摘要
一氧化氮(nitric oxide，NO)合酶(nitric oxide synthase，NOS)／NO与神经细胞分化密切相关。二甲基精氨酸二甲胺水解酶(dimethylarginine dimethylaminohydrolase，DDAH)能特异性水解内源性NOS抑制物非对称二甲基精氨酸(asymmetric dimethylarginine，ADMA)，上调NOS活性。DDAH广泛存在于各种细胞和心血管、神经系统组织中。目前认为其活性降低所致的ADMA代谢减少与多种心血管疾病的发生发展密切相关，是一个新的心血管疾病相关蛋白和药物防治靶点。新近研究发现，舌下运动神经损伤后DDAHmRNA的水平明显升高，同型半胱氨酸(Homocysteine，Hcy)可显著降低神经细胞DDAH的表达和活性并增加ADMA的水平，提示DDAH可能与神经系统发育或某些神经系统疾病的发生发展密切相关。
本论文将从分子细胞水平系统研究DDAH在神经细胞分化和凋亡中的作用，并探讨DDAH／ADMA通路是否介导3,4,5,6-四羟基(口山)酮和全反式维甲酸(all-trans retinoic acid，atRA)的抗凋亡作用。主要研究工作如下：
在神经生长因子(nerver growth factor，NGF)诱导PC12神经细胞分化的体外模型，利用基因转染技术沉默或过表达DDAH1基因，系统探讨DDAH1调节神经细胞分化的作用及分子机制。结果显示：NGF(50 ng／ml)能诱导神经细胞分化，增加NO水平、DDAH1、nNOS mRNA及蛋白表达，并呈时间依赖性；NGF降低DDAH2mRNA和蛋白表达；NGF对DDAH活性和ADMA水平无明显影响；L-NAME能抑制NGF诱导的神经细胞分化和NO水平，但不影响DDAH1及DDAH2的表达。沉默DDAH1可抑制NGF诱导的神经细胞分化及其标志性蛋白微管相关蛋白(microtubule-associatedprotein2，MAP2)的表达，同时抑制nNOS mRNA的表达；而过表达DDAH1诱导神经细胞分化，增加G_0／G_1期细胞百分比，增加nNOSmRNA的表达，L-NAME能抑制过表达DDAH1诱导的神经细胞分化。
(口山)酮是普遍存在于植物中的一类多酚类化合物。具有强效的抗氧化、抗炎作用。某些(口山)酮单体对血管内皮功能的保护作用与升高DDAH活性从而降低内源性ADMA水平有关。3,4,5，6-四羟基(口山)酮是我校药学院药物化学系合成的新型(口山)酮单体化合物。我室前期工已证明，3,4,5,6-四羟基(口山)酮对缺血心肌及血管内皮具有保护作用，其机制与抑制氧化应激有关。基于Aβ所致神经细胞凋亡与氧化应激密切相关及3,4,5,6-四羟基(口山)酮具有抗氧化特性，本实验采用Aβ_(25-35)诱导PC12神经细胞损伤建立阿尔茨海默病(Alzheimers disease，AD)的细胞模型，研究3,4,5,6-四羟基(口山)酮对Aβ_(25-35)所致PC12细胞凋亡的保护作用，并探讨其机制是否与DDAH／ADMA途径有关。结果显示：10μM Aβ_(25-35)与PC12神经细胞孵育48 h能显著增加细胞凋亡率；Aβ_(25-35)处理PC12细胞3 h后逐渐增加ROS水平和caspase-3活性，抗氧化剂PDTC(5μM)或caspase-3活性抑制剂DEVD-CHO(100μM)能显著抑制Aβ_(25-35)诱导的凋亡；(口山)酮(3，10或30μM)能显著抑制Aβ_(25-35)诱导的细胞凋亡，ROS水平和caspase-3活性的增加。Aβ_(25-35)处理PC12神经细胞6 h后能显著降低DDAH活性和增加ADMA水平，(口山)酮(3，10或30μM)能显著抑制Aβ_(25-35)诱导的上述作用。ADMA浓度和时间依赖性的诱导细胞凋亡；ADMA(10μM)处理PC12细胞3 h后逐渐增加ROS水平和caspase-3活性，抗氧化剂PDTC(5μM)或caspase-3活性抑制剂DEVD-CHO(100μM)能显著抑制ADMA诱导的细胞凋亡。
atRA在调控细胞生长、分化、凋亡等生命活动中起重要作用。atRA通过与维甲酸受体(retinoic acid receptor，RAR)和维甲类受体(retinoid X receptor，RXR)结合，从而启动一系列靶基因的转录。其中DDAH2的启动子区含有维甲酸反应元件。新近研究发现，在培养的内皮细胞atRA能上调DDAH2的表达。本研究将探讨CoCl_2诱导的PC12神经细胞调亡是否涉及DDAH／ADMA通路及atRA是否通过上调DDAH2表达抑制CoCl_2诱导的神经细胞凋亡。结果显示：125μM CoCl_2与PC12神经细胞孵育48 h能显著增加细胞凋亡率；CoCl_2处理PC12细胞3 h后逐渐增加ROS水平和caspase-3活性；atRA(0．1，1 or 10μM)能显著抑制CoCl_2诱导的细胞凋亡、ROS水平与caspase-3的活性的增加；抗氧化剂PDTC(5μM)或caspase-3活性抑制剂DEVD-CHO(100μM)能显著抑制CoCl_2诱导的凋亡。CoCl_2处理PC12神经细胞6 h后能显著降低DDAH活性和增加ADMA水平；8 h或12 h后分别显著降低DDAH2 mRNA或蛋白的表达；atRA(0．1，1 or 10μM)可显著抑制CoCl_2对DDAH活性、表达及ADMA水平的影响。在DDAH2基因被沉默的PC12神经细胞，atRA(0．1μM)不能抑制CoCl_2诱导的细胞凋亡；CoCl_2处理DDAH2基因被沉默的PC12神经细胞12 h后，atRA(0．1μM)不再抑制CoCl_2对ROS水平、caspase-3活性和ADMA水平的影响。
