西洛他唑对大鼠脑微血管内皮细胞缺血再灌注损伤的保护作用

目的研究西洛他唑对大鼠脑微血管内皮细胞缺血再灌注损伤的保护作用机制。方法以原代方法培养大鼠脑微血管内皮细胞并传代,将第3代传代细胞随机分为正常对照组、西洛他唑组、溶剂对照组和缺血再灌注模型组(再灌模型组)4组,对后3组大鼠建立脑微血管内皮细胞糖氧剥夺后复糖氧模型,模拟"缺血再灌注"过程,并对西洛他唑组和溶剂对照组在造模同时分别以终浓度1×10-5mmol/L西洛他唑〔二甲基亚砜(DMSO)为溶剂〕和0.05%(体积分数)DMSO进行干预。糖氧剥夺3 h复糖氧24 h后,测定各组细胞上清液中诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)水平及细胞内环磷腺苷酸(cAMP)水平,并以四唑盐(MTT)比色实验测定细胞活力。结果西洛他唑组与再灌模型组比较,eNOS水平明显提高,iNOS水平明显降低,cAMP水平及细胞存活率显著提高(均P<0.05)。结论西洛他唑对大鼠脑微血管内皮细胞的缺血再灌注损伤有保护作用,其作用机制可能与促进eNOS水平升高和iNOS水平降低有关。     

西洛他唑对大鼠皮层细胞糖氧剥离损伤的保护作用

目的探讨磷酸二酯酶抑制剂西洛他唑对糖氧剥离后大鼠皮层细胞培养的影响及作用机制。方法原代混合培养大鼠皮层细胞,建立糖氧剥离的细胞损伤模型模拟细胞"缺血损伤",然后进行干预。测定细胞培养上清中乳酸脱氢酶(LDH)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、神经元型一氧化氮合酶(nNOS)及诱导型一氧化氮合酶(i NOS)的含量;测定一氧化氮(NO)的分泌水平;测定细胞内环磷酸腺苷(cAMP)水平及四唑盐(MTT)比色试验测定细胞活力。结果西洛他唑组及依达拉奉组与糖氧剥离模型组比较,LDH、MDA漏出量显著减少(P均0.05),GSH-Px释放量明显升高(P均0.05),nNOS、i NOS的水平及NO的分泌量显著下降(P均0.05),细胞内cAMP水平明显升高(P均0.05);细胞存活率显著提高(P均0.05);西洛他唑与依达拉奉组比较,LDH、MDA漏出量及GSH-Px的释放量无差别,nNOS、i NOS和NO的水平明显降低(P均0.05),细胞内cAMP水平显著升高(P0.05);细胞存活率明显提高(P0.05)。结论西洛他唑对培养大鼠皮层细胞在糖氧剥离损伤中具有保护作用,其作用机制可能通过抗氧化、降低nNOS及i NOS的水平从而降低NO的分泌、升高细胞内cAMP水平来实现的。     

依达拉奉预处理对小鼠脑缺血再灌注损伤后皮质一氧化氮合酶表达的影响

目的探讨依达拉奉预处理对小鼠脑缺血再灌注(IR)损伤后皮质一氧化氮合酶(NOS)表达的影响。方法 48只健康ICR小鼠被分为假手术组、对照组和依达拉奉组。依达拉奉组和对照组分别给予依达拉奉3 mg/(kg.d)和同等体积的生理盐水腹腔注射共7 d,然后建立小鼠IR模型;缺血1 h、再灌注24 h时应用2,3,5-氯化三苯基四氮唑(TTC)染色法测量各组脑梗死体积,应用免疫组化法检测各组小鼠皮质神经元型、、诱导型和内皮型NOS(nNOS、iNOS、eNOS)阳性细胞数。结果与假手术组比较,对照组小鼠皮质nNOS、iNOS和eNOS阳性细胞数明显增多(均P<0.05);与对照组比较,依达拉奉组脑梗死体积明显缩小,皮质nNOS和iNOS阳性细胞数明显减少,eNOS阳性细胞数明显增多(均P<0.05)。结论依达拉奉预处理可以影响IR小鼠皮质nNOS、iNOS和eNOS的表达,发挥神经保护作用。     

