sPLA_2在神经炎症反应中的作用及其机制
|
摘要
研究背景
多种中枢神经系统功能障碍,比如脑感染,脑缺血,脑外伤,中风以及中枢神经系统退行性病变都涉及神经炎症反应。中枢神经系统炎症过程的一个重要特点就是促炎介质的生成和释放,花生四烯酸(AA)是其中的一类重要的促炎介质。花生四烯酸代谢途径最重要的酶促过程就是磷脂酶A2(PLA2)的水解。在病理条件下,PLA2将膜磷脂从Sn-2位脂酰基水解断裂,释放出花生四烯酸,AA经代谢途径下游的两种关键酶,环氧酶(COX)和脂氧酶(LOX)催化为前列腺素(PG)和白细胞三烯(LT)等类花生酸代谢产物,损伤脑细胞。PLA2是一类磷脂水解酶超家族,分布最广泛的两种亚型是胞质磷脂酶A2(cPLA2)和分泌型磷脂酶A2(sPLA2)。目前对于脂多糖(LPS)诱导的神经炎症过程中sPLA2的功能还不明了。乙酰葛根素是将葛根素经过结构改良获得的一种新型异黄酮类化合物,尽管我们已有的报道提示乙酰葛根素对脑缺血再灌注损伤具有一定的保护作用,但其保护作用机制尚不清楚。
研究目的
研究sPLA2在LPS刺激所致大鼠大脑皮层炎症以及星形胶质细胞炎症反应中的作用。重点研究了新型异黄酮类化合物乙酰葛根素的神经保护作用,并探讨其发挥神经保护作用的可能机制,为其发展为治疗神经炎症的具有自主知识产权的创新药物提供理论基础。
研究方法
本研究由两部分组成：
1.利用原代培养的大鼠星形胶质细胞来研究sPLA2-ⅡA在LPS诱导的神经炎症中的作用。采用RT-PCR来检测在原代培养大鼠星形胶质细胞和小胶质细胞中sPLA2-ⅡA mRNA的表达。以ELISA检测星形胶质细胞中PGE2浓度。Western blot检测AA代谢相关蛋白的表达。体内试验采用小鼠脑室注射LPS建立脑炎症损伤模型来研究sPLA2-ⅡA对小鼠大脑皮质炎症过程中MAPK-cPLA2α-PGE2通路的调节作用。采用ELISA来检测小鼠大脑皮质PGE2生成水平。Western blot来检测sPLA2-ⅡA, COX-2和mPGES-1蛋白表达水平以及ERK1/2, p38和cPLA2α的磷酸化水平。
2.采用高效液相色谱法(HPLC),对乙酰葛根素在大鼠体内的药动学过程进行了分析,同时利用原代培养的大鼠星形胶质细胞模拟体外炎症模型研究乙酰葛根素在LPS诱导类花生酸合成过程中的作用机制。MTT法检测乙酰葛根素对原代培养大鼠星形胶质细胞存活率的影响。以western blot及免疫荧光染色检测各蛋白表达水平,检测乙酰葛根素是否可以抑制group V sPLA2的蛋白水平以及阻断LPS所致ERK1/2/cPLA2α信号通路的激活。采用ELISA来检测细胞中PGE2和LTC4的释放量。花生四烯酸代谢途径的其他相关蛋白表达水平用western blot来检测。
研究结果
1.LPS能够引起原代培养的星形胶质细胞中sPLA2-ⅡA mRNA表达水平呈时间和剂量依赖性的增加；但在原代培养的小胶质细胞中没有这一现象。因此我们利用原代培养的星形胶质细胞来模拟体外炎症条件,来研究sPLA2-ⅡA在LPS诱导PGE2生物合成过程中的作用。在此实验中我们还引入了sPLA2-ⅡA特异性阻断剂SC-215,在星形胶质细胞中SC-215能够剂量依赖性的降低LPS诱导产生的PGE2水平。体内试验部分：脑室注射LPS时采用SC-215干预,可以使sPLA2-ⅡA的蛋白表达下降90%以上。与预期一致,PGE2的合成也在SC-215的干预下被明显抑制(下降7596以上)。在小鼠大脑皮质中,LPS能够引起时间依赖性的cPLA2α的磷酸化,ERK1／2的磷酸化要先于cPLA2α,并且在10min时便达到高峰。后续的磷酸化在30min时仍能检测到,在60min时回到基础水平。后续的实验结果显示,sPLA2-ⅡA缺失能使小鼠大脑皮质cPLA2α和ERKl／2的磷酸化水平被明显抑制,但对p38的磷酸化没有任何作用。小鼠脑室注射ERK1/2和cPLA2α的特异性阻断剂AACOCF3和U0126可以使脑组织PGE2水平分别下降3.5和4倍。COX-2在正常生理条件下的小鼠大脑皮质中没有表达,但其蛋白表达在LPS诱导后明显升高,sPLA2-ⅡA缺失又能降低这种表达。mPGES-1是PGE2生物合成过程末端的关键酶,LPS能够诱导使其酶活性升高,而sPLA2-IIA缺失能够阻断LPS的这种作用。
2.乙酰葛根素在大鼠体内代谢为葛根素,药动学过程符合二室模型,主要药动学参数为经灌胃给药葛根素AUC (0～∞)为(44.76±4.13)mg·L-1·h,经静脉注射给药AUC(0-∞)为(36.67±5.3)mg·L-1·h,与静脉给予乙酰葛根素后体内葛根素的暴露水平相比,灌胃给予葛根素后体内葛根素的暴露水平为48.12%(生物利用度为48.12%),经灌胃给药Cmax和tmax,t1/2分别为(12.07±0.15)μg/mL、(1±0.33)h.(2.52±0.21)h。乙酰葛根素对星形胶质细胞无细胞毒性。为了检测乙酰葛根素对大鼠星形胶质细胞炎症反应的影响,我们分析了LPS刺激后细胞上清中两种类花生酸,PGE2和LTC4的水平。结果表明,乙酰葛根素能够剂量依赖性的抑制LPS引起的PGE2和LTC4的释放。为探讨乙酰葛根素的保护作用是否涉及了group V sPLA2的改变,我们通过westernblot和免疫荧光检测了各组星形胶质细胞group V sPLA2的表达和活性。结果提示乙酰葛根素可呈剂量依赖性抑制LPS诱导的星形胶质细胞中group V sPLA2蛋白表达。cPLA2a是PGE2和LTC4产生的关键酶。前期的报道也提示,在多种刺激条件下,ERKl/2的磷酸化能够引起cPLA2α的磷酸化。转录因子NF-κB能够在转录水平上调节花生四烯酸代谢酶。为了研究乙酰葛根素的保护作用机制,我们用western blot检测了LPS刺激后星形胶质细胞中cPLA2α,ERK1/2及NF-κB p65的磷酸化水平。结果表明,乙酰葛根素可以降低LPS所致groupV sPLA2下游蛋白ERK1/2及的磷酸化以及cPLA2α的活化,并且抑制p65的磷酸化。众所周知COX-2和LOX-5分别是PGE2和LTC4生成的关键酶。MAPK-NF-κB能够调节COX-2和LOX-5的表达。我们下一步检测了乙酰葛根素对LPS诱导引起的COX-2及LOX-5的表达的影响。LPS分别引起COX-2和LOX-5蛋白水平4倍和2倍的增加,而乙酰葛根素预处理能够剂量依赖性地降低LPS的这种影响。研究结论
1.在原代培养的星形胶质细胞中,LPS诱导PGE2生物合成呈时间依赖性增多,并且伴随sPLA2-ⅡAmRNA呈时间依赖性增加,蛋白含量亦增加。sPLA2-ⅡA阻断剂SC-215可以阻断LPS的作用并且呈现剂量依赖性。体内实验结果与体外一致,提示sPLA2-ⅡA缺失能够改善LPS引起的神经炎症反应。采用p38和MEK1/MEK2的阻断剂,我们证明LPS诱导的cPLA2α的连续磷酸化和后续的PGE2的生物合成需要ERK1/2而不是p38的活化。体内试验进一步证实,sPLA2-ⅡA通过持续活化ERK1/2来调节cPLA2α的磷酸化。我们的实验结果也证明花生四烯酸代谢下游的两种关键酶COX-2和mPGES-1都受到SPLA2-ⅡA的调控。
2.高效液相色谱法可作为乙酰葛根素在大鼠体内药动学的检测手段。据文献报道,灌胃葛根素后葛根素的绝对生物利用度仅为5%-6%；本研究结果表明,灌胃乙酰葛根素后葛根素在大鼠体内的暴露水平得到显著提高(生物利用度提高至48.12%)。乙酰葛根素对急性实验性炎症模型具有较好的抗炎作用。乙酰葛根素的抗炎作用机制涉及到抑制炎症过程中炎症介质产生的上游信号分子,这可能是其发挥抗炎作用的部分机制。PGE2及LTC4合成的关键酶COX-2以及LOX-5在炎症的发生发展中发挥着重要的作用,而group V SPLA2及ERK1/2和转录因子NF-κB的活化也涉及其中。我们的实验表明,乙酰葛根素可通过抑制NF-κB及ERK1/2的活化,以及花生四烯酸代谢的关键酶如group V sPLA2, cPLA2α, COX-2及LOX-5的表达和活性从而减少炎性介质PGE2及LTC4的生成。
Background
Neuroinflammation is involved in various central nervous system (CNS) disorders, including brain infections, ischemia, trauma, stroke, and degenerative CNS diseases. One of the main characters of CNS inflammation is the generation of the pro-inflammatory mediators, such as arachidonic acid (AA). The main enzymatic process responsible for AA metabolism is catalyzed by phospholipase A2(PLA2). AA released mainly by PLA2from the Sn-2position of phospholipids is metabolized by cyclooxygenases (COX) and lipooxygenases (LOX) to its eicosanoid metabolites such as prostanoids (PG) and leukotrienes (LT) that damage brain cells. The most abundant isoforms of PLA2enzymes family include cytosolic PLA2α (cPLA2α) and secretory PLA2(sPLA2). However, the influence of sPLA2on endotoxin such as lipopolysaccharide (LPS) Induced neuroinflammation has not been reported. Acetylpuerarin is a newly modified isoflavone based on puerarin. Although current studies indicate that acetylpuerarin produces neuroprotection against ischemia-reperfusion injury, the precise mechanisms for its beneficial effects are still not fully understood.
