Roles of p53, c-Myc, Bcl-2, Bax and caspases in serum deprivation-induced neuronal apoptosis: a possible neuroprotective mechanism of basic fibroblast growth factor.

Using flow cytometric analysis, we examined the temporal changes of p53, c-Myc, Bcl-2, Bax expression in rat primary cortex neurons after serum deprivation. Activities of caspase-1 and caspase-3 were also measured. Serum deprivation induced apoptosis accompanied by a rapid down-regulation of p53, Bcl-2 and an up-regulation of c-Myc, Bax and caspase-3 activity. Pretreatment with basic fibroblast growth factor prevented the apoptosis with an attenuation of the changes of p53, Bcl-2, Bax levels and caspase-3 activity but had no effect on the change of c-Myc level. These results suggest that serum deprivation induces apoptosis through a signaling pathway involving p53, Bcl-2, Bax, c-Myc and caspase-3. The effect of the basic fibroblast growth factor against apoptosis may result from its capability of blocking the apoptosis cascade.     

碱性成纤维细胞生长因子对脑出血大鼠神经细胞凋亡和BaX、Bcl-2基因表达的影响

目的观察碱性成纤维细胞生长因子(bFGF)对大鼠脑出血后神经细胞凋亡及血肿周围大脑皮质和出血侧海马Bax、Bcl-2基因表达的影响。方法采用脑内注射胶原酶建立大鼠脑出血模型,应用TUNEL法测定大鼠大脑皮质、海马神经细胞凋亡数目。及应用采用半定量逆转录酶聚合链反应(RT-PCR),检测脑出血后血肿周围大脑皮质和出血侧海马的Bax mRNA、Bcl-2 mRNA表达。结果(1)bFGF组的皮质和海马神经细胞凋亡数比生理盐水(NS)对照组明显减少(P<0.05)。(2)bFGF组的血肿周围大脑皮质和出血侧海马Bax mRNA表达比NS对照组明显减少(P<0.05);而Bcl-2 mRNA表达比NS对照组明显增高(P<0.05)。结论bFGF提高大鼠脑出血后大脑组织和海马Bcl-2 mRNA的表达,降低Bax mRNA的表达,抑制脑出血后神经细胞凋亡。     

碱性成纤维细胞生长因子对癫痫大鼠海马神经元凋亡的影晌

目的探讨碱性成纤维细胞生长因子（bFGF）对致痫大鼠的神经保护作用及可能机制。方法给予戊四氮（PTZ）致痫大鼠每日腹腔注射bFGF（bFGF组，n=36）、生理盐水（NS组，n=36），分别于痫性发作后6h、12h、24h、48h、3d、5d处死取脑，切片进行bcl-2、caspase-3染色，用原位末端标记（TUNEL）方法检测海马神经元凋亡细胞。结果痫性发作6h后两组海马CA1、CA3区的bcl-2、caspase-3、TUNEL染色阳性表达差异无统计学意义（P〉0．05）；12～48h表达逐渐增强，bFGF组bcl-2、TUNEL的表达显著高于NS组，bFGF组caspase-3的表达显著低于NS组（P均〈0．01）；3d后表达减弱，bFGF组与NS组的差异无统计学意义（P〉0．05）。结论bFGF能显著减轻癫痫所致的海马神经元凋亡，提示可能通过调控bcl-2和caspase-3基因的表达发挥作用。     

脑缺血耐受与细胞凋亡及p53、p21、Bax表达关系的实验研究

目的　探讨大鼠局灶性缺血预处理的脑保护作用与细胞凋亡及凋亡相关因子p5 3、p2 1和Bax的关系。方法　采用线栓法阻塞大鼠大脑中动脉造成脑缺血预处理和致死性缺血模型 ,图像分析测算相对梗死体积 ,使用TUNEL染色标记神经细胞凋亡 ,免疫组化染色观察p5 3、p2 1和Bax蛋白表达。结果　与致死缺血组比较 ,缺血预处理组梗死体积减少 4 6 % (P <0 0 5 ) ,半暗区TUNEL阳性细胞数和p5 3阳性细胞数均明显降低 (均P <0 0 5 ) ,p2 1和Bax阳性细胞数无显著变化 (均P >0 0 5 )。结论　缺血预处理可能通过抑制严重缺血后p5 3表达 ,减轻神经细胞凋亡 ,发挥脑保护作用。p2 1和Bax在脑缺血耐受形成中可能不起重要作用。     