It is well known that nitric oxide (NO) synthase (NOS)/NO play an important role in neuronal differentiation. dimethylarginine dimethylaminohydrolase (DDAH), which widely expresses in different cell and tissue including cardiovascular and neuronal system is responsible for the degradation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS. Various studies have reported that endogenous ADMA is a key factor contributing to endothelial dysfunction and is associated with development of some cardiovascular diseases. And the reduced DDAH activity is believed to be the main cause of elevated ADMA levels, suggesting that the DDAH/ADMA pathway may be a new target for pervention and treatment of cardiovascular diseases. Recently, studies showed that DDAH mRNA in rat brain was up-regulated in axotomized motoneurons and the reduction of DDAH activity and elevation of ADMA level were abserved in Hcy-treated neuronal cells, suggesting that DDAH may be involved in neuronal development and some neuronal disease.
The thesis systematically investigated the role of DDAH in neuronal differentiation and apoptosis induced byβ-amyloid (Aβ) or cobalt chloride (CoCl_2), and explored the anti-apoptotic effects of 3,4,5,6-tetrahydroxyxanthone or all-trans retinoic acid (atRA) through improving the DDAH/ADMA pathway. The main research can be summarized as following.
By gene silencing and overexpression technology, we systemically investigate the regulatory role of DDAH1 in nerver growth factor (NGF) -induced differentiation of PC12. During the exposure of NGF (50 ng/ml) on PC12 cells, DDAH1 mRNA and protein expression increased progressively with time. By contrast, the reduction in DDAH2 mRNA and protein levels were observed. The ADMA level and DDAH activity did not changed during NGF-induced differentiation of PC12 cells. The differentiation was significantly diminished in DDAH1-depleted PC12 cells treated with NGF, concomitantly with the reduction of MAP2 (neuronal-cell-specific marker) and nNOS mRNA expression. Overexpression of DDAH1 inhibited cell cycle progression and induced neuronal differentiation, and increased the nNOS mRNA expression.
