海马神经元缺血/再灌注后自噬的表达及其作用

目的：研究自噬在大鼠海马神经元缺血缺氧/再灌注过程中的表达及自噬在神经元缺血缺氧/再灌注损伤中的作用。方法：原代培养的大鼠海马神经元经2 h的氧糖剥夺和不同时段的再灌注处理,MTT法检测细胞活性,透射电镜下检测自噬的特异性结构,免疫荧光化学法检测自噬特异性蛋白微管相关蛋白1轻链3（LC3B）的表达。应用自噬抑制剂3-甲基腺嘌呤（3-MA）检测神经元的活性。结果：经氧糖剥夺/再灌注后,海马神经元的活性比未经氧糖剥夺/再灌注组显著地降低。透射电镜和免疫荧光检测,未经氧糖剥夺/再灌注的神经元自噬的发生率极低,氧糖剥夺后和再灌注的不同时间段,均有自噬的发生。应用自噬抑制剂3-MA阻断自噬后,神经元的存活率显著降低。结论：缺血缺氧/再灌注能激活海马神经元的自噬,并可能在缺血缺氧/再灌注过程中起对抗损伤的作用。     

SIRT3在氧糖剥夺再灌注损伤小鼠神经元中的表达及意义

目的:通过建立体外脑缺血模型,探讨沉默信息因子3(SIRT3)在小鼠皮层神经元氧糖剥夺再灌注(OGD/R)损伤后的表达和意义。方法:C57BL/6J小鼠皮层神经元原代培养7天后,以氧糖剥夺不同时长(2 h、4 h、6 h、8 h)再灌注24 h作为观察时间点,利用细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)检测细胞活力;小鼠乳酸脱氢酶(LDH)试剂盒检测LDH释放;蛋白印迹法(Western blot WB)观察微管相关蛋白1轻链3(LC3-Ⅱ)、活化凋亡蛋白3(Cleaved caspase-3)、以及SIRT3的表达变化;免疫荧光下进一步观察LC3-II、SIRT3表达。结果:与正常组比,随着氧糖剥夺时间的延长,LDH释放量呈台阶式升高(P0.01),而神经元活性进展性下降(P0.01);蛋白印迹结果发现在缺血损伤后LC3-Ⅱ整体上调,并于OGD 4h达峰值,SIRT3分子表达趋势与LC3-Ⅱ相似均呈抛物线状,而Cleaved caspase-3整体上调;相应的,细胞免疫荧光结果显示缺血损伤后神经元胞体和突起中LC3呈点状高表达,与此同时SIRT3荧光强度亦增高。结论:神经元缺血时间越长损伤越重;LC3-Ⅱ和SIRT3表达呈现相似性;SIRT3可能通过调控线粒体自噬参与了拮抗神经元缺血损伤的作用。     

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。     

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。     

Beclin-1 shRNA对低氧诱导的SH-SY5Y细胞自噬的影响

目的：构建Beclin-1基因短发夹干扰RNA（shRNA）慢病毒载体,感染人SH-SY5Y细胞,观察沉默Beclin-1基因后低氧对SH-SY5Y细胞自噬的影响。方法：构建特异性靶向Beclin-1基因的shRNA慢病毒表达载体和阴性对照序列慢病毒载体;再将载体转染入SH-SY5Y细胞;RT-PCR检测Beclin-1的mRNA表达;Western blot检测Beclin-1蛋白表达;CCK-8法测定Beclin-1 shRNA对SH-SY5Y细胞活力的影响。再将空白对照、阴性对照、转染型三种细胞分别以21%常氧及5%低氧培养,Western blot检测各组细胞LC3蛋白表达;电镜观察自噬小体。结果：Beclin-1 shRNA能明显抑制SH-SY5Y细胞Beclin-1的mRNA及蛋白的表达;沉默Beclin-1基因后,Beclin-1 shRNA组细胞存活率与阴性对照组相比无差异;成功建立了稳定表达Beclin-1 shRNA的SH-SY5Y细胞。5%低氧处理后,与阴性对照组相比较,Beclin-1 shRNA组细胞中LC3Ⅱ/LC3Ⅰ比值下调,细胞内自噬小体数量减少。结论：慢病毒介导的Beclin-1shRNA对SH-SY5Y细胞的活力无影响,但可以抑制低氧诱导的自噬。     

