【作者】 仇志坤；

【导师】 徐江平； 李云峰；

【作者基本信息】 南方医科大学 ， 药理学， 2014， 博士

【摘要】 目的：创伤后应激障碍(Post-traumatic stress disorder, PTSD)属于焦虑症的特殊类型,指个体面临异常强烈精神应激后延迟发生的精神障碍。其临床表现多样化,主要包括创伤经历再体验、警觉增高致易激惹、回避行为和情感麻木等症状。现今PTSD具有发病率和患病率高,慢性病程,疗效差等特点。PTSD的发病机制较复杂。从神经生物学角度分析,涉及神经递质水平异常,下丘脑-垂体-肾上腺(Hypothalamic-pituitary-adrena, HPA)轴负反馈抑制增强,神经可塑性和神经类固醇调节异常。这些神经内分泌通路的失调可致使大脑结构和功能上的不正常从而引发一系列症状,例如闯入性再体验(闪回)和过度警觉。目前临床上缺乏PTSD特异性治疗药物。抗抑郁药五羟色胺(Serotonin,5-HT)重摄取抑制剂(Selective serotonin reuptake inhibitors, SSRIs)是治疗PTSD的首选药物,美国食品和药品管理局批准的仅有帕罗西汀和舍曲林,属于扩大适应症。但该类药物起效率不高(约60%),起效延迟(2-6周),并存在如胃肠道、性功能障碍等毒副反应。部分苯二氮卓类抗焦虑药也可用于来治疗PTSD。如咪达唑仑可破坏条件性惊恐记忆,达到PTSD行为的疗效。但该类药物存在依赖性和副作用大等缺点。因此,系统研究PTSD的新靶标新药物具有重要的民用和军事价值。转位蛋白18KDa (Translocator protein, TSPO)广泛分布于中枢和外周组织细胞线粒体外膜上。TSPO在脑内主要存在于胶质细胞,与配体结合可转运胆固醇进入线粒体膜内,促进神经类固醇合成。神经类固醇参与情绪和应激反应调节,对抑郁、焦虑等应激相关性精神系统疾病的治疗具有潜在价值。TSPO配体AC-5216(目前处于Ⅱ期临床阶段),在动物模型和临床治疗上具有抗焦虑,抗抑郁和抗惊恐疗效,而且没有肌松、镇静、戒断、耐受症状、损伤认知等安定样副反应作用。其作用机制是促进胆固醇跨膜并转运进入磷脂膜,增加孕烯醇酮及下游神经类固醇(e.g.孕酮、四氢孕酮等)合成。神经类固醇通过γ-氨基丁酸A受体(Gamma amino-butyric acid type A receptor, GABAA)受体上的神经类固醇作用位点增强GABA传导功能,改变神经元兴奋性,抑制海马-下丘脑-垂体-肾上腺(hippocampal-hypothalamic-pituitary-adrenal, HHPA)轴功能,调节情绪和应激反应。这些研究表明TSPO可作为PTSD治疗潜在靶标。然而,AC-5216具有难溶于水和结构优化空间较大的不足,因此生物利用度受到限制。据此,北京军事医学科学院毒物药物研究所药物化学研究室根据AC-5216的分子机构进行优化并定向合成系列水溶性衍生物,筛选证实新结构化合物YL-IPA08。YL-IPA08较之于AC-5216与TSPO结合亲和力更高,选择性更强,并已申请中国专利和美、日、欧盟国际专利。前期试验证实,YL-IPA08通过调节神经类固醇的合成具有抗焦虑和抗抑郁作用。尽管TSPO配体化合物AC-5216和YL-IPA08在抗应激性疾病中疗效显著,然而其在抗PTSD作用和机制中仍未见报道。针对此科学问题开展研究,阐明TSPO配体化合物抗PTSD作用和机制具有新颖性及应用价值。目前,多种模型应用于PTSD研究,其中包括时间依赖性敏化模型,短暂电击模型,单程延长应激模型,天敌模型和社交孤立模型。在本研究中应用小鼠短暂电击模型和大鼠时间依赖性敏化模型。短暂电击模型可体现临床上PTSD病态,如惊恐和焦虑行为。而时间依赖性敏化模型则具有表观有效性(模拟临床精神疾病状态),结构有效性(得到现今精神病理学理论支持),预测有效性(能预测临床前药物对PTSD患者的疗效)这几个特点。因此,以上这两个模型对研究在PTSD的药理学和生物学领域具有重要作用。综上分析,本研究应用AC-5216和YL-IPA08作用于短暂电击模型和时间依赖性敏化模型探讨TSPO配体药物在PTSD治疗中行为药效和机制。方法：(1)AC-5216和YL-IPA08对TSPO和中枢苯二氮卓受体(Central-type benzodiazepine receptor, CBR)的结合亲和力和对神经类固醇的水平调节放射性竞争配体结合试验研究AC-5216和YL-IPA08对大鼠小脑神经胶质细胞线粒体外膜TSPO和CBR结合亲和力。体外培养的星形胶质细胞系,通过酶联免疫吸附法(Enzyme-Linked ImmunoSorbent Assay, ELISA)测定给予AC-5216和YL-IPA08对细胞孕烯醇酮和孕酮含量,研究两者对神经类固醇如孕烯醇酮和孕酮的水平调节。(2)通过PTSD动物模型研究AC-5216与YL-IPA08的行为药效作用在小鼠短暂电击模型中,模型组小鼠在幽闭电击箱中接受2天各15次间断的、不可逃避的足底电击(0.8mA,间隔10s,持续10s)。正常对照组同样置于幽闭电击箱中,但不给予电击。训练完成之后的第2天开始给药,分为正常对照组,模型对照组,阳性对照药物舍曲林(15mg/kg p.o.)组,AC-5216组(0.03,0.1,0.3,1mg/kg p.o.)/YL-IPA08组(0.03,0.1,0.3,1,3mg/kg p.o.),1次/天。实验动物分别在电击后在相同的造模环境下进行环境重现实验,检测小鼠的僵住时间百分比。开场试验检测动物的自发活动。高架十字迷宫试验和爬梯试验,检测AC-5216和YL-IPA08抗PTSD行为药效。在时间依赖敏化模型中,大鼠造模(固定2h后,进行强迫游泳20min,恢复15min,暴露于麻醉乙醚中直至失去知觉。7天之后再次进行20min强迫游泳(方法如前)),正常对照组不给予任何刺激。训练完成之后的第2天开始给药,分为正常对照组,模型对照组,阳性对照药物舍曲林(15mg/kg p.o.)组,AC-5216组(0.03,0.1,0.3,1mg/kgp.o.)/YL-IPA08组(0.03,0.1,0.3,1,3mg/kg p.o.),1次/天。实验动物分别在造模后进行开场试验检测动物自发活动,环境重现实验和高架十字迷宫,检测AC-5216和YL-IPA08抗PTSD行为药效。(3)研究AC-5216与YL-IPA08抗PTSD作用机制根据AC-5216和YL-IPA08时间依赖性敏化模型中的最大效应剂量,采用TSPO配体PK11195拮抗AC-5216和YL-IPA08在时间依赖性敏化模型中的抗PTSD行为药效,观察AC-5216和YL-IPA08抗PTSD的行为药效是否通过TSPO所介导。提取PTSD动物模型(时间依赖性敏化模型)中大脑前额皮层,海马体和血样本,通过蛋白质免疫印迹研究AC-5216与YL-IPA08抗PTSD作用与大脑前额皮层和海马体TSPO表达相关性；通过放射性竞争配体结合试验研究AC-5216与YL-IPA08抗PTSD作用与大脑前额皮层,海马体和血小板中TSPO受体活性相关性；ELISA检测AC-5216与YL-IPA08抗PTSD作用与大脑前额皮层,海马体和血样本中孕酮和四氢孕酮的调节水平相关性；高效液相电化学色谱法分析AC-5216与YL-IPA08抗PTSD作用与前额皮层和海马体中5-HT,二羟苯乙酸(dihydroxy-phenyl acetic acid, DOPAC),多巴胺(dopamine, DA),5-羟吲哚乙酸(5-hydroxyindoleacetic acid,5-HIAA)和去甲肾上腺素(norepinephrine, NE)单胺递质水平变化的相关性。(4)统计分析结果采用SPSS13.0软件进行统计,各组计量数据均以平均值士标准误(Mean±S.E.M.)表示。体重结果采用重复性测量方差分析检测。结果多组间比较采用单因素方差分析(One-way ANOVA)进行分析,方差齐性时,组间两两比较采用LSD-t法；若方差不齐,将采用Welch法进行分析,多重比较采用Dunnett’s T3法。P<0.05表示差异具有统计学意义。