多巴胺D1和D2受体激动剂和拮抗剂对脑缺血/再灌注损伤的影响

实验应用开阔法、组织病理学方法、原位末端标记(in situ terminal deoxynucleotidyl transferase-metliated de-oxy-UTP mick end labeling,TUNEL)法及免疫组织化学等方法,探讨多巴胺D1、D2受体激动剂和拮抗剂对沙土鼠前脑缺血／再灌注损伤海马CA1区神经元凋亡及凋亡相关基因bcl-2、bax表达的影响。结果显示：前脑缺血5min可引起沙土鼠探索活动增加;再灌注3d,海马CA1区约95％的锥体细胞凋亡;再灌注7d,海马CA1区仅残存约2％—7％的存活锥体细胞;前脑缺血5min可抑制bcl-2的表达并诱导bax表达增高;预先应用D2受体激动剂培高利特可减轻缺血后沙土鼠行为学异常、抑制海马CA1区锥体细胞凋亡、提高锥体细胞存活数、显著诱导bcl-2的表达并抑制bax的表达。预先应用SKF38393、SCH23390及螺哌隆对以上结果无明显影响。实验结果提示,培高利特具有确切的脑保护作用,诱导bcl-2并抑制bax的表达可能是其脑保护作用机制之一。     

西酞普兰对慢性应激大鼠额叶皮质神经细胞bax mRNA、bcl-2 mRNA表达及凋亡的影响

目的:探讨西酞普兰对慢性应激大鼠的额叶皮质神经细胞bax、bcl-2 mRNA表达的影响。方法:取24只雄性SD大鼠随机分为对照组(不进行任何处理)、应激组(应激+生理盐水灌胃)、实验组(应激+西酞普兰灌胃),采用强迫游泳制造慢性应激模型(15 min/d,共4周),用原位杂交技术检测bax、bcl-2 mRNA表达情况,TUNEL法检测细胞凋亡,尼康图像分析(NIS DR)软件测量各指标阳性细胞数量。结果:应激组较对照组,大鼠额叶皮质bax mRNA阳性表达细胞明显增多(P0.01)、染色加深;bcl-2 mRNA阳性表达细胞明显减少(P0.01)且染色变浅,且TUNEL阳性细胞数量增多(P0.01),染色增强。而实验组较应激组大鼠,额叶皮质bax mRNA阳性表达细胞减少(P0.01)、染色变浅,bcl-2 mRNA阳性表达细胞明显增多(P0.05),染色加深,且TUNEL阳性细胞数量减少(P0.01),染色减弱。结论:大鼠额叶皮质神经细胞bax mRNA和bcl-2 mRNA的表达水平受到慢性应激的影响,导致细胞凋亡加剧,西酞普兰能够有效调控额叶皮质神经细胞bax和bcl-2 mRNA的表达水平,拮抗细胞凋亡,这可能是西酞普兰防治慢性应激引起的神经精神疾病的相关机制之一。     

大鼠脑创伤后caspase-3的过度表达与细胞凋亡的关系

目的:探讨大鼠脑创伤后海马神经组织中casepase-3表达及其在细胞凋亡中的机制。方法:雄性Wistar大鼠72只随机分成对照组和创伤组,用Marmarou方法造成大鼠重型弥漫性颅脑创伤,采用免疫组织化学检测海马CA1区神经细胞casepase-3蛋白表达情况,原位细胞DNA断裂检测末端标记(TUNEL)法观察大鼠海马CA1区神经细胞凋亡动态变化。同时行TUNEL与caspase-3双标染色。结果:对照组海马区神经细胞casepase-3未见明显表达,创伤组海马CA1区神经细胞casepase-3表达在伤后3小时开始升高,伤后3天达高峰(P0.01),伤后7天下降明显。对照组海马区未见TUNEL阳性细胞,创伤组海马区TUNEL阳性细胞伤后3小时开始增多,伤后3天达高峰(P0.01),伤后7天下降。可见创伤组TUNEL染色与caspase-3免疫染色双标阳性的细胞伤后6小时细胞数量逐渐增多,于伤后3天达高峰(P0.01),伤后7天双标阳性细胞数量下降。Casepase-3表达与TUNEL阳性细胞明显相关(P0.01)。结论:大鼠脑创伤后casepase-3的过度表达是影响大鼠脑创伤后神经细胞凋亡原因之一,抑制casepase-3活性表达对神经组织起保护作用。     