过表达CYLD对氧糖剥夺/复氧大鼠原代皮质神经元中NF-κB信号通路的影响

脑卒中是目前导致我国人口寿命缩短的最主要原因之一。在缺血性脑卒中的局灶性缺血/再灌注后,受损脑组织中存在多种复杂的病理生理机制,核因子-κB(nuclear factor kappaB, NF-κB)信号通路参与的炎症反应是重要机制之一。相关研究显示,头帕肿瘤综合征蛋白(cylindromatosis, CYLD)可以参与NF-κB信号通路的调节。在脑缺血/再灌注损伤中,上调CYLD表达水平,对氧糖剥夺/复氧后NF-κB信号通路是否存在影响?如何影响?尚未见明确报道,这需要我们进一步探究。该研究通过上调CYLD在SD大鼠原代皮质神经元中的表达水平,观察其对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation, OGD/R)后神经元中NF-κB信号通路的影响。使用过表达慢病毒感染体外培养的原代皮质神经元,采用免疫荧光实验鉴定神经元, Western blot及RT-qPCR验证CYLD的过表达情况, CCK-8实验检测细胞活力, Western blot检测p-IκBα的蛋白表达情况, RT-qPCR检测NF-κB p65的mRNA表达情况。结果显示,过表达CYLD慢病毒可有效提高神经元中CYLD的表达水平;过表达CYLD后,较对照组相比,神经元在氧糖剥夺/复氧处理后的活力有所增高, p-IκBα的表达水平有所下降,同时NF-κB p65的m RNA表达水平也明显降低。研究结果表明,在原代皮质神经元中过表达CYLD,能减轻氧糖剥夺/再复氧对神经元的损伤、抑制NF-κB信号通路的激活。     

高压氧预处理对兔脊髓缺血再灌注损伤的保护机制探讨

目的:探讨高压氧预处理对免脊髓缺血再灌注损伤的保护机制.方法:20只雄性新西兰大白兔,随机分为2组(每组n=10):对照组为常压空气组;HBO组为高压氧预处理组.采用肾下腹主动脉阻断法造成脊髓缺血再灌注损伤,观察两组再灌注后4h、12h、24h、48h时的神经功能评分;再灌注48 h时取出腰段脊髓组织(L5-7)测定脊髓抗氧化酶活性(SOD、CAT及GSH-px)及MAD含量.结果:再灌注4h、12h、24h、48h时,HBO组神经功能学评分均明显优于对照组(P<0.05).再灌注48h时,HBO组脊髓匀浆SOD、CAT及GSH-px活性明显高于对照组(P<0.05),脊髓匀浆MDA含量明显低于对照组(P<0.05).相关性分析发现,再灌注48 h时后肢运动神经功能学评分与脊髓SOD、CAT及GSH-px活性呈正相关(r=0.82,0.65,0.54,P<0.05),与脊髓MDA含量呈负相关(r=-0.69,P<0.05).结论:高压氧预处理对脊髓缺血再灌注损伤的保护机制可能与上调内源性抗氧化酶活性和清除自由基有关.     

肝细胞生长因子对氧糖剥夺/再灌注神经元的保护作用

本文旨在探讨肝细胞生长因子（hepatocyte growth factor,HGF）对神经元氧糖剥夺/再灌注损伤的影响。取原代培养12d的Sprague-Dawley大鼠大脑皮层神经元,无糖、无氧（95%N2＋5%CO2）孵育2h后,换含25mmol/L葡萄糖的培养液、常氧培养0-24h,以MTT比色法检测细胞活力、乳酸脱氢酶（lactate dehydrogenase,LDH）漏出率作为细胞损伤指标,建立体外氧糖剥夺/再灌注损伤细胞模型;用流式细胞仪和Hoechst33258染色分析细胞凋亡率;用RT-PCR和Western blot分别检测大鼠脑皮层神经元HGF受体c-Met mRNA和蛋白的表达。于氧糖剥夺2h/再灌注24h处理前2h,加入不同终浓度（5-120ng/mL）的HGF,观察HGF对皮层神经元的影响。结果显示,c-Met表达于皮层神经元,氧糖剥夺2h/再灌注24h后,c-Met mRNA和蛋白表达均显著上调,神经元细胞活力明显降低,LDH漏出率和细胞凋亡率显著增高。HGF预处理明显促进氧糖剥夺/再灌注损伤神经元的存活,降低LDH漏出率,最大效应剂量为80ng/mL。流式细胞术和Hoechst33258染色结果均显示,HGF（80ng/mL）显著降低氧糖剥夺/再灌注神经元的细胞凋亡率。此外,c-Met抑制剂SU11274（5μmol/L）完全阻断HGF的神经保护作用。结果表明,HGF对皮层神经元氧糖剥夺/再灌注损伤具有直接的保护作用,呈一定的剂量依赖关系,并能有效对抗神经元凋亡。     

