大鼠脊髓损伤早期Survivivn表达规律与细胞凋亡的关系

目的:探讨大鼠脊髓损伤早期不同时间段内脊髓组织中生存素(Survivin)表达变化规律和细胞凋亡的关系。方法:56只Wistar大鼠随机分两组:①假损伤组(对照组,n=8)与②损伤组(实验组,48只);假损伤组仅打开T10及部分T9、T11椎板,实验组均采用改良Allens法建立大鼠T10脊髓损伤模型,分别于伤后不同时间点(12h、24h、3d、5d、7d、10d)随机处死8只取材;分别用HE染色观察组织病理变化、免疫组织化学法观察脊髓组织中Survivin表达并对其进行定性定量分析和脱氧核糖末端转移酶介导的缺口末端标记法(TUNEL法)测定细胞凋亡指数,并进行相关性分析。结果:对照组大鼠在损伤及周边脊髓灰、白质中偶有Survivin蛋白表达、实验组12h有极少表达、3d时可见较多Survivin蛋白表达,然后开始下降;10d时与对照组比较仍有统计意义(P<0.05)。在脊髓损伤后对照组有少量细胞凋亡;而试验组12h就出现大量凋亡细胞,3d达高峰,5d和7d明显减少,10d仍有少量表达;实验组24h、3d、5d、7d、10d细胞凋亡和对照组相比差异均有统计学意义(P<0.05)。结论:脊髓损伤后Survivin表达呈上升趋势并有一定的规律性,与细胞凋亡成正相关,脊髓损伤后Survivin的表达升高参与了抑制神经细胞的凋亡,从而起到减轻脊髓损伤的作用。     

脂多糖致胎鼠脑白质损伤探讨

目的：观察胎鼠脑组织形态学和髓鞘碱性蛋白(MBP)的表达情况。方法：通过脂多糖复制孕鼠感染模型，利用免疫组化和HE染色等方法观察胎鼠脑组织。结果：(1)脂多糖可引起胎盘组织中性粒细胞浸润。(2)试验组胎鼠脑组织出现核仁不清的欠分化神经元，并有局灶性出血．髓鞘碱性蛋白染色较对照组弱。结论：脂多糖是制备胎鼠脑损伤的一种有效形式。(2)感染可引起髓鞘碱性蛋白表达减少，可能是白质损伤的重要原因。     

神经胶质细胞与慢性脑缺血致脑白质损伤的相关性研究进展

慢性脑缺血是由各种原因引起的脑血管狭窄和（或）低灌注,脑血流量长期、慢性供应不足,导致脑代谢障碍和功能衰退的一个病理过程,临床上主要表现为渐进性的学习记忆和空间辨别能力下降,甚至痴呆。脑白质损伤主要为脑白质疏松症,为脑室周围白质和深部白质区域弥散分布的斑点状或斑片状改变,在脑CT上为弥散低密度影,在脑MRIT2加权像上为双侧弥散性的高信号。     

细胞凋亡在脊髓损伤中的表达及意义

目的：探讨细胞凋亡在脊髓损伤发病机理中的作用。方法：采用Allen氏方法制作大鼠脊髓损伤模型，用HE染色及原位末端标记法，对大鼠脊髓损伤后不同时间脊髓组织中凋亡细胞的数目进行观察和分析，并作正常对照。结果：损伤后灰质和白质均检测到凋亡细胞，细胞凋亡发生在损伤后2h-4周，以3d时达到高峰，至4周时渐趋于正常。结论：大鼠脊髓损伤后神经细胞存在死亡和凋亡两种方式，对研究脊髓损伤的病理生理过程有重要意义。     

重型颅脑损伤后Pim-3的表达与细胞凋亡关系的研究

目的探讨颅脑损伤后Pim-3的表达及其与细胞凋亡的关系。方法 48只大鼠随机分为实验组与对照组。实验组大鼠制作重型颅脑损伤模型,对照组大鼠予以假手术处理,两组大鼠分别在伤后6、24、72h及7d免疫组化SP法检测Pim-3蛋白表达、TUNEL法检测细胞凋亡。结果重型颅脑损伤后大鼠脑组织Pim-3蛋白水平、细胞凋亡水平均较对照组明显升高,并在伤后24h达到高峰,7d后开始下降。结论 Pim-3可能在颅脑损伤后细胞凋亡中起重要作用。     

