延胡索乙素分别与URB597和URB602联合应用对CCI大鼠镇痛作用的研究

目的：观察延胡索乙素（1evo—tetrahydropalmatine，L—THP）分别与脂肪酸酰胺水解酶（fatty acid amide hydrolase，FAAH）、单酰甘油脂肪酶（monoacylglycerol lipase，MAGL）特异性抑制剂URB597、URB602分别联合应用对坐骨神经慢性压迫损伤（chronic constriction injury，CCI）所致大鼠神经病理性疼痛的影响。方法：成年健康雄性Wistar大鼠30只，体重180～220g，随机分为5组（，z=6）：假手术组、CCI组、延胡索乙素CCI组、延胡索乙素URB597CCI组、延胡索乙素URB602CCI组。假手术组仅暴露坐骨神经但不结扎，其余组均暴露并结扎坐骨神经。术毕即刻，延胡索乙素CCI组、延胡索乙素URB597CCI组和延胡索乙素URB602CCI组腹腔注射延胡索乙素40mg／（kg·d），连续14d；假手术组和CCI组给予等量生理盐水。术后11d开始，延胡索乙素URB597CCI组、延胡索乙素URB602CCI组手术切口局部分别注射URB597、URB6020．3mg／（kg·d），连续4d，假手术组、CCI组、延胡索乙素CCI组注射等量生理盐水。于术前1d、术后3d、7d、11d、12d、13d、14d测试各组大鼠机械痛阈（mechanical withdrawal threshold，MWT）和热痛阈（thermal withdrawal latency，TwL）。结果：与术前和假手术组比较，CCI组、延胡索乙素CCI组、延胡索乙素URB597CCI组和延胡索乙素URB602CCI组大鼠，术后3d开始各时间点机械痛阈和热痛阈显著降低（P〈0．5）；与CCI组比较，延胡索乙素CCI组、延胡索乙素URB597CCI组和延胡索乙素URB602CCI组大鼠术后3d、7d、11d、12d、13d、14d机械痛阈和热痛阈显著提高（P〈0．05）；与延胡索乙素CCI组比较，延胡索乙素URB597CCI组、延胡索乙素URB602CCI组大鼠，术后11d、12d、13d、14d机械痛阈和热痛阈明显提高（P〈0．05）；与延胡索乙素URB597CCI组比较，延胡索乙素URB602CCI组大鼠各时间点机械痛阈和热痛阈无显著性差异（P〉O．05）。结论：在CCI大鼠模型中，延胡索乙素可以产生良好的镇痛作用，当分别与URB597、URB602合用时可以增强其镇痛作用。

脂肪酸酰胺水解酶抑制剂URB597对人肝癌细胞MHCC97H生长和侵袭的抑制作用及机制研究

目的：观察脂肪酸酰胺水解酶抑制剂URB597对人肝癌高转移细胞MHCC97H细胞生长和侵袭性的抑制作用。方法：不同浓度URB597（1、5、10μmol/L）作用于MHCC97H细胞后不同时间,应用MTT法及流式细胞仪检测该种细胞生长活力及凋亡细胞数目的改变;应用Transwell实验检测该种细胞的运动能力及侵袭能力;应用蛋白质印迹法检测该种细胞内磷酸化Akt（p-Akt）和Akt表达量的变化。结果：上述3种浓度的URB597作用于该种细胞3-7d后,细胞活力明显降低,呈时间及剂量依赖性;10μmol/L URB597作用3-7d可显著增加凋亡细胞数目。URB597作用于细胞48h后,与对照组比较,5、10μmol/L URB597可显著抑制MHCC97H细胞侵袭能力（P〈0.01,P〈0.001）。3种浓度URB597作用于细胞24h后,5、10μmol/L URB597可显著下调细胞内p-Akt水平（P〈0.05,P〈0.01）。结论：URB597对体外生长的人肝癌细胞MHCC97H的生长和侵袭有明显抑制作用,该作用可能与其抑制磷酯酰肌醇-3激酶（PI3K）/Akt信号通路有关。