iNOS和OPN在EAE中动态表达及依达拉奉保护作用研究

目的研究诱导型一氧化氮合酶(iNOS)和骨桥蛋白(OPN)在实验性自身免疫性脑脊髓炎(EAE)中的动态表达以及依达拉奉治疗和保护作用机制的探讨。方法将EAE大鼠随机分为实验组和依达拉奉治疗组,根据发病时间又分为发病前组、高峰期组和缓解期组。光镜下观察脊髓HE染色炎性细胞浸润情况,观察免疫组化染色iNOS和OPN阳性细胞数目。结果依达拉奉治疗组与实验组比较,发病时间延迟、发病率降低及神经功能评分减低(P<0.05)。依达拉奉治疗组脊髓炎症细胞浸润较实验组减少。免疫组化染色iNOS和OPN阳性细胞在EAE发病前期上升,高峰期达到峰值,缓解期随疾病好转而下降,依达拉奉组均较同时期实验组阳性细胞表达数目减少(P<0.05)。结论依达拉奉可以减轻EAE大鼠临床发病程度和病理炎症损害,并降低不同发病时期iNOS和OPN表达程度。推测依达拉奉通过抗氧化和抗炎的双重机制发挥神经保护作用。     

依达拉奉对大鼠延髓缺血后神经元神经胶质细胞及微血管的影响

目的探索依达拉奉对大鼠延髓缺血后神经元、神经胶质细胞及微血管的影响。方法将Wistar大鼠分为假手术组、实验组、缺血对照组。其中实验组及缺血对照组,分别给予依达拉奉和生理盐水腹腔注射。标本采用HE染色、单宁酸氯化铁染色,对延髓内微血管密度、神经元计数、神经胶质细胞计数进行观察。结果实验组中神经元数量、微血管密度(MVD)减少的程度及神经胶质细胞增加的程度低于缺血对照组。结论在大鼠延髓缺血后依达拉奉治疗具有明显保护作用。     

依达拉奉对大鼠脑缺血后海马微血管密度NGF、BDNF表达的影响

目的探索依达拉奉对大鼠脑缺血后海马微血管密度、神经生长因子(NGF)、脑源性神经生长因子(BDNF)表达的影响。方法将Wistar大鼠分为假手术组、依达拉奉治疗组(以下简称治疗组)、缺血对照组。采用电凝右侧椎动脉和结扎双侧颈总动脉的方法制造大鼠脑缺血模型。分别给予治疗组和缺血对照组依达拉奉及等计量生理盐水腹腔注射。大鼠于相应时段灌注取材,每组标本分别采用单宁酸-氯化铁法染色微血管,免疫组织化学方法显示脑组织中的BDNF和NGF。光镜下观察脑组织中微血管密度、NGF和BDNF的变化,利用灰度值进行分析。结果微血管密度、NGF、BDNF灰度值治疗组均低于缺血对照组。结论 NGF、BDNF水平与微血管密度密切相关;依达拉奉可上调大鼠缺血脑组织中NGF、BDNF的表达,减轻脑组织损伤。     

依达拉奉对脑缺血再灌注大鼠海马一氧化氮的影响

目的研究依达拉奉对脑缺血再灌注大鼠海马一氧化氮(NO)产生的影响。方法大鼠脑缺血采用四血管阻断法,选择性测定电极检测的浓度。实验分为生理盐水组、依达拉奉组(Edaravone)和7-Nitroindazole(7-NI)组。结果依达拉奉和7-NI皆未影响大鼠的血压和海马血流量,均显著减少了缺血再灌注时海马内NO的产生(均P<0.001)。结论依达拉奉可能通过抑制神经型一氧化氮合酶(nNOS)或减少NO而起到神经保护作用。     