结果：(1)AC-5216和YL-IPA08对TSPO和CBR的亲和力和对神经类固醇的水平调节放射性竞争配体结合试验结果显示AC-5216在大鼠小脑组织TSPO和CBR亲和结合力ICs0分别为0.65士0.02nM和67.4±6.82nM; YL-IPA08则分别为0.23士0.04nM和706.18±10.18nM,提示与AC-5216相比,YL-IPA08与TSPO结合具有更高的亲和力和选择性,且与CBR几乎没有结合。ELISA结果显示AC-5216(1,2μM)(F=7.892, P=0.004)和YL-IPA08(1,2μM)(F=101.5,P=0.000)作用于星形胶质细胞2h后,培养液中孕烯醇酮水平均显著上调；同法检测AC-5216(1,2μM)(F=98.37, P=0.000)和YL-IPA08(1,2μM)(F=52.91, P=0.000)作用4h后,培养液中孕烯醇酮水平均显著上调；AC-5216(0.5,1,2μM)(F=22.41,P=0.000)和YL-IPA08(0.5,1,2μM)(F=23.97,P=0.000)作用2h后,培养液中孕酮水平均显著上调,同法检测AC-5216(0.5,1,2pM)(F=37.99, P=0.000)和YL-IPA08(0.5,1,2pM)(F=22.16, P=0.000)作用4h后,培养液中孕酮水平均显著上调。(2) PTSD动物模型(小鼠短暂电击模型和时间依赖性敏化模型)研究AC-5216与YL-IPA08抗PTSD的行为药效小鼠短暂电击模型结果显示,长期给予AC-5216(0.03,0.1,0.3,1mg/kg p.o.)(F=1.181,P=0.328,跨格数；F=1.025,P=0.418,站立次数；F=0.323,P=0.922,粪便颗粒数)和YL-IPA08(0.03,0.1,0.3,1,3mg/kg p.o.)(F=1.890, P=0.084,跨格数；F=0.342,P=0.932,站立次数；F=0.277,P=0.961,粪便颗粒数)均不影响实验小鼠以上指标,实验结果提示AC-5216和YL-IPA08均不影响实验动物自发活动和排便量。同时,在试验过程中AC-5216和YL-IPA08不影响小鼠体重变化(F=0.206, P=1.000, AC-5216; F=0.176, P=1.000, YL-IPA08).环境重现试验显示,与舍曲林(15mg/kg p.o.)一致, AC-5216(0.1,0.3mg/kg p.o.)(F=6.602,P=0.000)与YL-IPA08(0.3,1mg/kg p.o.)(F=7.554, P=0.000)在实验第十天能显著降低小鼠僵住时间；同样,AC-5216(0.1,0.3mg/kg p.o.)(F=11.87, P=0.000)与YL-IPA08(0.1,0.3,1mg/kg p.o.)(F=6.904, P=0.000)在实验第十七天也能显著降低小鼠僵住时间。实验结果提示AC-5216与YL-IPA08能显著抗PTSD引起的僵住行为作用。高架十字迷宫结果显示AC-5216(0.3mg/kg p.o.)(F=3.513, P=0.005)和YL-IPA08(1mg/kg p.o.)(F=2.318,P=0.035)能逆转电击小鼠在开臂停留的时间减少. AC-5216(0.1,0.3mg/kg p.o.)(F=2.462, P=0.033)和YL-IPA08(1mg/kg p.o.)(F=2.643,P=0.018)能逆转电击小鼠在开臂停留的次数减少。爬梯试验结果显示AC-5216(F=0.836, P=0.547)和YL-IPA08(F=0.827, P=0.568)不影响实验动物的爬梯数。该模型能降低小鼠的抬头数,AC-5216(0.1,0.3mg/kg p.o.)(F=4.504,P=0.002)和YL-IPA08(0.3,1mg/kg p.o.)(F=2.166, P=0.047)能逆转小鼠抬头数的减少。高架十字迷宫试验与爬梯试验结果提示AC-5216(0.1,0.3mg/kg p.o.)和YL-IPA08(1mg/kg p.o.)具有抗PTSD作用。大鼠时间依赖敏化模型结果显示,该模型不影响实验大鼠跨格数,站立次数和粪便颗粒数,长期给予AC-5216(0.03,0.1,0.3,1mg/kg p.o.)(F=0.735,P=0.623,跨格数；F=0.245,P=0.959,站立次数；F=0.388,P=0.884,粪便颗粒数)和YL-IPA08(0.03,0.1,0.3,1,3mg/kg p.o.)(F=1.176, P=0.327,跨格数；F=0.514,P=0.821,站立次数；F=0.479,P=0.847,粪便颗粒数)均不影响实验大鼠以上指标,实验结果提示该模型与AC-5216和YL-IPA08均不影响实验动物自发活动。同时,在试验过程中各处理组不影响大鼠体重变化(F=0.141,P=1.000,AC-5216; F=0.433, P0.000, YL-IPA08),电击显著延长大鼠的僵住时间,AC-5216(0.1,0.3mg/kg p.o.)(F=3.913, P=0.002)与YL-IPA08(0.3,1mg/kg p.o.)(F=33.77,P=0.000)能显著降低大鼠PTSD僵住时间,实验结果提示AC-5216与YL-IPA08能显著抗TDS大鼠PTSD僵住行为。高架十字迷宫结果显示AC-5216(F=0.216,P=0.968)与YL-IPA08(F=1.090, P=0.379)不影响实验动物的入臂时间,同时,AC-5216(F=1.281,P=0.298)与YL-IPA08(F=1.533,P=0.171)不影响实验动物的入臂次数。该模型能降低大鼠在开臂停留的时间与次数,AC-5216(0.1,0.3mg/kg p.o.)(F=2.423, P=0.036)和YL-IPA08(0.1,0.3mg/kg p.o.)(F=3.491,P=0.003)能逆转大鼠在开臂停留的时间减少。AC-5216(0.1,0.3mg/kg p.o.)(F=2.378,P=0.039)和YL-IPA08(0.1,0.3mg/kg p.o.)(F=3.916, P=0.001)能逆转大鼠在开臂停留的次数减少。高架十字迷宫试验结果提示舍曲林(15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.)和YL-IPA08(0.1,0.3mg/kg p.o.)具有抗PTSD作用。(3)研究AC-5216与YL-IPA08的抗PTSD作用机制TSPO拮抗剂PK11195(1,3mg/kg i.p.)拮抗AC-5216和YL-IPA08在时间依赖性敏化模型中抗PTSD行为药效研究。结果显示,PK11195拮抗AC-5216(F=0.352,P=0.954,跨格数；F=0.552,P=0.832,站立次数；F=0.206,P=0.993,粪便颗粒数)和YL-IPA08(F=0.263,P=0.982,跨格数；F=0.449,P=0.904,站立次数；F=0.155,P=0.998,粪便颗粒数)的行为药效的同时不影响实验大鼠跨格数,站立次数和粪便颗粒数。实验结果提示,PK11195拮抗作用不影响实验动物自发活动。惊恐环境重现试验显示,PK11195(3mg/kg i.p.)可拮抗AC-5216(0.3mg/kg p.o.)(F=4.552, P=0.000)与YL-IPA08(0.3mg/kg p.o.)(F=5.738,P=0.000)降低大鼠PTSD僵住时间的作用,而PK11195自身不影响大鼠僵住时间。PK11195(3mg/kgi.p.)能拮抗AC-5216(0.3mg/kg p.o.)和YL-IPA08(0.3mg/kgp.o.)逆转大鼠在开臂停留的时间(F=2.128, P=0.035, AC-5216; F=2.097, P=0.038, YL-IPA08)与次数(F=2.252, P=0.026, AC-5216; F=2.442,P=.016, YL-IPA08)的下降,而PK11195(1,3mg/kg p.o.)自身不影响大鼠在开臂停留的时间(F=0.244,P=0.987, AC-5216; F=0.310,P=0.970, YL-IPA08)与次数(F=0.702,P=0.705,AC-5216; F=0.898, P=0.531, YL-IPA08)。