外周血淋巴细胞CD40／CD40L表达与多发性肌炎／皮肌炎发病关系

目的:初步探讨协同刺激分子CD40/CD40L在多发性肌炎(PM)/皮肌炎(DM)发病机制中的作用。方法:采用流式细胞学检测PM/DM患者外周血淋巴细胞CD40、CD40L的表达,并与正常健康人对照。结果:CD40、CD40L在正常健康人外周血淋巴细胞几乎无表达,但在PM/DM患者表达增高,PM患者CD40平均荧光强度为10.24±4.15,CD40L平均荧光强度为1.35±0.77;DM患者CD40平均荧光强度为13.67±5.83,CD40L平均荧光强度为1.73±0.98。PM/DM患者比较差异无显著性(P>0.05)。结论:CD40/CD40L的表达可能参与了PM/DM的发病。     

实验性局灶性脑缺血再灌流后bcl—2蛋白的表达

目的探讨ｂｃｌ－２在大鼠局灶脑缺血再灌流损伤中的表达与缺血所致凋亡的关系。方法采用免疫组化方法观察ｂｃｌ－２蛋白在大鼠局灶性脑缺血再灌流后皮层和基底节区动态变化。结果ｂｃｌ－２蛋白在大脑中动脉阻塞２ｈ后，随再灌流时间延长其解剖分布不同。基底节区持续时间短，而皮层区持续时间较长，其中再灌流６ｈ，ｂｃｌ－２蛋白表达最显著。结论ｂｃｌ－２蛋白表达与神经细胞存活密切相关，可能是神经细胞自我保护机制之一，防止或减少细胞凋亡的发生     

Notch信号通路在大鼠局灶性脑缺血再灌注损伤恢复期的表达变化

目的探讨局灶性脑缺血再灌注损伤恢复期Notch信号通路的表达变化。方法构建SD大鼠大脑中动脉模型(MCAO)。将大鼠分为:对照组(12只),急性期组(24只),恢复期组(24只);急性期组分为3 d和7 d两个亚组,恢复期组分为15d和30d两个亚组。采用RT-PCR和Western blot测定Notch信号分子的mRNA和蛋白的表达变化。结果与对照组大鼠相比,脑缺血再灌注后第3天Delta-like 1、Delta-like 3、Delta-like 4和Jagged 1的mRNA表达水平明显升高(P 0. 05);第30天的表达水平显著低于对照组(P 0. 05)。Notch 1和Notch 2的mRNA表达水平在脑缺血再灌注后第3天显著升高(P 0. 05);第30天,Notch 1(P 0. 05)、Notch 2(P 0. 05)、Notch 3(P 0. 05)和Notch 4(P 0. 01)的mRNA表达水平均显著低于对照组。脑缺血再灌注后第3天Hes-1(P 0. 01)和Hey-1(P 0. 05)的表达水平较对照组均明显升高;第30天,Hes-1和Hey-1的表达水平低于对照组(P 0. 05)。NICD1和NICD2在大脑局灶性缺血后第3天半暗带区的表达水平显著上升(P 0. 05);第30天的表达水平显著下降(P 0. 05)。结论大鼠脑缺血再灌注损伤恢复期可能通过抑制Notch信号通路的表达,起到神经保护作用。     

大鼠短暂局灶性脑缺血再灌注后核转录因子-kB的表达

目的　研究核转录因子 - k B(NF- k B)在局灶性脑缺血再灌注中的动态表达规律及其作用。方法　采用线栓法建立大鼠局灶性脑缺血再灌注模型。应用细胞免疫组织化学法分析 NF- k B的移位 ,采用 Western- blot法检测脑组织中核 NF- k B的表达量。结果　局灶性脑缺血再灌注后 NF- k B明显从细胞浆移位于细胞核 ,核 NF- k B的表达量显著增加 (P<0 .0 1)。结论　局灶性脑缺血再灌注能够引起 NF- k B的表达增加 ,进一步产生炎症和免疫反应 ,从而参与了脑缺血再灌注损伤的发病机制     