脂肪酸酰胺水解酶抑制剂URB597诱导人肝癌细胞MHCC97H凋亡的作用及其机制研究

目的:观察脂肪酸酰胺水解酶(fatty acid amide hydrolase,FAAH)抑制剂URB597诱导人肝癌高转移细胞MHCC97H凋亡的作用,并探讨其机制。方法:给予不同浓度(1、5、10μmol/L)URB597与MHCC97H孵育,应用流式细胞仪检测细胞凋亡率。采用蛋白质印迹技术检测凋亡诱导因子(apoptosis inducing factor,AIF)在细胞核中的水平,及凋亡相关蛋白Bcl-2和Bax的表达水平。采用caspase 3试剂盒检测caspase 3的活性变化。结果:(1)不同浓度(1、5、10μmol/L)URB597作用细胞1、4、7d后,凋亡细胞数目明显增加,呈时间-剂量依赖性;(2)不同浓度URB597作用96h后细胞内caspase3活性明显升高,且细胞核中AIF水平显著增加;(3)10μmol/L URB597作用细胞96h后,与对照组相比,Bcl-2水平明显下调,Bax水平明显上调,Bcl-2/Bax比值显著下降。PI3K抑制剂LY294002亦可显著降低Bcl-2/Bax比值,但与10μmol/L URB597作用相比,对Bcl-2/Bax比值的影响程度较低。结论:URB597可通过促进人肝癌细胞凋亡抑制其增殖,该作用与其下调Bcl-2/Bax比值,影响线粒体通透性,诱导caspase依赖和非caspase依赖的细胞凋亡有关,且部分依赖于PI3K/Akt通路。

URB597 exerts neuroprotective effect against transient cerebral ischemic injury via restor⁃ing autophagy flux and inhibiting neuronal necroptosis in mice

OBJECTIVE URB597(KDS-4103)is a potent and selective inhibitor of the enzyme fatty acid amide hydrolase(FAAH)and can ele⁃vate the level of oleoylethanolamide(OEA),a naturally occurring endocannabinoid in the brain.However,the effect of URB597 on cerebral isch⁃emic injury in mice remains unclear.METHODS Focal cerebral ischemia was induced by middle cerebral artery occlusion for 30 min followed by reperfusion for 24 h in mice.To observe the dose-dependent effect,URB597(0.04-5.00 mg·kg-1,ip)was administered at the same time of reperfu⁃sion.To determine the time-dependent effect,URB597(1.00 mg·kg-1,ip)was administered as a single dose at 0,1,3 or 5 h after reperfusion.Twenty-four hours after brain ischemia,Beder⁃son scoring test and grip strength test were used to evaluate the neurological function;brain in⁃farct volume was assayed by 2,3,5-triphenyltetra⁃zolium chloride(TTC)staining or diffusion-weighted magnetic resonance imaging(MRI).Laser speckle imaging(LSI)technique was used to assay the regional cerebral blood flow(rCBF);NeuN immunofluorescence staining was used to observe the neuron survival in the penumbra.To further investigate the underlying mechanism,au⁃tophagy flux related proteins(LC3-Ⅱ,P62 and LAMP2)and necroptosis related proteins(pRIPK3 and pMLKL)were detected by Western blotting and immunofluorescence staining.RESULTS Twenty-four hours after brain ischemia,URB597 dose-dependently improved neurological func⁃tion and reduced brain infarct volume.The most effective dose was 1.00 mg·kg-1;the therapeutic time window was within 1 h after ischemic stroke.The protective effect is further confirmed by the results that post-ischemic treatment with URB597(1.00 mg·kg-1)significantly increased neurons survival,promoted autophagy flux and reduced cell necroptosis in cortical penumbra after cerebral I/R.CONCLUSION URB597 dose-and time-dependently exerts a neuroprotective effect against acute cerebral I/R injury.This neu⁃roprotective effect of URB597 may be associated with its restoration of autophagy flux and inhibi⁃tion of neuronal necroptosis in the cortical penumbra.