实验结果提示AC-5216与YL-IPA08抗PTSD作用是通过TSPO所介导的。蛋白质分子印迹结果显示,与正常对照组对比,模型组TSPO的表达水平显著下降,AC-5216(0.3mg/kg p.o.)(F=150.2, P=0.000,前额皮层；F=11.45,P=0.006,海马体)和YL-IPA08(0.3mg/kg p.o.)(F=18.69, P=0.002,前额皮层；F=12.66,P=0.005,海马体)能显著上调前额皮层和海马体TSPO表达。实验结果提示AC-5216和YL-IPA08抗PTSD作用与上调前额皮层和海马体TSPO表达相关。放射性竞争配体结合试验结果显示,与正常对照组对比,模型组TSPO的受体活性显著下降,AC-5216(0.3mg/kg p.o.)(F=15.63, P=0.0042,前额皮层；F=7.454,P=0.0236,海马体；F=6.825,P=0.0285,血小板)和YL-IPA08(0.3mg/kgp.o.)(F=11.99,P=0.0080,前额皮层；F=13.68,P=0.0058,海马体；F=11.24,P=0.0093,血小板)能显著上调前额皮层,海马体和血小板TSPO受体活性。实验结果提示AC-5216和YL-IPA08抗PTSD作用与上调前额皮层,海马体和血小板中TSPO受体活性相关。ELISA检测AC-5216和YL-IPA08抗PTSD作用与孕酮和四氢孕酮水平调节相关性。结果显示,在前额皮层,海马体和血清中,模型组孕酮和四氢孕酮含量显著下降。AC-5216(0.3,1mg/kg p.o.)和YL-IPA08(0.3,1mg/kg p.o.)能显著上调前额皮层(F=2.687, P=0.030, AC-5216; F=2.550, P=0.039, YL-IPA08),海马体(F=3.478, P=0.010, AC-5216; F=3.131, P=0.017, YL-IPA08)和血清(F=3.002,P=0.021, AC-5216; F=3.321, P=0.011, YL-IPA08)中四氢孕酮含量,但与前额皮层(F=3.985,P=0.003, AC-5216; F=2.802, P=0.022, YL-IPA08),海马体(F=3.171,P=0.012, AC-5216; F=2.379,P=0.045, YL-IPA08)和血清(F=2.722,P=0.025,AC-5216; F=2.925, P=0.018, YL-IPA08)中孕酮的调节水平无关。同法检测得PK11195(3mg/kg i.p.)可拮抗AC-5216(0.3mg/kg p.o.)(F=2.439, P=0.039,前额皮层；F=2.296,P=0.048,海马体；F=3.035,P=0.013,血清)和YL-IPA08(0.3mg/kgp.o.)(F=2.830,P=-0.018,前额皮层；F=3.587,P=0.005,海马体；F=3.318,P=0.007,血清)对四氢孕酮水平的上调,实验结果提示AC-5216和YL-IPA08抗PTSD作用与调节四氢孕酮水平上调有关,而这种调节是通过TSPO所介导的。高效液相电化学法检测AC-5216和YL-IPA08抗PTSD作用与单胺递质水平调节相关性。结果显示TDS模型组中5-HT水平下调,与舍曲林(15mg/kg p.o.)一致,AC-5216(1mg/kg p.o.)能逆转5-HT(F=3.663,P=0.007,前额皮层；F=2.473,P=0.046,海马体)水平下调。而对DOPAC(F=0.163,P=0.985,前额皮层；F=0.414,P=0.864,海马体),DA(F=0.604, P=0.725,前额皮层；F=0.591,P=0.735,海马体),5-HIAA(F=0.890, P=0.513,前额皮层；F=0.520,P=0.789,海马体)和NE(F=0.181,P=0.980,前额皮层；F=0.674,P=0.671,海马体)的水平无显著性影响；与舍曲林(15mg/kg p.o.)一致,YL-IPA08(1mg/kg p.o.)能逆转5-HT (F=2.457, P=0.045,前额皮层；F=2.605,P=0.034,海马体)水平下调。而对DOPAC(F=0.277, P=0.944,前额皮层；F=0.384,P=0.884,海马体),DA(F=0.157, P=0.986,前额皮层；F=0.474,P=0.823,海马体),5-HIAA(F=0.212, P=0.971,前额皮层；F=0.678,P=0.668,海马体)和NE(F=0.199, P=0.975,前额皮层；F=0.534,P=0.779,海马体)水平无显著性影响。试验结果提示AC-5216和YL-IPA08在TDS模型上的抗PTSD作用与上调5-HT水平有关。结论：YL-IPA08和AC-5216与TSPO均有高亲和力结合,而与CBR低亲和；YL-IPA08比AC-5216具有更高的配体亲和力和选择性,且易溶于水；YL-IPA08和AC-5216可上调星形胶质细胞中神经类固醇,如孕烯醇酮和孕酮的含量；首次发现TSPO配体YL-IPA08和AC-5216具有抗PTSD行为活性,且在抗PTSD有效剂量下显著升高前额皮层,海马和血清中四氢孕酮水平,上述作用可被TSPO拮抗剂完全阻断,提示TSPO及其下游的四氢孕酮介导了AC-5216和YL-IPA08的抗PTSD行为活性；5-HT可能也间接参与了AC-5216和YL-IPA08的抗PTSD作用。更多还原

【Abstract】 Objective:Post-traumatic stress disorder (PTSD) is a serious anxiety, which is the mental disorder that is associated with individual. The PTSD symptoms are various, including traumatic experience, avoidance behavior, and emotional numbing. PTSD is considered to be high incidence and prevalence, chronic and poor effect with great trauma care and concern.The etiology and pathogenesis of PTSD is complicated. The disorder is involved the abnormal neurotransmitters levels, the enhancement of the hypothalamic pitutary adrenal (HPA) axis in the negative feedkack inhibition and the abnormal of neural plasticity and neurosteroidogenesis. These dysregulation of the neuroendocrine pathways may result in the abnormal of brain structure and function and cause a series of symptoms, such as excessive vigilance and the intrusion, and experience (flashback).PTSD is lack of specific drugs treatment in clinic. In general, antidepressants selective serotonin reuptake inhibitors (SSRIs) are the first choice for treatment of PTSD, such as paroxetine and sertraline. But effective rate is not