聚合牛血红蛋白对缺血再灌注脑组织Na+/K+/-ATPase活性的影响

目的：观察聚合牛血红蛋白在缺血再灌注脑损伤中的治疗作用。方法：采用大鼠全脑缺血再灌注模型,观察缺血前后应用ＰＢＨｂ对脑组织Ｎａ＾＋Ｋ＾＋－ＡＴＰａｓｅ活性的影响。结果：与缺血再灌注组相比,所有白蛋白对照组的Ｎａ＾＋Ｋ＾＋－ＡＴＰａｓｅ活性未显示出明显差异,而所有ＰＢＨｂ组该酶活性下降程度减轻,其中脑复苏ＰＢＨｂ组比脑保护ＰＢＨｂ组效果更明显。结论：ＰＢＨｂ能通过提高Ｎａ＾＋Ｋ＾＋－ＡＴＰａｓｅ活性     

Remote ischemic postconditioning protects the brain from focal ischemia/reperfusion injury by inhibiting autophagy through the mTOR/p70S6K pathway

Objective: Remote ischemic postconditioning (RIPostC) has been recognized as an applicable strategy for protecting against cerebral ischemia/reperfusion (I/R) injury. This study was performed to examine the effect of RIPostC on cerebral I/R and to explore its underlying mechanism.

Methods: Healthy male SD rats (N = 36) were assigned randomly into 3 groups of 12 each: sham group, I/R model group and RIPostC group. Animal models were performed by filament insertion for 2 h with middle cerebral artery occlusion(MCAO) followed by 24 h of reperfusion. RIPostC was induced by 15 min occlusion of femoral arteries followed by 15 min of reperfusion for 3 cycles at the beginning of middle cerebral artery reperfusion. The neurological deficits, infarct size and brain edema were determined. Autophagy was examined by transmission electron microscopy (TEM). The protein levels of microtubule-associated protein light chain 3 (LC3-II), mammalian target of rapamycin (mTOR), serine/threonine kinase p70S6 kinase (p70S6K), and their phosphorylation (p-mTOR and p-p70S6K) in the brain tissue of the rats were determined by western blotting.

Results: Our results suggested that RIPostC significantly reduced I/R-induced brain injury, as exhibited by a significantly decreased infarct size, mitigated brain edema and improved neurological deficits. RIPostC also significantly reduced the LC3-II/LC3-I ratio and protein expression of Beclin 1. Much less severe neuronal injury and fewer autophagosomes were observed by TEM in the RIPostC group.

Conclusions: These results suggest that RIPostC attenuates cerebral I/R injury by inhibiting autophagy through the activation of the mTOR/p70S6K signaling pathway.     

大鼠局灶脑缺血病灶周围新血管生成的相关实验研究

目的本研究主要探讨大鼠局灶脑缺血病灶周围新血管形成现象,探讨外周血CD34+细胞和脑组织AC133抗原在新血管形成中的作用。方法将成年SD雄性大鼠随机分为:1.正常组,2.假手术组,3.动物模型组。分别取缺血再灌注1h、3h、6h、12h、24h、48h、3d、4d、7d、14d、28d共11个观察点。取前10个观察点外周血标本,采用流式细胞术检测单个核CD34+细胞平均荧光强度。采用免疫组化染色法检测48h、3d、4d、7d脑组织AC133抗原表达。对28d组进行脑血管荧光分布面积的检测。每个观察点5只大鼠,共65只大鼠,根据神经功能计分纳入实验。结果1.脑缺血再灌注28d梗死半球半暗带区域的血管荧光总面积较非梗死半球相似区域增加24.79%。2.大鼠脑缺血/再灌注3d外周血单个核CD34+细胞平均荧光强度明显降低,直到7d。14d恢复到基线水平。3.AC133抗原在再灌注4d梗死半球半暗带区域的血管内皮细胞表达阳性,3d、7d组无阳性表达。结论本研究证实大鼠脑缺血28天梗死半球半暗带区域的血管面积较非梗死半球增大。研究提示循环CD34+干细胞和AC133+细胞可能参与脑缺血损伤的血管修复和新血管形成。     