紫檀芪通过激活自噬缓解脊髓神经元细胞氧化应激损伤的作用及其机制

自噬在保护脊髓神经元细胞氧化应激损伤中具有重要的作用。紫檀芪(pterostilbene,PTE)是具有抗氧化作用的天然植物的提取物,但其对神经元细胞的作用及其机制尚不清楚。该文采用CCK-8分析PTE对大鼠原代脊髓神经元细胞的细胞毒性;不同浓度PTE作用神经元细胞24 h和48 h,透射电镜和Western blot检测微管相关蛋白轻链3-II(microtubule-associated protein 1 light chain3-II,MAP1LC3-II)、Beclin-1和P62蛋白质水平并分析自噬水平。PTE处理H2O2作用下的神经元细胞24 h,Western blot检测LC3-II水平,GFP-LC3转染观察自噬的数量。2′,7′-二氯二氢荧光素乙酰乙酸(2′,7′-dichloro-djhydrofl uorescein diacetate,DCFDA)和Mito SOX染色分析细胞活性氧(reactive oxygen species,ROS)水平,自噬相关基因5(autophagy related gene 5,ATG5)si RNA转染分析自噬在其中的作用。结果显示,20μmmol/L PTE对于神经元细胞无细胞毒性,PTE作用下神经元细胞中LC3-II、Beclin-1蛋白质水平呈剂量依懒性升高,而P62则呈剂量依懒性下降(P0.05)。PTE增加H2O2作用下的神经元细胞中LC3-II蛋白质水平(P0.05),自噬体数量增加,PTE可提高神经元细胞中自噬体数量。PTE明显降低神经元细胞中ROS水平,但ATG5 si RNA转染抑制自噬后显著逆转PTE的保护作用。该研究结果提示,PTE可能通过提高氧化应激状态下的脊髓神经元细胞自噬水平来抑制细胞ROS的产生。     

自噬对氧化应激引起的脊髓神经元细胞损伤的作用及其机制

氧化应激是脊髓损伤(spinal cord injury,SCI)后脊髓神经元细胞继发性损伤的重要机制,但是如何缓解氧化应激目前仍然不明确。该文培养SD(Sprague Dawley)大鼠原代脊髓神经元细胞,使用不同浓度的H-2O_2作用于神经元细胞12 h后,Western blot检测LC3-II、Beclin-1和P62蛋白质水平变化,分析自噬水平,电镜和绿色荧光蛋白标的记微管相关蛋白轻链3(green fluorescent proteinmicrotubule-associated protein 1 light chain 3,GFP-LC3)转染观察自噬的数量。ATG5 si RNA转染抑制自噬和雷帕霉素(rapamycin)促进细胞自噬,并使用CCK-8和TUNEL(terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling)染色分析自噬对氧化应激下神经元细胞的作用。结果显示,随着H-2O_2浓度增加,LC3-II和Beclin-1蛋白质水平显著升高,而P62蛋白质水平显著下降(P0.05)。透射电镜和共聚焦观察发现,10、50μmol/L H-2O_2可以增加神经元细胞中自噬体的数量。与对照组比较,H-2O_2明显抑制神经元细胞的活力(P0.05),ATG5 si RNA抑制自噬水平后,H-2O_2作用下的细胞活力进一步下降,而雷帕霉素促进自噬后却可以提高细胞的活力(P0.05)。TUNEL和膜联蛋白V/PI(annexin V/PI)染色结果发现,雷帕霉素可以抑制H-2O_2引起的神经元细胞凋亡(P0.05)。该研究结果提示,脊髓神经元细胞处于氧化应激状态下时,自噬代偿性激活并保护神经元细胞。     

Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis

Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increasedBcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.     