Objectives
The objective of the study is to investigate the role of sPLA2and anti-inflammatory effect of acetylpuerarin on eicosanoid signaling pathway.
Materials and Methods
This study includes two parts:
1. The role of sPLA2-IIA in LPS-induced neuroinflammation was investigated using primary rat astrocytes. RT-PCR analysis was performed to examine the mRNA level of sPLA2-IIA in primary rat astrocytes and microglia. ELISA-based assay was used to measure the PGE2concentration of the supernatant of astrocytes. Western blot determined the protein expression related to AA signaling. In vivo induced by the intracerebroventricle microinjection (i.c.v.) of LPS was employed to explore the regulation of MAPK-cPLA2a-PGE2pathways by sPLA2-IIA in mice cerebral cortex. ELISA-based assay was used to determine the PGE2concentration of the supernatant of mice cerebral cortex. Protein expression of sPLA2-IIA, phosphor-ERK1/2, phospho-p38, phospho-cPLA2a, COX-2and mPGES-1were evaluated by western blot.
2. The pharmacokinetic properties of puerarin after oral or i.v. dosing of acetylpuerarin was measured by HPLC analysis. We used primary rat astrocytes to simulate inflammatory situation to study the role of acetylpuerarin in LPS-stimulated route of eicosanoid biosynthetic pathway. We used MTT solution to examine the cytotoxicity of acetylpuerarin in primary rat astrocytes. Western blot and immunofluorescence staining were used to determine the protein level of group V sPLA2, PGE2and LTC4concentration was measured by using ELISA-based assay kit. Western blot examined the activation of ERK1/2, cPLA2a, NF-kB and protein expression of COX-2and LOX-5.
Results
1. LPS exposure led to a time-and dose-dependent increase of sPLA2-IIA mRNA expression in murine primary astrocytes but not induce increase of sPLA2-IIA mRNA or protein expression in the primary microglia, we used astrocytes to simulate similar inflammatory situation in vitro to study the role of sPLA2-IIA in LPS-stimulated route of PGE2biosynthetic pathway. To investigate the effect of sPLA2-IIA on PGE2release, a highly specific sPLA2-IIA inhibitor, SC-215, was presented to the medium of astrocytes together with LPS. SC-215treatment lowered the elevated PGE2level induced by LPS in a dose dependent manner in astrocytes. In vivo study we found that the addition of SC-215(1.218μg) with LPS (2.5μg) showed a nearly90%decrease in sPLA2-IIA expression. As expected, PGE2production were significantly inhibited (>75%) in the cortex of SC-215-treated mice at the same time point. Our study found that LPS caused a time-dependent induction of phospho-cPLA2α, phosphorylation of ERK1/2preceded that of cPLA2α and increased to a maximum at10minutes in LPS treated brain tissue. The residual phosphorylation was still detectable30minutes after stimulation and returned to baseline by60minutes. In subsequent experiments, changes in the activation of these downstream molecules in cortex after SC-215treatment were examined. The phosphorylation of cPLA2a at1hour and phosphorylation of ERK1/2at10minutes were significantly prevented in mice treated with SC-215prior to LPS challenge, but the induction of phospho-p38at10minutes were not affected upon SC-215pretreated and remained similar to LPS challenge values. Mice were i.c.v. injection of an inhibitor of cPLA2α activity (AACOCF3) or MEK1/MEK2inhibitor (U0126) and then exposed to LPS for1hour. AACOCF3and U0126infusion did decrease the brain concentrations of PGE2by3.5-and4-fold, respectively. The constitutive COX-1enzyme was expressed only at low levels in cortex and was not significantly altered by LPS treatment. In contrast, the COX-2enzyme was not expressed under basal conditions in mice cortex but was induced by LPS stimulation and was downregulated in the presence of SC-215. The mPGES-1is the final enzyme in the cascade to ultimately generate PGE2. In mice cortex, mPGES-1was found to be constitutively expressed and was upregulated upon LPS treatment, the change was significantly prevented by sPLA2-IIA inhibition.
2. The time-concentration curve of puerarin was accord with two-compartment model. The exposure profile of puerarin in plasma after i.g. administration acetylpuerarin were as follows: Cmax (12.07±0.15) μg/mL,Tmax (1±0.33) h, t1/2(2.52±0.21) h, AUC(0-∞)(44.76±4.13) mg·L-1·h. The AUC(0-∞) of puerarin after iv administration is (36.67±5.3) mg·L-1·h. The absolute bioavailability of puerarin is48.12%. Acetylpuerarin exerted no significant cytotoxicity on primary rat astrocytes. To examine the anti-inflammatory effect of acetylpuerarin on primary astrocytes, we analyzed the LPS-induced generation of PGE2and LTC4. The pretreatment of acetylpuerarin inhibited LPS-indcued production of PGE2and LTC4dose-dependently. To investigate the effect of acetylpuerarin on group V sPLA2, the protein level and immunoreactivity were examined by double immunofluorescence labeling and western blot. The results showed that group V sPLA2expression was noticeably enhanced by LPS stimulation, pretreatment with acetylpuerarin resulted in a dramatically dose-dependent decrease in group V sPLA2expression. To investigate the possible molecular mechanism by which acetylpuerarin prevents release of eicosanoids, we examined the LPS-induced phosphorylation and activation of cPLA2α, ERK1/2and p65subunit of NF-κB. Our data showed that acetylpuerarin partially blocked the induction of phosphorylation of NF-κB as well as phosphorylation of ERK1/2and prevents subsequent activation of cPLA2α in a time-dependent manner during LPS exposure and further decrease formation of inflammatory lipid mediators. Furthermore, stimulation of primary rat astrocytes with LPS caused approximately4-fold and2-fold increase in the protein levels of COX-2and LOX-5, respectively. Pre-incubation of astrocytes with acetylpuerarin prevented the LPS-induced protein expression of COX-2and LOX-5dose-dependently.
Conclusions
1. LPS evokes marked PGE2generation in primary astrocytes in a time-dependent fashion accompanied by release of sPLA2-IIA. The inhibition of sPLA2-IIA alleviates the release of PGE2following the neuroinflammation induced by LPS. ERK1/2, not p38is required for the sequential activation of cPLA2α and further release of PGE2induced by LPS. The elevated signaling of the sPLA2-IIA-ERK1/2-cPLA2a-COX-2-mPGES-1pathway contributes to PGE2overexpression and secretion in primary astrocytes and mice cortex.