脊髓损伤早期细胞凋亡的表达及意义

目的:观察大鼠急性脊髓损伤后不同时间细胞凋亡调控基因Bcl-2和Bax的表达。方法:Wistar大鼠56只随机分成7组,将6组制成急性脊髓损伤模型,另一组于伤后0h、4h、8h、24h、48h、72h对其运动功能进行评分,随后采集脊髓标本,分别进行HE染色、TENEL标记和免疫组化检测其凋亡调控基因Bcl-2和Bax的表达。结果:大鼠脊髓损伤后表现运动能力下降。TUNEL染色显示脊髓伤后4h出现凋亡阳性颗粒,伤后8h到达高峰。Bcl-2和Bax都在伤后4h可见阳性表达,Bcl-2表达在24h到达高峰,Bax的表达高峰出现在伤后8h。结论:大鼠脊髓损伤后,除存在细胞坏死外还存在神经细胞凋亡。细胞内Bcl-2与Bax的比例可能调节着凋亡的发生。     

丙泊酚对新生大鼠脑皮层星形胶质细胞凋亡的影响

目的研究丙泊酚对新生大鼠脑皮层星形胶质细胞凋亡的影响及其机制。方法原代分离和培养AST细胞,用GFAP免疫细胞化学鉴定。不同浓度丙泊酚(0、10、30、90μmol·L-1)作用AST细胞8 h。MMT法检测细胞生存率;Annexin V/PI双染检测细胞凋亡;Western blot法测定线粒体cyt-C漏出;分光光度计法检测caspase-3和caspase-9活性。结果分离培养出原代AST,不同浓度丙泊酚(0、10、30、90μmol·L~(-1))浓度依赖性地降低AST细胞生存率,诱导细胞线粒体cyt-C漏出,上调促凋亡蛋白caspase-3和caspase-9,诱发AST凋亡。结论丙泊酚在体外能诱发新生大鼠脑皮层星形胶质细胞凋亡,其机制可能与丙泊酚诱导线粒体cyt-C释放,激活线粒体凋亡通路有关。     

新生大鼠缺氧缺血性脑损伤时Bcl-2蛋白表达的研究

目的：通过研究新生大鼠脑缺血时bcl-2蛋白在海马区的表达特点，探讨bcl-2蛋白在缺血缺氧性脑损伤（HIBD）的作用。方法：56只7日龄新生SD大鼠随机分为1个对照组和7个实验组。给动物吸入含有92％氮气和8％氧气混合气体建立新生大鼠HIBD动物模型，分别在脑损伤后不同时间点（0．5、1、3、6、12、24、48、72h等）断头处死动物，取海马组织，用免疫组织化学方法检测海马脑神经细胞凋亡及bcl-2蛋白表达情况。结果：TUNEL染色的阳性细胞数与bcl—2蛋白阳性的细胞数在HIBD后的不同时间点出现分别有显著差异．海马区bcl-2蛋白阳性细胞在HIBD后立即出现，6h达到高峰，之后渐下降。结论：bel-2蛋白强表达于海马缺血区和缺血周边区的神经元。与神经元的存活或死亡有一定联系。     

芍药苷对新生大鼠缺氧缺血性脑损伤中脑细胞凋亡的影响

目的 观察芍药苷对新生大鼠缺氧缺血性脑损伤(HIBD)中脑细胞凋亡的影响,并探讨其脑保护的作用机制.方法 30只7日龄SD大鼠,随机均分成假手术组、缺氧缺血模型组、芍药苷低、中、高剂量治疗组.通过结扎左颈总动脉和吸入8%低氧混合气制备缺氧缺血性脑损伤(HIBD)动物模型.假手术组不进行缺氧缺血处理,各组新生鼠均于缺氧缺血后48 h处死,取大脑皮层及海马区组织进行HE染色及TUNEL法凋亡细胞检测.结果 假手术组大鼠的大脑皮层及海马区组织、细胞形态正常;缺氧缺血模型组的大脑皮层及海马区组织水肿,细胞周围间隙增宽,胞体肿胀变圆、变大、胞浆染色变浅;各芍药苷组的神经元细胞胞体肿胀及组织水肿情况较缺氧缺血模型组明显好转.假手术组的大脑皮层及海马CA1区未见凋亡细胞;缺氧缺血模型组的可见较多凋亡细胞;各芍药苷组的凋亡细胞较相应时点缺氧缺血模型组的减少,差异有统计学意义(P＜0.05).结论 芍药苷能减轻新生大鼠HIBD时的脑病理损伤,抑制大鼠HIBD时的脑细胞凋亡,对新生大鼠HIBD脑损伤具保护作用.     

