Metabolic fate of the new synthetic cannabinoid 7’N‐5F‐ADB in rat,human, and pooled human S9 studied by means of hyphenated high‐resolution mass spectrometry

New psychoactive substances (NPS) are an important issue in clinical/forensic toxicology. 7’N‐5F‐ADB, a synthetic cannabinoid derived from 5F‐ADB, appeared recently on the market. Up to now, no data about its mass spectral fragmentation pattern, metabolism, and thus suitable targets for toxicological urine screenings have been available. Therefore, the aim of this study was to elucidate the metabolic fate of 7’N‐5F‐ADB in rat, human, and pooled human S9 (pS9). The main human urinary excretion products, which can be used as targets for toxicological screening procedures, were identified by Orbitrap (OT)‐based liquid chromatography–high resolution‐tandem mass spectrometry (LC–HRMS/MS). In addition, possible differentiation of 7’N‐5F‐ADB and 5F‐ADB via LC–HRMS/MS was studied. Using the in vivo and in vitro models for metabolism studies, 36 metabolites were tentatively identified. 7’N‐5F‐ABD was extensively metabolized in rat and human with minor species differences observed. The unchanged parent compound could be found in human urine but metabolites were far more abundant. The most abundant ones were the hydrolyzed ester (M5), the hydrolyzed ester in combination with hydroxylation of the tertiary butyl part (M11), and the hydrolyzed ester in addition to glucuronidation (M30). Besides the parent compound, these metabolites should be used as targets for urine‐based toxicological screening procedures. Two urine‐paired human plasma samples contained mainly the parent compound (c = 205 μg/L, 157 μg/L) and, at a higher abundance, the compound after ester hydrolysis (M5). In pS9 incubations, the parent compound, M5, and M30 were detectable among others. Furthermore, a differentiation of both compounds was possible due to different retention times and fragmentation patterns.     

7‐keto‐DHEAmetabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field

7‐keto‐DHEA (3β‐hydroxy‐androst‐5‐ene‐7,17‐dione) is included in section S1 of the World Antidoping Agency (WADA) List of Prohibited Substances. The detection of its misuse in sports needs special attention, since it is naturally present in urine samples. The main goal of this study is to investigate the in vivo metabolism of 7‐keto‐DHEA after a single administration to healthy volunteers and to better describe the relationship between arimistane (androst‐5‐ene‐7,17‐dione) and 7‐keto‐DHEA after the application of the common routine procedures to detect anabolic steroids in WADA accredited antidoping laboratories. Free, glucuro‐, and sulpho‐conjugated steroids extracted from urine samples obtained before and after the administration of 7‐keto‐DHEA were analyzed by different gas chromatographic (GC)–mass spectrometric (MS) techniques. Gas chromatography coupled to tandem MS to study the effect on the endogenous steroid profile, coupled to isotope ratio mass spectrometry (IRMS) to investigate the potential formation of androgens derived from DHEA and coupled to high resolution accurate mass spectrometry (HRMS) to investigate new diagnostic metabolites. The analysis by IRMS confirmed that there is no formation of DHEA from 7‐keto‐DHEA. Ten proposed metabolites, not previously reported, were described. These include reduced and hydroxylated structures that are not considered part of the steroid profile in antidoping analyses. They showed considerable responses in all fractions analyzed. Some deoxidation reactions (including arimistane formation) were found and most probably can be linked to the sample preparation or instrumental analysis. This is important when interpreting the results after the application of procedures to detect steroids in urine currently used in antidoping laboratories. 7‐keto‐DHEA metabolism in humans for antidoping purposes was studied and unexpected results were found. This could lead to a misinterpretation of the data, depending on the procedure applied and the analytical instrumentation used.     