high (about60%). The onset latency will last from2-6weeks. Also, there are side-effects including sexual dysfunction, gastrointestinal tract, and suicide. Other anxiolyic drugs were used in the treatment of PTSD. Benzodiazepine, such as midazolam, can destroy the conditioned fear memory. But the drugs also have low efficiency. Therefore, it is important to explore the new target and treatment of PTSD.The translocator protein18KDa (TSPO) is distributed on the outer membrane of mitochondria in the central and peripheral tissue. TSPO transports the cholesterol into the mitochondrial membrane to promote the biosynthesis of neurosteroids. It had been found that TSPO is associated with mood and stress response. This target has potential value in the treatment of anxiety, depression and other stress-related psychiatric disorders. Reports have shown that TSPO is closely associated with PTSD. The TSPO ligand, such as AC-5216, has obvious antidepressant, anxiolytic and anti-panic effect in animal models without sedation, muscle relaxation tolerance and withdrawal symptoms. The mechanism may be that transport cholesterol into membrane phospholipids, which increased pregnenolone formation and its downstream neurosteroids, including progesterone and allopregnenolone. Neurosteroids enhanced the function of gamma amino-butyric acid type A receptor (GABAA) through GABAA receptors on the excitatory neurons, which inhibit the hippocampal hypothalamic pituitary adrenal (HHPA) axis function and regulation of emotion and stress response. It is indicated that TSPO can be used as a potential therapeutic target for PTSD.However, AC-5216is difficult to dissolve in water. Based on this situation, we synthesis its derivative YL-IPA08. It had been reported that YL-IPA08have higher affinity to TSPO than AC-5216and have antidepressant and anxiolytic effects by regulating neurosteroids synthesis. Also, we had applied the patent in this area. Although the anti-stress effect of AC-5216and YL-IPA08is obvious, there are few studies on anti-PTSD. It is important to clarify whether TSPO ligands have great value on anti-PTSD effect.The animal models (e.g. time-dependent sensitization (TDS) and inescapable electric foot shock, single prolonged stress (SPS), exposure to a predators, and social isolation) have been well established to resemble the clinical features of PTSD. The inescapable electric foot shock model and the TDS model were selected in the present study. The inescapable electric foot shock model based on the concept that animals exposed to foot shocks can represent pathophysiological process and core symptomatology of PTSD, including the freezing and anxiety-like behavior. Previous studies showed that animals experiencing a traumatic event, such as multiple foot shocks, rapidly form a strong phobia while a relatively mild aversive experience occurs. This phenomenon may correspond to the emergence of phobias in PTSD patients. In addition, the TDS model has proven to resemble the clinical condition with accurate face, construct, and predictive validity. Thus, both PTSD models represent important phenomenological and biological correlates of PTSD.In the present study, AC-5216and YL-IPA08were applied in inescapable electric foot shock model and TDS model to explore the anti-PTSD effect and mechanism of TSPO ligands.Methods:(1) The affinity of AC-5216and YL-IPA08to TSPO and the regulation of both the compounds on neurosteroidsWe cultured the astrocytes that were treated with