【作者】 相妍笑；

【导师】 张岫美；

【作者基本信息】 山东大学 ， 医学基础药理学， 2014， 博士

【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。更多还原

【Abstract】 BackgroundNeuroinflammation 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.ObjectivesThe objective of the study is to investigate the role of sPLA2and anti-inflammatory effect of acetylpuerarin on eicosanoid signaling pathway.Materials and MethodsThis 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.Results1. 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.Conclusions1. 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.更多还原