【作者】 李褒曼；

【导师】 彭亮；

【作者基本信息】 中国医科大学 ， 药理学， 2009， 博士

【摘要】 抗单、双相抑郁症药物对星形胶质细胞作用的研究前言单相和双相抑郁症均是常见的精神疾病,其中单相抑郁症就是通常所说的抑郁症,而双相抑郁症又称为躁狂抑郁症。目前临床上,治疗单相抑郁症的主要药物是以氟西汀为代表的五羟色胺特异性再摄取抑制剂（SSRIs）,治疗双相抑郁症的药物主要有碳酸锂、丙戊酸钠和卡马西平三种。但是,这些药物的确切药理机制还都有待进一步研究。磷脂酶A₂功能学上主要分为三类:分泌型磷脂酶A₂（sPLA₂）,非钙离子依赖型磷脂酶A₂（iPLA₂）,细胞溶质型磷脂酶A₂（cPLA₂）。它们能水解细胞膜磷脂中sn-2位点的酰酯键,产生溶血磷脂和游离的脂肪酸（例如花生四烯酸）。其中,cPLA₂的活化和花生四烯酸的释放,通过不同机制分别与多巴胺、谷氨酸、五羟色胺等受体发生联系,而这些作用又与第二信使水平的调控有关。cPLA_2a亚型是cPLA₂在星形胶质细胞中的主要表达类型。在整体实验中,以临床治疗相关浓度分别喂食大鼠锂盐、卡马西平六周或者丙戊酸30天后,每种药物都可以减少体内甘油磷脂脱酰基转变为花生四烯酸的过程,进而影响随后的再酰化进程。锂盐或卡马西平慢性处理六周可以减少大鼠脑内cPLA_2a的表达。五羟色胺（5-HT）是一种重要的神经递质,其受体有7种。5-HT₂受体分为5-HT_2A,5-HT_2B和5-HT_2C三种亚型,在原代培养的星形胶质细胞上均有表达。三种5-HT₂受体均为G（q/11）蛋白偶联受体,它们能活化磷脂酶C（PLC）,后者可分解磷脂酰肌醇二磷酸（PIP2）产生二脂酰甘油（DAG）和三磷酸肌醇（IP₃）,此反应导致细胞内游离钙离子浓度增加。在星形胶质细胞中,氟西汀可通过5-HT₂受体增加细胞内钙离子浓度。研究表明细胞外信号调控激酶（ERK）在中枢神经系统中发挥重要作用,例如在突触可塑性和记忆形成等方面起到关键作用。据报道在星形胶质细胞中,氟西汀刺激ERK_1/2磷酸化,从而诱导基因表达。Fos蛋白家族是一组转录因子,包括c-Fos、FosB、Fra-1、Fra-2和delta-FosB（为FosB的剪接变体）。很多磷酸化的蛋白激酶可以影响Fos蛋白表达,例如有丝分裂活化蛋白激酶（MAPK）、蛋白激酶A（PKA）、PKC。Fos蛋白可以影响蛋白稳定性、蛋白结合活力、一些转录因子的间接激活潜力等。原代培养的星形胶质细胞不含有五羟色胺转载体。本课题主要在原代培养的星形胶质细胞中,研究抗双相抑郁症药物和氟西汀对星形胶质细胞的作用及机制。即:①三种抗双相抑郁症药物对PLA₂的长期作用;②氟西汀是否通过5-HT_2B受体引起ERK磷酸化,进而影响转录因子的表达（以c-Fos和FosB为检测指标）。方法采用原代培养的小鼠星形胶质细胞。①分别用碳酸锂、丙戊酸钠、卡马西平和托吡酯处理细胞1-4周,用RT-PCR和免疫印迹方法检测药物对PLA₂三种亚型的作用;②在正常或经5-HT_2B受体siRNA干扰后的细胞中,氟西汀处理20分钟观察ERK_1/2磷酸化水平,再用氟西汀处理正常细胞1或4小时后,观察c-Fos和FosB mRNA或蛋白表达水平,并且检测这一作用是否可被5-HT_2B受体抑制剂SB204741,或ERK_1/2磷酸化抑制剂U0126所抑制。使用SPSS12.0软件进行统计学分析,多组资料用one-way ANOVA方法进行比较,P＜0.05表示差异具有统计学意义。结果1、三种抗双相抑郁症药物长期作用的酶特异性药物处理2周后,0.25 mM,0.5 mM和1mM碳酸锂,25μM和50μM卡马西平,100μM和1mM丙戊酸钠均可使cPLA_2amRNA和蛋白表达发生上调,仅持续时间不同。但是这三种药物对sPLA₂和iPLA₂表达没有影响。表明三种抗双相抑郁症药物长期作用具有cPLA_2a特异性。2、三种抗双相抑郁症药物对cPLA_2a调节具有药物特异性与卡马西平和丙戊酸钠一样,托吡酯也为抗癫痫药物,但没有抗双相抑郁症作用,100μM托吡酯对cPLA_2a表达1-4周没有作用。表明三种抗双相抑郁症药物对cPLA_2a长期作用具有抗双相抑郁症药物特异性。3、氟西汀通过5-HT_2B受体引起ERK_1/2磷酸化在正常培养的星形胶质细胞中,10μM氟西汀20分钟可以使ERK_1/2磷酸化水平增高。但在5-HT_2B受体成功沉默后的星形胶质细胞中,氟西汀的这一作用消失。氟西汀的作用还可被蛋白激酶C（PKC）抑制剂GF109203X和钙离子螯合剂所抑制。4、氟西汀可增加c-Fos和FosB表达10μM氟西汀在1小时和4小时分别增加c-Fos和FosB mRNA和蛋白表达水平。此作用可被SB204741和U0126抑制。表明氟西汀是通过5-HT_2B受体,引起ERK_1/2磷酸化后,进一步调节c-Fos和FosB表达。讨论1、三种抗双相抑郁症药物对cPLA_2a的调节低浓度的碳酸锂在研究时期内仅对cPLA_2a有上调作用,高浓度的碳酸锂作用长时间后有下调作用。长期处理影响表达水平的理论还在卡马西平和丙戊酸的结果中得到应证,二者高浓度（50μM和1 mM）的作用在处理2-3周后达到峰值,在4周后上调幅度消除或者迅速下降,然而二者低浓度（25μM和100μM）的作用在4周后更为显著,且发展的更慢。相反的是,无抗双相精神障碍疗效的托吡酯没有影响cPLA_2a的表达,并且三种抗双相精神障碍药物对iPLA₂和sPLA₂mRNA表达没有改变。以上观察（上调和下调）均表明了酶的特异性和药物的特异性,这些作用均有赖于药物的浓度和处理时间。在本实验中所使用的药物浓度与临床药物浓度具有相关性。在本实验中,cPLA_2a是PLA₂中唯一对磷脂底物（可转化为花生四烯酸）有特异作用的。在星形胶质细胞内,花生四烯酸以复杂的方式干扰细胞内游离钙离子浓度。钙离子在自身细胞内,以及在星形胶质细胞同神经元之间均有信号传导功能。并且,释放的花生四烯酸能进一步氧化为类花生酸类物质,转化为内源性大麻素或者在细胞膜上再酰化,此过程在过去被大大低估了。2、氟西汀对c-Fos和FosB的作用机制RNA沉默实验表明,在分化后的星形胶质细胞中氟西汀选择性地作用于5-HT_2B受体。氟西汀对ERK_1/2的作用与以前报道的氟西汀对细胞内钙离子的作用相似。在本实验中,我们发现氟西汀对c-Fos和FosB表达的上调作用可以被U0126抑制,提示在星形胶质细胞中氟西汀对基因的调控作用是由5-HT_2B受体引起的ERK磷酸化所介导的。此作用是否为五羟色胺再摄取抑制剂（SSRIs）治疗作用的关键,还有赖于检验其它SSRIs药物是否也刺激5-HT_2B受体。氟西汀和帕罗西汀有相似的治疗作用,近期实验我们还发现帕罗西汀诱导ERK磷酸化的浓度依赖性与氟西汀的作用完全一致。结论1、碳酸锂、丙戊酸钠和卡马西平三种抗双相精神障碍药物对cPLA_2amRNA和蛋白表达的长期调节存在共同趋势,并且这一作用具有cPLA_2a酶特异性及抗双相精神障碍类药物特异性。2、氟西汀通过5-HT_2B受体诱导c-Fos和FosB mRNA和蛋白表达增加,此过程依赖ERK_1/2磷酸化。更多还原