AC-5216and YL-IPA08(0.05,1,2μM)2h and4h and measured pregnenolone and progesterone in culture medium by enzyme linked immunosorbent assay (ELISA). (2) The anti-PTSD effects of AC-5216and YL-IPA08in behaviral testsWe explored the anti-PTSD effects of AC-5216and YL-IPA08by inescapable footshock model and TDS model. Briefly, after a5-min adaptation period, the mice were exposed to a total of15intermittent inescapable electric foot shocks (intensity:0.8mA, interval:10s, and duration:10s) delivered through the grid floor by an isolated shock generator. Control animals were placed in the same chamber for10min in the absence of electric foot shocks. Sertraline (15mg/kg p.o.), AC-5216(0.03,0.1,0.3, and1mg/kg p.o.) and YL-IPA08(0.03,0.1,0.3,1, and3mg/kg p.o.) were given once per day from3to17d.All the animals were exposed to reminders of the situation for5min on days5,10, and17without foot shocks, to measure the duration of contextual freezing behavior. This was achieved by placing the experimental animals in the same chamber where the foot shocks were delivered. In addition, we explore the spontaneous locoactivity by open field test and the PTSD behavior by the elevated plus maze test and the staircase test.In the TDS model, each rat was placed in a perspex restrainer for2h, with the tail-gate adjusted to keep the rat well contained without impairing limb circulation. Thereafter, rats were individually placed in a clear acrylic cylinder to perform a20-min forced swim. Following the15-min recuperation, rats were then exposed to ether vapors until loss of consciousness and were then removed. The animals were undisturbed for7days. Seven days after the initial stressor, the rats were exposed to a ’restress’session consisting of a20-min swim in clear acrylic cylinders. The control group rats were remained in a room adjacent to the TDS rats for the duration of TDS and were handled twice for several minutes. Sertraline (15mg/kg p.o.), AC-5216(0.03,0.1,0.3, and1mg/kg p.o.) and YL-IPA08(0.03,0.1,0.3,1, and3mg/kg p.o.) were given once per day from the second day. We explore the spontaneous locoactivity by open field test and the PTSD behavior by the contextual fear paradigm and the elevated plus maze test.(3) The anti-PTSD mechanism of AC-5216and YL-IPA08We explored the effects of TSPO receptor antagonist PK11195in the TDS model. The prefrontal cortex and hippocampus and blood samples were removed from the TDS model to explore the anti-PTSD mechanism of AC-5216and YL-IPA08. The expression changes of TSPO in prefrontal cortex and hippocampus were measured by western blotting; the activity of TSPO in prefrontal cortex, hippocampus and platelet were measured by binding assay; the progsterone and allopregnenolone in prefrontal cortex and hippocampus and blood samples was detect by ELISA; changes of monoamine levels (DOPAC (dihydroxy-phenyl acetic acid), DA (dopamine),5-HIAA (5-hydroxyindoleacetic acid), NE (norepinephrine)) in prefrontal cortex and hippocampus were analysis by high-performance liquid chromatography with electrochemical detection.