亚低温对大鼠局灶性脑缺血神经细胞凋亡及Bcl—2、Bax的影响

目的 研究不同时程的亚低温对大鼠大脑中动脉闭塞（MCAO）模型的神经细胞凋亡及Bcl-2、Bax的影响。方法 利用MCAO模型,缺血3小时后再灌注。缺血后即刻分别进行不同时程（0．5、1、3小时）的亚低温治疗,再灌注后24小时取脑切片作TUNEL染色及Bcl-2、Bax免疫组化染色。结果 （1）亚低温1、3小时组的凋亡细胞、Bax细胞数与对照组比较均明显降低（P＜0．05,P＜0．01）。（2）亚低温3小时组的Bcl－2细胞阳性率与对照组相比明显增加（P＜0．05）。（3）凋亡细胞阳性率与Bcl-2／Bax比值呈负相关关系（r＝－0．9759,P＜0．01）。结论 缺血性脑损伤的病理过程与细胞凋亡有关,Bcl-2／Bax比值能决定细胞凋亡的发生。亚低温能增强Bcl－2并降低Bax基因的表达,有抑制细胞凋亡的作用,是保护缺血神经元的重要方法之一。     

Bcl-2, Bax and Bcl-x expression in neuronal apoptosis: a study of mutant weaver and lurcher mice

We investigated the expression of the apoptosis modulating proteins Bcl-2, Bax and Bcl-x in the cerebellum of mutant lurcher and weaver mice. Lurcher Purkinje cells and weaver germinal (granule neuron progenitor) cells both die via apoptosis during the postnatal cerebellar development. No significant changes in the expression patterns were detected prior to the actual cell death process. Instead apoptotic lurcher Purkinje cells exhibited increased Bax and Bcl-x expression, while surviving cells had an expression pattern similar to that of healthy littermates. Increased Bax expression was also found in apoptotic weaver germinal cells, while no change of Bcl-x expression was detected. Bcl-2 was expressed at low levels in cerebellar neurons and no loss of Bcl-2 was evident. The observed expression patterns of Bcl-2, Bax and Bcl-x protein in apoptotic lurcher and weaver neurons support the hypothesis that the execution of neuronal apoptosis involves increased expression of Bax, which could represent a general mechanism in diverse neurodegenerative processes. Received: 13 July 1997 / Revised, accepted: 27 February 1998     

谷氨酸诱导转Bax基因PC12细胞凋亡及 神经生长因子的保护作用

目的探讨谷氨酸诱导转Bax基因PC12细胞凋亡及神经生长因子（NerveGrowthFactor,NGF）的保护作用。方法应用脂质体介导细胞转染方法进行Bax基因转染PC12细胞,结合流式细胞术和TUNEL法检测了谷氨酸处理前后转Bax基因PC12细胞的凋亡及NGF的保护作用。结果与对照组相比,谷氨酸处理转Bax基因PC12细胞凋亡峰增高,TUNEL阳性标记细胞增多;用NGF处理后该组细胞凋亡峰减低,TUNEL阳性标记细胞数减少。差异均有显著意义。结论谷氨酸处理转染Bax基因PC12细胞凋亡增加,NGF可以保护谷氨酸诱导的转染Bax基因PC12细胞凋亡。     

Effects of genistein on neuronal apoptosis, and expression of Bcl-2 and Bax proteins in the hippocampus of ovariectomized rats

Genistein is one of several isoflavones that has a structure similar to 17β-estradiol, has a strong antioxidant effect, and a high affinity to estrogen receptors. At 15 weeks after ovariectomy, the expression of Bcl-2 in the hippocampus of rats decreased and Bax expression increased, with an obvious upregulation of apoptosis. However, intraperitoneal injection of genistein or 17β-estradiol for 15 consecutive weeks from the second day after operation upregulated Bcl-2 protein expression downregulated Bax protein expression, and attenuated hippocampal neuron apoptosis. Our experimental findings indicate that long-term intervention with genistein can lead to a decrease in apoptosis in hippocampal neurons following ovariectomy, upregulate the expression of Bcl-2, and downregulate the expression of Bax. In addition, genistein and 17β-estradiol play equal anti-apoptotic and neuroprotective roles.     

Overexpression of nerve growth factor and basic fibroblast growth factor in AIDS dementia complex.

Although neurotrophic factors are currently considered as treatment for neurodegenerative diseases, little is still known about their presence in the central nervous system under pathological conditions. We investigated the expression of the neurotrophic molecules NGF, bFGF, BDNF and IGF-1 in brain tissue of patients suffering from AIDS dementia complex. In contrast to IGF-1 and BDNF, NGF and bFGF mRNA levels were significantly elevated. Strong NGF immunoreactivity was found in perivascular areas and was colocalized with infiltrating macrophages, whereas intense bFGF staining was found in cells with characteristic astrocytic morphology. These data suggest that the induction of NGF and bFGF alone appears to be insufficient as a compensatory mechanism to prevent ADC.     