2. The established method could be used for the analysis of pharmacokinetics of the acetylpuerarin in rats. According to the results in the literature, the absolute bioavailability of puerarin administered intragastricly in rats was only5%-6%, our results showed that the exposure profile of puerarin in plasma after i.g. administration acetylpurearin was markedly improved (the absolute bioavailability of puerarin was up to48.12%). The ability for acetylpuerarin to attenuate the expression of group V sPLA2and the activation of ERK1/2/cPLA2α loop and phospho-p65levels in astrocytes is an indication that acetylpuerarin can prevent the activation of signaling molecules and downstream pathways leading to eicosanoids generation. Acetylpuerarin significantly prevented the LPS-induced expression of COX-2and LOX-5in a dose-dependent manner. Thus, inhibition of COX-2and LOX-5appears to be responsible for the decreased biosynthesis of pro-inflammatory PGE2and LTC4.
多种中枢神经系统功能障碍,比如脑感染,脑缺血,脑外伤,中风以及中枢神经系统退行性病变都涉及神经炎症反应。中枢神经系统炎症过程的一个重要特点就是促炎介质的生成和释放,花生四烯酸(AA)是其中的一类重要的促炎介质。花生四烯酸代谢途径最重要的酶促过程就是磷脂酶A2(PLA2)的水解。在病理条件下,PLA2将膜磷脂从Sn-2位脂酰基水解断裂,释放出花生四烯酸,AA经代谢途径下游的两种关键酶,环氧酶(COX)和脂氧酶(LOX)催化为前列腺素(PG)和白细胞三烯(LT)等类花生酸代谢产物,损伤脑细胞。PLA2是一类磷脂水解酶超家族,分布最广泛的两种亚型是胞质磷脂酶A2(cPLA2)和分泌型磷脂酶A2(sPLA2)。目前对于脂多糖(LPS)诱导的神经炎症过程中sPLA2的功能还不明了。乙酰葛根素是将葛根素经过结构改良获得的一种新型异黄酮类化合物,尽管我们已有的报道提示乙酰葛根素对脑缺血再灌注损伤具有一定的保护作用,但其保护作用机制尚不清楚。
研究目的
研究sPLA2在LPS刺激所致大鼠大脑皮层炎症以及星形胶质细胞炎症反应中的作用。重点研究了新型异黄酮类化合物乙酰葛根素的神经保护作用,并探讨其发挥神经保护作用的可能机制,为其发展为治疗神经炎症的具有自主知识产权的创新药物提供理论基础。
研究方法
本研究由两部分组成：
1.利用原代培养的大鼠星形胶质细胞来研究sPLA2-ⅡA在LPS诱导的神经炎症中的作用。采用RT-PCR来检测在原代培养大鼠星形胶质细胞和小胶质细胞中sPLA2-ⅡA mRNA的表达。以ELISA检测星形胶质细胞中PGE2浓度。Western blot检测AA代谢相关蛋白的表达。体内试验采用小鼠脑室注射LPS建立脑炎症损伤模型来研究sPLA2-ⅡA对小鼠大脑皮质炎症过程中MAPK-cPLA2α-PGE2通路的调节作用。采用ELISA来检测小鼠大脑皮质PGE2生成水平。Western blot来检测sPLA2-ⅡA, COX-2和mPGES-1蛋白表达水平以及ERK1/2, p38和cPLA2α的磷酸化水平。
2.采用高效液相色谱法(HPLC),对乙酰葛根素在大鼠体内的药动学过程进行了分析,同时利用原代培养的大鼠星形胶质细胞模拟体外炎症模型研究乙酰葛根素在LPS诱导类花生酸合成过程中的作用机制。MTT法检测乙酰葛根素对原代培养大鼠星形胶质细胞存活率的影响。以western blot及免疫荧光染色检测各蛋白表达水平,检测乙酰葛根素是否可以抑制group V sPLA2的蛋白水平以及阻断LPS所致ERK1/2/cPLA2α信号通路的激活。采用ELISA来检测细胞中PGE2和LTC4的释放量。花生四烯酸代谢途径的其他相关蛋白表达水平用western blot来检测。
研究结果
1.LPS能够引起原代培养的星形胶质细胞中sPLA2-ⅡA mRNA表达水平呈时间和剂量依赖性的增加；但在原代培养的小胶质细胞中没有这一现象。因此我们利用原代培养的星形胶质细胞来模拟体外炎症条件,来研究sPLA2-ⅡA在LPS诱导PGE2生物合成过程中的作用。在此实验中我们还引入了sPLA2-ⅡA特异性阻断剂SC-215,在星形胶质细胞中SC-215能够剂量依赖性的降低LPS诱导产生的PGE2水平。体内试验部分：脑室注射LPS时采用SC-215干预,可以使sPLA2-ⅡA的蛋白表达下降90%以上。与预期一致,PGE2的合成也在SC-215的干预下被明显抑制(下降7596以上)。在小鼠大脑皮质中,LPS能够引起时间依赖性的cPLA2α的磷酸化,ERK1／2的磷酸化要先于cPLA2α,并且在10min时便达到高峰。后续的磷酸化在30min时仍能检测到,在60min时回到基础水平。后续的实验结果显示,sPLA2-ⅡA缺失能使小鼠大脑皮质cPLA2α和ERKl／2的磷酸化水平被明显抑制,但对p38的磷酸化没有任何作用。小鼠脑室注射ERK1/2和cPLA2α的特异性阻断剂AACOCF3和U0126可以使脑组织PGE2水平分别下降3.5和4倍。COX-2在正常生理条件下的小鼠大脑皮质中没有表达,但其蛋白表达在LPS诱导后明显升高,sPLA2-ⅡA缺失又能降低这种表达。mPGES-1是PGE2生物合成过程末端的关键酶,LPS能够诱导使其酶活性升高,而sPLA2-IIA缺失能够阻断LPS的这种作用。
2.乙酰葛根素在大鼠体内代谢为葛根素,药动学过程符合二室模型,主要药动学参数为经灌胃给药葛根素AUC (0～∞)为(44.76±4.13)mg·L-1·h,经静脉注射给药AUC(0-∞)为(36.67±5.3)mg·L-1·h,与静脉给予乙酰葛根素后体内葛根素的暴露水平相比,灌胃给予葛根素后体内葛根素的暴露水平为48.12%(生物利用度为48.12%),经灌胃给药Cmax和tmax,t1/2分别为(12.07±0.15)μg/mL、(1±0.33)h.(2.52±0.21)h。乙酰葛根素对星形胶质细胞无细胞毒性。为了检测乙酰葛根素对大鼠星形胶质细胞炎症反应的影响,我们分析了LPS刺激后细胞上清中两种类花生酸,PGE2和LTC4的水平。结果表明,乙酰葛根素能够剂量依赖性的抑制LPS引起的PGE2和LTC4的释放。为探讨乙酰葛根素的保护作用是否涉及了group V sPLA2的改变,我们通过westernblot和免疫荧光检测了各组星形胶质细胞group V sPLA2的表达和活性。结果提示乙酰葛根素可呈剂量依赖性抑制LPS诱导的星形胶质细胞中group V sPLA2蛋白表达。cPLA2a是PGE2和LTC4产生的关键酶。前期的报道也提示,在多种刺激条件下,ERKl/2的磷酸化能够引起cPLA2α的磷酸化。转录因子NF-κB能够在转录水平上调节花生四烯酸代谢酶。为了研究乙酰葛根素的保护作用机制,我们用western blot检测了LPS刺激后星形胶质细胞中cPLA2α,ERK1/2及NF-κB p65的磷酸化水平。结果表明,乙酰葛根素可以降低LPS所致groupV sPLA2下游蛋白ERK1/2及的磷酸化以及cPLA2α的活化,并且抑制p65的磷酸化。众所周知COX-2和LOX-5分别是PGE2和LTC4生成的关键酶。MAPK-NF-κB能够调节COX-2和LOX-5的表达。我们下一步检测了乙酰葛根素对LPS诱导引起的COX-2及LOX-5的表达的影响。LPS分别引起COX-2和LOX-5蛋白水平4倍和2倍的增加,而乙酰葛根素预处理能够剂量依赖性地降低LPS的这种影响。研究结论
1.在原代培养的星形胶质细胞中,LPS诱导PGE2生物合成呈时间依赖性增多,并且伴随sPLA2-ⅡAmRNA呈时间依赖性增加,蛋白含量亦增加。sPLA2-ⅡA阻断剂SC-215可以阻断LPS的作用并且呈现剂量依赖性。体内实验结果与体外一致,提示sPLA2-ⅡA缺失能够改善LPS引起的神经炎症反应。采用p38和MEK1/MEK2的阻断剂,我们证明LPS诱导的cPLA2α的连续磷酸化和后续的PGE2的生物合成需要ERK1/2而不是p38的活化。体内试验进一步证实,sPLA2-ⅡA通过持续活化ERK1/2来调节cPLA2α的磷酸化。我们的实验结果也证明花生四烯酸代谢下游的两种关键酶COX-2和mPGES-1都受到SPLA2-ⅡA的调控。
2.高效液相色谱法可作为乙酰葛根素在大鼠体内药动学的检测手段。据文献报道,灌胃葛根素后葛根素的绝对生物利用度仅为5%-6%；本研究结果表明,灌胃乙酰葛根素后葛根素在大鼠体内的暴露水平得到显著提高(生物利用度提高至48.12%)。乙酰葛根素对急性实验性炎症模型具有较好的抗炎作用。乙酰葛根素的抗炎作用机制涉及到抑制炎症过程中炎症介质产生的上游信号分子,这可能是其发挥抗炎作用的部分机制。PGE2及LTC4合成的关键酶COX-2以及LOX-5在炎症的发生发展中发挥着重要的作用,而group V SPLA2及ERK1/2和转录因子NF-κB的活化也涉及其中。我们的实验表明,乙酰葛根素可通过抑制NF-κB及ERK1/2的活化,以及花生四烯酸代谢的关键酶如group V sPLA2, cPLA2α, COX-2及LOX-5的表达和活性从而减少炎性介质PGE2及LTC4的生成。
Background
Neuroinflammation is involved in various central nervous system (CNS) disorders, including brain infections, ischemia, trauma, stroke, and degenerative CNS diseases. One of the main characters of CNS inflammation is the generation of the pro-inflammatory mediators, such as arachidonic acid (AA). The main enzymatic process responsible for AA metabolism is catalyzed by phospholipase A2(PLA2). AA released mainly by PLA2from the Sn-2position of phospholipids is metabolized by cyclooxygenases (COX) and lipooxygenases (LOX) to its eicosanoid metabolites such as prostanoids (PG) and leukotrienes (LT) that damage brain cells. The most abundant isoforms of PLA2enzymes family include cytosolic PLA2α (cPLA2α) and secretory PLA2(sPLA2). However, the influence of sPLA2on endotoxin such as lipopolysaccharide (LPS) Induced neuroinflammation has not been reported. Acetylpuerarin is a newly modified isoflavone based on puerarin. Although current studies indicate that acetylpuerarin produces neuroprotection against ischemia-reperfusion injury, the precise mechanisms for its beneficial effects are still not fully understood.
Objectives
The objective of the study is to investigate the role of sPLA2and anti-inflammatory effect of acetylpuerarin on eicosanoid signaling pathway.
Materials and Methods
This study includes two parts:
1. The role of sPLA2-IIA in LPS-induced neuroinflammation was investigated using primary rat astrocytes. RT-PCR analysis was performed to examine the mRNA level of sPLA2-IIA in primary rat astrocytes and microglia. ELISA-based assay was used to measure the PGE2concentration of the supernatant of astrocytes. Western blot determined the protein expression related to AA signaling. In vivo induced by the intracerebroventricle microinjection (i.c.v.) of LPS was employed to explore the regulation of MAPK-cPLA2a-PGE2pathways by sPLA2-IIA in mice cerebral cortex. ELISA-based assay was used to determine the PGE2concentration of the supernatant of mice cerebral cortex. Protein expression of sPLA2-IIA, phosphor-ERK1/2, phospho-p38, phospho-cPLA2a, COX-2and mPGES-1were evaluated by western blot.
2. The pharmacokinetic properties of puerarin after oral or i.v. dosing of acetylpuerarin was measured by HPLC analysis. We used primary rat astrocytes to simulate inflammatory situation to study the role of acetylpuerarin in LPS-stimulated route of eicosanoid biosynthetic pathway. We used MTT solution to examine the cytotoxicity of acetylpuerarin in primary rat astrocytes. Western blot and immunofluorescence staining were used to determine the protein level of group V sPLA2, PGE2and LTC4concentration was measured by using ELISA-based assay kit. Western blot examined the activation of ERK1/2, cPLA2a, NF-kB and protein expression of COX-2and LOX-5.
Results
1. LPS exposure led to a time-and dose-dependent increase of sPLA2-IIA mRNA expression in murine primary astrocytes but not induce increase of sPLA2-IIA mRNA or protein expression in the primary microglia, we used astrocytes to simulate similar inflammatory situation in vitro to study the role of sPLA2-IIA in LPS-stimulated route of PGE2biosynthetic pathway. To investigate the effect of sPLA2-IIA on PGE2release, a highly specific sPLA2-IIA inhibitor, SC-215, was presented to the medium of astrocytes together with LPS. SC-215treatment lowered the elevated PGE2level induced by LPS in a dose dependent manner in astrocytes. In vivo study we found that the addition of SC-215(1.218μg) with LPS (2.5μg) showed a nearly90%decrease in sPLA2-IIA expression. As expected, PGE2production were significantly inhibited (>75%) in the cortex of SC-215-treated mice at the same time point. Our study found that LPS caused a time-dependent induction of phospho-cPLA2α, phosphorylation of ERK1/2preceded that of cPLA2α and increased to a maximum at10minutes in LPS treated brain tissue. The residual phosphorylation was still detectable30minutes after stimulation and returned to baseline by60minutes. In subsequent experiments, changes in the activation of these downstream molecules in cortex after SC-215treatment were examined. The phosphorylation of cPLA2a at1hour and phosphorylation of ERK1/2at10minutes were significantly prevented in mice treated with SC-215prior to LPS challenge, but the induction of phospho-p38at10minutes were not affected upon SC-215pretreated and remained similar to LPS challenge values. Mice were i.c.v. injection of an inhibitor of cPLA2α activity (AACOCF3) or MEK1/MEK2inhibitor (U0126) and then exposed to LPS for1hour. AACOCF3and U0126infusion did decrease the brain concentrations of PGE2by3.5-and4-fold, respectively. The constitutive COX-1enzyme was expressed only at low levels in cortex and was not significantly altered by LPS treatment. In contrast, the COX-2enzyme was not expressed under basal conditions in mice cortex but was induced by LPS stimulation and was downregulated in the presence of SC-215. The mPGES-1is the final enzyme in the cascade to ultimately generate PGE2. In mice cortex, mPGES-1was found to be constitutively expressed and was upregulated upon LPS treatment, the change was significantly prevented by sPLA2-IIA inhibition.
2. The time-concentration curve of puerarin was accord with two-compartment model. The exposure profile of puerarin in plasma after i.g. administration acetylpuerarin were as follows: Cmax (12.07±0.15) μg/mL,Tmax (1±0.33) h, t1/2(2.52±0.21) h, AUC(0-∞)(44.76±4.13) mg·L-1·h. The AUC(0-∞) of puerarin after iv administration is (36.67±5.3) mg·L-1·h. The absolute bioavailability of puerarin is48.12%. Acetylpuerarin exerted no significant cytotoxicity on primary rat astrocytes. To examine the anti-inflammatory effect of acetylpuerarin on primary astrocytes, we analyzed the LPS-induced generation of PGE2and LTC4. The pretreatment of acetylpuerarin inhibited LPS-indcued production of PGE2and LTC4dose-dependently. To investigate the effect of acetylpuerarin on group V sPLA2, the protein level and immunoreactivity were examined by double immunofluorescence labeling and western blot. The results showed that group V sPLA2expression was noticeably enhanced by LPS stimulation, pretreatment with acetylpuerarin resulted in a dramatically dose-dependent decrease in group V sPLA2expression. To investigate the possible molecular mechanism by which acetylpuerarin prevents release of eicosanoids, we examined the LPS-induced phosphorylation and activation of cPLA2α, ERK1/2and p65subunit of NF-κB. Our data showed that acetylpuerarin partially blocked the induction of phosphorylation of NF-κB as well as phosphorylation of ERK1/2and prevents subsequent activation of cPLA2α in a time-dependent manner during LPS exposure and further decrease formation of inflammatory lipid mediators. Furthermore, stimulation of primary rat astrocytes with LPS caused approximately4-fold and2-fold increase in the protein levels of COX-2and LOX-5, respectively. Pre-incubation of astrocytes with acetylpuerarin prevented the LPS-induced protein expression of COX-2and LOX-5dose-dependently.