依达拉奉对大鼠延髓缺血后神经元凋亡微血管密度的影响

目的探索依达拉奉对大鼠延髓缺血后神经元数量及微血管密度的影响。方法将Wistar大鼠分为假手术组、实验组、缺血对照组。其中实验组及缺血对照组分别给予依达拉奉和生理盐水腹腔注射。标本采用单宁酸氯化铁染色、尼氏染色、Tunel染色,对延髓内微血管密度、神经元及凋亡、神经元计数进行观察。结果实验组中神经元、微血管密度(MVD)减少的程度及神经元凋亡数量均低于缺血对照组。结论依达拉奉在大鼠延髓缺血后具有明显保护作用。     

依达拉奉对大鼠延髓缺血后超氧化物歧化酶、丙二醛的影响

目的 探讨依达拉奉对大鼠延髓缺血中的影响.方法 将Wistar大鼠分为假手术组、依达拉奉干预组(干预组)、延髓缺血模型组(模型组),采用多点阻断脑动脉方法制造延髓缺血模型.依达拉奉干预组腹腔注射依达拉奉;延髓缺血模型组腹腔注射生理盐水.分别在实验7d、14d时取延髓,检测脑组织的含水量、SOD、MDA水平.结果 延髓缺血模型组脑组织SOD水平下降,脑组织MDA浓度先升高后降低;与延髓缺血模型组相比,干预组SOD下降的幅度小(P＜0.01),MDA浓度降低;干预组脑组织含水量明显低于延髓缺血模型组(P＜0.01).结论 依达拉奉有效清除自由基,对延髓缺血具有明显保护作用.     

依达拉奉和尼莫地平联合应用对脑缺血大鼠脑组织血管内皮生长因子表达的影响

目的 探讨依达拉奉与尼莫地平联合应用对脑缺血大鼠脑组织血管内皮生长因子(VEGF)表达的影响.方法 90只Wistar大鼠随机分为5组:正常对照组、脑缺血组、依达拉奉组、尼莫地平组和依达拉奉+尼莫地平组(依尼组),各组又分为缺血12 h、24 h、48 h亚组.用线栓法制作大鼠大脑中动脉堵塞(MCAO)脑缺血模型.制模后1 h,各药物干顶组分别注射依达拉奉3 mg/kg、尼莫地平2 mg/kg和依达拉奉+尼莫地平,正常对照组和脑缺血组大鼠分别腹腔注射生理盐水.各组大鼠分别于预定时间点处死,用逆转录-聚合酶链反应(RT-PCR)法检测脑组织VEGFmRNA水平.结果 各组缺血24 h、48 h亚组脑组织VEGF mRNA水平明显低于正常对照组(均P＜0.01),各药物干预组脑组织VEGF mRNA水平显著高于脑缺血组(均P＜0.01);依尼组脑组织VEGF mRNA水平又显著高于依达拉奉组和尼莫地平组(均P＜0.01).结论 依达拉奉和尼莫地平能上调缺血脑组织VEGF的表达水平,联合应用效果更好.     

Blockade of phosphodiesterase-III protects against oxygen-glucose deprivation in endothelial cells by upregulation of VE-cadherin