褪黑素对SD大鼠脑缺血再灌注损伤后c-fos表达的影响

目的探讨褪黑素在大鼠脑缺血再灌注损伤中的神经保护作用及可能机制。方法选取45只雄性SD大鼠,分为假手术组(5只)、脑缺血再灌注组(20只)、褪黑素干预组(20只);脑缺血再灌注组和褪黑素干预组根据时间点第6小时、第1天、第3天、第7天分为4个组,每组5只。采用Longa线栓法建立大鼠左侧大脑中动脉栓塞(MCAO)模型,采用HE染色检测脑组织的病理改变,TUNEL染色检测神经细胞的凋亡,免疫组织化学(免疫组化)法及蛋白质印迹法(Western Blotting)观察大鼠脑组织内c-fos表达情况。结果在脑缺血再灌注组的各时间点的HE染色显示,胶质细胞呈现程度不一的增生,神经元出现坏死;褪黑素干预能减轻脑缺血再灌注后胶质细胞增生及神经元的坏死。在TUNEL染色凋亡检测中,脑缺血再灌注组各时间点的神经细胞凋亡升高;褪黑素干预组各时间点的细胞凋亡数低于脑缺血再灌注组(P <0.05)。在免疫组化及蛋白质印迹检测中,脑缺血再灌注组c-fos表达增加,在第1天时达到高峰,之后表达逐步降低;在褪黑素干预组,c-fos表达趋势与缺血再灌注组一致,但表达水平比缺血再灌注组相应时间点低,差异有统计学意义(P <0.05)。结论褪黑素能够减轻脑缺血再灌注后神经元的损伤,降低c-fos的表达,表明褪黑素可能通过调控c-fos的表达在脑缺血再灌注中发挥神经保护作用。     

大鼠局灶性缺血再灌注后大脑皮质ADM及其mRNA的表达

目的探讨肾上腺髓质素（ADM）与脑缺血再灌注损伤的关系。方法采用线栓法制成大鼠大脑中动脉缺血再灌注模型，阻断血流2h进行再灌注。应用免疫组织化学法和RT—PCR法检测不同时间段大鼠局灶性脑缺血再灌注后大脑皮质ADM及其mRNA的表达，并进行动态观察。结果正常大鼠大脑皮质有ADM及其mR—NA的表达，假手术组ADM及其mRNA表达略高于正常组，P〉0．05；大鼠脑缺血再灌注后大脑皮质ADM及其mRNA过表达，与正常对照组及假手术组相比差异显著，P〈0．05。动态观察发现，脑缺血2h再灌注2h，大脑皮质ADM及其mRNA即表达，再灌注22h达高峰，至1w仍明显多于正常对照组，P〈0．05。结论脑缺血再灌注后ADM及其mRNA呈现规律性过表达。     

L-NAME对大鼠局灶性脑缺血灌注损伤Fos蛋白表达的影响

目的观察一氧化氮含量的变化对缺血再灌注损伤后Fos蛋白表达的影响。方法采用线拴法制作大鼠局灶性脑缺血再灌注损伤模型,利用NADPH组化和Fos蛋白免疫组化双标技术研究NOS抑制剂L－NAME对大鼠局灶性脑缺血再灌注损伤脑皮层Fos蛋白表达的影响。结果缺血60min再灌注3h后损伤侧脑组织皮质一氧化氮合酶阳性神经元较正常增多并深染,Fos蛋白表达增加,L－NAME（3mg／kg）治疗组脑皮质神经元Fos蛋白的表达量较对照组减少,L－NAME（10mg/kg）治疗组脑皮质神经元Fos蛋白的表达量较对照组明显减少,同时也可见给予L－NAME后脑组织皮质内NOS阳性神经元无论在数量上还是在细胞着色、胞体突起均明显减少。结论c－fos基因表达也可能部分参与了NO的致神经细胞损伤过程。     