Conclusions
1. LPS evokes marked PGE2generation in primary astrocytes in a time-dependent fashion accompanied by release of sPLA2-IIA. The inhibition of sPLA2-IIA alleviates the release of PGE2following the neuroinflammation induced by LPS. ERK1/2, not p38is required for the sequential activation of cPLA2α and further release of PGE2induced by LPS. The elevated signaling of the sPLA2-IIA-ERK1/2-cPLA2a-COX-2-mPGES-1pathway contributes to PGE2overexpression and secretion in primary astrocytes and mice cortex.
2. The established method could be used for the analysis of pharmacokinetics of the acetylpuerarin in rats. According to the results in the literature, the absolute bioavailability of puerarin administered intragastricly in rats was only5%-6%, our results showed that the exposure profile of puerarin in plasma after i.g. administration acetylpurearin was markedly improved (the absolute bioavailability of puerarin was up to48.12%). The ability for acetylpuerarin to attenuate the expression of group V sPLA2and the activation of ERK1/2/cPLA2α loop and phospho-p65levels in astrocytes is an indication that acetylpuerarin can prevent the activation of signaling molecules and downstream pathways leading to eicosanoids generation. Acetylpuerarin significantly prevented the LPS-induced expression of COX-2and LOX-5in a dose-dependent manner. Thus, inhibition of COX-2and LOX-5appears to be responsible for the decreased biosynthesis of pro-inflammatory PGE2and LTC4.
引文
1. Shirai Y, Ito M (2004) Specific differential expression of phospholipase A2 subtypes in rat cerebellum [J]. J Neurocytol 33:297-307.
2. Burke JE, Dennis EA (2009) Phospholipase A2 biochemistry [J]. Cardiovasc Drugs Ther 23:49-59.
3. Janssen MJ, van de Wiel WA, Beiboer SH, van Kampen MD, Verheij HM, et al. (1999) Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K [J]. Protein Eng 12:497-503.
4. Edwards SH, Thompson D, Baker SF, Wood SP, Wilton DC (2002) The crystal structure of the H48Q active site mutant of human group IIA secreted phospholipase A2 at 1.5 A resolution provides an insight into the catalytic mechanism [J]. Biochemistry 41:15468-15476.
5. Niessen HW, Krijnen PA, Visser CA, Meijer CJ, Erik Hack C (2003) Type Ⅱ secretory phospholipase A2 in cardiovascular disease:a mediator in atherosclerosis and ischemic damage to cardiomyocytes [J]? Cardiovasc Res 60:68-77.
6. Ghosh M, Tucker DE, Burchett SA, Leslie CC (2006) Properties of the Group Ⅳ phospholipase A2 family [J]. Prog Lipid Res 45:487-510.
7. Shimizu T, Ohto T, Kita Y (2006) Cytosolic phospholipase A2:biochemical properties and physiological roles [J]. IUBMB Life 58:328-333.
8. Niknami M, Patel M, Witting PK, Dong Q (2009) Molecules in focus: cytosolic phospholipase A2-alpha [J]. Int J Biochem Cell Biol 41:994-997.
9. Ghosh M, Loper R, Ghomashchi F, Tucker DE, Bonventre JV, et al. (2007) Function, activity, and membrane targeting of cytosolic phospholipase A(2)zeta in mouse lung fibroblasts [J]. J Biol Chem 282:11676-11686.
10. Pavicevic Z, Leslie CC, Malik KU (2008) cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells [J]. J Lipid Res 49:724-737.
11. Tian W, Wijewickrama GT, Kim JH, Das S, Tun MP, et al. (2008) Mechanism of regulation of group IVA phospholipase A2 activity by Ser727 phosphorylation [J]. J Biol Chem 283:3960-3971.
12. Leu BH, Schmidt JT (2008) Arachidonic acid as a retrograde signal controlling growth and dynamics of retinotectal arbors [J]. Dev Neurobiol 68:18-30.
13. Xu J, Chalimoniuk M, Shu Y, Simonyi A, Sun AY, et al. (2003) Prostaglandin E2 production in astrocytes:regulation by cytokines, extracellular ATP, and oxidative agents [J]. Prostaglandins Leukot Essent Fatty Acids 69:437-448.
14. Wang Z, Ramanadham S, Ma ZA, Bao S, Mancuso DJ, et al. (2005) Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase Ⅱbeta expressed in pancreatic islet beta-cells [J]. J Biol Chem 280:6840-6849.
15. Farooqui AA, Yang HC, Hirashima Y, Horrocks LA (1999) Determination of plasmalogen-selective phospholipase A2 activity by radiochemical and fluorometric assay procedures [J]. Methods Mol Biol 109:39-47.
16. Green JT, Orr SK, Bazinet RP (2008) The emerging role of group VI calcium-independent phospholipase A2 in releasing docosahexaenoic acid from brain phospholipids [J]. J Lipid Res 49:939-944.
1. Polazzi E, Contestabile A (2002) Reciprocal interactions between microglia and neurons: from survival to neuropathology [J]. Rev Neurosci 13:221-242.
2. Sairanen T, Ristimaki A, Karjalainen-Lindsberg ML, Paetau A, Kaste M, et al. (1998) Cyclooxygenase-2 is induced globally in infarcted human brain [J]. Ann Neurol 43:738-747.
3. Dore S, Otsuka T, Mito T, Sugo N, Hand T, et al. (2003) Neuronal overexpression of cyclooxygenase-2 increases cerebral infarction [J]. Ann Neurol 54: 155-162.
4. Vidensky S, Zhang Y, hand T, Goellner J, Shaffer A, et al. (2003) Neuronal overexpression of COX-2 results in dominant production of PGE2 and altered fever response [J]. Neuromolecular Med 3:15-28.
5. Dennis EA (1997) The growing phospholipase A2 superfamily of signal transduction enzymes [J]. Trends Biochem Sci 22:1-2.
6. Masuda S, Murakami M, Komiyama K, Ishihara M, Ishikawa Y, et al. (2005) Various secretory phospholipase A2 enzymes are expressed in rheumatoid arthritis and augment prostaglandin production in cultured synovial cells [J]. FEBS J 272:655-672.
7. Chalbot S, Zetterberg H, Blennow K, Fladby T, Grundke-Iqbal I, et al. (2009) Cerebrospinal fluid secretory Ca2+-dependent phospholipase A2 activity is increased in Alzheimer disease [J]. Clin Chem 55:2171-2179.
8. Yagami T, Ueda K, Asakura K, Hata S, Kuroda T, et al. (2002) Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis [J]. Mol Pharmacol 61:114-126.
9. Murakami M, Kambe T, Shimbara S, Kudo I (1999) Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways [J]. J Biol Chem 274:3103-3115.
10. Ding J, Yu JZ, Li QY, Wang X, Lu CZ, et al. (2009) Rho kinase inhibitor Fasudil induces neuroprotection and neurogenesis partially through astrocyte-derived G-CSF [J]. Brain Behav Immun 23:1083-1088.
11. Lin CH, Cheng FC, Lu YZ, Chu LF, Wang CH, et al. (2006) Protection of ischemic brain cells is dependent on astrocyte-derived growth factors and their receptors [J]. Exp Neurol 201:225-233.
12. Briones TL, Woods J, Wadowska M, Rogozinska M, Nguyen M (2006) Astrocytic changes in the hippocampus and functional recovery after cerebral ischemia are facilitated by rehabilitation training [J]. Behav Brain Res 171: 17-25.
13. Hayakawa K, Nakano T, Irie K, Higuchi S, Fujioka M, et al. (2010) Inhibition of reactive astrocytes with fluorocitrate retards neurovascular remodeling and recovery after focal cerebral ischemia in mice [J]. J Cereb Blood Flow Metab 30:871-882.
14. Xu J, Chalimoniuk M, Shu Y, Simonyi A, Sun AY, et al. (2003) Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents [J]. Prostaglandins Leukot Essent Fatty Acids 69:437-448.
15. Zhou H, Lapointe BM, Clark SR, Zbytnuik L, Kubes P (2006) A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide [J]. J Immunol 177:8103-8110.
16. Johnson RW, Gheusi G, Segreti S, Dantzer R, Kelley KW (1997) C3H/HeJ mice are refractory to lipopolysaccharide in the brain [J]. Brain Res 752:219-226.
