Quantification of anandamide,oleoylethanolamide and palmitoylethanolamide in rodent brain tissue using high performance liquid chromatographyelectrospray mass spectroscopy

Reported concentrations for endocannabinoids and related lipids in biological tissues can vary greatly; therefore, methods used to quantify these compounds need to be validated. This report describes a method to quantify anandamide （AEA）, oleoylethanolamide （OEA） and palmitoylethanolamide （PEA） from rodent brain tissue. Analytes were exwacted using acetonitfile without further sample clean up, resolved on a C 18 reverse-phase column using a gradient mobile phase and detected using eleclmspmy ionization in positive selected ion monitoring mode on a single quadrupole mass spectrometer. The method produced high recovery rates for AEA, OEA and PEA, ranging from 98.1% to 106.2%, 98.5% to 102.2% and 85.4% to 89.5%, respectively. The method resulted in adequate sensitivity with a lower limit of quantification for AEA, OEA

Regulatory effects of anandamide on intracellular Ca^(2+) concentration increase in trigeminal ganglion neurons

Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neu- ~ ~ ~ 2＋ ~ ~ 2＋ rotransmlsslon by decreasing Ca reflux through high voltage-gated Ca channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca2＋ influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca2＋ concentration by the cannabinoid receptor type 1 agonist anandamide, and its underlying mechanisms. Using whole cell voltage-damp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca2＋ influx in a dose-dependent manner, which then triggered an increase of intracellular Ca2＋ concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca2＋concentration by anandamide. This result showed that anandamide increased intracellu- lar Ca2＋ concentration and inhibited high voltage-gated Ca2＋ channels through different signal transduction pathways.

Endocannabinoids Anandamide and Its Cannabinoid Receptors in Liver Fibrosis after Murine Schistosomiasis

This study examined endogenous carmabinoid （ECB）-anandamide （AEA） and its cannabinoid receptors （CBR） in mice liver with the development ofschistosomajaponicum. Mice were infected with schistosoma by means of pasting the cercaria onto their abdomens. Liver fibrosis was pathologically confirmed nine weeks after the infection. High performance liquid chromatography （HPLC） was employed to determine the concentration of AEA in the plasma of mice. Immunofluo-rescence was used to detect the expression of CBR1 and CBR2 in liver tissue. Morphological examination showed typical pathological changes, with worm tubercles of schistosoma deposited in the liver tissue, fibrosis around the worm tubercles and infiltration or soakage of inflammatory cells. Also, CBR1 and CBR2 were present in hepatocytes and hepatic sinusoids of the two groups, but they were obviously enhanced in the schistosoma-infected mice. However, the average optical density of CBR1 in the negative control and fibrosis group was 13.28±7.32 and 30.55±7.78, and CBR2 were 28.13±6.42 and 52.29±4.24 （P〈0.05）. The levels of AEA in the fibrosis group were significantly increased as compared with those of the control group. The concentrations of AEA were （0.37±0.07） and （5.67±1.34） ng/mL （P〈0.05）. It is concluded that the expression of endocannabinoids AEA and its cannabinoid receptor CBR were significantly increased in schistosoma-infected mice. Endogenous endocannabinoids may be involved in the development of schistosoma-induced liver fibrosis.

The Endocannabinoid,Anandamide,Induces Cannabinoid Receptor-Independent Cell Death in Renal Proximal Tubule Cells

