Temporary and partial inhibition of platelets by SM-20302 prevents coronary artery thrombosis in a chronic canine model

A cDNA clone coding for the guinea pig leukotriene B₄ (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein–Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B₄ (K_d value of 0.4 nM) and were expressed at high levels (B_max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [³H]leukotriene B₄ specific binding at the recombinant guinea pig BLT receptor is leukotriene B₄>20-OH-leukotriene B₄>12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen-1-oic acid)>12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen-1-oic acid)>20-COOH-leukotriene B₄>U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol)leukotriene C₄=leukotriene D₄=leukotriene E₄. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B₄ and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca²⁺ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of G₁₆ was required to achieve a robust Ca²⁺ driven response. Leukotriene B₄ was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B₄ analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B₄ receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.     

几种抗炎药对白三烯B4生物合成的影响

白三烯B₄为花生四烯酸5-脂氧酶代谢产物,是炎症反应中的重要介质。目前抑制白三烯B₄生物合成的药物尚不多见。本文建立了测定白细胞来源白三烯B₄的反相高效液相色谱法,并初步探讨了几种抗炎药对白三烯B₄生物合成的影响。结果表明阿斯匹林、消炎痛、炎痛喜康、氢化可的松及麝香第一色带对白三烯B₄生物合成几无影响,而氟灭酸可明显抑制白三烯B₄生成,提示它可能是5-脂氧酶抑制剂。     

UPLC法测定维生素B4片的含量

目的:建立UPLC法测定维生素B4片中维生素B4的含量的方法.方法:采用ACQUITY UPLC BEH Shield RP18(100 mm×2.1 mm,1.7 μm)色谱柱,流动相为0.02 mol·L-1磷酸二氢钾溶液-甲醇(90:10)(加0.5%的三乙胺,用H3PO4调pH至为4.0),流速为0.3 ml·min-1,柱温为50℃,检测波长为262 nm.结果:维生素B4在2.79～139.50 μg· ml-1浓度范围内,线性关系良好(r=0.999 8),平均回收率为100.2%,RSD为0.3%(n=9).结论:本方法简便准确,重复性好,可用维生素B4片的质量控制.     

Connective tissue mast cells are the target of formaldehyde to induce tracheal hyperresponsiveness in rats: Putative role of leukotriene B4 and nitric oxide

Formaldehyde (FA) exposure induces upper airways irritation and respiratory abnormalities, but its mechanisms are not understood. Since mast cells are widely distributed in the airways, we hypothesized that FA might modify the airways reactivity by mechanism involving their activation. Tracheal rings of rats were incubated with Dulbecco's modified medium culture containing FA (0.1 ppm) in 96-well plastic microplates in a humid atmosphere. After 30 min, 6 h, and 24–72 h, the rings were suspended in an organ bath and dose–response curve to methacholine (MCh) were determined. Incubation with FA caused a transient tracheal hyperresponsiveness to MCh that was independent from tracheal epithelium integrity. Connective tissue mast cell depletion caused by compound 48/80 or mast cell activation by the allergic reaction, before exposure of tracheal rings to FA prevented the increased responsiveness to MCh. LTB₄ concentrations were increased in the culture medium of tracheas incubated with FA for 48 h, whereas the LTB₄-receptor antagonist MK886 (1 μM) added before FA exposure rendered the tracheal rings normoreactive to MCh. In addition, FA exposure did not cause hyperresponsiveness in tracheal segments incubated with l-arginine (1 μM).     

HPLC法测定复方吡拉西坦脑蛋白水解物片中维生素B1、B2和B6含量

摘 要 目的:建立HPLC法测定复方吡拉西坦脑蛋白水解物片中维生素B₁、维生素B₂和维生素B₆含量的方法。方法: 采用Insteril ODS-3色谱柱（250 mm×4.6 mm,5 μm）,流动相：0.01 mol·L^-1庚烷磺酸钠（含0.25%三乙胺,用冰醋酸调节pH至3.8)-甲醇（75∶25）,柱温30℃,检测波长为280 nm,流速：1.0 ml·min^-1。结果: 维生素B₁、维生素B₂、维生素B₆分别在3.98～99.40 μg·ml^-1（r=0.999 7）、4.08～101.91μg·ml^-1（r=0.999 9）、2.08～52.00 μg·ml^-1（r=0.999 9）范围内线性关系良好,平均回收率分别为99.18%、99.53%、99.27%,RSD分别为0.60%、0.67%、0.71%(n=9)。结论:本法简便、快速、准确,可用于复方吡拉西坦脑蛋白水解物片中维生素B₁、维生素B₂和维生素B₆的含量测定     

Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheral blood mononuclear cells