17. Di Santo E, Alonzi T, Fattori E, Poli V, Ciliberto G, et al. (1996) Overexpression of interleukin-6 in the central nervous system of transgenic mice increases central but not systemic proinflammatory cytokine production [J]. Brain Res 740:239-244.
18. Kawano T, Anrather J, Zhou P, Park L, Wang G, et al. (2006) Prostaglandin E2 EP1 receptors:downstream effectors of COX-2 neurotoxicity [J]. Nat Med 12: 225-229.
19.张斌,魏欣冰,高鹏,见文成,王姿颖,et al. (2006)脑星形胶质细胞的纯化培养[J].山东大学学报(医学版)44：3.
20. Bonventre JV, Huang Z, Taheri MR, O'Leary E, Li E, et al. (1997) Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2 [J]. Nature 390:622-625.
21. Pavicevic Z, Leslie CC, Malik KU (2008) cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells [J]. J Lipid Res 49:724-737.
22. Tian W, Wijewickrama GT, Kim JH, Das S, Tun MP, et al. (2008) Mechanism of regulation of group IV A phospholipase A2 activity by Ser727 phosphorylation [J]. J Biol Chem 283:3960-3971.
23. Dieter P, Kolada A, Kamionka S, Schadow A, Kaszkin M (2002) Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by Group Ⅳ cytosolic phospholipase A2, but not by Group V and Group IIA secretory phospholipase A2 [J]. Cell Signal 14: 199-204.
24. Cannon TR, Mann GE, Baydoun AR (1998) Mechanisms of acute vasodilator response to bacterial lipopolysaccharide in the rat coronary microcirculation [J]. Br J Pharmacol 123:637-644.
25. Takakuwa T, Endo S, Nakae H, Kikichi M, Inada K, et al. (1994) PAF acetylhydrolase and arachidonic acid metabolite levels in patients with sepsis [J]. Res Commun Chem Pathol Pharmacol 84:283-290.
26. Saito O, Svensson CI, Buczynski MW, Wegner K, Hua XY, et al. (2010) Spinal glial TLR4-mediated nociception and production of prostaglandin E(2) and TNF [J]. Br J Pharmacol 160:1754-1764.
27. Kwiatkoski M, Soriano RN, Araujo RM, Azevedo LU, Batalhao ME, et al. (2013) Hydrogen sulfide inhibits preoptic prostaglandin E2 production during endotoxemia [J]. Exp Neurol 240:88-95.
28. Wu YZ, Medjane S, Chabot S, Kubrusly FS, Raw I, et al. (2003) Surfactant protein-A and phosphatidylglycerol suppress type IIA phospholipase A2 synthesis via nuclear factor-kappaB [J]. Am J Respir Crit Care Med 168: 692-699.
29. Fonteh AN, Atsumi G, LaPorte T, Chilton FH (2000) Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells [J]. J Immunol 165:2773-2782.
30. Blom M, Tool AT, Wever PC, Wolbink GJ, Brouwer MC, et al. (1998) Human eosinophils express, relative to other circulating leukocytes, large amounts of secretory 14-kD phospholipase A2 [J]. Blood 91:3037-3043.
31. Clark JD, Lin LL, Kriz RW, Ramesha CS, Sultzman LA, et al. (1991) A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP [J]. Cell 65: 1043-1051.
32. Sharp JD, White DL, Chiou XG, Goodson T, Gamboa GC, et al. (1991) Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2 [J]. J Biol Chem 266:14850-14853.
33. Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, et al. (1993) cPLA2 is phosphorylated and activated by MAP kinase [J]. Cell 72:269-278.
34. Bosetti F, Weerasinghe GR (2003) The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse [J]. J Neurochem 87:1471-1477.
1. Bolognesi ML, Minarini A, Rosini M, Tumiatti V, Melchiorre C (2008) From dual binding site acetylcholinesterase inhibitors to multi-target-directed ligands (MTDLs):a step forward in the treatment of Alzheimer's disease [J]. Mini Rev Med Chem 8:960-967.
2. Bolognesi ML, Banzi R, Bartolini M, Cavalli A, Tarozzi A, et al. (2007) Novel class of quinone-bearing polyamines as multi-target-directed ligands to combat Alzheimer's disease [J]. J Med Chem 50:4882-4897.
3. Meyer MC, Rastogi P, Beckett CS, McHowat J (2005) Phospholipase A2 inhibitors as potential anti-inflammatory agents [J]. Curr Pharm Des 11:1301-1312.
4. Serhan CN (2000) Preventing injury from within, using selective cPLA2 inhibitors [J]. Nat Immunol 1:13-15.
5. Balsinde J, Dennis EA (1996) Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages [J]. J Biol Chem 271:6758-6765.
6. Bonventre JV, Huang Z, Taheri MR, O'Leary E, Li E, et al. (1997) Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2 [J]. Nature 390:622-625.
7. Palumbo S, Toscano CD, Parente L, Weigert R, Bosetti F (2011) Time-dependent changes in the brain arachidonic acid cascade during cuprizone-induced demyelination and remyelination [J]. Prostaglandins Leukot Essent Fatty Acids 85:29-35.
8. Wu B, Liu M, Liu H, Li W, Tan S, et al. (2007) Meta-analysis of traditional Chinese patent medicine for ischemic stroke [J]. Stroke 38:1973-1979.
9. Feng ZQ, Wang YY, Guo ZR, Chu FM, Sun PY (2010) The synthesis of puerarin derivatives and their protective effect on the myocardial ischemia and reperfusion injury [J]. J Asian Nat Prod Res 12:843-850.
10. Liu R, Wei XB, Zhang XM (2007) Effects of acetylpuerarin on hippocampal neurons and intracellular free calcium subjected to oxygen-glucose deprivation/reperfusion in primary culture [J]. Brain Res 1147:95-104.
11. Hou L, Wei Xb, al. e (2004) Protective effects of Compounds N-2211 on focal brain ischemica-reperfusion injury in rats [J]. Qilu Pharm Affaris (Chinese) 3: 3.
12. XM.Li, Wei X, al. e (2005) Effect of acetylpuerarin on NO level and NOS activity in brain tissue and serum of focal cerebral ischemia reperfusion injury rats [J]. Chin Pharm J 11:4.
13.郭东艳,杨大坚,陈士林等(2008)葛根素及其衍生物在大鼠体内的药代动力学研究[J].陕西中医学院学报31：3.
14.张斌,魏欣冰,高鹏,见文成,王姿颖,et al. (2006)脑星形胶质细胞的纯化培养[J].山东大学学报(医学版)44：3.
15. Pavicevic Z, Leslie CC, Malik KU (2008) cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells [J]. J Lipid Res 49:724-737.
16. Diaz-Munoz MD, Osma-Garcia IC, Cacheiro-Llaguno C, Fresno M, Iniguez MA (2010) Coordinated up-regulation of cyclooxygenase-2 and microsomal prostaglandin E synthase 1 transcription by nuclear factor kappa B and early growth response-1 in macrophages [J]. Cell Signal 22:1427-1436.
17. Niranjan R (2014) The role of inflammatory and oxidative stress mechanisms in the pathogenesis of Parkinson's disease:focus on astrocytes [J]. Mol Neurobiol 49:28-38.
18. Murakami M, Kudo I, Inoue K (1993) Molecular nature of phospholipases A2 involved in prostaglandin 12 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2 [J]. J Biol Chem 268:839-844.
19. Fonteh AN, Atsumi G, LaPorte T, Chilton FH (2000) Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells [J]. J Immunol 165:2773-2782.
20. Satake Y, Diaz BL, Balestrieri B, Lam BK, Kanaoka Y, et al. (2004) Role of group V phospholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption [J]. J Biol Chem 279:16488-16494.
21. Uozumi N, Kume K, Nagase T, Nakatani N, Ishii S, et al. (1997) Role of cytosolic phospholipase A2 in allergic response and parturition [J]. Nature 390: 618-622.
22. Balestrieri B, Hsu V W, Gilbert H, Leslie CC, Han WK, et al. (2006) Group V secretory phospholipase A2 translocates to the phagosome after zymosan stimulation of mouse peritoneal macrophages and regulates phagocytosis [J]. J Biol Chem 281:6691-6698.
23. Denkers EY, Butcher BA, Del Rio L, Kim L (2004) Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-signaling cascades during intracellular Toxoplasma gondii infection [J]. Immunol Rev 201: 191-205.
24. Qi HY, Shelhamer JH (2005) Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharide-stimulated macrophages [J]. J Biol Chem 280: 38969-38975.