Correlation between chromosome damage and apoptosis induced by fludarabine and idarubicin in normal human lymphocytes

Fludarabine (FLU, a fluorinated purine analog) and idarubicin (IDA, a DNA-topoisomerase II poison) are frequently used in cancer chemotherapy. The effects of these drugs on cultured normal human lymphocytes were studied to establish the possible involvement of chromosome damage in the apoptotic program. Chromosome aberrations (CA) were evaluated in first division metaphases and the apoptotic process was measured by morphological and electrophoretical techniques. The percentage of abnormal cells was increased from the doses of FLU 1.0 microg/ml and IDA 0.005 microg/ml (P<0.0001) with an important decrease in the mitotic index (MI) for the highest doses assayed. A significant dose-dependent induction of abnormal cells was observed for both drugs. An increase of apoptotic cells was found at 5.0 and 10.0 microg/ml of FLU (P<0.001) while IDA activated apoptosis at 0.05 microg/ml (P<0.01) and markedly from 0.1 microg/ml (P<0.001). These increments were dose dependent. Apoptotic cell morphology was associated with DNA fragmentation at the highest doses. The increased induction of abnormal cells and the decreased MI were in correlation with the apoptotic index for FLU and IDA, suggesting the role of CA in drug-induced cell death.     

热、放射诱导的SHG-44细胞凋亡与Fas/FasL、Caspase-3表达关系

目的 了解热疗联合放射对SHG-44细胞凋亡的影响及凋亡相关基因Fas／FasL和Caspase-3的变化。方法 采用流式细胞仪(FCM)检测对照组、单纯放射(2Gy)组、放射(2Gy) 42℃加热(加热时问40分钟)组细胞凋亡的改变,并用RT-PCR法检测Fas／FasL和Caspase-3的表达。结果 空白对照组、单纯放射组、放射 热疗组凋亡比率分别为20．2％、29．3％和59．1％;Fas在三个实验组中均表达,但FasL和Caspase-3仅在单纯放射组、放射 热疗组中表达。结论 凋亡是热杀灭脑胶质瘤细胞SHG-44的主要方式,凋亡与Fas／FasL、Caspase-3有关。     

三叶青黄酮诱导肺癌SPC-A-1细胞凋亡与cleaved-caspase-3表达的关系

目的：观察三叶青黄酮诱导肺癌SPC-A-1细胞凋亡作用和对cleaved-caspase-3表达的影响,探讨其抗肺癌的可能作用机制。方法：体外培养SPC-A-1细胞,以不同剂量三叶青黄酮进行处理,未给予三叶青黄酮组作为对照组。采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（MTT）比色分析法,观察三叶青黄酮对SPC-A-1细胞增殖抑制作用,计算半数抑制浓度（IC₅₀）;采用流式细胞仪和TUNEL染色检测三叶青黄酮对SPC-A-1细胞凋亡的影响;分别采用免疫细胞分析和蛋白印记分析（Western blot）检测三叶青黄酮对SPC-A-1细胞cleaved-caspase-3表达的影响。结果：三叶青黄酮对SPC-A-1细胞增殖具有明显抑制作用,呈剂量和时间依赖性,并诱导SPC-A-1细胞凋亡,与对照组比较,差异具有统计学意义（P<0.05或P<0.01）;三叶青黄酮显著增加SPC-A-1细胞cleaved-caspase-3表达水平,与对照组比较,差异具有统计学意义（P<0.05或P<0.01）。结论：三叶青黄酮可显著抑制SPC-A-1细胞增殖,并诱导其凋亡,机制可能与增强cleaved-caspase-3表达有关。     

