人参皂苷Rc、Re、Rf和Rg1对药物代谢酶CYP1A1活性诱导作用研究

目的研究人参皂苷Rc、Re、Rf和Rg1能否激活h AhR受体信号通路诱导CYP1A1基因及蛋白表达。方法利用前期实验室构建的p GL4.17-CYP1A1报告基因质粒、pc DNA3.1-h AhR表达质粒与pRL-TK内参质粒共转染Hep G2细胞,检测人参皂苷Rc、Re、Rf和Rg1对AhR的转录激活效应;并利用Real-time PCR及Western blot技术对人参皂苷Rc、Re、Rf和Rg1的不同浓度、不同时间处理组进行mRNA及蛋白的检测。结果报告基因模型检测结果显示,人参皂苷Rc、Re、Rf和Rg1对AhR具有转录激活作用,其中人参皂苷Re与Rf对AhR转录激活效应明显;同时不同浓度人参皂苷Rc、Re、Rf和Rg1均能上调CYP1A1 mRNA与蛋白表达水平。结论人参皂苷Rc、Re、Rf和Rg1可以诱导CYP1A1mRNA与蛋白质水平的表达,这种诱导作用可能与上述皂苷成分激活AhR并提高其对CYP1A1的转录活性有关。     

基于药物代谢酶CYP3A4的乌头碱配伍人参皂苷、甘草苷的减毒机制研究

目的 考察乌头碱配伍人参皂苷Rb₁、甘草苷后对HepG2药物代谢酶（Cytochrome P450,CYP450）中3A4亚型的报告基因荧光活性、mRNA转录及蛋白翻译水平的影响。方法 将pGLuc-CYP3A4报告基因质粒与pcDNA3.1-hPXR表达质粒共转染HepG2细胞,检测乌头类生物碱、人参皂苷和甘草的单体成分对CYP3A4的激活效应;并利用实时荧光定量PCR（qRT-PCR）及Western blotting技术检测人参皂苷Rb₁、甘草苷与乌头碱对CYP3A4 mRNA及蛋白水平的影响。结果 报告基因模型检测结果显示,与对照组比较,乌头碱、新乌头碱、次乌头碱和乙酰乌头碱能下调CYP3A4报告基因荧光强度（P<0.05）,其中乌头碱下调能力最强,人参皂苷Rb₁、Rc、Re、Rg1以及甘草苷、异甘草苷、甘草素和甘草酸均能上调报告基因的荧光强度（P<0.05、0.01）,其中人参皂苷Rb₁和甘草苷上调能力最明显;同时乌头碱能下调CYP3A4 mRNA与蛋白表达水平（P<0.05、0.01）,人参皂苷Rb₁和甘草苷能逆转乌头碱下调CYP3A4的能力（P<0.05、0.01）。结论 人参皂苷Rb₁、甘草苷与乌头碱配伍后可上调CYP3A4的表达,减少乌头碱在体内蓄积时间,起到减毒的作用。     