25. Boyanovsky BB, Li X, Shridas P, Sunkara M, Morris AJ, et al. (2010) Bioactive products generated by group V sPLA(2) hydrolysis of LDL activate macrophages to secrete pro-inflammatory cytokines [J]. Cytokine 50:50-57.
26. Tanabe T, Tohnai N (2002) Cyclooxygenase isozymes and their gene structures and expression [J]. Prostaglandins Other Lipid Mediat 68-69:95-114.
27. Radmark O, Samuelsson B (2009) 5-Lipoxygenase: mechanisms of regulation [J]. J Lipid Res 50 Suppl:S40-45.
28. Russell ST, Tisdale MJ (2009) Mechanism of attenuation by beta-hydroxy-beta-methylbutyrate of muscle protein degradation induced by lipopolysaccharide [J]. Mol Cell Biochem 330:171-179.
2. Burke JE, Dennis EA (2009) Phospholipase A2 biochemistry [J]. Cardiovasc Drugs Ther 23:49-59.
3. Janssen MJ, van de Wiel WA, Beiboer SH, van Kampen MD, Verheij HM, et al. (1999) Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K [J]. Protein Eng 12:497-503.
4. Edwards SH, Thompson D, Baker SF, Wood SP, Wilton DC (2002) The crystal structure of the H48Q active site mutant of human group IIA secreted phospholipase A2 at 1.5 A resolution provides an insight into the catalytic mechanism [J]. Biochemistry 41:15468-15476.
5. Niessen HW, Krijnen PA, Visser CA, Meijer CJ, Erik Hack C (2003) Type Ⅱ secretory phospholipase A2 in cardiovascular disease:a mediator in atherosclerosis and ischemic damage to cardiomyocytes [J]? Cardiovasc Res 60:68-77.
6. Ghosh M, Tucker DE, Burchett SA, Leslie CC (2006) Properties of the Group Ⅳ phospholipase A2 family [J]. Prog Lipid Res 45:487-510.
7. Shimizu T, Ohto T, Kita Y (2006) Cytosolic phospholipase A2:biochemical properties and physiological roles [J]. IUBMB Life 58:328-333.
8. Niknami M, Patel M, Witting PK, Dong Q (2009) Molecules in focus: cytosolic phospholipase A2-alpha [J]. Int J Biochem Cell Biol 41:994-997.
9. Ghosh M, Loper R, Ghomashchi F, Tucker DE, Bonventre JV, et al. (2007) Function, activity, and membrane targeting of cytosolic phospholipase A(2)zeta in mouse lung fibroblasts [J]. J Biol Chem 282:11676-11686.
10. Pavicevic Z, Leslie CC, Malik KU (2008) cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells [J]. J Lipid Res 49:724-737.
11. Tian W, Wijewickrama GT, Kim JH, Das S, Tun MP, et al. (2008) Mechanism of regulation of group IVA phospholipase A2 activity by Ser727 phosphorylation [J]. J Biol Chem 283:3960-3971.
12. Leu BH, Schmidt JT (2008) Arachidonic acid as a retrograde signal controlling growth and dynamics of retinotectal arbors [J]. Dev Neurobiol 68:18-30.
13. Xu J, Chalimoniuk M, Shu Y, Simonyi A, Sun AY, et al. (2003) Prostaglandin E2 production in astrocytes:regulation by cytokines, extracellular ATP, and oxidative agents [J]. Prostaglandins Leukot Essent Fatty Acids 69:437-448.
14. Wang Z, Ramanadham S, Ma ZA, Bao S, Mancuso DJ, et al. (2005) Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase Ⅱbeta expressed in pancreatic islet beta-cells [J]. J Biol Chem 280:6840-6849.
15. Farooqui AA, Yang HC, Hirashima Y, Horrocks LA (1999) Determination of plasmalogen-selective phospholipase A2 activity by radiochemical and fluorometric assay procedures [J]. Methods Mol Biol 109:39-47.
16. Green JT, Orr SK, Bazinet RP (2008) The emerging role of group VI calcium-independent phospholipase A2 in releasing docosahexaenoic acid from brain phospholipids [J]. J Lipid Res 49:939-944.
1. Polazzi E, Contestabile A (2002) Reciprocal interactions between microglia and neurons: from survival to neuropathology [J]. Rev Neurosci 13:221-242.
2. Sairanen T, Ristimaki A, Karjalainen-Lindsberg ML, Paetau A, Kaste M, et al. (1998) Cyclooxygenase-2 is induced globally in infarcted human brain [J]. Ann Neurol 43:738-747.
3. Dore S, Otsuka T, Mito T, Sugo N, Hand T, et al. (2003) Neuronal overexpression of cyclooxygenase-2 increases cerebral infarction [J]. Ann Neurol 54: 155-162.
4. Vidensky S, Zhang Y, hand T, Goellner J, Shaffer A, et al. (2003) Neuronal overexpression of COX-2 results in dominant production of PGE2 and altered fever response [J]. Neuromolecular Med 3:15-28.
5. Dennis EA (1997) The growing phospholipase A2 superfamily of signal transduction enzymes [J]. Trends Biochem Sci 22:1-2.
6. Masuda S, Murakami M, Komiyama K, Ishihara M, Ishikawa Y, et al. (2005) Various secretory phospholipase A2 enzymes are expressed in rheumatoid arthritis and augment prostaglandin production in cultured synovial cells [J]. FEBS J 272:655-672.
7. Chalbot S, Zetterberg H, Blennow K, Fladby T, Grundke-Iqbal I, et al. (2009) Cerebrospinal fluid secretory Ca2+-dependent phospholipase A2 activity is increased in Alzheimer disease [J]. Clin Chem 55:2171-2179.
8. Yagami T, Ueda K, Asakura K, Hata S, Kuroda T, et al. (2002) Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis [J]. Mol Pharmacol 61:114-126.
9. Murakami M, Kambe T, Shimbara S, Kudo I (1999) Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways [J]. J Biol Chem 274:3103-3115.
10. Ding J, Yu JZ, Li QY, Wang X, Lu CZ, et al. (2009) Rho kinase inhibitor Fasudil induces neuroprotection and neurogenesis partially through astrocyte-derived G-CSF [J]. Brain Behav Immun 23:1083-1088.
11. Lin CH, Cheng FC, Lu YZ, Chu LF, Wang CH, et al. (2006) Protection of ischemic brain cells is dependent on astrocyte-derived growth factors and their receptors [J]. Exp Neurol 201:225-233.
12. Briones TL, Woods J, Wadowska M, Rogozinska M, Nguyen M (2006) Astrocytic changes in the hippocampus and functional recovery after cerebral ischemia are facilitated by rehabilitation training [J]. Behav Brain Res 171: 17-25.
13. Hayakawa K, Nakano T, Irie K, Higuchi S, Fujioka M, et al. (2010) Inhibition of reactive astrocytes with fluorocitrate retards neurovascular remodeling and recovery after focal cerebral ischemia in mice [J]. J Cereb Blood Flow Metab 30:871-882.
14. Xu J, Chalimoniuk M, Shu Y, Simonyi A, Sun AY, et al. (2003) Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents [J]. Prostaglandins Leukot Essent Fatty Acids 69:437-448.
15. Zhou H, Lapointe BM, Clark SR, Zbytnuik L, Kubes P (2006) A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide [J]. J Immunol 177:8103-8110.
16. Johnson RW, Gheusi G, Segreti S, Dantzer R, Kelley KW (1997) C3H/HeJ mice are refractory to lipopolysaccharide in the brain [J]. Brain Res 752:219-226.
17. Di Santo E, Alonzi T, Fattori E, Poli V, Ciliberto G, et al. (1996) Overexpression of interleukin-6 in the central nervous system of transgenic mice increases central but not systemic proinflammatory cytokine production [J]. Brain Res 740:239-244.
18. Kawano T, Anrather J, Zhou P, Park L, Wang G, et al. (2006) Prostaglandin E2 EP1 receptors:downstream effectors of COX-2 neurotoxicity [J]. Nat Med 12: 225-229.
19.张斌,魏欣冰,高鹏,见文成,王姿颖,et al. (2006)脑星形胶质细胞的纯化培养[J].山东大学学报(医学版)44：3.
20. Bonventre JV, Huang Z, Taheri MR, O'Leary E, Li E, et al. (1997) Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2 [J]. Nature 390:622-625.
21. Pavicevic Z, Leslie CC, Malik KU (2008) cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells [J]. J Lipid Res 49:724-737.