【Abstract】 IntroductionUnipolar depression and bipolar depression are both common mental disorders, and unipolar depression is usually called depression,and bipolar depression is also called manic-depressive illness.Clinically,anti-unipolar depression drugs are mainly serotonin-specific reuptake inhibitors（SSRIs）,fluoxetine as represented,and lithium salts（Li⁺）,valproic acid（VPA）,and carbamaze-pine（CBZ）are the three classical anti-bipolar drugs.But the accurate pharmacological mechanisms of these drugs have not been clear and need to be further investigated.In brain tissue,phospholipases A₂（PLA₂）mainly includes three subtypes: secretory phospholipase A₂（sPLA₂）,cytosolic phospholipase A₂（cPLA₂）,and calcium-independent phospholipase A₂（iPLA₂）.These PLA₂ could specifically hydrolyze the acyl ester bond at the sn-2 position of glycerol in membrane phospholipids to produce lysophospholipids and free fatty acids（such as arachidonic acid,AA）.cPLA₂ activity and arachidonic acid release is linked to dopamine, glutamate,serotonin receptors through different coupling mechanisms.These mechanisms modulate the release of arachidonic acid and levels of second messengers in brain tissue.The majority of cPLA₂ in astrocytes is cPLA_2a.In intact brain, scientists have established that after 6 weeks（Li⁺ and CBZ）or 30 days（VPA）of treatment in rats with doses leading to therapeutically relevant drug levels,each drug decreased the in vivo turnover of arachidonic acid by deacylation from glycerophospholipids followed by re-acylation.Chronic treatment with Li⁺ for 6 weeks decreased the expression of cPLA_2ain rat brain.Serotonin[5-hydroxytryptamine（5-HT）]is an important neurotransmitter in the CNS that has seven subtypes of receptors termed 5-HT₁ through 5-HT₇.5-HT₂ receptor comprises of 5-HT_2A,5-HT_2Band 5-HT_2Creceptors which all express on astrocytes.All three 5-HT₂ receptors are Gq/11 protein-coupled and their stimulation activates phospholipase C（PLC）,generating diacylglycerol（DAG）and inositol 1,4,5-trisphosphate（IP3）by hydrolysis of phosphatidyl-inositol 4,5-bisphosphate（PIP2） and leading to an increase of free cytosolic calcium concentration[Ca²⁺]_i.Acute exposure to fluoxetine similarly increases[Ca²⁺]_i in astrocytes by a direct 5-HT₂ receptor action.Recently some reports shows that extracellular-signal regulated kinases（ERK） plays an important role in CNS,for example,in synaptic plasticity and memory formation.Fluoxetine causes ERK phosphorylation,followed by inducing expressions of certain genes.The Fos family of transcription factors includes c-Fos,FosB,Fra-1 and Fra-2 as well as smaller FosB（splice variants of FosB）.The activity of all Fos family members is modulated by different