide overproduction via endothelial NADPH oxidase gp91(phox). Therefore, increasing endogenous levels of CuZn-SOD in CA may provide a novel cardioprotective strategy for maintaining coronary perfusion under conditions of H/R.     

Cutting edge: infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox

The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-). However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.     

Role of components of the phagocytic NADPH oxidase in oxygen sensing.

It has been hypothesized that O(2) sensing in type I cells of the carotid body and erythropoietin (EPO)-producing cells of the kidney involves protein components identical to the NADPH oxidase system responsible for the respiratory burst of phagocytes. In the present study, we evaluated O(2) sensing in mice with null mutant genotypes for two components of the phagocytic oxidase. Whole body plethysmography was used to study unanesthetized, unrestrained mice. When exposed to an acute hypoxic stimulus, gp91(phox)-null mutant and wild-type mice increased their minute ventilation by similar amounts. In contrast, p47(phox)-null mutant mice demonstrated increases in minute ventilation in response to hypoxia that exceeded that of their wild-type counterparts: 98.0 +/- 18.0 vs. 20.0 +/- 13.0% (n = 11, P = 0.003). In vitro recordings of carotid sinus nerve (CSN) activity demonstrated that resting (basal) neural activity was marginally elevated in p47(phox)-null mutant mice. With hypoxic challenge, mean CSN discharge was 1.5-fold greater in p47(phox)-null mutant than in wild-type mice: 109.61 +/- 13.29 vs. 72.54 +/- 7.65 impulses/s (n = 8 and 7, respectively, P = 0.026). Consequently, the hypoxia-evoked CSN discharge (stimulus-basal) was approximately 58% larger in p47(phox)-null mutant mice. Quantities of EPO mRNA in kidney were similar in gp91(phox)- and p47(phox)-null mutant mice and their respective wild-type controls exposed to hypobaric hypoxia for 72 h. These findings confirm the previous observation that absence of the gp91(phox) component of the phagocytic NADPH oxidase does not alter the O(2)-sensing mechanism of the carotid body. However, absence of the p47(phox) component significantly potentiates ventilatory and chemoreceptor responses to hypoxia. O(2) sensing in EPO-producing cells of the kidney appears to be independent of the gp91(phox) and p47(phox) components of the phagocytic NADPH oxidase.     

Gp91phox contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide (O2-*) levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in O2-* levels between adventitial cells and medial SMC in aorta from gp91phox-deficient (gp91phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91phox KO and wild-type FB to account for these observations. Because gp91phox is associated with membranes, we examined NADPH-stimulated O2-. production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91phox in FB, antisense to gp91phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91phox did not alter expression of the gp91phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91phox contributes to NADPH oxidase function in vascular FB, but not SMC.     

Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.     

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.