(4) Statical analysisUnless otherwise specified, statistical analysis was performed using SPSS13.0. All data were presented as the means±S.E.M. The statistical significance of experimental observations was determined by one-way analysis of variance (ANOVA). The body weight was determined by repeated measure analysis. The statical significances between two groups were analyzed by LSD t-test or Welch analysis followed by Dunnett’s T3test. For all tests, differences with P<0.05were considered significant.Results:(1) The affinity of AC-5216and YL-IPA08to TSPO and the regulation of both the compounds on neurosteroidsBinding assay showed that affinity IC50of AC-5216in TSPO and CBR of the outer mitochondrial membrane of glial cells were0.65±0.02nM and67.4±6.82nM, respectively; affinity IC50of YL-IPA08in TSPO and CBR of the outer mitochondrial membrane of glial cells were0.23±0.04nM and706.18±10.18nM. The results indicated that AC-5216and YL-IPA08have high affinity to TSPO, but low affinity to CBR in the mitochondrial outer membrane of glial cells. We determine the concentration of pregnenolone and progesterone that treated by AC-5216(0.5,1,2μM) and YL-IPA08(0.5,1,2μM) in astrocytes in the culture by ELISA. The result showed that after2h treatment of AC-5216and YL-IPA08, pregnenolone (F=7.892, P=0.004, AC-5216; F=101.5, P=0.000, YL-IPA08) and progesterone (F=22.41, P=0.000, AC-5216; F=23.97,P=0.000, YL-IPA08) levels were significantly increased. After4h treatment of AC-5216and YL-IPA08, pregnenolone (F=98.37, P=0.000, AC-5216; F=52.91, P=0.000, YL-IPA08) and progesterone (F=37.99,P=0.000, AC-5216; F=22.16,P=0.000, YL-IPA08) levels were significantly increased. The results suggested that pregnenolone and progesterone levels were upregulated by AC-5216and YL-IPA08.(2) The anti-PTSD effects of AC-5216and YL-IPA08in behaviral testThe footshock model in mice showed that AC-5216(0.03,0.1,0.3,1mg/kg p.o.) and YL-IPA08(0.03,0.1,0.3,1,3mg/kg p.o.) does not affect the number of crossings (F=1.181, P=0.328, AC-5216; F=1.890, P=0.084, YL-IPA08), rears (F=1.025, P=0.418, AC-5216; F=0.342, P=0.932, YL-IPA08) and fecal pellets (F=0.323, P=0.922, AC-5216; F=0.277, P=0.961, YL-IPA08). It is indicated that AC-5216and YL-IPA08do not affect the locomotor activity in mice.The body weight was not affected by AC-5216(F=0.206, P=1.000) and YL-IPA08(F=0.176, P=1.000).The footshock significantly increased the freezing time, sertraline (15mg/kg p.o.) significantly reduce the freezing time, AC-5216and YL-IPA08do not reduce the freezing time on the5th day (F=2.618, P=0.025, AC-5216; F=4.592, P=0.000, YL-IPA08); on the10th day (F=6.602, P=0.000, AC-5216; F=6.456, P=0.000), YL-IPA08), sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(0.3,1mg/kg p.o.) significantly reduce the freezing time; on the17th day (F=11.868, P=0.000, AC-5216; F=6.904,.P=0.000, YL-IPA08), sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(0.1,0.3,1mg/kg p.o.) significantly reduce the freezing time. The results suggested that the fear induced by PTSD was allivated by AC-5216and YL-IPA08in mice. The elevated plus maze test showed that total time spent in (F=0.929,P=0.779, AC-5216; F=1.868, P=0.088, YL-IPA08) and the entries into (F=0.532, P=0.490, AC-5216; F=0.570, P=0.778, YL-IPA08) the arms were not different between the groups. The time spent in (F=3.513, P=0.005, AC-5216; F=2.318,P=0.035, YL-IPA08) and the entries into (F=2.462, P0.033, AC-5216; F=2.643,P=0.018, YL-IPA08) the open arms was reduced in the model group, but both of the parameters were reversed by sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(1mg/kg p.o.). The staircase test showed that there were no differences among the groups in steps climbing (F=0.836, P=0.547, AC-5216; F=0.827, P=0.568, YL-IPA08). The model can reduce the number of rears (F=4.504, P=0.002, AC-5216; F=2.166,P=0.047, YL-IPA08), but the reduction can be reversed by sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(0.3,1mg/kg p.o.). The elevated plus maze test and staircase test results suggest sertraline (15mg/kg p.o.), AC-5216(0.1,0.3,1mg/kg p.o.) and YL-IPA08(1mg/kg p.o.) has the anti-PTSD effect. The results suggested that PTSD was allivated by AC-5216and YL-IPA08in rats.The effect of AC-5216and YL-IPA08in the TDS model showed that AC-5216(0.03,0.1,0.3,1mg/kg p.o.) and YL-IPA08(0.03,0.1,0.3,1, and3mg/kg p.o.) does not affect the number of crossings (F=0.735, P=0.623, AC-5216; F=1.176, P=0.327, YL-IPA08), rears (F=0.245, P=0.959, AC-5216; F=0.514, P=0.821, YL-IPA08) and fecal pellets (F=0.388, P=0.884, AC-5216; F=0.479, P=0.847, YL-IPA08). It is indicated that AC-5216and YL-IPA08do not affect the locomotor activity in rats. AC-5216(F=0.141,P=1.000) and YL-IPA08(F=0.433,P=1.000) also do not affect the body weight in rats. Contextual fear paradigm showed that the model group significantly increased the freezing time, sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.)(F=3.913, P=0.002) and YL-IPA08(0.1,0.3mg/kg p.o.)(F=33.77, P=0.000) significantly reduce the freezing time. The results suggested that the fear induced by PTSD can be allivated by AC-5216and YL-IPA08in rats. The elevated plus maze test showed that total time spent in (F=1.281, P=0.298, AC-5216; F=1.090, P=0.379, YL-IPA08) and the entries into (F=0.216,P=0.986, AC-5216; F=1.533, P=0.171, YL-IP08) the arms were not different between the groups. The time spent in (F=2.423, P=0.036, AC-5216; F=3.491, P=0.003, YL-IPA08) and the entries into (F=2.378, P=0.039, AC-5216; F=3.916,P=0.001, YL-IPA08) the open arms was reduced in the model group, but both of the parameters were reversed by sertraline (15mg/kg p.o.), AC-5216(0.1,0.3mg/kg p.o.) and YL-IPA08(0.1,0.3mg/kg p.o.). The results suggested that the PTSD behaviors were allivated by AC-5216and YL-IPA08in rats.(3) The anti-PTSD mechanism of AC-5216and YL-IPA08The effect of AC-5216and YL-IPA08was antagonized by PK11195in the on the time dependent sensitization model. The results showed that PK11195(1,3mg/kg i.p.) does not affect the number of crossings (F=0.352, P=0.954, AC-5216; F=0.263, P=0.982, YL-IPA08), rears (F=0.552, P=0.832AC-5216; F=0.449, P=0.904, YL-IPA08) and fecal pellets (F=0.206, P=0.993, AC-5216; F=0.155, P=0.998, YL-IPA08). It is indicated that PK11195(1,3mg/kg, i.p.) did not affect the locomotor activity. Contextual fear paradigm showed that the reduction of the freezing time by AC-5216(0.3mg/kg p.o.)(F=4.552, P=0.000) and YL-IPA08(0.3mg/kg p.o.)(F=5.738, P=0.000) were antagonized by PK11195(3mg/kg, i.p.) and the elevated plus maze test showed that total time spent in (F=0.244, P=0.987, AC-5216; F=0.310, P=0.970, YL-IPA08) and the entries into (F=0.702, P=0.705, AC-5216; F=0.898, P=0.531, YL-IPA08) the arms were not different between the groups. The time (F=2.128, P=0.035, AC-5216; F=2.097, P=0.038, YL-IPA08) spent in and the entries (F=2.252, P=0.026, AC-5216; F=2.442, P=0.016, YL-IPA08) into the open arms was increased by AC-5216(0.3mg/kg p.o.) and YL-IPA08(0.3mg/kg p.o.), which were antagonized by PK11195(3mg/kg, i.p.). The results suggested that AC-5216and YL-IPA08allivated PTSD by TSPO.Western blot showed that the expression of TSPO is down-regulated in the prefrontal cortex and hippocampus of the TDS group. The down-regulated TSPO is reversed by administration of AC-5216(0.3mg/kg p.o.)(F=150.2, P=0.000, prefrontal cortex; F=11.45, P=0.006, hippocampus) and YL-IPA08(0.3mg/kg p.o.)