冠心病外周血白细胞介素7表达对免疫应答的影响及可能机制分析

目的探讨冠心病外周血白细胞介素7(IL-7)表达对免疫应答的影响及可能机制。方法选择冠心病患者82例,根据诊断分为稳定性心绞痛组22例,不稳定性心绞痛组39例,急性心肌梗死组21例;同期选取健康志愿者20例为正常对照组。ELISA法检测血清IL-7、高敏C反应蛋白(hs-CRP)、B型钠尿肽(BNP)水平;流式细胞术检测T细胞免疫球蛋白黏蛋白分子3(Tim-3)水平。分离外周血单核细胞,重组IL-7刺激12 h为刺激者,采用重组IL-7刺激为未刺激者。检测Tim-3水平;Western blot检测信号转导和转录激活因子5(STAT-5)、磷酸化STAT-5(p-STAT-5)、细胞因子信号转导负调控因子3(Socs-3)蛋白表达水平。结果急性心肌梗死组血清IL-7、hs-CRP水平明显高于正常对照组(P<0.01);稳定性心绞痛组和不稳定性心绞痛组血清IL-7、hs-CRP水平与正常对照组比较,差异无统计学意义(P>0.05)。稳定性心绞痛组、不稳定性心绞痛组、急性心肌梗死组血清BNP明显高于正常对照组(P<0.05)。急性心肌梗死组Tim-3 CD4T细胞、Tim-3 CD8T细胞明显高于稳定性心绞痛组、不稳定性心绞痛组、正常对照组(P<0.05)。正常对照组和急性心肌梗死组刺激者Tim-3 CD8T细胞均明显高于未刺激者[(2.61±0.87)%vs(0.88±0.30)%、(4.80±1.53)%vs(2.15±0.69)%,P<0.05]。急性心肌梗死组刺激者和未刺激者Tim-3 CD4T细胞和Tim-3 CD8T细胞明显高于正常对照组(P<0.01)。刺激者p-STAT-5、Socs-3蛋白相对表达量明显高于未刺激者(P<0.05),两者STAT-5表达比较无显著差异(P>0.05)。结论急性心肌梗死患者外周血IL-7水平明显升高;IL-7可通过上调Tim-3表达,在冠心病发生、发展中发挥抑制炎性应答的作用。     

P2X7受体在非小细胞肺癌中的表达及其临床意义*

的?探讨非小细胞肺癌（NSCLC）患者癌组织中P2X7R的表达及其临床意义。方法?回顾性分析2012年1月—2014年1月华中科技大学同济医学院附属武汉中心医院收治的NSCLC患者118例癌组织标本。采用免疫组织化学法检测NSCLC组织标本中P2X7R的表达,根据免疫组织化学结果将患者分为P2X7R高、低表达组。采用Kaplan-Meier曲线和Log-rank χ2检验比较P2X7R高、低表达组无瘤生存率和总生存率的差异;采用Cox比例风险模型分析影响NSCLC患者无瘤生存率和总生存率的独立危险因素。 结果?72例（61.0%）患者癌组织中P2X7R高表达。不同TNM分期和有无淋巴结转移患者的P2X7R蛋白高表达率比较,差异有统计学意义（P?<0.05）。P2X7R高表达组5年无瘤生存率低于P2X7R低表达组（18.1% VS 25.1%）（P?<0.05）;P2X7R高表达组5年总生存率低于P2X7R低表达组（23.3% VS 30.1%）（P?<0.05）。单因素Cox比例风险模型分析显示,P2X7R表达、TNM分期及淋巴结转移是影响NSCLC患者无瘤生存率的危险因素（P?<0.05）。多因素Cox比例风险模型分析表明,P2X7R表达[OlR=2.082（95% CI：1.079,3.747）,P?=0.001]及TNM分期[OlR=3.210（95% CI：0.587,6.448）,P?=0.028]是影响NSCLC患者无瘤生存率的危险因素。单因素Cox比例风险模型回归分析显示,P2X7R表达、TNM分期和淋巴结转移是影响NSCLC患者总生存率的危险因素（P?<0.05）。多因素Cox比例风险模型回归分析表明,P2X7R表达[OlR=2.893（95%CI：1.582,4.562）,P?=0.002]及TNM分期[OlR=1.910（95%CI：0.587,2.449）,P?=0.038]是影响NSCLC患者总生存率的危险因素。结论?P2X7R表达可作为影响NSCLC预后的新分子标志物。     

Fractalkine 通过激活 Wnt/β-catenin 信号通路抑制脂多糖诱导的巨噬细胞M1型极化

目的 研究Fractalkine（CX3CL1,FKN）对脂多糖（LPS）诱导的RAW264.7巨噬细胞免疫应答的作用机制。方法 体外培养RAW264.7细胞,使用慢病毒技术构建过表达FKN的稳定细胞珠。细胞分为8组：（1）空白对照组;（2）LPS组,细胞给予脂多糖（1 μg/mL,12 h）;（3）ICG-001组,细胞给与Wnt/β-catenin信号通路抑制剂ICG-001（10 μmol/mL,48 h）;（4）过表达FKN组;（5）ICG-001+LPS组,细胞给与ICG-001（10 μmol/mL预处理36 h）后,加入脂多糖（1 μg/mL,12 h）;（6）过表达FKN+LPS组,过表达 FKN细胞给与脂多糖（1 μg/mL,12 h）;（7）过表达FKN+ICG-001组,过表达FKN细胞给予ICG-001（10 μmol/mL,48 h）;（8）过表达 FKN+ICG-001+LPS 组,过表达 FKN 细胞给与 ICG-001（10 μmol/mL 预处理 36 h）后,加入脂多糖（1 μg/mL,12 h）;应用 CCK-8细胞增殖实验检测ICG-001作用于巨噬细胞中的安全浓度;应用酶联免疫吸附（ELISA）实验检测巨噬细胞上清液中M1型极化因子TNF-α和IL-6的含量;应用蛋白质免疫印记（WB）实验检测巨噬细胞中FKN、Wnt/β-catenin通路关键因子Wnt-4和β-catenin、M1型极化因子iNOS、TNF-α和IL-6的蛋白表达水平;应用免疫荧光（IF）实验检测巨噬细胞中M1型极化因子IL-6蛋白的定位。结果 过表达FKN的RAW264.7细胞中FKN的蛋白水平较空白对照组明显升高（P<0.01）。CCK-8实验显示ICG-001作用于RAW264.7细胞48 h的IC50为10 μmol/mL。与LPS组相比,ICG-001+LPS组上清液中TNF-α和IL-6的分泌量以及细胞内TNF-α、IL-6和iNOS蛋白含量均升高（P<0.05）,而细胞内FKN、Wnt-4和β-catenin蛋白含量均显著降低（P<0.01）;EXFKN+LPS组上清液中TNF-α和IL-6的分泌量以及细胞内TNF-α、IL-6和iNOS蛋白含量均显著降低（P<0.01）,而细胞内FKN、Wnt-4和β-catenin蛋白含量均显著升高（P<0.01）。与LPS组相比,ICG-001+LPS组中IL-6在细胞质中的定位增强,而EXFKN+LPS组中IL-6在细胞质中的定位受到抑制。结论 过表达FKN通过激活Wnt/β-catenin信号通路抑制脂多糖诱导的巨噬细胞M1型极化。     

代谢型谷氨酸受体7对人胚胎神经干细胞增殖的影响

目的 研究代谢型谷氨酸受体7(mGluR7)对人胚胎神经干细胞增殖的影响。方法 利用MTT比色法分析mGluR7过表达载体、mGluR7siRNA在不同时间对体外的人胚胎NSCs细胞活力的影响, 神经球测量方法分析mGluR7对人神经球生长的影响, 流式细胞术分析mGluR7对人NSCs细胞周期及细胞凋亡的影响。结果 MTT结果表明在处理24 h, 48 h和72 h后, mGluR7显著增强人NSCs的细胞活力, 增殖细胞数量显著增多, 神经球直径显著增大, mGluR7siRNA抑制人神经球的增殖。过表达mGluR7后, 与对照组相比G1/G0期和细胞比率显著下降, S期细胞比率显著上升, mGluR7过表达促进了细胞G1到S期转化, 沉默mGluR7将人NSCs细胞阻滞在G1/G0期。应用流式细胞术分析过表达mGluR7对人NSCs细胞凋亡的影响, 转染48 h后, 早凋晚凋均显著下降, 沉默mGluR7能够显著诱导人NSCs的早凋和晚凋。结论 mGluR7可促进体外培养的人胚胎皮质NSCs增殖。     

胆汁酸通过p-STAT3-SOCS3途径调节下丘脑神经元食欲及抑制信号肽表达

目的 探索胆汁酸牛磺石胆酸或鹅去氧胆酸对小鼠下丘脑GT1-7细胞食欲抑制信号肽表达的影响及可能的作用途径。方法 将实验分为3组,每组3个平行样本。空白对照组：仅给予含10%胎牛血清高糖培养基培养GT1-7细胞。胆汁酸牛磺石胆酸处理组：分别采用10 nmol/L,100 nmol/L,1 μmol/L和10 μmol/L浓度的胆汁酸牛磺石胆酸处理GT1-7细胞12、24和48 h。鹅去氧胆酸处理组：分别采用10 nmol/L,100 nmol/L,1 μmol/L和10 μmol/L浓度的鹅去氧胆酸处理GT1-7细胞12、24和48 h。采用Real-time PCR 检测GT1-7细胞食欲抑制信号肽阿黑皮素原（POMC）mRNA表达水平;ELISA法检测POMC剪切产物抑制食欲肽α-促黑素细胞激素水平。采用Western blot检测G蛋白偶联胆汁酸受体（TGR5）和法呢醇X受体表达以及磷酸化信号转导转录激活因子3（p-STAT3）,磷酸化蛋白激酶（p-AKT）,细胞因子信号传导抑制因子3（SOCS3）蛋白表达量。结果 Western blot结果显示,下丘脑神经元GT1-7细胞能表达TGR5和FXR两种胆汁酸受体,且表达量受胆汁酸调节。Real-time PCR检测结果显示,10 μmol/L tLCA或鹅去氧胆酸处理GT1-7细胞24 h后POMC mRNA表达水平上升,ElISA结果也发现10 μmol/L 胆汁酸牛磺石胆酸或鹅去氧胆酸处理GT1-7细胞24 h后具有抑制食欲作用的POMC衍生肽[α]-MSH释放增多。Western blot结果显示,胆汁酸处理可上调GT1-7细胞p-STAT3和p-AKT蛋白表达,且调控STAT3信号通路的SOCS3蛋白表达也有增加。结论 胆汁酸牛磺石胆酸或鹅去氧胆酸通过GT1-7细胞胆汁酸受体TGR5和FXR促进POMC mRNA表达,增加[α]-促黑素细胞激素水平,提示胆汁酸具有通过抑制食欲信号肽表达调节食欲的潜在作用,该作用是通过p-AKT和p-STAT3 -SOCS3 信号通路实现。     

盐酸右美托咪定对老年大鼠术后认知功能及P2X7受体表达的影响

目的 观察盐酸右美托咪定对术后大鼠认知功能和海马区炎症反应的影响,探讨P2X7 受体在其中的作用机制。方法 成年雄性SD大鼠60只,随机分为3组：对照组(A组,n=20)、手术组(B组,n=20)、右美组(C组,n=20),B组和C组大鼠在腹腔注射2%戊巴比妥钠(50mg/kg)后施行脾切除术,A组大鼠仅仅开腹后关腹缝合,不进行脾切除术。C组在手术前进行尾静脉穿刺,于麻醉后经尾静脉给予右美托咪定初始计量10μg/kg (超过1min),随后以10μg/kg/h的速度输注;B组尾静脉穿刺后输注同等体积的生理盐水。手术结束后2周,在各组存活大鼠中随机选取 10只大鼠行 Morris 水迷宫实验观察认知功能的变化,使用酶联免疫吸附( ELISA) 法检测大鼠海马组织中IL-1β、TNF-α浓度,Western blot 法检测海马组织中 P2X7 的表达。 结果 与A组相比,B组大鼠找到水下平台的潜伏期明显延长,空间探索实验中跨越原平台次数明显减少,海马区IL-1β、TNF-α含量增高,P2X7表达明显上调,差异具有统计学意义(P<0.05)。与B组相比,C组大鼠找到水下平台的潜伏期明显延长,跨越原平台次数明显减少,海马区IL-1β、TNF-α含量增高,P2X7表达明显上调,差异具有统计学意义(P<0.05)。A组与C组大鼠相比,各项指标差异无统计学意义(P>0.05)。结论 盐酸右美托咪定可改善手术后引起的认知功能障碍,可能与盐酸右美托咪定抑制 P2X7 受体活性,降低海马区炎症反应有关。