Quercetin protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead

Quercetin, a flavonoid, effectively improved the lead-induced histology changes including structure damage and leukocyte infiltration in rat kidney. The present study was designed to explore the protective mechanism of quercetin against lead-induced oxidative DNA damage and apoptosis in rat kidney. We found that quercetin markedly decreased the ROS level and lowered the GSH/GSSG ratio in the kidney of lead-treated rat. The increase of 8-hydroxydeoxyguanosine level in the kidney of lead-treated rat was effectively suppressed by quercetin. Furthermore, quercetin markedly restored Cu/Zn-SOD, CAT and GPx activities in the kidney of lead-treated rat. TUNEL assay showed that lead-induced apoptosis in rat kidney was significantly inhibited by quercetin, which might be attributed to its antioxidant property. In conclusion, these results suggested that quercetin could protect the rat kidney against lead-induced injury by improving renal function, attenuating histopathologic changes, reducing ROS production, renewing the activities of antioxidant enzymes, decreasing DNA oxidative damage and apoptosis.     

Quercetin protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead

Quercetin, a flavonoid, effectively improved the lead-induced histology changes including structure damage and leukocyte infiltration in rat kidney. The present study was designed to explore the protective mechanism of quercetin against lead-induced oxidative DNA damage and apoptosis in rat kidney. We found that quercetin markedly decreased the ROS level and lowered the GSH/GSSG ratio in the kidney of lead-treated rat. The increase of 8-hydroxydeoxyguanosine level in the kidney of lead-treated rat was effectively suppressed by quercetin. Furthermore, quercetin markedly restored Cu/Zn-SOD, CAT and GPx activities in the kidney of lead-treated rat. TUNEL assay showed that lead-induced apoptosis in rat kidney was significantly inhibited by quercetin, which might be attributed to its antioxidant property. In conclusion, these results suggested that quercetin could protect the rat kidney against lead-induced injury by improving renal function, attenuating histopathologic changes, reducing ROS production, renewing the activities of antioxidant enzymes, decreasing DNA oxidative damage and apoptosis.     

Correlation between catalepsy and dopamine decrease in the rat striatum induced by neuroleptics.

The time-course of the development of catalepsy was well correlated with that of a decrease in dopamine in the corpus striatum in rats after the subcutaneous administration of 10 mg/kg haloperidol. The striatal dopamine was decreased by a subcutaneous injection of haloperidol in the same dose-dependent manner as the catalepsy was intensified with increasing doses of the drug. By repeated administration of haloperidol or trifluperidol, the catalepsy in rats was intensified, and the dopamine decrease in the rat striatum was extended through 10 days treatment. However, by a repeated administration of ID-4708, a new butyrophenone compound, the intensity of catalepsy and the dopamine decrease induced by its initial administration was little altered. An oral administration of 100 mg/kg chlorpromazine produced moderate catalepsy and decreased the striatal dopamine to 80% of the value for control animals. No catalepsy or only slight catalepsy was observed after the administration of thioridazine, clozapine and sulpiride, and a decrease of dopamine in the striatum did not occur.     

Gypenoside attenuates white matter lesions induced by chronic cerebral hypoperfusion in rats

