LC-MS/MS法测定苦参提取物中苦参碱和氧化苦参碱的质量浓度

摘要 目的：探讨液相色谱/串联质谱法(Liquid chromatography-tandem mass spectrometry,LC-MS/MS)测定苦参提取物中苦参碱和氧化苦参碱的质量浓度。方法：验证LC-MS/MS测定苦参提取物中苦参碱、氧化苦参碱的可行性与有效性,并判定检出限、重复性、稳定性、加样回收率。结果绘制苦参碱、氧化苦参碱的检出标准曲线。结果：LC-MS/MS检测苦参提取物中苦参碱与氧化苦参碱的一级质谱图和二级质谱图显示,苦参碱和氧化苦参碱的相对分子量为：248.36、246.36。色谱图显示苦参碱与氧化苦参碱无干扰色谱峰,各成分及内标峰形良好,表明该方法专属性强。在5 μg/L~500 μg/L浓度范围内,苦参碱、氧化苦参碱均呈现良好的线性关系,线性方程分别为y=6.10x-118.29和y=18.29x-14.22,检出限均为0.1 μg/L。LC-MS/MS法测定苦参提取物中苦参碱、氧化苦参碱的重复性、稳定性、加样回收率RSD均<5 %。结论：LC-MS/MS法测定苦参提取物中苦参碱和氧化苦参碱的质量浓度具有可行性、高灵敏度、高特异性等优点,可用于苦参提取物的质量控制与评价,并为后续他人的研究提供关键试验参考条件。     

黄檀素对心肌缺血/再灌注损伤的治疗作用及机制研究

摘要 目的：探讨黄檀素（DAL）对小鼠心肌缺血/再灌注（MI/R）损伤心肌的治疗作用及其作用机制。方法：选择成年雄性C57 BL/6J小鼠随机分为假手术组（Sham）、模型组（MI/R）、地尔硫组（Diltiazem）和DAL低、中、高剂量组（10、30、90 mg/kg/d）,每组10只。结扎小鼠冠状动脉左前降支（LAD）,缺血30 min,再灌注1 h建立小鼠MI/R损伤模型。术后第1天起,Sham组、MI/R组小鼠均灌胃等体积生理盐水,Diltiazem组、DAL各剂量组小鼠灌胃相应药液,每日1次,连续7 d。给药结束后检测小鼠血清中肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）活性及肿瘤坏死因-α（TNF-α）、白细胞介素-6（IL-6）含量;检测小鼠心肌组织中超氧化歧化酶（SOD）活性和丙二醛（MDA）含量;苏木精-伊红染色（HE）检测心肌损伤病理形态;Western Blot检测心肌组织中Akt和P-Akt的蛋白水平表达变化;超声检测左室舒张末内径（ESD）、左室舒张末容积（EDV）、射血分数（EF）和短轴缩短率（FS）。结果：DAL可以减轻小鼠血清中CK-MB、LDH活性及TNF-α、IL-6含量;升高小鼠心肌组织中SOD活性,减少MDA生成;增加p-Akt的蛋白水平表达,减轻心肌组织病理损伤,改善心脏功能。结论：DAL可以通过增加Akt磷酸化促进心肌细胞存活,减轻心肌组织病理损伤,进而抑制小鼠MI/R损伤,改善心脏功能,最终发挥心肌治疗作用。     