Nerve growth factor affects focal cerebral cortical neuronal Bcl-2 and Bax expression in a mouse model of oxyhemoglobin-induced subarachnoid hemorrhage★

BACKGROUND:Studies have demonstrated that oxyhemoglobin(OxyHb)can induce brain cell apoptosis in vivo. OBJECTIVE:To observe the effects of exogenous nerve growth factor(NGF)on cerebral cortical neuronal Bcl-2 and Bax expression in mice with OxyHb-induced subarachnoid hemorrhage. DESIGN,TIME AND SETTING:A completely randomized grouping,controlled animal experiment was performed at the Experimental Center for Biomedicine,College of Medicine,Xi’an Jiaotong University between February and April 2005. MATERIALS:...     

木犀草素对大鼠缺血性脑组织上调Nrf2、Bcl-2下调Bax的表达

目的探讨木犀草素对缺血性脑组织的保护机制。方法用线栓法制备大鼠大脑中动脉闭塞(MCAO)模型。实验采用Western blot和RT-q PCR观察Nrf2、Bcl2、Bax蛋白和基因表达变化,比较各组脑梗死体积、患侧脑水肿和神经功能缺损评分。结果木犀草素可显著改善神经功能缺失,减轻脑水肿,减小脑梗死体积(P0.05)。木犀草素干预后,能明显诱导保护性基因Nrf2的表达,升高Bcl2的表达,降低Bax的表达(P0.05)。结论 Nrf2、Bcl2和Bax可能是木犀草素的作用靶点,木犀草素可能是通过诱导Nrf2的表达、升高Bcl2的表达,降低Bax的表达起到了脑保护作用。     

Adenovirus-mediated gene transfer of basic fibroblast growth factor promotes the survival of primary-cultured rat neuronal cells

We constructed two replication-deficient recombinant adenovirus vectors coding human basic fibroblast growth factor (bFGF), one with and one without the interleukin-2 (IL-2) secretory signal sequence and examined their neurotrophic effects on primary neuronal cells in vitro. The primary neuronal cells were successfully infected at a high efficiency with the adenovirus vectors. bFGF protein was detected in the culture medium of the neurons infected with both these vectors. The cells infected with the bFGF-expressing adenovirus containing the IL-2 signal sequence showed 2- to 10-fold higher levels of secretion levels than cells infected with the native bFGF-expressing adenovirus alone. Both bFGF-expressing vectors augmented the survival of primary neuronal cells in an in vitro culture, compared with a mock infection or control virus infection. Notably, the cells infected with the bFGF-expressing adenovirus containing the IL-2 signal sequence were markedly enhanced cell survival in the early phase of the culture, compared with the control cells and even those infected with the bFGF-expressing adenovirus without the IL-2 signal sequence. However, in the late phase of neuronal culture, neither viral vector could support the cell survival. In contrast the co-infection of the bFGF-expressing vector with a Bcl-xL-expressing vector was extremely effective on neuronal survival.     

Induction of basic fibroblast growth factor in Alzheimer's disease pathology.

Alzheimer's disease (AD) and its hallmark, plaques, may be due to an imbalance of trophic support. It has been suggested that plaque biogenesis may involve a growth factor which induces sprouting of neurites to form plaques. Thus, we studied the distribution of basic fibroblast growth factor (bFGF), in the hippocampus from AD brain and in rodent brain after entorhinal ablation. Both cases have a partial deafferentation of the hippocampus. The strongest bFGF immunoreactivity in AD was shown in plaques of the dentate gyrus. Rodent brains showed a lesion-induced increase of bFGF in the dentate gyrus, primarily localized to astrocytes. Our results indicate that bFGF may serve an important biological function in plaques and possibly attract neurites.     