URB597对anandamide水解作用的抑制可以减少大鼠尼古丁渴求行为的再现—与大麻CB1受体拮抗剂利莫那班的比较研究

目的:吸烟在全世界引起了严重的健康问题,全球约5百万早产儿死于吸烟。此外,吸烟是一种以戒断后反复复吸为特点的慢性复发性疾病。尼古丁是烟草中导致人类成瘾的主要成分,可以使啮齿类动物形成持续的自身给药行为和条件性位置偏爱(CPP)。大量数据表明内源性大麻系统在尼古丁成瘾神经机制中有重要的作用。阻断CB1受体减少了尼古丁自身给药,线索诱导的尼古丁渴求的再现,以及尼古丁诱导的CPP。然而,最近的数据提示URB597通过抑制脂肪酸酰胺水解酶(fat acid amide hydrolase,FAAH)增强anandamide的传递,进而减少尼古丁关联行为,如尼古丁自身给药行为的获得和再现,尼古丁诱导大鼠CPP的表达和再现(Scherma et al.,2008)。CB1受体拮抗剂利莫他班对尼古丁自身给药累进比率(progressive ratio,PR),尼古丁点燃诱导的复吸,以及URB597对线索诱导尼古丁渴求再现的作用尚没有研究报道。本研究旨在填补这一空白。方法:大鼠在形成稳定的尼古丁累进自身给药行为后,观察利莫他班急性(0.3-3mg·kg-1,ip)或反复给药(1mg·kg-1,ip)对尼古丁转换点(breaking point)的作用。该测试完成后,所有的大鼠进行消退训练。我们比较了自身给药行为消退后URB597不可逆地选择性抑制FAAH和选择性CB1受体拮抗剂利莫他班两种干预对线索诱导的和尼古丁点燃(0.15mg·kg-1,sc)诱导的尼古丁复吸行为再现的作用。结果:利莫他班(0.3-3mg·kg-1)剂量依赖性的减少了尼古丁的转换点,1mg·kg-1的利莫他班的作用稳定。在尼古丁给药行为消退后,尼古丁点燃和尼古丁相关线索可以再现尼古丁自身给药行为。利莫他班(1mg·kg-1)和URB597(0.3 and 1mg·kg-1)显著减少了这种自身给药行为的再现。结论:本研究结果显示DB1受体的完整性是尼古丁自身给药行为动机所必需的,利莫他班可以稳定的减少这种行为。利莫他班阻断CB1受体或者通过抑制FAAH活性增强anandamide水平都减少了尼古丁渴求行为的再现。URB597的这些作用提示anadamide可能在尼古丁渴求中起抑制性的作用;这个假设还需要进一步研究介导尼古丁渴求的多种内源性大麻物质的功能。既然利莫他班在戒烟和复吸的使用上受精神副作用的限制,我们的结果提示更精确的靶相调节大麻系统可能可以预防戒烟者的复吸。

Pharmacological manipulation of cannabinoid neurotransmission reduces neuroinflammation associated with normal aging

We have previously demonstrated that antagonism of glutamate NMDA receptors or activation of endocannabinoid receptors could reduce experimentally induced neuroinflammation within the hippocampus of young rats. In the current study, we investigated whether pharmacological manipulation of glutamate or endocannabinoid neurotransmission could reduce naturally-occurring neuroinflammation within the hippocampus of aged rats. We investigated whether UCM707, an inhibitor of endocannabinoid re-uptake, WIN- 55,212-2, an endocannabinoid receptor agonist, and URB597, an inhibitor of endocannabinoid catabolism, or memantine, a non-competitive, low-affinity, inhibitor of the open NMDA receptor channel, could reduce the number of MHC II-IR microglia within the hippocampus. All of the drugs, except URB597, reduced the number of reactive microglia, as compared to vehicle treated rats. The current results suggest potential pharmacological approaches that may mitigate the pathological consequences of chronic brain inflammation associated with numerous neurodegenerative diseases.