大鼠脑创伤后caspase-3的过度表达与细胞凋亡的关系

目的：探讨大鼠脑创伤后海马神经组织中casepase-3表达及其在细胞凋亡中的机制。方法：雄性Wistar大鼠72只随机分成对照组和创伤组。用Marmarou方法造成大鼠重型弥漫性颅脑创伤,采用免疫组织化学检测海马CA1区神经细胞casepase-3蛋白表达情况,原位细胞DNA断裂检测末端标记（TUNEL）法观察大鼠海马CA1区神经细胞凋亡动态变化。同时行TUNEL与caspase-3双标染色。结果：对照组海马区神经细胞casepase-3未见明显表达,创伤组海马CA1区神经细胞casepase-3表达在伤后3小时开始升高,伤后3天达高峰（P〈0．01）,伤后7天下降明显。对照组海马区未见TUNEL阳性细胞,创伤组海马区TUNEL阳性细胞伤后3小时开始增多,伤后3天达高峰（P〈0．01）,伤后7天下降。可见创伤组TUNEL染色与caspase-3免疫染色双标阳性的细胞伤后6小时细胞数量逐渐增多,于伤后3天达高峰（P〈0．01）,伤后7天双标阳性细胞数量下降。Casepase-3表达与TUNEL阳性细胞明显相关（P〈0．01）。结论：大鼠脑创伤后casepase-3的过度表达是影响大鼠脑创伤后神经细胞凋亡原因之一,抑制casepase-3活性表达对神经组织起保护作用。     

阻断缝隙连接对脑缺血后海马迟发性神经元凋亡的影响

目的探讨阻断缝隙连接(gap junction)通讯对大鼠局灶性脑缺血后海马迟发性神经元死亡(delayed neuronal death,DND)及Bcl-2蛋白表达的影响。方法术前2h左侧脑室注射缝隙连接阻断剂甘珀酸(carbenoxolone,CBX),对照组左侧脑室注射生理盐水,颈内动脉插线法制备大鼠大脑中动脉缺血再灌注模型,采用DNA原位末端标记TUNEL技术及免疫荧光技术,观察阻断缝隙连接对大鼠局灶性脑缺血3d后海马迟发性神经元死亡及BCL-2蛋白表达的影响。结果不给予缝隙连接阻断剂,大脑中动脉缺血模型有45%的大鼠在术后3d出现海马迟发性神经元死亡;用甘珀酸阻断缝隙连接后,30%的大鼠出现海马迟发性神经元死亡,其发生率明显减小(P<0.01);与对照组相比,干预组Bcl-2蛋白的表达较高(P<0.01),两组Bcl-2蛋白的表达均高于假手术组(P<0.01)。结论阻断缝隙连接通讯可以减少局灶性脑缺血后海马迟发性神经元死亡的发生率,Bcl-2参与了局灶性脑缺血后海马神经元凋亡的调节。     

大肠杆菌诱导U937细胞凋亡过程中bcl-2和bax基因的表达

目的研究bcl-2和bax在大肠杆菌诱导U937细胞凋亡中的作用。方法U937细胞的凋亡用AnnexinV-FITC/PI染色流式细胞仪技术测定。Bcl-2和bax基因表达用RT-PCR法测定。结果当细胞与细菌浓度比分别为0,1:5,1:10,1:20,1:50及1:100时作用30min均可诱导U937细胞凋亡,凋亡率分别为3·16%±0·90%,9·46%±0·84%,17·90%±1·41%,35·59%±3·76%,38·35%±7·12%和55·07%±5·82%,呈浓度依赖性。在细胞凋亡过程中bcl-2和bax的表达均呈现趋势变化,bax表达逐渐增强,bcl-2表达逐渐减弱。结论E.coli可诱导U937细胞凋亡;其机制涉及bcl-2表达下调和bax表达上调。     

bc1-2和bax及NF-kB在白藜芦醇诱导肝癌细胞凋亡中的作用

目的探讨白藜芦醇诱导肝癌细胞凋亡的途径.方法白藜芦醇体外处理HepG2肝癌细胞24 h后,以免疫组化检测凋亡调控基因bcl-2和bax及NF-kB的表达.结果白藜芦醇处理组HepG2细胞bcl-2的阳性积分和NF-kB的阳性细胞密度均明显低于对照组(P＜0.01);而bax阳性积分明显高于对照组(P＜0.01).结论白藜芦醇能下调HepG2细胞bcl-2基因的表达,上调bax的表达,同时抑制NF-kB的活化,这可能是其诱导HepG2细胞凋亡的途径之一.     

caspase-3、bax、bcl-2及c-kit在中华大蟾蜍脊髓损伤后的表达变化

目的：探究高等动物脊髓损伤修复困难的原因。方法：利用免疫组化方法检验中华大蟾蜍脊髓损伤后凋亡相关基因及c—kit的表达。结果：①损伤后促凋亡因子（easpase-3及bax）随时间变化的规律为先升高再降低;②抑制凋亡因子bcl-2表达规律为先降低后升高;③bcl-2／bax比值的变化趋势与easpase-3的相反,bel-2/bax比值的升高可能抑制caspase-3表达;④c—kit表达呈现先增高再降低的规律,c-kit表达出现峰值时bcl-2表达增加,bax减少。结论：这些差异可能能够促进蟾蜍神经损伤后修复,也是低等脊椎动物较高等脊椎动物再生能力强的原因之一,为哺乳类脊髓损伤后修复方法研究提供一定的理论依据。     

西红花苷对急性高海拔低氧条件大鼠脑海马PGC-1α表达及凋亡的影响

本研究探讨西红花苷提前干预用药对急性高海拔低氧条件下大鼠脑海马组织的影响。以西红花苷药效机制为研究内容,通过组织病理及透射电镜超微结构形态学观察,RT-PCR和Western blotting检测PGC-1α、TFAM、Bcl-2、Bax、Caspase-3在mRNA和蛋白水平表达,TUNEL凋亡检测海马神经元凋亡水平。研究发现,在低氧模型组海马组织在第1天、第3天、第5天、第7天细胞结构有不同程度紊乱,线粒体损伤,以第1天、第3天最为显著,而西红花苷组线粒体损伤明显改善;PGC-1α、TFAM、Bcl-2相对表达量比较发现西红花苷组在mRNA和蛋白水平表达在第1天、第3天和第5天明显高于低氧模型组(p0.05),而第7天两组相对表达量差别无统计学意义(p0.05);比较Bax、Caspase-3在第1天、第3天和第5天m RNA和蛋白水平相对表达量发现西红花组相明显低于低氧模型组(p0.05),而第7天两组相对表达量差别无统计学意义(p0.05);TUNEL检测发现脑海马CA1区神经元凋亡率与低氧模型比较西红花苷组在第1天、第3天、第5天比较均较低(p0.05),表明西红花苷通过抗线粒体损伤及凋亡具有保护急性高海拔低氧大鼠脑海马的药理作用,为研究高原病的防治提供理论依据。     

HBO治疗对急性CO中毒后海马不同分区神经细胞凋亡的作用研究

目的:通过研究高压氧(HBO)治疗急性CO中毒大鼠海马不同分区神经细胞凋亡情况,探讨HBO治疗急性CO中毒的应用及机理。方法:利用雄性SD大鼠,建立急性CO中毒模型。应用免疫组织化学以及免疫荧光的方法,测定在染毒和CO中毒HBO治疗后1 d、3 d、7 d、14 d和21d Bcl-2、caspase-3、Neu N、BAX和MMP-9的表达水平的变化。结果:海马CA3区神经细胞对急性CO中毒与HBO治疗比CA1和CA2区更加敏感;急性CO中毒后,海马各区神经细胞凋亡程度随1 d、3 d、7 d、14 d和21 d时间延长而加重;BAX、caspase-3和Bcl-2等凋亡相关因子的表达水平与MMP-9的变化趋势一致:在1d开始增多,3d达到最大值,7d开始减少,14 d与21 d与正常组类似;CO中毒大鼠进行HBO治疗后,海马各区MMP-9、BAX、caspase-3和Bcl-2的表达水平明显降低;且HBO治疗7 d后,海马各区这些凋亡相关因子的表达降低最为明显。结论:海马CA3区神经细胞对急性CO中毒及HBO治疗敏感;海马神经细胞凋亡可能与神经细胞表达MMP-9降解神经细胞周围的基质,表达BAX、caspase-3和Bcl-2等凋亡相关因子促进凋亡发生有关;HBO治疗可降低MMP-9以及BAX、caspase-3和Bcl-2等凋亡因子的表达,抑制神经细胞的凋亡;HBO治疗7d对神经细胞凋亡的抑制作用最明显。     

法舒地尔对急性心肌梗死大鼠心肌组织中Rho激酶的影响及心肌保护作用(英文)

目的:分析急性心肌梗死(AMI)后大鼠心肌组织Rho激酶表达的变化及心肌细胞凋亡情况,观察法舒地尔对急性心肌梗死(AMI)后大鼠心肌组织Rho激酶表达的影响,探讨法舒地尔对心梗后心肌的保护作用。方法:选取雄性Wistar大鼠,随机分为三组:治疗组、AMI组、假手术组。治疗组及AMI组均结扎左前降支(LAD)制作AMI模型;假手术组只在其LAD下穿线不结扎。治疗组给予法舒地尔5mg/kg,腹腔注射,每日两次;对照组和假手术组给予等量生理盐水。1周后,EvensBlue及NBT双染色确定缺血面积及梗死面积,RT-PCR法测定rho激酶mRNA的表达,DNA断裂的原位末端标记法(T UNEL法)检测缺血区心肌细胞凋亡指数(AI),免疫组化测定凋亡相关蛋白bcl-2及bax表达的变化。结果:1周后,AMI组与假手术组相比,AMI组大鼠Rho激酶mRNA表达增加(P0.01),凋亡相关蛋白bax表达增加(P0.01),bcl-2表达减少(P0.01),AI明显增加(P0.01)。治疗组与AMI组相比,梗死面积显著减小(P0.05),Rho激酶mRNA及bax表达显著减少,AI显著降低,bcl-2表达显著增加(均P0.01)。结论:大鼠AMI后,心肌组织中Rho激酶的表达增加,心肌细胞凋亡指数增加,连续应用法舒地尔1周能有效减少心肌细胞凋亡指数,起到心肌保护的作用。     

Ethyl Pyruvate Attenuates Early Brain Injury Following Subarachnoid Hemorrhage in the Endovascular Perforation Rabbit Model Possibly Via Anti-inflammation and Inhibition of JNK Signaling Pathway

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is the main cause to poor outcomes of SAH patients, and early inflammation plays an important role in the acute pathophysiological events. It has been demonstrated that ethyl pyruvate (EP) has anti-inflammatory and neuroprotective effects in various critical diseases, however, the role of EP on EBI following SAH remains to be elucidated. Our study aimed to evaluate the effects of EP on EBI following SAH in the endovascular perforation rabbit model. All rabbits were randomly divided into three groups: sham, SAH?+?Vehicle (equal volume) and SAH?+?EP (30?mg/kg/day). MRI was performed to estimate the reliability of the EBI at 24 and 72?h after SAH. Neurological scores were recorded to evaluate the neurological deficit, ELISA kit was used to measure the level of tumor necrosis factor-α (TNF-α), and western blot was used to detect the expression of TNF-α, tJNK, pJNK, bax and bcl-2 at 24 and 72?h after SAH. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Fluoro-jade B (FJB) staining were used to detect neuronal apoptosis and neurodegeneration respectively, meanwhile hematoxylin and eosin (H&E) staining was used to assess the degree of vasospasm. Our results demonstrated that EP alleviated brain tissue injury (characterized by diffusion weighted imaging and T2 sequence in MRI scan), and significantly improved neurological scores at 72?h after SAH. EP decreased the level of TNF-α and downregulated pJNK/tJNK and bax/bcl-2 in cerebral cortex and hippocampus effectively both at 24 and 72?h after SAH. Furthermore, EP reduced TUNEL and FJB positive cells significantly. In conclusion, the present study supported that EP afforded neuroprotective effects possibly via reducing TNF-α expression and inhibition of the JNK signaling pathway. Therefore, EP may be a potent therapeutic agent to attenuate EBI following SAH.     

Alterations in the cellular distribution of bcl-2, bcl-x and bax in the adult rat substantia nigra following striatal 6-hydroxydopamine lesions

The proteins of the bcl-2 family play an important role during apoptosis and may also regulate cell death in response to oxidative stress, which has been implicated in Parkinson's disease. In this study we examined the localization of the pro-apoptotic protein bax, and the anti-apoptotic proteins bcl-2 and bcl-x_L in the substantia nigra (SN) of the adult rat and their response to oxidative stress caused by striatal injections of 6-hydroxydopamine (6-OHDA). Our data show that bcl-2, bcl-x and bax proteins are present in the SN. Bcl-2 and bax are localized primarily in neurons including all those positive for tyrosine hydroxylase (TH). The intraneuronal distribution of bcl-2 and bax were different. Bcl-2 was diffuse throughout the cell while bax was localized in well-defined structures around the nucleus and within processes. Bcl-x staining in neurons was weak, though it was strongly expressed in GFAP-positive astrocytes. 6-OHDA injections, which resulted in loss of dopamine neurons between 7–14 days post-lesion, altered the distribution of bax, bcl-2 and bcl-x proteins in the SN. Bcl-2 and bax were decreased in the TH-positive cells of the SN from 3 to 14 days post-lesion and many TH-positive neurons were bcl-2 negative. Neuronal bcl-x was initially unchanged after lesion, but increased in astrocytes between 3–7 days post-lesion before the increase in GFAP immunoreactivity, which was detectable at days 10–14. While the neuronal distribution of bcl-2 and bcl-x does not change following lesion, bax became evenly distributed thought the soma. Morphological features of apoptosis, including TUNEL labeling and chromatin condensation was not observed. These data suggest that striatal 6-OHDA lesions do not result in classical apoptosis in the SN of the adult rat, even though there are changes in the content and distribution of members of the bcl-2 family of proteins.     

瘦素对小鼠脑缺血/再灌注损伤神经元凋亡的影响

目的：研究Leptin在脑缺血性损伤神经元凋亡中的作用及其机制。方法：将75只雄性昆明小鼠完全随机分成3组,即假手术组、缺血/再灌注模型组、Leptin干预组;通过大脑中动脉栓塞（MCAO）复制小鼠局灶性脑缺血再灌注损伤模型,Leptin干预组在缺血0 min腹腔注射Leptin（1μg/g体重）,TUNEL染色检测神经元凋亡,RT-PCR检测凋亡相关基因bcl-2和caspase-3 mRNA表达,免疫组化凋亡相关基因bcl-2和caspase-3蛋白水平的表达。结果：模型组脑缺血中心区神经元以坏死为主,与假手术组相比,其半影区神经元凋亡数量显著增多、促凋亡基因cas-pase-3和抑凋亡基因bcl-2的mRNA和蛋白表达水平均显著升高（P<0.01）;与模型组比较,Leptin干预组半影区凋亡神经元数量显著减少、caspase-3 mRNA和蛋白表达水平显著降低（P<0.01）,抑凋亡基因bcl-2 mRNA和蛋白表达水平显著升高（P<0.01）。结论：Leptin能够通过上调抑凋亡基因bcl-2表达,下调促凋亡基因caspase-3表达抑制神经元凋亡,在脑缺血性损伤中发挥神经保护作用。     

Epigenetic modification involved in benzene-induced apoptosis through regulating apoptosis-related genes expression

Benzene is an established haematotoxic and genotoxic carcinogen. DNA methyltransferase inhibitor, 5-aza (5-aza-2'-eoxycytidine) and histone deacetylase inhibitor, TSA (trichostatin A) are two kinds of key epigenetic modification reagents. Although apoptosis has been considered as the key cytotoxicity mechanism, the effects of these epigenetic reagents on benzene-induced apoptosis have not been reported. In this study, BMCs (bone marrow cells) from rats were incubated with benzene and then with either 5-aza, TSA alone or the combination of the two drugs. Apoptosis and mRNA expression were detected by annexin V/PI (propidium iodide) staining assay and real-time PCR, respectively. Results showed that benzene caused cell apoptosis accompanied with bcl-2 mRNA decrease, caspase-3 and bax mRNA increase. Moreover, benzene-induced apoptosis and the decrease of bcl-2 mRNA were both reversed by both 5-aza and TSA, but the role of TSA was significantly larger than 5-aza. More interestingly, these increases in benzene-induced caspase-3 and bax mRNA expression were obviously suppressed by 5-aza but not by TSA. In conclusion, 5-aza inhibited benzene-induced apoptosis through down-regulating of caspase-3 and bax and up-regulating bcl-2 mRNA expression, whereas the effect of TSA on apoptosis dominatingly affected bcl-2 mRNA expression, and 5-aza together with TSA had no synergic effect on benzene-induced apoptosis.     

Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells

lwTRODUCTIONHeparin is a polysuifated glycosaminoglycanwith a high negatbe charge. Heparin is synthesized in various tissues, especially in the lha, 1ung,and gut. In addition to its allti-coagulant activityheparin is known to have anti-hypertensive[1], auiinflammatory[2], and antiproliferative effects. Be-sides, heparin inhibits leukocyte rol1ing and its adhe-sion to endothelium, its aggregation, degranulation,and the generation of superoxide anion by actndingncotrophils[3~51. Heparin and …