22. Tian W, Wijewickrama GT, Kim JH, Das S, Tun MP, et al. (2008) Mechanism of regulation of group IV A phospholipase A2 activity by Ser727 phosphorylation [J]. J Biol Chem 283:3960-3971.
23. Dieter P, Kolada A, Kamionka S, Schadow A, Kaszkin M (2002) Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by Group Ⅳ cytosolic phospholipase A2, but not by Group V and Group IIA secretory phospholipase A2 [J]. Cell Signal 14: 199-204.
24. Cannon TR, Mann GE, Baydoun AR (1998) Mechanisms of acute vasodilator response to bacterial lipopolysaccharide in the rat coronary microcirculation [J]. Br J Pharmacol 123:637-644.
25. Takakuwa T, Endo S, Nakae H, Kikichi M, Inada K, et al. (1994) PAF acetylhydrolase and arachidonic acid metabolite levels in patients with sepsis [J]. Res Commun Chem Pathol Pharmacol 84:283-290.
26. Saito O, Svensson CI, Buczynski MW, Wegner K, Hua XY, et al. (2010) Spinal glial TLR4-mediated nociception and production of prostaglandin E(2) and TNF [J]. Br J Pharmacol 160:1754-1764.
27. Kwiatkoski M, Soriano RN, Araujo RM, Azevedo LU, Batalhao ME, et al. (2013) Hydrogen sulfide inhibits preoptic prostaglandin E2 production during endotoxemia [J]. Exp Neurol 240:88-95.
28. Wu YZ, Medjane S, Chabot S, Kubrusly FS, Raw I, et al. (2003) Surfactant protein-A and phosphatidylglycerol suppress type IIA phospholipase A2 synthesis via nuclear factor-kappaB [J]. Am J Respir Crit Care Med 168: 692-699.
29. Fonteh AN, Atsumi G, LaPorte T, Chilton FH (2000) Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells [J]. J Immunol 165:2773-2782.
30. Blom M, Tool AT, Wever PC, Wolbink GJ, Brouwer MC, et al. (1998) Human eosinophils express, relative to other circulating leukocytes, large amounts of secretory 14-kD phospholipase A2 [J]. Blood 91:3037-3043.
31. Clark JD, Lin LL, Kriz RW, Ramesha CS, Sultzman LA, et al. (1991) A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP [J]. Cell 65: 1043-1051.
32. Sharp JD, White DL, Chiou XG, Goodson T, Gamboa GC, et al. (1991) Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2 [J]. J Biol Chem 266:14850-14853.
33. Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, et al. (1993) cPLA2 is phosphorylated and activated by MAP kinase [J]. Cell 72:269-278.
34. Bosetti F, Weerasinghe GR (2003) The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse [J]. J Neurochem 87:1471-1477.
1. Bolognesi ML, Minarini A, Rosini M, Tumiatti V, Melchiorre C (2008) From dual binding site acetylcholinesterase inhibitors to multi-target-directed ligands (MTDLs):a step forward in the treatment of Alzheimer's disease [J]. Mini Rev Med Chem 8:960-967.
2. Bolognesi ML, Banzi R, Bartolini M, Cavalli A, Tarozzi A, et al. (2007) Novel class of quinone-bearing polyamines as multi-target-directed ligands to combat Alzheimer's disease [J]. J Med Chem 50:4882-4897.
3. Meyer MC, Rastogi P, Beckett CS, McHowat J (2005) Phospholipase A2 inhibitors as potential anti-inflammatory agents [J]. Curr Pharm Des 11:1301-1312.
4. Serhan CN (2000) Preventing injury from within, using selective cPLA2 inhibitors [J]. Nat Immunol 1:13-15.
5. Balsinde J, Dennis EA (1996) Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages [J]. J Biol Chem 271:6758-6765.
6. Bonventre JV, Huang Z, Taheri MR, O'Leary E, Li E, et al. (1997) Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2 [J]. Nature 390:622-625.
7. Palumbo S, Toscano CD, Parente L, Weigert R, Bosetti F (2011) Time-dependent changes in the brain arachidonic acid cascade during cuprizone-induced demyelination and remyelination [J]. Prostaglandins Leukot Essent Fatty Acids 85:29-35.
8. Wu B, Liu M, Liu H, Li W, Tan S, et al. (2007) Meta-analysis of traditional Chinese patent medicine for ischemic stroke [J]. Stroke 38:1973-1979.
9. Feng ZQ, Wang YY, Guo ZR, Chu FM, Sun PY (2010) The synthesis of puerarin derivatives and their protective effect on the myocardial ischemia and reperfusion injury [J]. J Asian Nat Prod Res 12:843-850.
10. Liu R, Wei XB, Zhang XM (2007) Effects of acetylpuerarin on hippocampal neurons and intracellular free calcium subjected to oxygen-glucose deprivation/reperfusion in primary culture [J]. Brain Res 1147:95-104.
11. Hou L, Wei Xb, al. e (2004) Protective effects of Compounds N-2211 on focal brain ischemica-reperfusion injury in rats [J]. Qilu Pharm Affaris (Chinese) 3: 3.
12. XM.Li, Wei X, al. e (2005) Effect of acetylpuerarin on NO level and NOS activity in brain tissue and serum of focal cerebral ischemia reperfusion injury rats [J]. Chin Pharm J 11:4.
13.郭东艳,杨大坚,陈士林等(2008)葛根素及其衍生物在大鼠体内的药代动力学研究[J].陕西中医学院学报31：3.
14.张斌,魏欣冰,高鹏,见文成,王姿颖,et al. (2006)脑星形胶质细胞的纯化培养[J].山东大学学报(医学版)44：3.
15. Pavicevic Z, Leslie CC, Malik KU (2008) cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells [J]. J Lipid Res 49:724-737.
16. Diaz-Munoz MD, Osma-Garcia IC, Cacheiro-Llaguno C, Fresno M, Iniguez MA (2010) Coordinated up-regulation of cyclooxygenase-2 and microsomal prostaglandin E synthase 1 transcription by nuclear factor kappa B and early growth response-1 in macrophages [J]. Cell Signal 22:1427-1436.
17. Niranjan R (2014) The role of inflammatory and oxidative stress mechanisms in the pathogenesis of Parkinson's disease:focus on astrocytes [J]. Mol Neurobiol 49:28-38.
18. Murakami M, Kudo I, Inoue K (1993) Molecular nature of phospholipases A2 involved in prostaglandin 12 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2 [J]. J Biol Chem 268:839-844.
19. Fonteh AN, Atsumi G, LaPorte T, Chilton FH (2000) Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells [J]. J Immunol 165:2773-2782.
20. Satake Y, Diaz BL, Balestrieri B, Lam BK, Kanaoka Y, et al. (2004) Role of group V phospholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption [J]. J Biol Chem 279:16488-16494.
21. Uozumi N, Kume K, Nagase T, Nakatani N, Ishii S, et al. (1997) Role of cytosolic phospholipase A2 in allergic response and parturition [J]. Nature 390: 618-622.
22. Balestrieri B, Hsu V W, Gilbert H, Leslie CC, Han WK, et al. (2006) Group V secretory phospholipase A2 translocates to the phagosome after zymosan stimulation of mouse peritoneal macrophages and regulates phagocytosis [J]. J Biol Chem 281:6691-6698.
23. Denkers EY, Butcher BA, Del Rio L, Kim L (2004) Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-signaling cascades during intracellular Toxoplasma gondii infection [J]. Immunol Rev 201: 191-205.
24. Qi HY, Shelhamer JH (2005) Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharide-stimulated macrophages [J]. J Biol Chem 280: 38969-38975.
25. Boyanovsky BB, Li X, Shridas P, Sunkara M, Morris AJ, et al. (2010) Bioactive products generated by group V sPLA(2) hydrolysis of LDL activate macrophages to secrete pro-inflammatory cytokines [J]. Cytokine 50:50-57.
26. Tanabe T, Tohnai N (2002) Cyclooxygenase isozymes and their gene structures and expression [J]. Prostaglandins Other Lipid Mediat 68-69:95-114.
27. Radmark O, Samuelsson B (2009) 5-Lipoxygenase: mechanisms of regulation [J]. J Lipid Res 50 Suppl:S40-45.
28. Russell ST, Tisdale MJ (2009) Mechanism of attenuation by beta-hydroxy-beta-methylbutyrate of muscle protein degradation induced by lipopolysaccharide [J]. Mol Cell Biochem 330:171-179.