应用人肝细胞研究人参皂苷Rb1和Rg1、丹参素钠、冰片对CYP450药物代谢酶1A2、2B6和3A4的体外诱导作用

目的 使用原代培养的人肝细胞研究人参皂苷Rb₁和Rg₁、丹参素钠、冰片对细胞色素P450酶（CYP450）的诱导作用。方法 分别将3批次的冷冻原代人肝贴壁细胞进行接种培养,使用人参皂苷Rb₁和Rg₁、丹参素钠、冰片（30 μmol/L）对CYP1A2、CYP2B6和CYP3A4进行诱导,实时荧光定量PCR（qRT-PCR）法测定CYP450酶mRNA表达水平,评价4种单体成分对P450酶的诱导作用。阳性对照为20 μmol/L利福平（CYP3A4诱导剂）、50 μmol/L奥美拉唑钠（CYP1A2诱导剂）和1 mmol/L苯巴比妥钠（CYP2B6诱导剂）;阴性对照组为10 μmol/L红霉素。冰片（30 μmol/L）与利福平联合给药,观察冰片对利福平的CYP1A2、CYP2B6和CYP3A4诱导作用的影响。结果 3批次人肝细胞的诱导结果显示,阳性诱导剂奥美拉唑、苯巴比妥、利福平分别显著诱导CYP1A2、CYP2B6、CYP3A4的表达,阴性对照红霉素对CYP1A2、CYP2B6及CYP3A4的mRNA表达水平未见明显影响,人参皂苷Rb₁和Rg₁、丹参素钠、冰片在30 μmol/L浓度给药时,对3批次人肝细胞的CYP1A2、CYP2B6和CYP3A4 mRNA表达水平未见明显影响;30 μmol/L冰片与利福平联合给药,与利福平单独给药比较,3批次肝细胞的CYP2B6、CYP3A4 mRNA表达水平均减少,对CYP3A4的mRNA表达水平影响尤为显著。结论 人参皂苷Rb₁和Rg₁、丹参素钠、冰片在30 μmol/L浓度给药时,对3批次人肝细胞的CYP1A2、CYP2B6和CYP3A4均没有诱导作用。冰片与利福平联合用药时,可以阻断利福平对CYP2B6和CYP3A4的诱导作用,对CYP3A4的影响尤为显著。     

人参皂苷Rg1及其肠内菌代谢产物Rh1对小鼠免疫细胞功能的影响

目的初探人参皂苷Rg₁及其肠内菌代谢产物Rh₁对正常小鼠免疫功能的影响。方法用Rh₁与Rg₁分别处理脾T细胞,B细胞及腹腔巨噬细胞(Mφ);MTT比色法测T和B细胞增殖能力;中性红比色法测Mφ的吞噬功能;Griess法测Mφ释放NO的水平。结果Rh₁能促进脾细胞增殖、下调Con A诱导的T细胞增殖;Rh₁与Rg₁对LPS诱导的B细胞增殖均无明显作用;Rg₁和Rh₁能提高Mφ的吞噬能力和促进NO的释放。结论Rg₁及其代谢产物Rh₁可共同作用于T细胞和Mφ而产生免疫调节作用。     

基于细胞膜色谱技术的人参皂苷及五味子木脂素与血管内皮生长因子受体的作用分析

目的 运用细胞膜色谱（CMC）技术检测分析人参皂苷与五味子木脂素中15种成分与血管内皮生长因子（VEGF）受体的作用。方法 运用Western blotting法检测筛选VEGF受体高表达细胞株;构建VEGF受体CMC,并建立ECV304/CMC online-LC检测方法,以索拉菲尼作为阳性药,对人参皂苷Rb₁、Rb₂、Rb₃、Rc、Rd、Rg₁、Rg₂、Rg₃、Rh₁、Ro、F₂,五味子甲素,五味子乙素,五味子醇甲,五味子醇乙进行VEGF受体结合强度的检测分析;CCK-8法检测筛选出的成分（5、10 μg/mL）对ECV304细胞的促增殖作用。结果 Western blotting结果显示,ECV304细胞中VEGF受体高表达;人参皂苷Rb₁、Rb₂、Rb₃、Rc、Rd、Rg₃、Rh₁、F₂、五味子甲素、五味子乙素与VEGF受体CMC柱有结合作用;CCK-8法检测以上成分对ECV304细胞的活力影响发现,人参皂苷Rb₂能促进细胞的增殖。结论 人参皂苷Rb₂能与VEGF受体结合,且显著促进ECV304细胞增殖。     