We recently reported that a phosphodiesterase-III inhibitor, cilostazol, prevented the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tissue plasminogen activator (tPA) and that it reversed tPA-induced cell damage by protecting the neurovascular unit, particularly endothelial cells. However, the mechanisms of cilostazol action are still not clearly defined. The adheren junction (AJ) protein, VE-cadherin, is a known mediator of endothelial barrier sealing and maintenance. Therefore, we tested whether cilostazol might promote expression of adhesion molecules in endothelial cells, thereby preventing deterioration of endothelial barrier functions. Human brain microvascular endothelial cells were exposed to 6-h oxygen-glucose deprivation (OGD). We compared cilostazol with aspirin treatments and examined 2 representative AJ proteins: VE-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1). A protein kinase A (PKA) inhibitor, LY294002 (a PI3-K inhibitor), db-cAMP, and RP-cAMPS were used to assess the roles of cAMP, PKA, and PI3-K signaling, respectively, in cilostazol-induced responses. Cilostazol and db-cAMP prevented OGD-stress injury in endothelial cells by promoting VE-cadherin expression, but not PECAM-1. Aspirin did not prevent cell damage. P13-K inhibition by LY294002 had no influence on the effects of cilostazol, but inhibition of cAMP/PKA with PKA inhibitor and Rp-cAMPS suppressed cilostazol-induced inhibition of cell damage and promotion of VE-cadherin expression. In contrast, OGD stress had no detectable effects on VEGF, VEGF receptor, or angiopoietin-1 levels. Cilostazol promotes VE-cadherin expression through cAMP/PKA-dependent pathways in brain endothelial cells; thus, cilostazol effects on adhesion molecule signaling may provide protection against OGD stress in endothelial cells.     

Cilostazol attenuates ischemia–reperfusion-induced blood–brain barrier dysfunction enhanced by advanced glycation endproducts via transforming growth factor-β1 signaling

We investigated the effects of cilostazol, a selective inhibitor of phosphodiesterase 3, on blood–brain barrier (BBB) integrity against ischemia–reperfusion injury enhanced by advanced glycation endproducts (AGEs). We used in vitro BBB models with primarily cultured BBB-related cells from rats (brain capillary endothelial cells, astrocytes and pericytes), and subjected cells to either normoxia or 3-h oxygen glucose deprivation (OGD)/24-h reoxygenation with or without AGEs. Treatment of AGEs did not affect the transendothelial electrical resistance (TEER) in the BBB model under normoxia, but there was a significant decrease in TEER under 3-h OGD/24-h reoxygenation conditions with AGEs. Cilostazol inhibited decreases in TEER induced by 3-h OGD/24-h reoxygenation with AGEs. Immunocytochemical and Western blot analyses showed that AGEs reduced the expression of claudin-5, the main functional protein of tight junctions (TJs). In contrast, cilostazol increased the expression of claudin-5 under 3-h OGD/24-h reoxygenation with AGEs. Furthermore, while AGEs increased the production of extracellular transforming growth factor (TGF)-β1, cilostazol inhibited the production of extracellular TGF-β1 and restored the integrity of TJs. Thus, we found that AGEs enhanced ischemia–reperfusion injury, which mainly included decreases in the expression of proteins comprising TJs through the production of TGF-β1. Cilostazol appeared to limit ischemia–reperfusion injury with AGEs by improving the TJ proteins and inhibiting TGF-β1 signaling.     

LncRNA SNHG12 ameliorates brain microvascular endothelial cell injury by targeting miR-199a

Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation(OGD/R) insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pc DNA-control, pc DNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.     

The rapid decrease in astrocyte-associated dystroglycan expression by focal cerebral ischemia is protease-dependent.

During focal cerebral ischemia, the detachment of astrocytes from the microvascular basal lamina is not completely explained by known integrin receptor expression changes. Here, the impact of experimental ischemia (oxygen-glucose deprivation (OGD)) on dystroglycan expression by murine endothelial cells and astrocytes grown on vascular matrix laminin, perlecan, or collagen and the impact of middle cerebral artery occlusion on alphabeta-dystroglycan within cerebral microvessels of the nonhuman primate were examined. Dystroglycan was expressed on all cerebral microvessels in cortical gray and white matter, and the striatum. Astrocyte adhesion to basal lamina proteins was managed in part by alpha-dystroglycan, while ischemia significantly reduced expression of dystroglycan both in vivo and in vitro. Furthermore, dystroglycan and integrin alpha6beta4 expressions on astrocyte end-feet decreased in parallel both in vivo and in vitro. The rapid loss of astrocyte dystroglycan during OGD appears protease-dependent, involving an matrix metalloproteinase-like activity. This may explain the rapid detachment of astrocytes from the microvascular basal lamina during ischemic injury, which could contribute to significant changes in microvascular integrity.     