Preconditioning Stimuli Induce Autophagy via Sphingosine Kinase 2 in Mouse Cortical Neurons

Sphingosine kinase 2 (SPK2) and autophagy are both involved in brain preconditioning, but whether preconditioning-induced SPK2 up-regulation and autophagy activation are linked mechanistically remains to be elucidated. In this study, we used in vitro and in vivo models to explore the role of SPK2-mediated autophagy in isoflurane and hypoxic preconditioning. In primary mouse cortical neurons, both isoflurane and hypoxic preconditioning induced autophagy. Isoflurane and hypoxic preconditioning protected against subsequent oxygen glucose deprivation or glutamate injury, whereas pretreatment with autophagy inhibitors (3-methyladenine or KU55933) abolished preconditioning-induced tolerance. Pretreatment with SPK2 inhibitors (ABC294640 and SKI-II) or SPK2 knockdown prevented preconditioning-induced autophagy. Isoflurane also induced autophagy in mouse in vivo as shown by Western blots for LC3 and p62, LC3 immunostaining, and electron microscopy. Isoflurane-induced autophagy in mice lacking the SPK1 isoform (SPK1^−/−), but not in SPK2^−/− mice. Sphingosine 1-phosphate and the sphingosine 1-phosphate receptor agonist FTY720 did not protect against oxygen glucose deprivation in cultured neurons and did not alter the expression of LC3 and p62, suggesting that SPK2-mediated autophagy and protections are not S1P-dependent. Beclin 1 knockdown abolished preconditioning-induced autophagy, and SPK2 inhibitors abolished isoflurane-induced disruption of the Beclin 1/Bcl-2 association. These results strongly indicate that autophagy is involved in isoflurane preconditioning both in vivo and in vitro and that SPK2 contributes to preconditioning-induced autophagy, possibly by disrupting the Beclin 1/Bcl-2 interaction.     

Preconditioning with hyperbaric oxygen induces tolerance against oxidative injury via increased expression of heme oxygenase-1 in primary cultured spinal cord neurons

Hyperbaric oxygen (HBO) preconditioning can induce ischemic tolerance in the spinal cord. The effect can be attenuated by the administration of an oxygen free radical scavenger or by inhibition of antioxidant enzymes. However, the mechanism underlying HBO preconditioning of neurons against ischemic injury remains enigmatic. Therefore, in the present study primary cultured spinal cord neurons were treated with HBO and then subjected to a hydrogen peroxide (H(2)O(2)) insult. The results show that H(2)O(2) stimulation of the cultured spinal neurons caused severe DNA damage and decreased cell viability, and that these neurons were well protected against damage after a single exposure to HBO preconditioning (0.35 MPa, 98% O(2), 37 degrees C, 2 h). The protective effect started 4 h after pretreatment and lasted for at least 24 h. The cultured neurons after HBO treatment also exhibited increased heme oxygenase-1 (HO-1) expression at both the protein and mRNA levels, which paralleled the protective effect of HBO. Treatment with tin-mesoporphyrin IX (SnMP), a specific HO-1 inhibitor, before HBO pretreatment abolished the HBO-induced adaptive protection noted in the cultured spinal neurons. In conclusion, HBO preconditioning can protect primary cultured spinal cord neurons against oxidative stress, and the upregulation of HO-1 expression plays an essential role in HBO induced preconditioning effect.     

高压氧预处理对急性减压所致的大鼠肺组织细胞凋亡及Bcl-2/Bax表达的影响

目的:探讨高压氧预处理对减压病大鼠肺组织细胞凋亡的影响相关蛋白表达的影响。方法:雄性SD大鼠24只,随机分为3组,正常对照组(NC group)、HBO预处理组(HBOP group)、减压组(DCS group),每组8只。连续进行HBO预处理5天后进行减压病模型制备,取左侧肺组织进行湿干重比值测定,右侧肺组织用于病理实验;HE染色观察肺组织病理学改变,免疫组织化学法标记Bcl-2、Bax、Caspase-3与MMP-9阳性细胞表达,并对bcl-2/bax值进行分析。结果:减压组肺组织Bax、Caspase-3与MMP-9阳性细胞数明显增加(P0.05),而Bcl-2阳性细胞表达减少(P0.05);高压氧预处理组与减压组相比,Bax、Caspase-3与MMP-9阳性细胞数明显减少(P0.05),而Bcl-2阳性细胞表达增加(P0.05);大鼠肺组织减压组与高压氧预处理组Bcl-2/Bax值较对照组明显降低(P0.05);与减压组相比,高压氧预处理组明显升高(P0.05)。结论:HBO预处理可以减轻减压对肺组织的病理损伤,减轻肺泡和支气管上皮细胞的变性坏死,抑制细胞凋亡,从而起到对减压病的保护作用。     