Astrogliosis in culture. IV. Effects of basic fibroblast growth factor

Previous studies have shown that the mechanical wounding of 3-week-old cultured rat astrocytes results in cell proliferation and hypertrophy resembling astrocyte responses to a brain injury in vivo. We now report the effects of basic fibroblast growth factor (bFGF) and an anti-bFGF antibody on astrocyte morphology, proliferation, and migration following in vitro wounding of confluent secondary cultures. Addition of bFGF (20 ng/ml) to wounded cultures induced morphological changes characteristic of differentiation in wounded and nonwounded areas of the culture. Combined treatment with bFGF and an anti-bFGF antibody (100 μg/ml) prevented this effect. Astrocyte proliferation along the edges of a scratch wound was at maximum 24 hr after wounding in cells growing in Eagle's minimum essential medium (EMEM) containing 10% serum. Low serum concentration and treatment with dibutyryl cyclic adenosine monophosphate (dbc-AMP) reduced injury-associated astrocyte proliferation. Addition of bFGF to cultures in EMEM with serum increased astrocyte proliferation at 18 and 24 hr after wounding. This effect was reduced considerably by treatment of cultures with bFGF in combination with an anti-bFGF antibody. The combined treatment and the antibody alone reduced cell division to a level lower than in control cultures. Twenty-four hr following wounding, astrocytes along the edges of the wound exhibited extension of thick, flat processes into the wound area. At 3 and 5 days after wounding, a bodily migration of astrocytes into the wounded area was observed. Addition of bFGF significantly increased astrocyte migration 1 day after wounding, with maximum effect on day 3 and no subsequent increase on day 5. A combination of bFGF and anti-bFGF antibody as well as the antibody alone reduced astrocyte migration to a level lower than in controls. Immunohistochemical localization and isoform pattern of bFGF in astrocytes did not change with dbc-AMP treatment or wounding. We conclude that mechanically wounded confluent astrocytes respond to bFGF added to the culture medium by enhancing cell division, differentiation, and migration. In addition, the results of the antibody treatment also suggest a role for endogenous bFGF in astrocyte proliferation and migration elicited by wounding in vitro. These results support the notion that in vivo, both bFGF released by injury and endogenous bFGF synthesized by astrocytes, contribute to the cellular responses that lead to astrogliosis. © 1995 Wiley-Liss, Inc.     

Protective effects of basic fibroblast growth factor against hippocampal neuronal damage following cerebral ischemia in the gerbil

We examined the effects of treatment with basic fibroblast growth factor (b-FGF) on hippocampal CA1 neuronal damage following 3 min of forebrain ischemia in the gerbil. Continuous infusion of b-FGF (24 or 240 ng/day over 4 days) using an implanted osmotic minipump into the lateral ventricle prevented CA1 neuronal damage in a dose-dependent manner.     

Effect of basic fibroblast growth factor and danshen on bcl-2 and p53 mRNA expression in the brain of rats exposed to repeated,high, positive acceleration （＋Gz）

BACKGROUND： Both animal experiments and clinical studies have shown that basic fibroblast growth factor （bFGF） and danshen （Salvia miltiorrhiza） can exhibit protective effects on ischemia-reperfusion cerebral injury.
OBJECTIVE： To test whether bFGF and danshen can protect cerebral injury induced by exposure to repeated, high, positive acceleration （＋Gz） in an animal model and to analyze the possible mechanisms.
DESIGN, TIME AND SETTING： Randomized controlled animal study. The experiment was performed at the Research Center for Molecular Biology, Air-force General Hospital of Chinese PLA from April to August 2000.
MATERIALS： A total of 20 clean grade, healthy, Sprague Dawley rats of both genders, weighing （200 ± 15） g, were provided by our experimental animal center. Rats were randomly divided into 5 groups： the control group, ＋Gz exposure group, bFGF group, danshen group, and saline group, with 4 animals per group. bFGF （Beijing Bailuyuan Biotechnology Co. Ltd.） and danshen solution （Shanghai Zhongxi Pharmaceutical Co. Ltd.） were used.
METHODS： All rats were fixed on a rotary arm of a centrifugal apparatus （2 m in radius） with their heads oriented towards the center of the apparatus. Except for rats in the control group, the value of ＋Gz exposure was ＋14 Gz with an acceleration rate of 1.5 G/s. The peak force lasted for 45 seconds. ＋Gz exposure was performed three times with intervals of 30 minutes. Rats in the control group received the same ＋Gz procedure, but the G value was ＋1 Gz. Rats in bFGF group and danshen group were intraperitoneally injected with 100 μg/kg bFGF or 15 g/kg danshen solution, respectively, at 30 minutes prior to centrifugation and immediately after centrifugation. Rats in saline group were injected with the same volume of saline. Six hours after exposure, rats were decapitated. One hemisphere was preserved in liquid nitrogen for RNA extraction and the other was processed for apoptosis detection.
MAIN OUTCOME MEASURES： mRNA     

Developmental expression of the basic fibroblast growth factor gene in rat brain.

Basic fibroblast growth factor (bFGF) is a trophic factor for a variety of neuronal/glial cell populations. The RNase protection assay, with a cRNA complementary to the coding region of bFGF mRNA, was used to investigate the brain distribution and developmental regulation of bFGF mRNA expression. In adult rats bFGF mRNA is distributed throughout the brain, the highest levels being observed in cerebral cortex, hippocampus and spinal cord. The levels of bFGF mRNA in all the brain structures are low in newborn rats, increase thereafter to reach a peak of expression around postnatal day 21. bFGF mRNA levels are significantly different between various brain structures during the first and second postnatal week. Adult and aged rats (Fisher 344) express the same levels of bFGF mRNA in the various brain regions. The onset of bFGF mRNA expression suggests that this growth factor is important for the maturation as well as for the maintenance of different cell populations of the central nervous system.