Background: The endocannabinoid (EC) system is well characterized in the central nervous system but scarcely studied in peripheral organs. In this paper, we newly identify the effect of the EC anandamide (AEA) upon renal proximal tubule cells. Methods: Measurement of lactate dehydrogenase (LDH) release after treatment of primary renal proximal tubule cells (RPTEC) and renal carcinoma cell line (Caki-1) with AEA, arachidonic acid (AA), ethanolamide (EtAm), EC receptor CB1 antagonist (AM251), CB2 receptor antagonist (SR144528), TRPV1 receptor antagonist (capsazepine), degradation enzyme fatty acid amide hydrolase (FAAH) antagonist (URB597), antioxidants GSH-EE;Trolox, GSH depletor BSO, membrane cholesterol depletor (MCD), apoptosis inhibitor zVAD, necroptosis inhibitor Nec-1 or ferroptosis inhibitor Fer-1. Western blot and qRT-PCR analysis plus determination of reactive oxygen species (ROS) via H2-DCFDA were performed. Histology for EC enzymes, N-acetylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) and FAAH, as well as the determination of physiological levels of ECs in human and rat renal tissue via liquid chromatography were conducted. Results: AEA both dose- and time-dependently induces cell death in RPTEC and Caki-1 within hours, characterized by cell blebbing, not influenced by blocking the described EC receptors by AM251, SR144528, capsazepine or FAAH by URB597 or MCD. Cell death is mediated via ROS. There is no difference found in the histology of the enzymes FAAH and NAPE-PLD in human renal tissue with interstitial nephritis. Blocking of apoptotic, necroptotic or ferroptotic cell death does not lead to a reduction in LDH release in vitro. Conclusion: The endocannabinoid anandamide induces cell death in renal proximal tubule cell in a time- and dose-dependent manner. This pathway is mediated via ROS and is independent of cannabinoid receptors, membrane cholesterol or FAAH activity.

Cannabinoids and endocannabinoids as therapeutics for nervous system disorders:preclinical models and clinical studies

Cannabinoids are lipophilic substances derived from Cannabis sativa that can exert a variety of effects in the human body.They have been studied in cellular and animal models as well as in human clinical trials for their therapeutic benefits in several human diseases.Some of these include central nervous system(CNS)diseases and dysfunctions such as forms of epilepsy,multiple sclerosis,Parkinson’s disease,pain and neuropsychiatric disorders.In addition,the endogenously produced cannabinoid lipids,endocannabinoids,are critical for normal CNS function,and if controlled or modified,may represent an additional therapeutic avenue for CNS diseases.This review discusses in vitro cellular,ex vivo tissue and in vivo animal model studies on cannabinoids and their utility as therapeutics in multiple CNS pathologies.In addition,the review provides an overview on the use of cannabinoids in human clinical trials for a variety of CNS diseases.Cannabinoids and endocannabinoids hold promise for use as disease modifiers and therapeutic agents for the prevention or treatment of neurodegenerative diseases and neurological disorders.

Medicinal cannabis products for the treatment of acute pain

For thousands of years,medicinal cannabis has been used for pain treatment,but its use for pain management is still controversial.Meta-analysis of the literature has shown contrasting results on the addition of cannabinoids to opioids compared with placebo/other active agents to reduce pain.Clinical studies are mainly focused on medicinal cannabis use in chronic pain management,for which the analgesic effect has been proven in many studies.This review focuses on the potential use of medical cannabis for acute pain management in preclinical studies,studies on healthy subjects and the few pioneering studies in the clinical setting.

内源性大麻素对人肺癌A549细胞增殖的影响及机制初探

目的观察N-araehidonoyl ethanolamide（Anandamide，AEA）对A549细胞的作用并且探讨AEA的作用机制。方法倒置显微镜观察细胞的形态学变化，并用MTT法考察细胞增殖情况。结果AEA引起A549细胞死亡，且呈现剂量依赖性，IC50为（50．90±11．27）wmol·L^-1。A549细胞上表达有辣椒素受体（vanilloidreceptor1，VRl），AEA对A549的作用，不可以被VRl特异性的拮抗剂Capsazepine（-10μmol·L^-1）和RutheniumRea（-10μmol·L^-1）所拮抗。但AEA的作用可以被阿司匹林所减轻（P〈0．05或P〈0．01）。结论①AEA引起A549细胞死亡并不依赖VRl。②AEA的环氧化酶代谢产物Prostaglandin-ethanolamide（SPG-EAs）有可能参与该过程。