(F=18.69, P=0.002, prefrontal cortex; F=12.66, P=0.005, hippocampus). It is indicated that the anti-PTSD effect of AC-5216and YL-IPA08is associated with up-regulation of TSPO expression.Binding assay showed that the activity of TSPO receptor is down-regulated in the prefrontal cortex, hippocampus and platelet of the TDS group. The down-regulated TSPO receptor activcty is reversed by administration of AC-5216(0.3mg/kg p.o.)(F=15.63, P=0.0042, prefrontal cortex; F=7.454, P=0.0236, hippocampus; F=6.825, P=0.0285, platelet) and YL-IPA08(0.3mg/kg p.o.)(F=11.99, P=0.0080, prefrontal cortex; F=13.68,P=0.0058, hippocampus; F=11.24, P=0.0093, platelet). It is indicated that the anti-PTSD effect of AC-5216and YL-IPA08is associated with up-regulation of TSPO receptor activity.ELISA showed that progesterone and allopregnenolone was reduced in the prefrontal cortex, hippocampus and serum in the TDS model group. However, AC-5216(0.3,1mg/kg p.o.) and YL-IPA08(0.3,1mg/kg p.o.) reversed the down-regulation of allopregnenolone in the prefrontal cortex (F=2.687,P=0.030, AC-5216; F=2.550, P=0.039, YL-IPA08), hippocampus (F=3.478, P=0.010, AC-5216; F=3.131, P=0.017, YL-IPA08) and serum (F=3.002, P=0.021, AC-5216; F=3.321,P=0.011, YL-IPA08), but not the progesterone in the prefrontal cortex (F=3.985, P=0.003, AC-5216; F=2.802,P=0.022, YL-IPA08), hippocampus (F=3.171, P=0.012, AC-5216; F=2.379, P=0.045, YL-IPA08) and serum (F=2.722, P=0.025, AC-5216; F=2.925, P=0.018, YL-IPA08). It also found that the effect of AC-5216and YL-IPA08were antagonized by PK11195(3mg/kg, i.p.) in the prefrontal cortex (F=2.439, P=0.039, AC-5216; F=2.830, P=0.018, YL-IPA08), hippocampus (F=2.296, P=0.048; F=3.587, P=0.005, YL-IPA08) and serum (F=3.035, P=0.013, AC-5216; F=3.318,P=0.007, YL-IPA08) of allopregnenolone. The result indicated that the anti-PTSD effect of AC-5216and YL-IPA08was associated with up-regulation of allopregnenolone.HPLC-ECD showed that5-HT was reduced in the TDS model group, sertraline (15mg/kg p.o.), AC-5216(1mg/kg p.o.) can reverse the down-regulation of5-HT (F=3.663, P=0.007, prefrontal cortex; F=2.473, P=0.046, hippocampus), but no effect on DOPAC (F=0.163, P=0.985, prefrontal cortex; F=0.414, P=0.864, hippocampus), DA (F=0.604, P=0.725, prefrontal cortex; F=0.591, P=0.735, hippocampus),5-HIAA (F=0.890, P=0.513, prefrontal cortex; F=0.520, P=0.789, hippocampus) and NE (F=0.181, P=0.980, prefrontal cortex; F=0.674, P=0.671, hippocampus). YL-IPA08(1mg/kg p.o.) can reverse the down-regulation of5-HT (F=2.457, P=0.045, prefrontal cortex; F=2.605, P=0.034, hippocampus), but no effect on DOPAC (F=0.277, P=0.944, prefrontal cortex; F=0.384, P=0.884, hippocampus), DA (F=0.157, P=0.986, prefrontal cortex; F=0.474, P=0.823, hippocampus),5-HIAA (F=0.199, P=0.975, prefrontal cortex; F=0.678, P=0.668, hippocampus) and NE (F=0.317, P=0.924, prefrontal cortex; F=0.534, P=0.779, hippocampus). It is indicated that anti-PTSD effects of AC-5216and YL-IPA08was associated with the up-regulation of5-HT.Conclusion:AC-5216and YL-IPA08have high affinity to TSPO, but low affinity to CBR. In addition, YL-IPA08has higher affinity to TSPO than AC-5216and dissolved in water. Neurosteriods levels were pregulated by AC-5216and YL-IPA08. AC-5216and YL-IPA08have anti-PTSD effect. The anti-PTSD effect of AC-5216and YL-IPA08was associated with up-regulation of allopregnenolone and the regulation was mediated by TSPO. The anti-PTSD effects of AC-5216and YL-IPA08was associated with the up-regulation of5-HT.更多还原