HPLC法测定注射用益气复脉（冻干）中的4种低含量人参皂苷

目的 建立一种同时测定注射用益气复脉（冻干）（YQFM）中4种皂苷（人参皂苷Rf、人参皂苷Rc、人参皂苷Ro和人参皂苷Rg₅）含量的HPLC法。方法 用Pro Elut PLS固相萃取柱预先处理样品,然后采用Waters Symmetry C₁₈色谱柱（250 mm×4.6 mm,5 μm）,以乙腈-0.05%磷酸水溶液为流动相进行梯度洗脱,体积流量1.0 mL/min,柱温26℃,检测波长203 nm,进样量10 μL。结果 人参皂苷Rf（0.008 96~0.224 mg/mL）,人参皂苷Rc（0.009 84~0.246 mg/mL）,人参皂苷Ro（0.010 06~0.251 5 mg/mL）,人参皂苷Rg₅（0.002 61~0.026 1 mg/mL）线性良好;准确度试验（n=6）中,人参皂苷Rf、人参皂苷Rc、人参皂苷Ro和人参皂苷Rg₅的平均回收率分别为93.9%、91.8%、97.4%和87.8%;20批YQFM中人参皂苷Rf、人参皂苷Rc、人参皂苷Ro和人参皂苷Rg₅平均含量分别为0.233 3（0.195 2~0.271 1 mg/g）、0.438 4（0.325 8~0.497 1 mg/g）、0.739 6（0.615 8~0.910 8 mg/g）、0.066 1 mg/g（0.038 2~0.087 8 mg/g）。结论 所建立的方法简单可靠,可用于测定YQFM中人参皂苷Rf、人参皂苷Rc、人参皂苷Ro和人参皂苷Rg₅ 4种皂苷的含量测定。     

人参皂苷Rg1对H2O2诱导的HaCaT细胞氧化损伤保护作用研究

目的 观察人参皂苷Rg₁对H₂O₂诱导的HaCaT细胞氧化损伤保护作用,并探讨其机制。方法 体外培养HaCaT细胞, H₂O₂诱导细胞建立氧化应激损伤模型,分为空白组、H₂O₂损伤组、人参皂苷Rg₁保护组。细胞增殖与毒性检测试剂盒(CCK-8)检测细胞存活率,Hochest染色法检测细胞凋亡情况,活性氧检测试剂盒测定细胞活性氧(ROS)水平,Western blot检测细胞中caspase-3、caspase-6、caspase-8、GAPDH蛋白表达。结果 H₂O₂诱导HaCaT细胞半数抑制浓度为100 μg?mL^-1;与H₂O₂损伤组比较,5、10和15mg?L^-1人参皂苷Rg₁预处理后,HaCaT细胞存活率明显升高(P<0.05),细胞核皱缩损伤状态明显改善,细胞凋亡数量显著减少。同时,人参皂苷Rg₁预处理可显著降低HaCaT细胞ROS水平,下调凋亡相关标志蛋白-活化型caspase-3、caspase-6、caspase-8蛋白表达水平。结论 人参皂苷Rg₁对H₂O₂诱导的HaCaT细胞氧化应激损伤具有一定的保护作用,其机制可能与增强细胞清除自由基能力及抑制凋亡相关。     

基于近红外光谱分析技术的注射用益气复脉(冻干)红参醇提过程在线监测

目的 利用近红外光谱分析技术建立注射用益气复脉（冻干）主要原料红参醇提过程中3种单体皂苷——人参皂苷Rg₁、Re和Rb₁的定量模型,实现提取过程中关键指标的快速检测。方法 在线采集红参醇提过程的近红外光谱,以超高效液相色谱（UPLC）法测定提取过程药液中人参皂苷Rg₁、Re和Rb₁的量为参考值,采用偏最小二乘法建立光谱与测定值之间的定量校正模型,进而对提取过程进行在线分析。结果 人参皂苷Rg₁和Re的建模波段均为9 403.7～7 498.3 cm^-1和6 102～5 446.3 cm^-1组合波段;人参皂苷Rb₁的建模波段为5 774.1～5 446.3 cm^-1。人参皂苷Rg₁、Re、Rb₁定量模型的交叉验证决定系数（R²）分别为99.40、99.44、99.41,交叉验证均方根误差分别为5.18、2.77、11.00。结论 所建立的3种单体皂苷定量模型预测性能良好,能够有效测定红参醇提过程中人参皂苷Rg₁、Re和Rb₁的量。     