Palmitoylethanolamide对脑缺血再灌注损伤作用的研究进展

脑缺血再灌注损伤（cerebral ischemia reperfusion）是指颅内动脉狭窄或闭塞,导致局部血流量降低或停止一段时间,脑血流恢复后,反而引起脑组织损伤加重的现象。十六酰胺乙醇（Palmitoylethanolamide,PEA）是酰基乙醇胺类（N-acyle-thanolamines,NAEs）家族的一员,因为其结构与内源性大麻素anandamide相似而引起研究者广泛的兴趣,

Endocannabinoid Signaling in Modulating Periimplantation Events

Cannabinoids have long been suspected associating with abnormal fetal growth and outcome. However, the molecular mechanisms in which they are involved had long been obscure for several decades. Only recently, after the identification of two types of cannabinoid receptors （CB1-R and CB2-R） and the following discoveries of their corresponding endogenous ligands, the mystery behind those seemingly facts began gradually unveiled. Through a series of landmark research, it is now indicated that the endocannabinoid signaling via the ligand-receptor interaction plays an important role in modulating early development of preimplantation embryo and synchronizing embryo development with uterine receptivity for implantation. Current data suggest that the physiological functions their metabolic pathways are potentially very of endocannabinoid signaling as well as exited areas to be explored further. This review will first introduce the reproductive functions of endocannabinoid signaling from epidemiological and molecular background, then focus on its reciprocal interactions between the embryo and maternal tissues, as well as related metabolic aspects in regards to implantation. It is hoped that further investigation of this physiologically fundamental signaling will generate more exciting information elucidating the complexity of implantation thus lead to a better control of human reproduction.

内源性大麻素诱导人肝癌细胞MHCC97H凋亡中Caspase-3的作用

目的 检测内源性大麻素（AEA）是否诱导人肝癌高转移细胞株MHCC97H细胞凋亡、以及Caspase-3在细胞凋亡过程中的作用.方法 分别于AEA（100 μmol/L）作用细胞前后,应用Annexin V Binding方法 、流式细胞仪检测细胞凋亡率;荧光活性检测试剂盒检测细胞Caspase-3活性;半定量逆转录-聚合酶链反应（RT-PCR）方法 ,检测Caspase-3、bcl-2及bax的mRNA表达.结果 AEA作用细胞3、6、12、24 h,细胞凋亡率分别为（14.13±2.17）%、（27.04±3.21）%、（34.98±7.74）%及（81.35±9.71）%,其中24 h的细胞凋亡率与对照组（12.72±0.44）%比较,差异有统计学意义（P〈0.01）.AEA作用细胞24 h,细胞的Caspase-3活性由142.0±5.8增加至226.0±6.4（P〈0.01）;Caspase-3 mRNA的表达由0.24±0.13升高至0.54±0.32（P〈0.05）;如预先加入Caspase-3抑制剂（Z-VAD-FMK）后再加入AEA,则细胞的Caspase-3活性不再升高90.0±4.3.RT-PCR方法 未检测到bcl-2 mRNA及bax mRNA的表达.结论 AEA对人肝癌高转移细胞MHCC97H具有凋亡诱导作用,Caspase-3参与了凋亡的调控.

CYP4X1的结构、分布、表达调控及功能的研究进展

CYP4X1是CYP4新的亚家族成员,也是重要的Orphans CYPs成员之一。其核苷酸序列同源性分析显示CYP4X1结构在跨物种间是高度保守的,这预示着它具有重要的生物学功能。CYP4X1广泛存在于人体各组织中,尤其选择性在脑内高表达,提示其可能在神经血管功能中扮演关键作用。CYP4X1的表达存在昼夜节律调节、明显的性别差异和年龄差异以及外源物可诱导其表达。CYP4X1重要的生物学功能之一是代谢内源性大麻素Anandamide生成唯一的单加成产物14,15-EET-EA以及参与脂肪的代谢。EET-EAs可能发挥着EETs相似的生物学功能。此外,CYP4X1与肿瘤的分级有关,其可能成为肿瘤治疗的潜在药物靶标。