13例SCN2A 基因突变相关儿童神经系统疾病表型分析

摘要 目的：总结并分析SCN2A基因突变引起的儿童神经系统疾病相关表型谱特点。方法：采用回顾性研究,收集2018年6月至2021年6月在上海交通大学医学院附属上海儿童医学中心神经内科诊治的患儿,并经二代基因测序检测,纳入SCN2A基因突变者,研究并总结患儿神经系统临床表型特点。结果：共纳入13例SCN2A突变患儿,包括新生突变9例和遗传性突变4例。其中11例患儿伴有癫痫发作,发作年龄为1日龄~1岁11月龄,4例在新生儿期起病 （36%）,1~3 月龄起病2例（18%）,4~12月龄起病2例（18%）,1岁后起病3例（27%）;发作类型中强直阵挛发作、痉挛发作、局灶性发作均各有4例（36%）,阵挛发作1例（9%）。另有2例无癫痫发作的患儿,1例表现为全面性发育迟缓,另一例表现为发育迟缓合并孤独症谱系疾病。11例癫痫患儿中,丛集性发作患儿10例。遗传性突变4例患儿中2例智力、运动发育正常;9例新生突变的患儿中8例伴有运动、智力发育落后,1例发育正常。11例癫痫患儿表型中良性家族性新生儿癫痫1例,新生儿惊厥2例,婴儿痉挛症2例,不能分类的早发性癫痫性脑病3例,儿童期起病的癫痫性脑病2例,热厥附加症1例。结论：SCN2A基因突变引起的儿童神经系统疾病以癫痫表现居多、癫痫表型谱广,少数表现为不伴癫痫发作的发育迟缓和孤独症谱系疾病。     

炙甘草汤通过抗氧化抑制博莱霉素诱导的小鼠肺纤维化

摘要 目的：探讨炙甘草汤对特发性肺纤维化（Idiopathic pulmonary fibrosis,IPF）小鼠纤维化相关指标的影响,挖掘炙甘草汤治疗IPF的机制。方法：将60只SPF级ICR小鼠随机分为空白组、模型组、吡菲尼酮组和炙甘草汤组,除空白组外,其余组采用气管滴注博莱霉素（5 mg/kg)方法复制IPF小鼠模型,并给予相应的药物治疗。空白组和模型组小鼠灌胃生理盐水,吡菲尼酮组和炙甘草汤组小鼠分别灌胃吡菲尼酮(50 mg/kg)和炙甘草汤（25.4 g/kg）,各组均连续给药4周后取材,记录各组小鼠的死亡情况,计算各组肺系数;观察肺组织切片病理变化;碱水解法检测肺组织羟脯氨酸（HYP）含量;比色法检测肺组织丙二醛（MDA）含量、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）的活性;免疫组化、荧光定量PCR检测α-SMA、COL1A蛋白和mRNA的表达水平。结果：炙甘草汤组小鼠死亡数减少,肺系数显著降低（P＜0.01）,炎性细胞浸润和胶原沉积面积大量减少,肺泡结构逐渐修复,HYP、MDA含量降低（P＜0.01）,SOD活性（P＜0.05）和GSH-Px活性（P＜0.01）显著增强;α-SMA、COL1A蛋白和mRNA表达均降低（P＜0.01）。结论：炙甘草汤通过抑制氧化应激反应,从而抑制成纤维细胞活化,减少细胞质基质沉积,从而减缓IPF疾病进程。     

SiO2NPs通过诱导氧化应激激活Fas/FasL通路促进TM4细胞凋亡

目的 探讨纳米二氧化硅（silicon dioxide nanoparticles,SiO₂NPs）对小鼠睾丸支持细胞（TM4）的毒性作用及其分子机制。方法 将TM4细胞暴露于不同浓度的SiO₂NPs（0、1、10、100 mg/L）培养液24 h,处理结束后,采用光学显微镜和CCK-8法检测小鼠睾丸支持细胞的形态和活性。利用荧光探针DCFH-DA检测细胞内活性氧（ROS）水平,MDA和SOD试剂盒检测细胞内的丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。利用Annexin V-FITC/PI凋亡试剂盒分析TM4细胞的凋亡水平,免疫印迹法检测Fas、FasL、Caspase-8、Caspase-3、Bax和Bcl-2等细胞凋亡信号分子的蛋白质表达水平。结果 研究发现,SiO₂NPs呈剂量依赖性地抑制TM4细胞增殖,降低细胞存活率和数量,并影响细胞的形态结构。此外,SiO₂NPs诱导TM4细胞产生过量ROS,引起脂质过氧化产物MDA含量以及抗氧化酶SOD活性增加导致氧化应激。进一步研究显示,SiO₂NPs显著增加TM4细胞凋亡水平,并激活Fas/FasL死亡受体介导的细胞凋亡信号通路。有意思的是,抗氧化剂NAC可以通过降低氧化应激水平有效缓解SiO₂NPs引起的TM4细胞损伤和细胞凋亡。结论 综上,SiO₂NPs通过诱导氧化应激激活Fas/FasL信号通路促进TM4细胞凋亡。     