小鼠局灶性脑缺血再灌注后IL-33及其受体的时程表达

目的检测白细胞介素-33(IL-33)及其膜受体ST2和可溶型受体sST2在小鼠局灶性脑缺血再灌注后不同时程的表达特征。方法利用线栓法闭塞大脑中动脉(MCAO)30 min诱导建立小鼠可逆性局灶性脑缺血再灌注损伤模型,通过半定量RT-PCR检测脑缺血再灌注后6 h、24 h和3 d缺血脑组织中IL-33及其膜受体ST2、凋亡相关蛋白Caspase-8和Caspase-3的mRNA表达水平,并通过免疫组织化学染色观察了IL-33在不同缺血脑区(运动皮质、感觉皮质、海马和纹状体)的时程表达情况;ELISA法检测了小鼠MCAO模型再灌注后不同时间点血清中IL-33及其可溶型受体sST2的表达水平。结果 IL-33 mRNA在缺血后6 h和3 d表达减少,但在24 h无明显改变;凋亡相关蛋白Caspase-3和Caspase-8在缺血后3个时间点均显著增高,且Caspase-3在6 h和3 d的mRNA表达水平较24 h高;ST2 mRNA在缺血后6 h无减少,但在24 h和3 d有明显减少;除了MCAO 24 h组运动皮质和纹状体阳性染色增加外,IL-33阳性细胞数在缺血后不同时程各脑区均有不同程度减少;缺血后外周血中IL-33的表达量无明显升高或降低,而sST2的表达水平在缺血后6 h即已显著升高。结论脑缺血再灌注后IL-33/ST2信号通路被下调,其与sST2表达增多的效应发挥和神经元凋亡有关。     

大鼠局灶性脑缺血再灌后转录因子ATF4的表达变化

目的观察鼠脑缺血再灌注后ATF4mRNA及蛋白的表达变化。方法制备SD大鼠大脑中动脉闭塞模型,RT-PCR法、免疫组化染色分别测定鼠脑缺血半暗带区再灌注后不同时相ATF4mRNA及蛋白的表达变化。结果模型组ATF4mRNA与蛋白于再灌注后1h表达增加;ATF4mRNA表达于再灌注后6h达高峰,其蛋白表达于再灌注后24h达高峰。结论鼠脑缺血再灌注可诱导ATF4在缺血半暗带区表达,提示ATF4可能在脑缺血再灌注损伤中发挥重要作用。     

局灶脑缺血再灌流后ICAM-1表达与白细胞浸润

目的　探讨大鼠局灶性脑缺血再灌流后细胞间粘附分子 - 1(ICAM- 1)表达规律与白细胞浸润。方法　采用大鼠 MCA线栓闭塞 /再通法建立局灶性缺血 /再灌流模型 ,动态观察 ICAM- 1、髓过氧化物酶 (MPO)变化情况。结果　 (1)鼠脑缺血 /再灌流后 MCA供血区皮质及梗死周边区的微血管 ICAM- 1表达于再灌流 6 h已增强 ,36～ 48h达高峰 ,以后逐渐减弱 ,但第 7天表达仍强于假手术组。(2 )坏死周边区神经元也表达 ICAM- 1。(3)再灌流 6 h出现白细胞浸润 ,浸润高峰在 2 4～ 36 h,6 d后恢复正常。结论　 (1)缺血再灌流使微血管表达 ICAM- 1上调 ,同时也伴随坏死周边区的神经元表达 ICAM- 1。 (2 )白细胞浸润与 ICAM- 1表达规律同步。     

bFGF对大鼠局灶性缺血再灌注脑组织的保护作用

目的探讨bFGF对大鼠脑缺血再灌注损伤后神经细胞凋亡和脑组织中SOD、MDA含量变化的影响。方法应用线栓法制作大鼠局灶性脑缺血再灌注模型,大脑中动脉阻塞1h再灌注损伤24h,检测假手术组、缺血再灌注组和bFGF组的凋亡细胞数和脑组织中SOD、MDA的含量。结果假手术组偶见凋亡细胞,缺血再灌注组和bFGF组凋亡细胞数分别是26.35±5.67和18.65±5.91,与缺血再灌注组相比,bFGF组缺血区皮质凋亡神经元明显减少(P<0.05)。SOD,MDA测定结果显示三组间比较,具有显著性差异(P<0.01和P<0.05)。结论抗氧化作用可能是bFGF减少大鼠脑缺血再灌注损伤后细胞凋亡的分子机制之一。     