kinases,i.e.,MAPK,PKA or PKC,and then Fos protein influences protein stability,DNA-binding activity and the transactivating potential of the transcription factors.In primary cultured astrocytes with dibutyryl cyclic AMP（dBcAMP）,there is no serotonin transporter.In the present work,we studied of fluoxetine and anti-bipolar drugs:①the regulation of cPLA₂ by chronic treatment with three anti-bipolar drugs;②whether fluoxetine-mediated 5-HT_2Breceptor stimulation in astrocytes caused by ERK phosphorylation,followed by effecting the expressions of transcription factors （c-Fos and FosB）.MethodsPrimary cultured of mouse astrocytes.①After treating with lithium carbonate, valproate sodium and carbamazepine for 1-4 weeks,measuring the mRNA and protein expressions of three PLA₂ subtypes by RT-PCR and Western blot;②In astrocytes control or RNA interfered with 5-HT_2Breceptor siRNA,determining the phosphorylation level of ERK_1/2with fluoxetine for 20 min,then checking the mRNA and protein expressions of c-Fos and FosB with fluoxetine for 1 or 4 hours,in the absence or presence of SB204741(5-HT_2Breceptor antagonist)or U0126（an inhibitor of ERK phosphorylation）.The results are analysised with one-way ANOVA by SPSS12.0 software.P＜0.05 indicates the statically significant difference.Results1、The chronic effect of three anti-bipolar drugs is enzyme-specificAfter 2 weeks,lithium carbonate（0.25 mM,0.5 mM and 1 mM）,carbamazepine （25μM and 50μM）,valproate sodium（100μM and 1 mM）could upregulate the mRNA and protein of cPLA_2a,only the time duration was different.But these drugs had non-function to sPLA₂ and iPLA₂,indicating the prolonged treatment with three bipolar drugs having cPLA_2aenzyme-specificity.2、The chronic effect of anti-bipolar drugs to cPLA_2ais drug-specificTopiramate,an anticonvulsant as well as CBZ and VPA,but,which has no anti-bipolar effect.The expression of cPLA_2acould not be regulated by 100μM topiramate for 1-4 weeks.Suggesting the chronic regulation of cPLA_2awith three bipolar drugs being drug-specificity.3、ERK phosphorylation induced by fluoxetine via 5-HT_2BreceptorIn common cultured astrocytes,fluoxetine could increase the phosphorylation level of ERK_1/2in 20 min,but this function was abolished in astrocytes RNA interfered with 5-HT_2Breceptor siRNA.And ERK phosphorylation induced by fluoxetine could be inhibited by GF109203X（PKC inhibition）or BAPTA/AM(a chelator of intracellular Ca²⁺).4、The expression of c-Fos and FosB increased by fluoxetine10μM fluoxetine could respectively increase the mRNA and protein expressions of c-Fos and FosB,which could be inhibited by SB204741 or U0126.Indicating the regulation of c-Fos and FosB by fluoxetine via 5-HT_2Breceptor,and dependenting on ERK phosphorylation mediated by fluoxetine.Discussion 1、The expression of PLA₂ regulated by three anti-bipolar drugsLower