Cerebral white matter lesions (WMLs) are frequently observed in vascular dementia and Alzheimer's disease and are believed to be responsible for cognitive dysfunction. The cerebral WMLs are most likely caused by chronic cerebral hypoperfusion and can be experimentally induced by permanent bilateral common carotid artery occlusion (BCCAO) in rats. Previous studies found the involvement of oxidative stress and astrocytic activation in the cerebral WMLs of BCCAO rats. Gypenoside (GP), a pure component extracted from the Gyrostemma pentaphyllum Makino, a widely reputed medicinal plants in China, has been reported to have some neuroprotective effects via anti-oxidative stress and anti-inflammatory mechanisms. In the present study, we investigated the protective effect of GP against cerebral WMLs and the underlying mechanisms for its inhibition of cognitive decline in BCCAO rats. Adult male Sprague-Dawley rats were orally administered daily doses of 200 and 400 mg/kg GP for 33 days after BCCAO, and spatial learning and memory were assessed using the Morris water maze. Following behavioral testing, oxygen free radical levels and antioxidative capability were measured biochemically. The levels of lipid peroxidation and oxidative DNA damage were also assessed by immunohistochemical staining for 4-hydroxynonenal and 8-hydroxy-2′-deoxyguanosine, respectively. Activated astrocytes were also assessed by immunohistochemical staining and Western blotting with GFAP antibodies. The morphological changes were stained with Klüver-Barrera. Rats receiving 400 mg/kg GP per day performed significantly better in tests for spatial learning and memory than saline-treated rats. GP 400 mg/kg per day were found to markedly scavenge oxygen free radicals, enhance antioxidant abilities, decrease lipid peroxide production and oxidative DNA damage, and inhibit the astrocytic activation in corpus callosum and optic tract in BCCAO rats. However, GP 200 mg/kg per day had no significant effects. GP may have therapeutic potential for treating dementia induced by chronic cerebral hypoperfusion and further evaluation is warranted.     

Neuroprotective activities of catalpol against CaMKII-dependent apoptosis induced by LPS in PC12 cells

Background and Purpose

Neurodegenerative diseases present progressive neurological disorder induced by cell death or apoptosis. Catalpol, an iridoid glucoside isolated from the root of Rehmannia glutinosa Libosch, is present in a wide range of plant families. Although catalpol is an effective anti-apoptotic agent in LPS-induced neurodegeneration, the underlying mechanism has not been established. Here we have identified some of the mechanisms involved the prevention by catalpol of apoptosis induced by LPS in an experimental model of neurodegeneration in vitro.

Experimental Approach

Apoptosis was induced by adding LPS (80 ng·mL⁻¹) to pheochromocytoma (PC12) cells, pretreated with catalpol for 12 h. We measured intracellular reactive oxygen species (ROS), apoptosis and intracellular calcium concentration ( [Ca²⁺]i) by flow cytometry or laser confocal scanning microscopy. We also analysed the protein expression of Bcl-2, Bax and Ca²⁺-calmodulin-dependent protein kinase II (CaMKII)-dependent apoptosis signal-regulating kinase-1 (ASK-1)/JNK/p38 signalling pathway in PC12 cells by Western blot.

Key Results

Catalpol stimulated expression of Bcl-2 and inhibited the expression of Bax. Catalpol also attenuated the increase in Ca²⁺ concentration induced by LPS in PC12 cells and down-regulated CaMK phosphorylation. The CaMKII-dependent ASK-1/JNK/p38 signalling cascade was blocked by catalpol. All these changes were accompanied by a decrease of apoptosis induced by LPS in PC12 cells.

Conclusions and Implications

The data presented here provide new mechanistic insights into the links between the CaMKII-dependent ASK-1/JNK/p38 signalling pathway and the protective effect of catalpol on apoptosis induced by LPS in PC12 cells.     

Gambogic acid alleviates inflammation and apoptosis and protects the blood-milk barrier in mastitis induced by LPS

Mastitis is one of the most common diseases among dairy cows. There is still much debate worldwide as to whether antibiotic therapy should be given to dairy cows, or if natural products should be taken as a substitute for antibacterial therapy. As the antibiotic treatment leads to the bacterial resistance and drug residue in milk, introducing natural products for mastitis is becoming a trend. This study investigates the mechanisms of the protective effects of the natural product gambogic acid (GA) in lipopolysaccharide (LPS)-induced mastitis. For in vitro treatments, it was found that GA reduced IL-6, TNF-α, and IL-1β levels by inhibiting the phosphorylation of proteins in the nuclear factor κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathway. GA also maintained a stable membrane mitochondrial potential and inhibited the overproduction of reactive oxygen species, which protected the cells from apoptosis. On the other hand, in vivo treatments with GA were found to reduce pathological symptoms markedly, and protected the blood-milk barrier from damage induced by LPS. The results demonstrate that GA plays a vital role in suppressing inflammation, alleviating the apoptosis effect, and protecting the blood-milk barrier in mastitis induced by LPS. Thus, these results suggest that the natural product GA plays a potential role in mastitis treatment.