Hydrogen sulfide protects spinal cord and induces autophagy via miR-30c in a rat model of spinal cord ischemia-reperfusion injury

Background

Hydrogen sulfide (H2S), a novel gaseous mediator, has been recognized as an important neuromodulator and neuroprotective agent in the nervous system. The present study was undertaken to study the effects of exogenous H2S on ischemia/reperfusion (I/R) injury of spinal cord and the underlying mechanisms.

Methods

The effects of exogenous H2S on I/R injury were examined by using assessment of hind motor function, spinal cord infarct zone by Triphenyltetrazolium chloride (TTC) staining. Autophagy was evaluated by expressions of Microtubule associated protein 1 light chain 3 (LC3) and Beclin-1 which were determined by using Quantitative Real-Time PCR and Western blotting, respectively.

Results

Compared to I/R injury groups, H₂S pretreatment had reduced spinal cord infarct zone, improved hind motor function in rats. Quantitative Real-Time PCR or Western blotting results showed that H2S pretreatment also downregulated miR-30c expression and upregulated Beclin-1 and LC3II expression in spinal cord. In vitro, miR-30c was showed to exert negative effect on Beclin-1 expression by targeting its 3’UTR in SY-SH-5Y cells treated with Oxygen, Glucose Deprivation (OGD). In rat model of I/R injury, pretreatment of pre-miR-30c or 3-MA (an inhibitor for autophagy) can abrogated spinal cord protective effect of H2S.

Conclusion

H2S protects spinal cord and induces autophagy via miR-30c in a rat model of spinal cord hemia-reperfusion injury.     

Hyperbaric Oxygen Preconditioning Induces Tolerance against Oxidative Injury and Oxygen-Glucose Deprivation by Up-Regulating Heat Shock Protein 32 in Rat Spinal Neurons

Objective

Hyperbaric oxygen (HBO) preconditioning (HBO-PC) has been testified to have protective effects on spinal cord injury (SCI). However, the mechanisms remain enigmatic. The present study aimed to explore the effects of HBO-PC on primary rat spinal neurons against oxidative injury and oxygen-glucose deprivation (OGD) and the relationship with heat shock proteins (HSPs).

Methods

Primary rat spinal neurons after 7 days of culture were used in this study. HSPs were detected in rat spinal neurons following a single exposure to HBO at different time points by Western blot. Using lactate dehydrogenase release assay and cell counting kit-8 assay, the injuries induced by hydrogen peroxide (H2O2) insult or OGD were determined and compared among neurons treated with HBO-PC with or without HSP inhibitors.

Results

The results of Western blot showed that HSP27, HSP70 and HSP90 have a slight but not significant increase in primary neurons following HBO exposure. However, HSP32 expression significantly increased and reached highest at 12 h following HBO exposure. HBO-PC significantly increased the cell viability and decreased the medium lactate dehydrogenase content in cultures treated with H2O2 or OGD. Pretreatment with zinc protoporphyrin IX, a specific inhibitor of HSP32, significantly blocked the protective effects of HBO-PC.

Conclusions

These results suggest that HBO-PC could protect rat spinal neurons in vitro against oxidative injury and OGD mostly by up-regulating of HSP32 expression.     