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway

Electroacupuncture(EA) has anti-oxidative and anti-inflammatory actions,but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion(I/R) injury involves modulation of the extracellular regulated kinase 1/2(ERK1/2) signaling pathway is unclear.Middle cerebral artery occlusion(MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours.A 30-minute period of EA stimulation was applied to both Baihui(DU20) and Dazhui(DU14) acupoints in each rat(10 mm EA penetration depth,continuous wave with a frequency of 3 Hz,and a current intensity of 1–3 m A) when reperfusion was initiated.EA significantly reduced infarct volume,alleviated neuronal injury,and improved neurological function in rats with MCAO.Furthermore,high m RNA expression of Bax and low m RNA expression of Bcl-2 induced by MCAO was prevented by EA.EA substantially restored total glutathione reductase(GR),glutathione(GSH) and glutathione peroxidase(GSH-Px) levels.Additionally,Nrf2 and glutamylcysteine synthetase(GCS) expression levels were markedly increased by EA.Interestingly,the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059(a specific MEK inhibitor).Collectively,our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA.Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.     

经鼻给予VEGF治疗脑缺血/再灌注大鼠的量效关系

目的探讨经鼻给予血管内皮生长因子(VEGF)治疗脑缺血/再灌注损伤大鼠的量效关系。方法48只SD大鼠随机分为四组:低剂量组(100μg/mL)、中剂量组(200μg/mL)、高剂量组(500μg/mL)及盐水对照组(n=12)。通过阻塞大脑中动脉制作大鼠局灶性脑缺血90 m in再灌注损伤模型。缺血后1d、7d和14 d行神经功能评价,14 d动物被麻醉,行组织学检查,应用图像分析系统计算梗死体积、评价血管形成。结果与对照组相比,经鼻给予中剂量VEGF,可明显降低梗死体积,改善神经功能(P<0.01);而低和高剂量组对比于对照组,不能降低脑缺血后大鼠脑梗死体积和改善神经功能(P>0.05)。与对照组相比,经鼻给予中和高剂量VEGF,可增加缺血后脑表面血管形成(P<0.01);而低剂量组对比于对照组,不能促进脑缺血后血管生成(P>0.05)。结论经鼻给予中剂量VEGF可有效降低脑缺血/再灌注损伤大鼠梗死体积,改善神经功能,增加血管密度。因此经鼻给予中等剂量(200μg/mL)VEGF是治疗脑缺血/再灌注损伤的最佳剂量,其可用于进一步评价VEGF作用的有效实验剂量。     

Resveratrol attenuates brain damage in permanent focal cerebral ischemia via activation of PI3K/Akt signaling pathway in rats

ABSTRACT

Objective: The aim of this study was to evaluate the potential molecular mechanism of resveratrol (RSV) that attenuates brain damage from focal cerebral ischemia.

Methods and materials: To investigate whether phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway was involved in RSV anti-inflammatory and neuroprotective properties. Middle cerebral artery occlusion (MCAO) animal model was used in this study. Adult male Sprague–Dawley (SD) rats underwent MCAO, and then received treatment with RSV or vehicle after the onset of ischemia. PI3K inhibitor LY294002 was injected intracerebroventricularly to inhibit the PI3K/Akt signaling pathway. Neurological deficit scores and cerebral water content were assessed 24 h after MCAO. The inflammatory factors interleukin (IL)-1β, tumor necrosis factor (TNFα), and cyclooxygenase-2 (COX2) mRNA level were examined by real-time PCR. The enzymatic activity of myeloperoxidase (MPO) was measured 24 h after MCAO. The protein expression of phospho-Akt and COX2 in ischemic brain were determined by western blot.