concentrations of lithium carbonate led within the period studied only to an up-regulation,but it is possible that longer exposure times might have resulted in a down-regulation.The concept that the length of the treatment period may affect the response is supported by the findings with carbamazepine and valproic acid where the effect of higher concentrations（50μM;1 mM）peaked after 2-3 weeks of treatment and was abolished or greatly reduced after 4 weeks,whereas that to the lower concentration（25μM;100μM）was pronounced after 4 weeks,but also developed more slowly.In contrast,topiramate,which has no anti-bipolar effect had no effect on cPLA_2aexpression,and mRNA expression of iPLA₂ and sPLA₂ was unaltered by Li+, VPA,and CBZ.Thus,the observed effects（up-regulation and down-regulation）show both enzyme specificity and drug specificity,and it was in a systematic fashion dependent upon drug concentration and the length of the treatment period.The concentrations used in the present study are probably pharmacologically relevant.In the present context,it is of interest that cPLA_2ais the only PLA₂ that has specificity for phospholipid substrates containing arachidonic acid（AA）.Moreover,the released arachidonic acid can be further oxidized to eicosanoids,converted to endocannabinoids or re-acylated in the membrane,a process that in the past has been quantitatively greatly underestimated.2、The mechanism of c-Fos and FosB expression regulated by fluoxetineRNA interference experiments showed that fluoxetine in cultures of well-differentiated astrocytes exclusively acts on 5-HT_2Breceptors.The potency of fluoxetine in the induction of ERK_1/2transactivation was similar to that previously reported for its effect on[Ca²⁺]i and in agreement with its affinity for the 5-HT_2B receptor.In the present work,we demonstrated that the upregulation of c-Fos and FosB expression by fluoxetine in astrocytes was inhibited by U0126,suggesting that the effects of fluoxetine on gene regulation in astrocytes were induced by 5-HT_2B receptor-mediated ERK_1/2phosphorylation.Whether or not such an effect might be of importance for the therapeutic effect of serotonin-specific uptake inhibitors（SSRIs）, depends upon whether other SSRIs also stimulate 5-HT_2Breceptors at realistic extracellular brain concentrations.Fluoxetine and paroxetine have approximately similar therapeutic potency and recent preliminary experiments have shown that the concentration dependence for paroxetine-mediated ERK phosphorylation is virtually identical to that found for fluoxetine.Conclusion1、The expression of cPLA_2amRNA and protein regulated by lithium carbonate, CBZ and VPA had similar trend,and this function was anti-bipolar drug-specific and enzyme-specific.2、Fluoxetine up-regulated the expression of c-Fos and FosB by stimulation of 5-HT_2Breceptor and ERK phosphorylation.更多还原