Xanthones are polyphenol compounds existing in many plants which have potent anti-inflammation and anti-oxidation effects. 3,4,5,6-tetrahydroxyxanthone is synthesized by the department of chemical pharmacy. The previous work of our lab has shown that 3,4,5,6-tetrahydroxyxanthone has protective effects on endothelium and myocardium by reduction of ROS and ADMA production and enhancement of DDAH activity. According to the oxidative stress is invovled in the apoptosis induced by Aβand the anti-apoptotic effect of 3,4,5,6-tetrahydroxyxanthone, we studied the relationship between anti-apoptotic effect of xanthone on Aβ_(25-35)-treated PC 12 cells and DDAH/ADMA pathway. Treatment with Aβ_(25-35) (10μM) for 48 h induced apoptosis of PC12; After 3 hours of incubation with Aβ_(25-35) (10μM), the intracellular ROS level and caspase-3 activity were increased; Pretreatment with PDTC (5μM) or DEVD-CHO (100μM) significantly attenuated the apoptosis induced by Aβ_(25-35) (10μM); Pretreatment with xanthone (3, 10 or 30μM) significantly attenuated the apoptosis elicited by Aβ; Xanthone treatment significantly inhibited the elevated ROS level and caspase-3 activity induced by Aβ_(25-35) (10μM for up to 48 hours). Incubation of PC12 with Aβ_(25-35) for 6 hours led to decrease in the DDAH activity and increase in ADMA level; Preincubation of PC 12 with xanthone (3, 10 or 30μM) significantly inhibited the decreased DDAH activity and elevated ADMA induced by Aβ_(25-35). ADMA induced apoptosis of PC12 in time and concentration-dependent way; After 3 hours of incubation with ADMA (10μM), the intracellular ROS level and caspase-3 activity were increased; Pretreatment with PDTC (5μM) or DEVD-CHO (100μM) significantly attenuated the apoptosis elicited by ADMA (10μM); PDTC or DEVD-CHO itself had no effect on cell apoptosis.
AtRA has a wide range of biological processes, such as cell growth, differentiation and apoptosis. It was reported that atRA regulated gene transcription via binding with retinoic acid receptor (RAR) and retinoid X receptor (RXR). Recent study showed that atRA induced the expression of DDAH2 in endothelial cells. Therefore, in the present study we investigated the role of DDAH/ADMA pathway in CoCl_2-induced apoptosis of PC12 cells and studied the relationship between anti-apoptotic effect of atRA on CoCl_2-treated PC 12 cells and DDAH/ADMA pathway. Treatment with CoCl_2 (125μM) for 48 h induced apoptosis of PC12; After 3 hours of incubation with CoCl_2 (125μM), the intracellular ROS level and caspase-3 activity were increased; Pretreatment with PDTC (5μM) or DEVD-CHO (100μM) significantly attenuated the apoptosis induced by CoCl_2 (125μM); Pretreatment with atRA (0.1, 1 or 10μM) significantly attenuated the apoptosis elicited by CoCl_2; AtRA treatment significantly inhibited the elevated ROS level and caspase-3 activity induced by CoCl_2. Incubation of PC 12 with CoCl_2 for 6 hours led to decrease in the DDAH activity, and the decreased levels of DDAH2 mRNA and protein also reached significance after 8 hours and 12 hours of incubation, respectively. However, the level of ADMA in the medium was elevated after 6 hours of incubation with CoCl_2. Preincubation of PC12 with atRA (0.1, 1 or 10μM) significantly inhibited the reduction of DDAH activity and DDAH2 expression, and elevation of ADMA induced by CoCl_2. The inhibitory effects of atRA (0.1μM) on cell apoptosis were blocked in the cells depleted of DDAH2; The inhibitory effects of atRA (0.1μM) on ROS production, caspase-3 activity and ADMA accumulation also were attenuated after 12 hours of incubation with CoCl_2.
引文
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