ALPL缺陷加速高脂诱导小鼠肝内脂肪沉积

摘要 目的：明确肝/骨/肾型碱性磷酸酶基因（liver/bone/kidney alkaline phosphatase,ALPL）在高脂诱导肝内脂肪沉积的作用。方法：利用野生型（WT）和ALPL敲除小鼠（ALPL^+/-）,给予高脂饮食8周诱导脂肪肝模型,检测小鼠肝内脂肪沉积和血清中葡糖糖、甘油三脂及胆固醇含量,并应用RT-PCR、Western blotting和免疫荧光染色检测肝组织中脂肪酸生成和转运相关基因表达变化。结果：ALPL^+/-组在正常饮食条件下较WT组肝内脂肪沉积无明显变化,而血清中葡萄糖和胆固醇含量增加;高脂条件下,敲除ALPL小鼠肝内脂肪沉积明显增加,且伴随血清中甘油三脂含量增加。RT-PCR和Western blotting结果显示,在高脂诱导下ALPL^+/-小鼠肝组织中关键脂肪酸生成基因ACC1、ACC2和 PPAR-γ,及脂肪酸生成基因LPL表达明显增加。此外,免疫荧光染色结果显示高脂诱导下敲除ALPL小鼠肝组织中的PPAR-γ阳性肝细胞明显增加。结论：ALPL敲除促进肝内脂肪酸生成和转运,加速高脂诱导小鼠肝内脂肪沉积,为阐明脂肪肝病变分子机制提供理论支持。     

lp-PLA2、RBP与冠心病病变程度相关性及其疾病诱发的危险因素分析

摘要 目的：研究脂蛋白磷脂酶A2（lp-PLA2）、视黄醇结合蛋白（RBP）与冠心病（CAD）病变程度的相关性及冠心病病变发生的危险因素。方法：以2019年12月至2021年12月在本院诊治的冠心病患者140例作为研究对象,所有患者均进行冠状动脉造影检查,并根据冠状动脉侧支循环形成情况进行分级。所有患者都给予血清lp-PLA2、RBP检测并进行相关性、危险因素分析。结果：在140例患者中,冠状动脉侧支循环未形成40例（对照组）,冠状动脉侧支循环形成100例（研究组）,研究组中Ⅰ级40例,Ⅱ级38例,Ⅲ级22例。研究组的血清lp-PLA2、RBP含量都高于对照组（P<0.05）;不同分级患者的血清lp-PLA2、RBP含量对比也有明显差异（P<0.05）。在冠心病患者中,Spearsman相关分析显示血清lp-PLA2、RBP含量与侧支循环形成分级存在正相关性（P<0.05）。logistic回归分析显示血清lp-PLA2、RBP含量均为影响冠心病患者侧支循环形成分级的危险因素（P<0.05）。ROC曲线分析显示血清lp-PLA2、RBP含量预测冠心病患者侧支循环分级的曲线下面积为0.891、0.805。结论：随着冠状动脉侧支循环形成,冠心病患者的血清lp-PLA2、RBP含量明显增加,lp-PLA2、RBP与侧支循环形成分级存在相关性,也是影响侧支循环分级的危险因素,也可预测侧支循环分级状况。     

APOE4通过Calpain/p35-p25/Cdk5增加tau蛋白磷酸化

摘要 目的：探究载脂蛋白APOE4对小鼠海马组织中tau蛋白磷酸化的作用。方法：采用6月龄人载脂蛋白APOE3,APOE4转基因纯合小鼠,用Western Blot检测小鼠海马组织中tau蛋白的磷酸化程度及Calpain蛋白、p35/25、CDK5等蛋白表达水平。使用脑立体定位术向小鼠侧脑室注射Ca²⁺螯合剂EGTA或二甲基亚砜DMSO两次,给药时间间隔4小时,第二次给药结束后两小时内处死小鼠。检测海马中Calpain蛋白、CDK5、p35/25及tau蛋白的磷酸化的变化情况。结果：①与野生型小鼠和APOE3-TR小鼠相比,APOE4-TR小鼠海马中tau蛋白在Ser396,Thr181及Thr231位点的磷酸化均显著性增高,同时Calpain2、p35/25和CDK5的表达水平也增加。②使用Ca²⁺螯合剂EGTA后,与对照DMSO给药组相比,Ca²⁺螯合剂EGTA给药组小鼠海马组织中tau蛋白在Ser396位的磷酸化显著下降,但未检测到tau蛋白在Thr181及Thr231位点的磷酸发生显著性变化,同时Calpain 2蛋白、p35/25和CDK5的表达水平降低。结论：人载脂蛋白APOE4引起小鼠海马tau蛋白磷酸化异常增高,并且可能是通过Calpain/p35-p25/CDK5信号通路调控tau蛋白Ser396位点磷酸化。     

青蒿素经PI3K/Akt通路改善突触可塑性减轻糖尿病小鼠认知障碍

目的 本研究旨在阐明青蒿素对II型糖尿病（T2DM）小鼠认知功能障碍的改善作用及其机制。方法 C57BL/6J小鼠单次腹腔注射STZ（100 mg/kg）后联合高脂饲料喂养建立T2DM模型。T2DM小鼠随后腹腔注射青蒿素（40 mg/kg/d）或等体积溶剂。干预4周后，新物体识别、Y迷宫和Morris水迷宫实验检测小鼠的学习和记忆能力。蛋白质印迹法（Western blot）检测海马PI3K、Akt、磷酸化Akt、SYN和PSD-95蛋白的表达。透射电镜观察海马CA1区突触密度和突触超微结构改变。结果 与模型组相比，青蒿素干预组T2DM小鼠的认知功能显著改善，海马中PI3K和磷酸化Akt水平升高，SYN和PSD-95蛋白表达增加，CA1区神经元丢失减少。此外，青蒿素干预组小鼠CA1区的突触密度、PSD-95和突触界面曲率增加，突触间隙宽度减小。结论 青蒿素可能通过激活海马PI3K/Akt途径增强突触可塑性，从而减轻T2DM小鼠认知功能障碍；青蒿素有望成为治疗糖尿病性认知功能障碍的新型药物。     

老年心力衰竭患者BNP、LVEDD、LVEF水平与心脏功能的关系

摘要 目的：探究老年心力衰竭患者的脑利钠肽（Brain natriuretic peptide,BNP）、左室舒张末径（Left ventricular end diastolic diameter,LVEDD）、左室射血分数（Left ventricular ejection fraction,LVEF）水平与心脏功能的关系。方法：选择2019年3月-2020年12月于我院接受治疗的150例老年心力衰竭患者,按照其BNP水平将其分为A（BNP水平<94 pg/mL,43例）、B（BNP水平94~349.9 pg/mL,40例）、C（BNP水平350~988.9 pg/mL,44例）、D（BNP水平≥989 pg/mL,23例）4组,对比4组患者LVEDD、LVEF水平、血同型半胱氨酸（Homocysteine,HCY）水平,对比4组患者不同心功能分级比率,对比4组患者随访2个月心脏不良事件发生率,最后分析BNP、LVEDD和LVEF与心力衰竭患者心功能分级相关性。结果：A、B、C、D四组患者LVEDD、HCY呈递增趋势,LVEF呈递减趋势,C、D两组患者的LVEDD、HCY水平明显高于A、B两组（P<0.05）,C、D两组LVEF水平明显低于A、B两组患者（P<0.05）;A、B、C、D四组患者心功能分级逐渐加重,A组患者I级70例,II级27例,B组患者II级60例,III级29例,C组III级68例,IV级20例,D组III级3例,IV级43例,各组间对比心功能分级差异具有统计学意义（P<0.05）;A、B、C、D四组患者心脏不良事件发生率分别为4.12 %、11.24 %、26.14 %,43.48 %,不良事件发生率逐渐升高,差异具有统计学意义（P＜0.05）;BNP、LVEDD与心功能分级呈正相关（r=0.878、0.564,P<0.05）,LVEF与心功能分级呈负相关（r=0.781,P<0.05）。结论：BNP、LVEDD与LVEF指标可以作为心力衰竭评估指标,能够对心力衰竭患者心脏功能及预后进行评估。     

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.     

Endocannabinoids and Inflammatory Response in Periodontal Ligament Cells

Endocannabinoids are associated with multiple regulatory functions in several tissues. The main endocannabinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG), have been detected in the gingival crevicular fluid of periodontitis patients, but the association between periodontal disease or human periodontal ligament cells (hPdLCs) and endocannabinoids still remain unclear. The aim of the present study was to examine the effects of AEA and 2-AG on the proliferation/viability and cytokine/chemokine production of hPdLCs in the presence/absence of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS). The proliferation/viability of hPdLCs was measured using 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT)-assay. Interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) levels were examined at gene expression and protein level by real-time PCR and ELISA, respectively. AEA and 2-AG did not reveal any significant effects on proliferation/viability of hPdLCs in the absence of P. gingivalis LPS. However, hPdLCs viability was significantly increased by 10–20 µM AEA in the presence of P. gingivalis LPS (1 µg/ml). In the absence of P. gingivalis LPS, AEA and 2-AG did not exhibit any significant effect on the expression of IL-8 and MCP-1 expression in hPdLCs, whereas IL-6 expression was slightly enhanced by 10 µM 2-AG and not affected by AEA. In P.gingivalis LPS stimulated hPdLCs, 10 µM AEA down-regulated gene-expression and protein production of IL-6, IL-8, and MCP-1. In contrast, 10 µM 2-AG had an opposite effect and induced a significant up-regulation of gene and protein expression of IL-6 and IL-8 (P<0.05) as well as gene-expression of MCP-1 in P. gingivalis LPS stimulated hPdLCs. Our data suggest that AEA appears to have an anti-inflammatory and immune suppressive effect on hPdLCs’ host response to P.gingivalis LPS, whereas 2-AG appears to promote detrimental inflammatory processes. In conclusion, AEA and 2-AG might play an important role in the modulation of periodontal inflammation.     

Endocannabinoids Are Expressed in Bone Marrow Stromal Niches and Play a Role in Interactions of Hematopoietic Stem and Progenitor Cells with the Bone Marrow Microenvironment

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB₁ and CB₂. The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB₁ receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10⁷ cells), and 2-AG (75.2 ng/10⁷ cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10⁷ cells) and 2-AG (98.8 ng/10⁷ cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB₁ agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1^−/− showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)^−/− mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1^−/−, as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.     

Leptin Levels Are Negatively Correlated with 2-Arachidonoylglycerol in the Cerebrospinal Fluid of Patients with Osteoarthritis

Background

There is compelling evidence in humans that peripheral endocannabinoid signaling is disrupted in obesity. However, little is known about the corresponding central signaling. Here, we have investigated the relationship between gender, leptin, body mass index (BMI) and levels of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the serum and cerebrospinal fluid (CSF) of primarily overweight to obese patients with osteoarthritis.

Methodology/Principal Findings

Patients (20 females, 15 males, age range 44-78 years, BMI range 24-42) undergoing total knee arthroplasty for end-stage osteoarthritis were recruited for the study. Endocannabinoids were quantified by liquid chromatography – mass spectrometry. AEA and 2-AG levels in the serum and CSF did not correlate with either age or BMI. However, 2-AG levels in the CSF, but not serum, correlated negatively with CSF leptin levels (Spearman’s ρ -0.48, P=0.0076, n=30). No such correlations were observed for AEA and leptin.

Conclusions/Significance

In the patient sample investigated, there is a negative association between 2-AG and leptin levels in the CSF. This is consistent with pre-clinical studies in animals, demonstrating that leptin controls the levels of hypothalamic endocannabinoids that regulate feeding behavior.     

Targeting brain and peripheral plasticity of the lipidome in acute kainic acid-induced epileptic seizures in mice via quantitative mass spectrometry

Epilepsy is a highly common chronic neurological disorder, manifested in many different types, affecting ~ 1% of the worldwide human population. The molecular mechanisms of epileptogenesis have not yet been clarified, and pharmacoresistance exhibited by 30–40% of epilepsy patients remains a major obstacle in medical care. Growing evidence indicates a role of lipid signalling pathways in epileptogenesis, thus lipid signals emerge as potential biomarkers for the onset and evolving course of the epileptic disorder, as well as potential therapeutic agents and targets. For this purpose, we applied a lipidomic strategy to unravel lipid alterations in brain regions, periphery tissues and plasma that are specific for acute epileptic seizures in mice at 1 h after seizure induction by systemic kainic acid injection as compared to vehicle controls. Specifically, levels of (i) selected phospholipids and sphingomyelins, (ii) the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), and the endocannabinoid-related compounds oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), (iii) arachidonic acid (AA), (iv) selected eicosanoids, and (v) fatty acyl content of lipidome were determined in pulverized tissues from six brain regions of kainic acid induced epileptic seizure models and vehicle controls: hypothalamus, hippocampus, thalamus, striatum, cerebellum and cerebral cortex, and from peripheral organs, such as heart and lungs, and in plasma. Alterations in lipid levels after acute epileptic seizures as compared to non-seizure controls were found to be brain region- and periphery tissue-specific, including specific plasma lipid correlates, highlighting their value as marker candidates in translational research studies, and/or drug discovery and response monitoring.     

Opposing actions of endocannabinoids on cholangiocarcinoma growth is via the differential activation of Notch signaling

The endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) have opposing effects on cholangiocarcinoma growth. Implicated in cancer, Notch signaling requires the γ-secretase complex for activation. The aims of this study were to determine if the opposing effects of endocannabinoids depend on the differential activation of the Notch receptors and to demonstrate that the differential activation of these receptors are due to presenilin 1 containing- and presenilin 2 containing-γ-secretase complexes. Mz-ChA-1 cells were treated with AEA or 2-AG. Notch receptor expression, activation, and nuclear translocation were determined. Specific roles for Notch 1 and 2 on cannabinoid-induced effects were determined by transient transfection of Notch 1 or 2 shRNA vectors before stimulation with AEA or 2-AG. Expression of presenilin 1 and 2 was determined after AEA or 2-AG treatment, and the involvement of presenilin 1 and 2 in the cannabinoid-induced effects was demonstrated in cell lines with low presenilin 1 or 2 expression. Antiproliferative effects of AEA required increased Notch 1 mRNA, activation, and nuclear translocation, whereas the growth-promoting effects induced by 2-AG required increased Notch 2 mRNA expression, activation, and nuclear translocation. AEA increased presenilin 1 expression and recruitment into the γ-secretase complex, whereas 2-AG increased expression and recruitment of presenilin 2. The development of novel therapeutic strategies aimed at modulating the endocannabinoid system or mimicking the mode of action of AEA on Notch signaling pathways would prove beneficial for cholangiocarcinoma management.     

Pharmacokinetics of Sublingual Versus Oral Estradiol in Transgender Women

ObjectiveTo investigate the pharmacokinetics of 17β-estradiol (E2) administered orally versus those of 17β-E2 administered sublingually in transgender women.MethodsSingle doses of 17β-E2 were administered orally (1 mg) to 10 transgender women and then sublingually (1 mg) after a 1-week washout period. Blood samples were collected at baseline (0 hour) and at 1, 2, 3, 4, 6, and 8 hours after dosing. The samples were frozen and analyzed using liquid chromatography mass spectrometry (LC-MS/MS) and immunoassay.ResultsThe results demonstrated that sublingual E2 had a significantly higher peak serum E2 concentration of 144 pg/mL, measured using LC-MS/MS, compared with an oral E2 concentration of 35 pg/mL, measured using LC-MS/MS (P = .003). Sublingual E2 peaked at 1 hour and oral E2 peaked at 8 hours, as measured using LC-MS/MS. The area under the curve (AUC) (0-8 hours) for sublingual E2, measured using LC-MS/MS, was 1.8-fold higher than the AUC (0-8 hours) for oral E2, measured using LC-MS/MS. Additionally, sublingual E2 was found to have an increased E2-to-estrone ratio at all time points (1.1 ± 1.0 vs 0.7 ± 0.4, P ≤ .0001), the clinical significance of which is unclear.ConclusionOral E2 administered sublingually has a different pharmacokinetic profile, with higher serum E2 levels and AUC (0-8 hours) than traditionally administered oral E2. Multidaily dosing may be necessary to suppress testosterone levels with sublingual E2. The appropriate dosing, efficacy, and safety of sublingual E2, compared with those of other E2 preparations, are unknown.     

Endocannabinoids measurement in human saliva as potential biomarker of obesity

Background

The discovery of the endocannabinoid system and of its role in the regulation of energy balance has significantly advanced our understanding of the physiopathological mechanisms leading to obesity and type 2 diabetes. New knowledge on the role of this system in humans has been acquired by measuring blood endocannabinoids. Here we explored endocannabinoids and related N-acylethanolamines in saliva and verified their changes in relation to body weight status and in response to a meal or to body weight loss.

Methodology/Principal Findings

Fasting plasma and salivary endocannabinoids and N-acylethanolamines were measured through liquid mass spectrometry in 12 normal weight and 12 obese, insulin-resistant subjects. Salivary endocannabinoids and N-acylethanolamines were evaluated in the same cohort before and after the consumption of a meal. Changes in salivary endocannabinoids and N-acylethanolamines after body weight loss were investigated in a second group of 12 obese subjects following a 12-weeks lifestyle intervention program. The levels of mRNAs coding for enzymes regulating the metabolism of endocannabinoids, N-acylethanolamines and of cannabinoid type 1 (CB₁) receptor, alongside endocannabinoids and N-acylethanolamines content, were assessed in human salivary glands.The endocannabinoids 2-arachidonoylglycerol (2-AG), N-arachidonoylethanolamide (anandamide, AEA), and the N-acylethanolamines (oleoylethanolamide, OEA and palmitoylethanolamide, PEA) were quantifiable in saliva and their levels were significantly higher in obese than in normal weight subjects. Fasting salivary AEA and OEA directly correlated with BMI, waist circumference and fasting insulin. Salivary endocannabinoids and N-acylethanolamines did not change in response to a meal. CB₁ receptors, ligands and enzymes were expressed in the salivary glands. Finally, a body weight loss of 5.3% obtained after a 12-weeks lifestyle program significantly decreased salivary AEA levels.

Conclusions/Significance

Endocannabinoids and N-acylethanolamines are quantifiable in saliva and their levels correlate with obesity but not with feeding status. Body weight loss significantly decreases salivary AEA, which might represent a useful biomarker in obesity.     

The endocannabinoid system in neurodegeneration

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed.     

Mass‐Spectrometric Identification of Anandamide and 2‐Arachidonoylglycerol in Nematodes

The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, and Pelodera strongyloides) produce endocannabinoids; i.e., anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG). In this study, AEA and 2‐AG were identified as endogenous products from nematodes by using electrospray‐ionization ion‐trap MS/MS (ESI‐IT‐MS) experiments operated in the positive‐ionization mode. Endocannabinoids were identified by product ion scan and concentrations were measured by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Both AEA and 2‐AG were identified in all of the nematode samples, even though these species lack known cannabinoid receptors. Neither AEA nor 2‐AG were detected in the fat‐3 mutant of C. elegans, which lacks the necessary enzyme to produce arachidonic acid, the fatty acid precursor of these endocannabinoids.