HPLC法测定人参三七颗粒中人参皂苷Rg1、Re、Rb1和三七皂苷R1的含量

摘 要 目的: 建立同时测定人参三七颗粒中人参皂苷Rg₁、Re、Rb₁和三七皂苷R₁含量的方法。方法: 采用HPLC法,色谱柱为Sunfire C₁₈（150 mm×4.6 mm,5 μm）分析柱,流动相以乙腈-水梯度洗脱;检测波长为203 nm;柱温30℃;流速1.0 ml·min^-1。结果:人参皂苷Rg₁,Re,Rb₁和三七皂苷R₁之间有较好的分离度,4种成分在线性范围内与峰面积之间线性关系良好,人参皂苷Rg₁、Re、Rb₁和三七皂苷R₁加样回收率分别为99.83%,97.84%,98.43%,97.34%,RSD分别为2.08%,1.66%,1.73%和1.42%（n=5）。结论:本方法可同时测定人参三七颗粒中的人参皂苷Rg₁,Re,Rb₁和三七皂苷R₁含量。     

UFLC法测定复方丹参片中三七皂苷R1、人参皂苷Rg1及Rb1

目的 探索建立超快速液相色谱（UFLC）法测定复方丹参片中三七皂苷R₁、人参皂苷Rg₁及Rb₁。方法 采用SHIMADZU Shim-pack XR-ODS Ⅲ（2.0 mm×75 mm, 1.6 μm）色谱柱;以乙腈-水作为流动相进行梯度洗脱;体积流量为0.4 mL/min;检测波长为203 nm;进样体积为3 μL。结果 三七皂苷R₁、人参皂苷Rg₁及Rb₁分别在0.025 7～0.257 0、0.101 2～1.012 0、0.104 4～1.044 0 μg与峰面积呈良好的线性关系,平均加样回收率分别为96.7%、98.1%、98.8%。结论 本方法在15min内可以将三七皂苷R₁、人参皂苷Rg₁及Rb₁有效分离,节省了大量人力和流动相的消耗,为中药的质量控制技术提供参考方法。     

Preferential induction of CYP1A1 and CYP1B1 in CCSP-positive cells.

Both benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands of aryl hydrocarbon receptors (AhR). Although animal studies indicate that both compounds induce pathological changes in the peripheral lung, the specific cell type involved remains unclear. Clara cells, expressing Clara cell specific protein (CCSP) and abundant in cytochrome P450, are nonciliated bronchiolar epithelial cells in the peripheral lung. Here we explore the hypothesis that CCSP-positive Clara cells are highly responsive to AhR ligands and are the primary cell type involved in BaP- and TCDD-induced toxicities. The responsiveness to AhR ligands was evaluated by measuring the respective mRNA and protein levels of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) using real-time RT-PCR and immunocytochemistry assays. Two in vitro models were used: primary cultures of human small airway epithelial (SAE) cells and rat lung slice cultures. In the presence of calcium, human SAE cells differentiated into CCSP-positive cells. BaP- and TCDD-induced mRNA and protein levels of CYP1A1 and CYP1B1 levels were significantly elevated in CCSP-positive cell cultures. Similarly, AhR mRNA and protein levels were increased in CCSP-positive cell cultures, as determined by real-time RT-PCR and Western blot analysis. When rat lung slice cultures were treated with BaP or TCDD for 24 h, CYP1A1 and CYP1B1 proteins were strongly induced in Clara cells. These results indicate that, in the peripheral lung of both rats and humans, CCSP-positive cells (Clara cells) may be more sensitive to AhR ligands than other cell types.     

有机阴离子转运多肽1B1的遗传药理学进展

人体存在多种类型的药物转运体,对于药物的吸收、分布和排泄起重要作用。参与药物跨膜转运的转运体功能受影响,将可能导致诸多临床药物的疗效、毒副作用甚至药物相互作用的发生。在各种影响因素中,遗传多态性所起的作用最为重要,可导致基因表达和蛋白功能发生改变。目前,阐明转运体基因的多态性以及基因型与表型之间的相互关系已成为应用遗传信息指导临床个体化用药的必要步骤。本文就肝脏有机阴离子转运多肽1B1（OATP1B1[OATP-C],编码基因SLCO1B1）基因多态性对药代动力学和药效动力学的影响及其临床意义等方面的进展作一综述。     

Sulfation of resveratrol in human liver: Evidence of a major role for the sulfotransferases SULT1A1 and SULT1E1

Sulfation of resveratrol, a polyphenolic compound present in grapes and wine with anticancer and cardioprotective activities, was studied in human liver cytosol. In the presence of 3′-phosphoadenosine-5′-phosphosulfate, three metabolites (M1–3) whose structures were identified by mass spectrometry and NMR as trans-resveratrol-3-O-sulfate, trans-resveratrol-4′-O-sulfate, and trans-resveratrol-3-O-4′-O-disulfate, respectively. The kinetics of M1 formation in human liver cytosol exhibited an pattern of substrate inhibition with a K_i of 21.3?±?8.73?µM and a V_max/K_m of 1.63?±?0.41?µL?min^?1mg^?1 protein. Formation of M2 and M3 showed sigmoidal kinetics with about 56-fold higher V_max/K_m values for M3 than for M2 (2.23?±?0.14 and 0.04?±?0.01?µL?min^?1?mg^?1). Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 is almost exclusively catalysed by SULT1A1 and only to a minor extent by SULT 1A2, 1A3 and 1E1, whereas M2 is selectively formed by SULT1A2. M3 is mainly catalysed by SULT1A2 and 1A3. In conclusion, the results elucidate the enzymatic pathways of resveratrol in human liver, which must be considered in humans following oral uptake of dietary resveratrol.     

Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium.

Endometriosis is a debilitating disease estimated to affect 10% of reproductive-age women and characterized by the growth of endometrial tissue outside of the uterus. The present study characterizes a human endometrial explant culture model for studying the direct effects of TCDD exposure by assessing the expression of CYP1A1 and CYP1B1 mRNA (Northern blotting), protein (Western blotting), and activity (7-ethoxyresorufin-O-deethylase; EROD) in explants cultured with and without TCDD. Explants were obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. The explants were cultured with 10 nM estradiol (E(2)) or 1 nM E(2) plus 500 nM progesterone (P(4)) with or without TCDD (first 24 h). The expression of CYP1A1 and CYP1B1 mRNA was greatest with 10 nM TCDD and increased up to 72 h after initial exposure. EROD activity increased up to 120 h. Explants from a secretory phase biopsy became reorganized in culture and formed a new epithelial membrane, while maintaining basic endometrial morphology and viability for up to 120 h. At 24 h, TCDD significantly increased CYP1A1 and CYP1B1 mRNA, and at 72 h, TCDD significantly increased EROD activity and CYP1B1 protein compared to explants cultured without TCDD for similar times. CYP1B1 protein also exhibited substantial constitutive expression that was similar in uncultured biopsies, where CYP1B1 protein was immunolocalized in the cytoplasm of epithelial glands, with only occasional patches of protein in the surface epithelial membrane. In explants cultured with and without TCDD exposure, CYP1B1 protein was localized in the cytoplasm of the new surface epithelial membrane and glands closest to the surface. CYP1A1 protein was not detected in uncultured biopsies or explants. Both younger age (age 30 and under) and proliferative phase were associated with higher TCDD-induced EROD activity in specimens treated with E(2):P(4). No significant endometriosis-related differences were observed for any of the biomarkers, but the detection of disease-specific change was limited by small sample size and variability in tissue-cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.     

Potent inhibition of human cytochrome P450 1B1 by tetramethoxystilbene

Human cytochrome P450 1B1 (CYP1B1) is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17β-estradiol (E2) to 4-hydroxy E2 by CYP1B1 has been postulated to be an important factor in mammary carcinogenesis. The inhibition of recombinant human CYP1B1 by 2,2′,4,6′-tetramethoxystilbene (TMS) was investigated using either the Escherichia coli membranes of recombinant human CYP1B1 coexpressed with human NADPH-P450 reductase or using purified enzyme. 2,2′,4,6′-TMS showed potent and selective inhibition of ethoxyresorufin O-deethylation (EROD) activity of CYP1B1 with IC₅₀ values of 2 nM. 2,2′,4,6′-TMS exhibited 175-fold selectivity for CYP1B1 over CYP1A1 (IC₅₀, 350 nM) and 85-fold selectivity for CYP1B1 over CYP1A2 (IC₅₀, 170 nM). However, inhibition of human NADPH-P450 reductase activity by 2,2′,4,6′-TMS was negligible. The modes of inhibition by 2,2′,4,6′-TMS were noncompetitive for CYP1A1 and CYP1B1. Moreover, 2,2′,4,6′-TMS significantly suppressed EROD activity and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 or CYP1B1 gene expression in human tumor cells such as HepG2 and MCF-10A. Taken together, our results indicate that 2,2′,4,6′-TMS is a potently selective inhibitor of human CYP1B1 as well as a suppressor of CYP1B1 expression and may be a valuable tool for determining enzyme properties of human CYP1B1.     

甲型H1N1流感疫苗不良事件监测及评估

目的:分析甲型H1N1流感疫苗的接种情况,及时总结接种的监测与评估,为开展甲型H1N1流感防控提供参考。方法:对国内外2009年接种甲型H1N1流感疫苗的不良反应监测报告进行分析。结果:甲型H1N1流感疫苗被认为与季节性流感疫苗总体安全性近似。结论:在甲型H1N1流感大流行的背景下,接种该疫苗利大于弊。     

甲流期间469例发热病例血常规分析

目的:了解发热病人的血常规特点,做好甲流期间感染性疾病的预诊断。方法:选取我门诊2009年5月～12月期间发热分诊处接诊的病例469例,分析其体温和血常规的关系。结果:血常规分析显示,甲流期间发热患者随着体温逐渐升高,中性粒细胞百分比(NC)增高的比例显著升高,白细胞计数(WBC)和淋巴细胞百分比(LC)变化无统计学意义。结论:甲流期间,发热患者体温越高,细菌感染的趋势越大,而WBC和LC变化无特异性。     

Pharmacological characterization of A-131701, a novel α1-adrenoceptor antagonist selective for α1A- and α1D-compared to α1B-adrenoceptors

New compounds selective for α_1A-adrenoceptors in the prostate may offer enhanced efficacy for benign prostatic hyperplasia (BPH), with fewer side effects than current treatment. A-131701 (3-[2-((3aR,9bR)-cis-6-methoxy-2,3,3a,4,5,9b,hexahydro-[1H]-benz[e]isoindol-2-yl)ethyl]pyrido[3′,4′:4,5]thieno [3,2-d]pyrimidine-2,4(1H,3H)-dione), from a novel class of benz[e]isolindole pyridothienopyrimidines and pyridothienopyrazines, is selective for α_1a- and α_1d-adrenoceptors in radioligand binding studies (0.22 nM at α_1a-, 0.97 nM at α_1d-) compared to α_1b-sites (2.5 nM) and in isolated tissue bioassays (pA₂ values of 8.9–9.0 for α_1A-receptors in rat vas deferens or canine prostate strips, 9.1 at α_1D-sites (rat aorta)), compared to 7.9 at α_1B-sites (rat spleen). A-131701 also potently blocked radioligand binding to α₁-adrenoceptors in canine and human prostatic membranes, but was considerably weaker at α₂-adrenoceptors. In isoflurane-anesthetized dogs, A-131701 antagonized epinephrine-induced increases in intraurethral pressure (IUP) with a pseudo-pA₂ value of 8.17. In spontaneously hypertensive rats, A-131701 caused transient decreases in mean arterial blood pressure (MABP) and transient tachycardia. The area under the curve (AUC₀→₆₀ min) for the hypotensive response was dose-related, with a log index value for A-131701 of 5.33, suggesting a selectivity of >600-fold comparing IUP to MABP effects. In pentobarbital-anesthetized dogs, A-131701 was more potent in blocking phenylephrine (PHE)-induced increases in IUP (pseudo-pA₂ = 8.0) compared to concurrently measured MABP (pseudo-pA₂ = 7.2), or sixfold selective. Doses greater than 1,000 nmol/kg i.v. of A-131701 were required to lower blood pressure by 10 mm Hg in these dogs (pED₁₀ =. 5.57), indicating a uroselectivity ratio of >250, superior to doxazosin, terazosin, or tamsulosin. Thus, A-131701 is selective for α_1A- and α_1D- vs. α_1B-adrenoceptors in vitro, and prostatic function vs. blood pressure effects in vivo, which may provide therapeutic advantages in the treatment of BPH. Drug Dev. Res. 44:140–162, 1998. © 1998 Wiley-Liss, Inc.