丹参酮ⅡA通过抑制TLR4/NF-κB途径减轻糖氧剥夺对大鼠脑微血管内皮细胞的炎症损伤

目的 探讨丹参酮ⅡA对大鼠脑微血管内皮细胞糖氧剥夺炎症损伤的影响及其机制。方法 原代培养雄性SD大鼠脑微血管内皮细胞，分为对照组、糖氧剥夺组和丹参酮ⅡA组。对照组用DMEM培养，糖氧剥夺组细胞用无糖DMEM培养，丹参酮ⅡA组用在无糖DMEM基础上加用含8 μg/ml丹参酮ⅡA培养。利用CCK8检测细胞活力，酶联免疫吸附试验检测细胞炎症因子IL-6和TNF-α水平；实时PCR和免疫印迹法检测细胞TLR4和NF-κB mRNA和蛋白表达水平。结果 与对照组相比，糖氧剥夺组细胞活力明显降低（P<0.05），IL-6和TNF-α水平明显增加（P<0.05），TLR4和NF-κB mRNA和蛋白表达均明显增加（P<0.05）；丹参酮ⅡA明显抑制糖氧剥夺作用（P<0.05），但未恢复到对照组水平（P<0.05）。结论 丹参酮ⅡA可减轻糖氧剥夺对大鼠脑微血管内皮细胞的炎症损伤，其作用机制可能与抑制TLR4-NF-κB信号通路有关     

MicroRNA-143-3p对缺糖缺氧的大鼠脑微血管内皮细胞的保护作用及机制

目的 探讨MicroRNA-143-3p(miR-143-3p)对缺糖缺氧的大鼠脑微血管内皮细胞的保护作用及机制。方法 建立大鼠脑微血管内皮细胞(rBMECs)缺糖缺氧(OGD)损伤模型,分为正常培养组和OGD组,qRT-PCR检测各组不同时间rBMECs中miR-143-3p的相对表达水平; 干扰rBMECs中miR-143-3p表达的实验将细胞分为正常培养组、OGD组、NC inhibitor+OGD组和miR-143-3p inhibitor+OGD组,qRT-PCR检测各组细胞中miR-143-3p相对表达水平; MTT、流式细胞术分别检测各组细胞的增殖、周期和凋亡情况。结果 与正常培养组比较,OGD组OGD处理2、4、6、8和10 h后rBMECs中miR-143-3p相对表达水平明显上调,且呈明显上升趋势; 转染miR-143-3p inhibitor能够显著降低OGD条件下rBMECs中miR-143-3p相对表达水平; 与正常培养组比较,OGD组细胞的增殖能力、周期转化能力显著降低,而细胞凋亡比例显著增加; 与NC inhibitor+OGD组比较,miR-143-3p inhibitor+OGD组细胞的增殖能力、周期转化能力显著增强,而细胞凋亡比例显著降低。结论 OGD处理显著抑制rBMECs的增殖、周期转化、促进凋亡; 干扰miR-143-3p表达对OGD条件下的rBMECs具有保护作用。     

Brain-Derived Glia Maturation Factor b Participates in Lung Injury Induced by Acute Cerebral Ischemia by Increasing ROS in Endothelial Cells

Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia(ACI), we established a middle cerebral artery occlusion(MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor b(GMFB) based on quantitative analysis of the global rat serum proteome.Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was overexpressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation(OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium(CM) after OGD.We then used the CM to culture pulmonary microvascular endothelial cells(PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover,ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells.In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI.     

Brain-Derived Glia Maturation Factor β Participates in Lung Injury Induced by Acute Cerebral Ischemia by Increasing ROS in Endothelial Cells

Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia (ACI), we established a middle cerebral artery occlusion (MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor β (GMFB) based on quantitative analysis of the global rat serum proteome. Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was over-expressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation (OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium (CM) after OGD. We then used the CM to culture pulmonary microvascular endothelial cells (PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover, ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells. In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI.