Effect of radix Salviae miltiorrhizae on the gene expression of nitric oxide synthase in ischemic rat brains.

The effect of Radix Salviae Miltiorrhizae (RSM) on the gene expression of nitric oxide synthase (NOS) in rat brains during ischemia was studied with in situ hybridization and the results were analyzed with IBAS 2000 Image Analysis System. It was found that NOS gene expression of cerebral cortex and caudate-putamen was markedly increased in 24 hours in ischemia group (P < 0.01). In RSM-treated rats, although the NOS gene expression of ischemic side was also increased as compared with contralateral cortex and caudate-putamen, it was significantly lower in RSM-treated rats than those of the controls (P < 0.05, P < 0.01). The present study indicates that RSM can partly inhibit NOS gene expression of cerebral cortex and caudate-putamen during ischemia. This may be one of the protective mechanisms of RSM on cerebral ischemia.     

CHANGES OF ENDOTHELIN-1 GENE EXPRESSION IN RAT BRAINS DURING ISCHEMIA AND ISCHEMIC REPERFUSION

ＣＨＡＮＧＥＳＯＦＥＮＤＯＴＨＥＬＩＮ－１ＧＥＮＥＥＸＰＲＥＳＳＩＯＮＩＮＲＡＴＢＲＡＩＮＳＤＵＲＩＮＧＩＳＣＨＥＭＩＡＡＮＤＩＳＣＨＥＭＩＣＲＥＰＥＲＦＵＳＩＯＮＷｕＷｅｉｐｉｎｇ（吴卫平）；ＫｕａｎｇＰｅｉｇｅｎ（匡培根）ａｎｄＬｉＺｈｅｎｚｈｏ...     

The effect of radix Salviae Miltiorrhizae (RSM) on substance P in cerebral ischemia--animal experiment.

The levels of substance P (SP) in rat brains were assayed in 64 rats. Bilateral common carotid artery ligation was done in 49 rats. Half an hour before ligation, 25 rats were given 10 g/kg of RSM; 24 rats were given the same volume of normal saline as controls. Sham operation was done in 15 rats. Half an hour and 3 hours after cerebral ischemia, the rats were quickly decapitated. SP concentration was assayed in the cerebral cortex, caudate nucleus and brain stem. In saline-treated animals, the SP level of caudate nucleus at 3-hour group was significantly decreased as compared with the 0.5-hour group and sham-operated group respectively. No significances were found among RSM-treated groups and sham-operated groups. The SP levels were shown: brain stem greater than caudate nucleus greater than cerebral cortex. The preliminary results suggest that SP may be involved in the pathophysiologic procedures of cerebral ischemia and RSM may attenuate the dysfunction of SP during cerebral ischemia.     

大鼠局灶性脑缺血再灌注脑内p47phox表达变化的研究

目的通过观察大鼠局灶性脑缺血再灌注后p47phox基因在脑内表达动态变化,探讨p47phox在局灶性脑缺血再灌注后对脑缺血损害的作用及机制,可能为p47phox基因作为脑卒中的候选基因提供理论依据。方法采用逆转录聚合酶链反应(RT-PCR)法检测大鼠局灶性脑缺血再灌注后脑缺血再灌注组和假手术组再灌后第1、3、6、12和24h和正常对照组p47phox基因在脑内的表达。结果正常对照组和假手术组大鼠脑皮质内有少量p47phox mRNA表达,组内各时间点比较无统计学意义(P>0.05);脑缺血再灌注组,缺血再灌注1h后p47phox mRNA表达增加,至3h达高峰(与假手术组和正常对照组相比差异显著,P<0.01)。结论p47phox可能参与了脑缺血再灌注后损伤,并在其中发挥了重要作用。     

肾上腺髓质素对大鼠局灶性脑缺血再灌注后脑组织的保护作用

目的探讨肾上腺髓质素与脑缺血再灌注损伤的关系。方法采用栓线法制成大鼠大脑中动脉缺血再灌注模型，阻断血流2h后进行再灌注。应用免痘组织化学染色法拴测不同时间段大鼠局灶性脑缺血再灌注后肾上腺髓质素的表达情况，并进行动态观察。结果正常大鼠脑内即有肾上腺髓质表达，假手术后肾上腺髓质素表达略有增加，但与正常对照组相比无明显差异，P〉0．05；大鼠脑缺血再灌注后肾上腺髓质素免疫阳性细胞增多，与正常对照组及假手术组相比差异显著，均P＜0．05；大鼠脑缺血再灌注后缺血侧既缺血对侧肾上腺髓质素免疫阳性知胞均增多，但以缺血侧区域增多最为明显，P〈0．05。动态观察发现，脑缺血再灌注2h后，肾上腺髓质素免痘阳性细胞即增多，缺血再灌注6h迭高峰，至1周左右仍明显增多。结论脑缺血再灌注后肾上腺髓质表达增强。     

The effect of radix Salviae miltiorrhizae on vasoactive intestinal peptide in cerebral ischemia: an animal experiment

The levels of vasoactive intestinal peptide (VIP) in three regions of rat brain were assayed in 62 rats. Bilateral common carotid artery ligation was done in 50 rats. Half an hour before ligation 26 rats were given 10 g/kg of Radix Salviae Miltiorrhizae (RSM); 24 rats were given same volume of normal saline as controls. A sham operation was done in 12 rats. Half an hour (n = 30) and 3 hours (n = 32) after operation, the rats were quickly decapitated. VIP levels were assayed in cerebral cortex, hippocampus and caudate nucleus. In salin-treated animals, VIP levels of cerebral cortex and caudate nucleus at 3 hour group were significantly decreased compared with the sham-operated group. No significant difference was found between RSM-treated and sham-operated groups. The preliminary results suggest that VIP may be involved in the pathophysiological procedures of cerebral ischemia and RSM may attenuate the dysfunction of VIP during cerebral ischemia.     

Effect of radix Salviae miltiorrhizae on extracellular adenosine and evaluation of its protective efficacy in ischemic reperfusion rat--microdialysis, HPLC and histopathologic studies.

The effects of Radix Salviae Miltiorrhizae (RSM) on extracellular adenosine (Ade) and its metabolites, i.e. inosine, hypoxanthine and xanthine, were studied with microdialysis and HPLC techniques during cerebral ischemia-reperfusion induced by 4-vessel occlusion in rat brain. Histological examination of hippocampus was performed 6 h after reperfusion. ECF (extracellular fluid) adenosine and its metabolites were compared between the controls (n = 6) and RSM-treated rats (n = 6). Basal level of Ade and its metabolites release were not greatly affected by pretreatment with RSM, and no significant difference as compared with the sham-operated (n = 6). Ade and its metabolites were dramatically increased after ischemia, and decreased near basal-level and its metabolites remained high at the end of reperfusion. In the RSM-treated animals, the tendency of changes of Ade and its metabolites was just the same as in the controls, but the magnitudes of changes were significantly lower at some different time points. In sham-operated animals, no changes were observed at different time points both during ischemia (30 min.) and reperfusion (60 min.). Histopathological findings demonstrated that RSM pretreatment results in better histologic preservation of the pyramidal cells in the postischemic reperfusion CA1 sector both qualitatively and quantitatively. These results indicated that RSM protects against cerebral ischemia reperfusion injury.     

糖尿病小鼠局灶性脑缺血再灌注损伤中脑周细胞的变化

目的 探讨糖尿病高血糖状态下局灶脑缺血再灌注损伤中脑周细胞的变化特点.方法 采用四氧嘧啶一次性腹腔注射制备1型糖尿病高血糖C57BL/6小鼠模型,以线栓法为基础使大脑中动脉缺血建立局灶性脑缺血再灌注小鼠模型,使用行为学、组织化学、qRT-PCR及Western blot等方法,对比观察假手术组(空白对照组)、正常血糖脑缺血再灌注组(缺血组)、糖尿病高血糖脑缺血再灌注组(高糖并缺血组)大脑皮质区周细胞数量改变和α-平滑肌肌动蛋白(α-SMA)的相对表达情况.结果 免疫组化及TTC染色显示,与空白对照组比较,缺血组缺血1h再灌注24 h后脑组织中α-SMA阳性细胞数量明显增多,梗死脑组织水肿明显(P＜0.05);高糖并缺血组再灌注24 h,α-SMA阳性细胞数目较同期缺血组更多,梗死区域水肿极严重(P＜0.05);qRT-PCR和Western blot分别显示高糖并缺血组α-SMA蛋白对应基因、α-SMA蛋白的相对表达量均高于缺血组(P＜0.05).结论 糖尿病高血糖与缺血再灌注两种损伤机制同时作用于脑组织时,使脑组织损伤更严重,损伤区域α-SMA的表达增强,周细胞数量明显增加.     

超短波与丹参对脑缺血再灌注损伤大鼠脑保护作用的比较研究

目的:观察并比较超短波与丹参对大鼠局灶性脑缺血再灌注损伤的保护作用并探讨两者作用机制和有无协同作用.方法:用线栓法制备一侧大脑中动脉栓塞-再灌注大鼠模型,造成大鼠右脑缺血2 h再灌注24 h,采用5级评分法评定神经功能缺损程度来筛选病例.造模后,大鼠分成假手术组,对照组,超短波组,丹参组及超短波与丹参合用组.所有大鼠均于再灌注24h后断头取脑,分别观察脑含水量、缺血侧脑组织的超氧化物歧化酶(SOD)和丙二醛(MDA)含量.结果:超短波治疗、丹参治疗及二者合用治疗均能减轻大鼠缺血侧脑含水量(P＜0.05),提高抗氧化酶SOD含量(P＜0.05),降低自由基产物MDA的含量(P＜0.05),3个治疗组之间差异无显著性意义.结论:超短波治疗与丹参治疗对大鼠局灶性脑缺血再灌注损伤具有神经保护作用,此作用可能与减轻脑水肿,升高SOD,降低MDA有关,二者疗效比较未见明显差异.     

大鼠急性脑缺血后Hephaestine表达变化的研究

目的　研究大鼠急性局灶性脑缺血后皮层和纹状体hephaestine表达的变化。 方法　用线栓法制备大鼠急性大脑中动脉阻塞 (MCAO)再灌注模型 ,在再灌注后的不同时间点应用免疫组化及图像分析检测皮层和纹状体的表达。结果　MCAO再灌注后大鼠出现大脑中动脉梗塞的神经系统损害体征 ,TTC染色有白色梗死区。hep haestine在正常大鼠的皮层、纹状体有表达 ,在急性脑缺血再灌注后 12h缺血侧皮层、纹状体表达明显增加并持续到再灌注后 4 8h ,在 2 4h达到高峰 (P <0 .0 1) ,至 1周时表达较正常明显减少 (P <0 .0 1)。结论　大鼠急性脑缺血再灌注后hephaestine的表达出现明显的变化 ,在急性脑缺血的病理生理变化中可能起着重要的作用。     

缺血再灌注后皮层神经元内蛋白酶体活性变化与延迟性神经元死亡的关系

目的探讨缺血再灌注后大鼠皮层神经元内蛋白酶体活性改变与神经元延迟性死亡的关系。方法采用20min全脑缺血大鼠模型。将50只Wistar大鼠按照再灌注时间分为5组：假手术组、0．5h恢复组、4h恢复组、24h恢复组及72h恢复组,每组10只大鼠。以Sue-llvy-aline为底物测定蛋白酶体的活性;采用HE染色在光镜下观察缺血再灌注后神经元的死亡。应用免疫组织化学法结合激光扫描共聚焦显微镜观察缺血再灌注后蛋白酶体在细胞内的分布。结果假手术组蛋白酶体活性[吸光度（A）值]为54602±1602,缺血再灌注0．5h后蛋白酶体活性为42036±1465（与假手术组比较,P〈0．01）,虽然4h后其活性一过性恢复到47536±2532（P〈0．05）,但24h后则下降为45450±649（P〈0．01）,再灌注后72h其活性为43108±995（P〈0．01）。HE染色显示,缺血再灌注72h后,可见皮层神经元部分死亡。激光共聚焦扫描显微镜显示缺血再灌注24h后,皮层神经元的胞核与胞质内的蛋白酶体都有减少,72h后死亡神经元胞核内的蛋白酶体几乎全部消失,周围的胞质内只有少量蛋白酶体。结论全脑缺血再灌注后,蛋白酶体活性下降是导致神经元延迟性死亡的一个重要因素。     

远隔后适应对大鼠脑组织中淀粉样前体蛋白表达的影响

目的研究远隔缺血后适应对大鼠脑缺血再灌注损伤后存在于神经元和神经胶质细胞中的淀粉样前体蛋白(β-amyloid precursor protein,β-APP)的影响。方法应用线栓法制作大鼠大脑中动脉闭塞缺血模型,缺血即刻和再灌注即刻分别给予肢体缺血后适应。应用双侧股动脉夹闭10 min/放开10 min,反复3次进行肢体缺血后适应。实验分为7组:假手术组,单纯缺血再灌注组(4 h、24 h组),缺血再灌注+缺血即刻远隔缺血后适应组(4、24 h组),缺血再灌注+再灌注即刻远隔缺血后适应组(4 h、24 h组),每组4只大鼠。采用免疫荧光方法观察β-APP蛋白表达的部位;采用免疫印迹方法研究β-APP的蛋白表达水平。结果脑缺血组大鼠缺血脑组织β-APP蛋白表达在缺血后不同时间点与假手术组比较差异无统计学意义。但是,可以看出β-APP在大鼠缺血后4 h无升高,缺血后24 h开始升高。给予远隔缺血后适应组与单纯缺血组比较,24 h时缺血即刻给予远隔后适应组,β-APP蛋白水平显著低于对照组(P<0.05)。结论远隔缺血后适应可能会通过降低脑缺血后β-APP蛋白表达水平,从而减轻脑缺血损伤。     

米诺环素对大鼠脑缺血半暗带脑血流量及内皮素-1蛋白表达的影响

目的：探讨米诺环素对大鼠脑缺血再灌注损伤后缺血半暗带脑血流量及内皮素-1（endothclin-1,ET-1）蛋白表达的影响。方法采用线栓法制作大鼠脑缺血再灌注损伤模型,将35只 SD 大鼠随机分为假手术组（n＝10）、模型组（n＝15）及米诺环素组（n＝15）。再灌注24 h 后,采用 Longa 法评估大鼠神经功能,激光多普勒血流仪监测大鼠脑缺血半暗带局部脑血流量,免疫组织化学法观察缺血侧皮质 ET-1蛋白的分布情况,再灌注6、24 h 时放射免疫法定量测定 ET-1蛋白的水平。结果同假手术组相比,模型组大鼠神经功能评分增加（P＜0．05）;缺血半暗带脑血流量明显减少（P ＜0．05）,ET-1蛋白表达显著增加（P ＜0．05）,米诺环素组较模型组大鼠神经功能评分明显降低（P＜0．05）,脑缺血半暗带局部脑血流量增加（P＜0．05）,ET-1蛋白的表达显著降低（P＜0．05）。结论米诺环素可增加大鼠脑缺血后局部脑血流量,抑制 ET-1蛋白的表达,减轻大鼠神经功能损害,从而发挥神经保护作用。     

局灶性脑缺血再灌注大鼠IL—1Ra mRNA的表达

目的　观察脑缺血再灌注后白细胞介素 1受体拮抗剂 (IL 1Ra)mRNA的表达。②方法　采用线栓法制备SD大鼠局灶性大脑中动脉阻塞 (MCAO)动物模型 ,应用RT PCR方法观察脑缺血再灌注对大鼠IL 1RamRNA表达的影响。③结果　MCAO模型大鼠缺血侧大脑皮质再灌注后 12 ,2 4h的IL 1RamRNA表达与正常对照组比较明显增加 (F =39.84,q=9.0 3,15 .91,P <0 .0 0 1) ;缺血侧纹状体缺血再灌注后 12h的IL 1RamRNA表达与正常对照组比较明显增加 (F =7.37,q=6 .5 4,P <0 .0 0 1)。④结论　脑缺血后内源性IL 1RamRNA表达增加 ,可能是机体对缺血性损伤的自我保护机制。     

肾上腺髓质素对大鼠局灶性脑缺血再灌注后脑组织的保护作用

目的 探讨肾上腺髓质素在脑缺血再灌注大鼠脑内的表达情况及其对再灌注损伤的保护作用.方法 采用拴线法制成大鼠大脑中动脉缺血再灌注模型,阻断血流2 h后进行再灌注.应用免疫组织化学染色法检测不同时间点大鼠局灶性脑缺血再灌注后肾上腺髓质素的表达情况,并进行动态观察.结果 正常对照组大鼠脑内即有肾上腺髓质素表达,假手术组术后肾上腺髓质素表达略有增加,但与正常对照组相比差异无显著性意义(P＞0.05);大鼠脑缺血再灌注后肾上腺髓质素免疫阳性细胞增多,与正常对照组及假手术组相比差异均有显著性意义(P＜0.05);大鼠脑缺血再灌注后缺血侧和缺血对侧肾上腺髓质素免疫阳性细胞均增多,但以缺血侧区域增多最为明显.动态观察发现,脑缺血再灌注2 h后,肾上腺髓质素免疫阳性细胞即增多,缺血再灌注6 h达高峰,至1周仍明显增多.结论 脑缺血再灌注后肾上腺髓质素表达增强,对脑缺血再灌注损伤具有保护作用.     

蕨麻正丁醇提取物对卵巢缺血再灌注损伤大鼠内皮素-1的影响

目的探讨蕨麻正丁醇(Pa)对急性卵巢缺血再灌注损伤大鼠血清及卵巢组织中内皮素-1(ET-1)的影响。方法 Wistar雌性大鼠60只,随机分为6组:假手术组(C组)、缺血24 h组(I24h)、缺血再灌注24 h组(R24h组)、缺血再灌注48 h组(R48h组)、给予Pa提取物预处理+缺血再灌注24 h组(Pa+R24h组)和Pa提取物预处理+缺血再灌注48 h组(Pa+R48h组)。酶联免疫吸附试验测定法检测各组血清ET-1水平;反转录聚合酶链反应、Western blot法检测各组大鼠卵巢组织中ET-1 mRNA和蛋白的表达。结果与C组比较,I24h组、R24h组和R48h组大鼠血清ET-1水平、卵巢组织中ET-1 mRNA及蛋白表达均显著升高(P<0.05);与I24h组比较,R24h组、R48h组大鼠血清ET-1水平、卵巢组织中ET-1 mRNA和蛋白表达均显著升高,差异有统计学意义(P<0.05)。而Pa+R24h组和Pa+R48h组大鼠血清ET-1水平、卵巢组织中ET-1 mRNA和蛋白表达与R24h组、R48h组比较显著下降,差异有统计学意义(P<0.05)。结论 ET-1在大鼠卵巢缺血再灌注损伤过程中表达上调,Pa提取物进行干预可降低ET-1的表达。     

DFMO对缺血再灌流后大鼠皮质、海马中ODC mRNA表达的影响

目的：探讨DFMO抑制ODC活性的作用机制。方法：将40只SD雄性大鼠随机分为缺血对照组和DFMO治疗组，复制大鼠全脑缺血再灌流模型前30min用DFMO治疗处理，用RT-PCR方法检测2组大鼠皮质、海马中2，4，6，8h ODC mRNA的表达，比较其变化情况。结果：治疗组大鼠皮质、海马中ODC mRNA的表达在缺血再灌流2，4，6h均较缺血对照组明显降低（分别P<0.05，P<0.01，P<0.01），8h无明显差异（P>0.05）。结论：DFMO抑制了脑缺血再灌流后大鼠皮质、海马中ODC mRNA 的表达，可能是其抑制ODC活性的原因之一。     

葛根素对脑缺血-再灌注损伤细胞凋亡和p53表达的影响

目的 观察葛根素对预处理后大鼠局灶性脑缺血时海马区凋亡细胞和p53蛋白表达的影响.方法 利用大脑中动脉栓线阻断法制作大鼠局灶性脑缺血2h后再灌注损伤模型.72只SD大鼠随机分为假手术组（S组）、缺血再灌注组（IR组）和葛根素组（P组）.依术后处死动物时间不同,每组分为3个亚组（n=8）.用免疫组化法检测p53蛋白和TUNEL法检测凋亡细胞的表达.结果 P组P2h组p53蛋白表达增高,P24h组显著增高,P72h组p53蛋白表达有所下降,但仍低于IR组（P＜0.01）;P组凋亡细胞数显著低于相应IR组（P＜0.01）.结论 葛根素对大鼠局灶性脑缺血再灌注损伤具有保护作用,其作用机制可能与葛根素抗细胞凋亡、下调p53蛋白有关.     

Protective effect of radix Salviae miltiorrhizae on apoptosis of neurons during focal cerebral ischemia and reperfusion injury.

We have found that Radix Salviae Miltiorrhizae (RSM) plays a protective role in ischemic brain injury, which attracted us to investigate the effect of RSM on apoptosis of neurons during cerebral ischemia and reperfusion. The apoptotic cells in ischemic brains at different reperfusion intervals were tested with the method of TdT-mediated dUTP-DIG nick end labeling (TUNEL), and the effect of RSM on the apoptosis of neurons was studied in left middle cerebral artery (LMCA) occlusion in rat models (n = 18). The results showed that few scattered apoptotic cells were observed in right cerebral hemisphere after LMCA occlusion and reperfusion, and that a lot of apoptotic cells were found in left ischemic cerebral cortex and caudoputamen at 12 h reperfusion, and they reached peak at 24-48 h reperfusion. However, in rats pretreated with RSM, the number of apoptotic cells in left cortex and caudoputamen reduced significantly and the neuronal damage was much milder at 24 h reperfusion as compared with those of saline-treated rats. From this study, we conclude that administration of RSM can reduce the apoptotic of neurons induced by cerebral ischemia and reperfusion and afford significant cerebroprotection in the model of focal cerebral ischemia and reperfusion.     

一氧化氮对大鼠局灶性脑缺血再灌注损伤的影响

目的观察L-精氨酸(L-Arg)和氨基胍(AG)对大鼠局灶性脑缺血组织中一氧化氮(NO)含量的影响,探讨NO对脑缺血再灌注损伤的作用及机制。方法将60只大鼠随机分为假手术组、模型组、L-Arg组和AG组,每组15只。L-Arg组、AG组、模型组和假手术组用线栓法建立大鼠局灶性脑缺血(MCAO)模型后,L-Arg组按500mg·kg-1腹腔注射1mLL-Arg注射液;AG组按100mg·kg-1术后腹腔注射1mLAG注射液;模型组和假手术组术后腹腔注射1mL灭菌生理盐水。观察缺血再灌注后12、24、72h大鼠行为学改变,血清中NO浓度和脑内一氧化氮合酶(NOS)分布的变化。结果与模型组相比,L-Arg组在缺血再灌注12h行为学评分显著降低(P<0.05),AG组在缺血再灌注24h行为学评分显著降低(P<0.05);AG组在缺血再灌注12h行为学评分高于L-Arg组(P<0.05)。与假手术组相比,模型组、L-Arg组和AG组缺血再灌注12、24、72h的NO含量均显著增加(P<0.05);与模型组相比,L-Arg组缺血再灌注12h的N0含量显著升高(P<0.05),AG组缺血再灌注24h的NO含量显著降低(P<0.05)。缺血再灌注12和24h,AG组的NO含量均显著低于L-Arg组(P<0.05);与假手术组相比,缺血再灌注12、24、72h的模型组、L-Arg组和AG组的iNOS阳性细胞均显著增加(P<0.05);与模型组相比,缺血再灌注12、24、72h的L-Arg组iNOS阳性细胞数差别无统计学意义(P>0.05);但AG组在3个时间点的iNOS阳性细胞数均显著低于模型组(P<0.05);AG组在3个时间点的iNOS阳性细胞数均低于L-Arg组(P<0.05)。结论脑缺血再灌注损伤后脑组织内NO和NOS的表达随时间动态变化,且NO在参与缺血性脑损伤过程中可能具有双重作用。L-Arg、AG通过不同的作用机制对大鼠局灶性脑缺血再灌注损伤具有保护作用。