Results: RSV significantly reduced neurological deficit scores, cerebral water content and the enzymatic activity of MPO, all of which were abolished by LY294002 administration. Real-time PCR showed that RSV significantly suppressed the upregulation of the inflammatory factors IL-1β, TNFα, COX2 mRNA levels. RSV significantly inhibited upregulated the protein expression of COX2 24 h after MCAO, which effect was abolished by LY294002 administration.

Conclusion: RSV attenuated ischemic brain damage induced by cerebral artery occlusion mainly through PI3K/Akt signaling pathway.

Abbreviation: MCAO: Middle cerebral artery occlusion; RSV: resveratrol; PI3K/Akt: phosphatidylinositol 3-kinase/Akt; TNF: tumor necrosis factor; COX2: cyclooxygenase-2; MPO: myeloperoxidase; IL: interleukin.     

大鼠局灶性脑缺血再灌注后早期生长反应基因-1 mRNA的表达

目的探讨早期生长反应基因-1( early growth response gene-1,Egr-1)mRNA在大鼠局灶性脑缺血再灌注后的表达情况。方法取10只健康雄性SD大鼠,体重200 ～250 g,应用原位杂交和RT-PCR方法检测大鼠局灶性脑缺血再灌注后Egr-1 mRNA的表达情况。结果 (1)原位杂交结果:假手术组神经元及胶质细胞为轻度阳性表达。缺血2 h再灌注2 h后,缺血侧Egr-1 mRNA细胞阳性表达明显增强。再灌注4 h时Egr-1 mRNA细胞阳性表达最高,再灌注22 h Egr-1 mRNA细胞阳性表达下降,至166 h时下降更加明显,但仍显著高于假手术组。(2)RT-PCR结果:大鼠脑缺血再灌注后缺血侧Egr-1 mRNA的表达明显高于假手术组,P<0.01。动态观察发现,脑缺血2h再灌注2 h后,Egr-1 mRNA即高表达,缺血2 h再灌注4 h达高峰,再灌注46 h已明显下降,但仍高于假手术组,P<0.01,随缺血再灌注时间延长,Egr-1 mRNA表达又逐渐增多,至再灌注166 h其表达亦明显高于假手术组,P<0.01。结论缺血再灌注后Egr-1 mRNA有规律性表达。     

MicroRNA-670 aggravates cerebral ischemia/reperfusion injury via the Yap pathway

Apoptosis is an important programmed cell death process involved in ischemia/reperfusion injury. MicroRNAs are considered to play an important role in the molecular mechanism underlying the regulation of cerebral ischemia and reperfusion injury. However, whether miR-670 can regulate cell growth and death in cerebral ischemia/reperfusion and the underlying mechanism are poorly understood. In this study, we established mouse models of transient middle artery occlusion and Neuro 2 a cell models of oxygen-glucose deprivation and reoxygenation to investigate the potential molecular mechanism by which miR-670 exhibits its effects during cerebral ischemia/reperfusion injury both in vitro and in vivo. Our results showed that after ischemia/reperfusion injury, miR-670 expression was obviously increased. After miR-670 expression was inhibited with an miR-670 antagomir, cerebral ischemia/reperfusion injury-induced neuronal death was obviously reduced. When miR-670 overexpression was induced by an miR-670 agomir, neuronal apoptosis was increased. In addition, we also found that miR-670 could promote Yap degradation via phosphorylation and worsen neuronal apoptosis and neurological deficits. Inhibition of miR-670 reduced neurological impairments after cerebral ischemia/reperfusion injury. These results suggest that microRNA-670 aggravates cerebral ischemia/reperfusion injury through the Yap pathway, which may be a potential target for treatment of cerebral ischemia/reperfusion injury. The present study was approved by the Institutional Animal Care and Use Committee of China Medical University on February 27, 2017(IRB No. 2017 PS035 K).