脊髓自噬功能激活与大鼠2型糖尿病神经病理性疼痛的关系

目的：探讨脊髓自噬功能与大鼠2型糖尿病神经病理性疼痛（DNP）的关系。方法：雄性SD大鼠（42只）高糖高脂饲养8周,腹腔单次注射链脲佐菌素（STZ）制备大鼠2型糖尿病模型。两周后检测机械缩足阈值（MWT）和热缩足潜伏期（TWL）,降至基础值80%以下者为2型糖尿病神经病理性疼痛大鼠,记为DNP组（24只）;未降至基础值80%以下者为2型糖尿病无神经病理性疼痛大鼠,记为DA组（18只）。另取18只大鼠为对照（control,C）组,普通饲料喂养。于确定DA与DNP分组后的第3、7和14天,测定机械缩足阈值（MWT）和热缩足潜伏期（TWL）,并在行为学检测结束后各组随机取6只大鼠处死,取L4~L6脊髓膨大,采用Western blot法检测自噬特异性蛋白微管相关蛋白1（Beclin-1）、微管相关蛋白1轻链3（LC3）和P62的表达。另取6只7 d DNP组大鼠采用免疫荧光双染法检测脊髓背角P62与小胶质细胞、星形胶质细胞、神经元的共表达情况。结果：连续8周喂养高糖高脂饲料的SD大鼠的血浆胰岛素水平升高,胰岛素敏感指数下调,表明出现胰岛素抵抗;在腹腔注射STZ后,血糖升高达到2型糖尿病诊断标准（≥16.7 mmol/L）;与C组、DA组比较,DNP组大鼠在第3、7和14天时MWT降低,TWL缩短,并且脊髓背角LC3-Ⅱ、Beclin-1表达上调,P62表达下降（P<0.05）。免疫荧光双染色显示,P62在脊髓背角表达,主要与神经元共存,少量与小胶质细胞共存,几乎不与星形胶质细胞共表达。结论：2型糖尿病神经病理性疼痛大鼠脊髓LC3-Ⅱ、Beclin-1和P62表达的改变提示脊髓自噬功能激活;脊髓背角中神经元自噬激活在2型糖尿病大鼠DNP的发生和发展起着关键作用。     

高压氧预处理对大鼠局灶性脑缺血再灌注损伤的保护作用

目的:研究高压氧预处理对大鼠脑缺血再灌注损伤的保护作用。方法:36只SD大鼠随机分为假手术组、模型组及高压氧预处理组,每组12只。高压氧预处理组大鼠在造模前5天给予高压氧预处理。采用线栓法建立大鼠脑缺血再灌注模型,观察高压氧预处理对脑缺血再灌注损伤大鼠神经功能缺损评分、脑梗死面积的影响,检测大鼠缺血脑组织COX-2 mRNA和蛋白的表达以及IL-1β、TNF-α、MDA的含量。结果:高压氧预处理可明显改善脑缺血再灌注大鼠神经功能缺损评分,减少脑梗死面积,降低COX-2m RNA和蛋白表达量,抑制IL-1β、TNF-α的表达,降低MDA水平。结论:高压氧预处理对大鼠脑缺血再灌注损伤具有明显的保护作用,其机制可能与抑制IL-1β、TNF-α、COX-2的表达以及减弱脂质过氧化反应有关。     

Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy

The hippocampal CA1 region is sensitive to hypoxic and ischemic injury but can be protected by ischemic preconditioning (IPC). However, the mechanism through which IPC protects hippocampal CA1 neurons is still under investigation. Additionally, the role of autophagy in determining the fate of hippocampal neurons is unclear. Here, we examined whether IPC induced autophagy to alleviate hippocampal CA1 neuronal death in vitro and in vivo with oxygen glucose deprivation (OGD) and bilateral carotid artery occlusion (BCCAO) models. Survival of hippocampal neurons increased from 51.5% ± 6.3% in the non-IPC group (55 min of OGD) to 77.3% ± 7.9% in the IPC group (15 min of OGD, followed by 55 min of OGD 24 h later). The number of hippocampal CA1 layer neurons increased from 182 ± 26 cells/mm² in the non-IPC group (20 min of BCCAO) to 278 ± 55 cells/mm² in the IPC group (1 min × 3 BCCAO, followed by 20 min of BCCAO 24 h later). Akt phosphorylation and microtubule-associated protein light chain 3 (LC3)-II/LC3-I expression were increased in the preconditioning group. Moreover, the protective effects of IPC were abolished only by inhibiting the activity of autophagy, but not by blocking the activation of Akt in vitro. Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group. The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA. In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC. These data suggested that IPC may attenuate ischemic injury in hippocampal CA1 neurons through induction of Akt-independent autophagy.