Mycotoxins are secondary metabolites having a high cytotoxic potential. They are produced by molds and released in food and feed. To date, the mechanisms underlying the mycotoxin-induced cytotoxicity have not been fully clarified. The induction of oxidative stress, as a possible mechanism, has been postulated. This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB₁ (0, 5 and 20 μg/ml) and FB₁ (0, 35 and 70 μg/ml). Reactive oxygen metabolites (ROM), intracellular thiols (SH), malondialdehyde (MDA) and gene expression of cytoplasmic superoxide dismutase (SOD) and glutathione peroxidase (GSHPX-1) were measured on PBMC after incubation. The highest concentration of AFB₁ and all concentrations of FB₁ caused an increase (p < 0.05) of intracellular ROM without any time dependent effect. Intracellular SH decreased with 20 μgAFB₁/ml (p < 0.05) and the effect was particularly marked after 7 days of exposure. Intracellular SH were not affected by FB₁ even though a lower (p < 0.05) SH level after 2 days exposure than after 7 days was observed. MDA increased (p < 0.05) in AFB₁ or FB₁ treated PBMC. The exposure to FB₁ for 7 days increased MDA (p < 0.05) only in cells treated with 70 μg/ml. Exposure of PBMC to AFB₁ reduced SOD mRNA while FB₁ decreased both SOD and GSHPX-1 mRNA abundance. These results demonstrate that, even though by different mechanisms, AFB₁ and FB₁ may induce cytotoxicity through an impairment of the oxidative status of PBMC.     

高效液相色谱法测定赖氨肌醇维B12口服溶液中维生素B12及肌醇的含量

目的:建立赖氨肌醇维B12口服溶液中维生素B12和肌醇的含量测定方法.方法:采用高效液相色谱法,紫外及示差折光检测器,ODS柱和氨基柱分别测定维生素B12和肌醇含量.流动相分别为乙腈-0.05 mol·L-1磷酸二氢钾溶液(13∶87)用磷酸调节pH至3.0,乙腈-水(70∶30);检测波长分别为 361 nm 和 210 nm;流量:1.0 mL·min-1;进样体积:20 μL.结果:维生素B12和肌醇浓度分别在1.52～6.08 μg·mL-1和5.02～20.06 mg·mL-1范围内与峰面积呈良好的线性关系,平均回收率(n=9)分别为99.5%(RSD=0.76%)和99.4%(RSD=1.39%).结论:本法专属性强,结果准确,重现性好,适用于赖氨肌醇维B12口服溶液中维生素B12和肌醇的含量测定.     

高效液相色谱-光化学衍生法测定湖南产莲子中黄曲霉毒素G1、G2、B1、B2

目的对不同来源的湖南产莲子中黄曲霉毒素G_1、G_2、B_2、B_1进行高效液相色谱-光化学衍生法测定。方法采用高效液相色谱–光化学衍生法,采用岛津GL Inertsil ODS-3色谱柱(250 mm×4.6 mm,5μm),流动相为甲醇–乙腈–水(35∶13∶52),柱温35℃;光化学衍生器(254 nm)激发波长λ_(ex)=360 nm,发射波长λ_(ex)=450 nm。结果黄曲霉毒素G_1、G_2、B_2、B_1分别在6.7～33.3、11.4～56.8、10.3～51.5、4.1～20.3 pg线性关系良好;平均回收率分别为98.07%、97.72%、96.11%、99.52%,RSD值分别为1.69%、1.40%、2.72%、1.34%(n=6)。9批莲子样品中,有6批未检出黄曲霉毒素,来自于农贸市场农户自存的2批次检出黄曲霉毒素B_1,实验室塑料袋包装储存1年的1批检出黄曲霉毒素B_1、B_2,但质量分数均低于法定标准限量。结论不同来源湖南产莲子中黄曲霉毒素质量分数均低于《中国药典》2020年版规定限量。该法测定结果准确、重复性良好,可为完善莲子安全性控制和质量标准提升提供实验依据。     

高效液相色谱-荧光检测法测定沉香中黄曲霉毒素B1、B2、G1、G2

目的建立高效液相色谱–光化学衍生–荧光检测法测定沉香药材中黄曲霉毒素B1、B2、G1、G2。方法采用高效液相色谱法,通过免疫亲和柱提取和净化,荧光检测器检测。Agilent Zorbax Ecilpse Plus C18色谱柱(250 mm×4.6 mm,5μm);流动相:甲醇–水(45∶55);体积流量:0.8 m L/min;柱温:30℃;进样盘温度:4℃;荧光激发波长为360 nm,发射波长为450 nm。结果黄曲霉毒素B1、B2、G1、G2分别在9.3~74.4、3.0~24.0、9.3~74.4、3.5~28.0 pg线性关系良好,r均大于0.998 0;检测限分别为1.86、0.60、1.86、0.70 pg,定量限分别为7.44、2.40、7.44、2.80 pg。平均回收率分别为78%、92%、82%、99%,RSD值分别为4.4%、3.0%、4.3%、2.8%。结论所建立的方法结果准确、重复性、稳定性均良好,可用于沉香药材中黄曲霉毒素的质量控制。     

The endocannabinoid anandamide inhibits kinin B1 receptor sensitization through cannabinoid CB1 receptor stimulation in human umbilical vein

The possible inhibition of kinin B₁ receptor up-regulation by arachidonoylethanolamide (anandamide) was evaluated in isolated human umbilical vein. Anandamide and its metabolically stable analogue, R-N-(2-Hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (R-(+)-methanandamide), produced a selective and dose-dependent inhibition of kinin B₁ receptor-sensitized contractile responses. The inhibitory effect of anandamide on B₁ receptor-sensitized responses failed to be modified either by 5-biphenyl-4-ylmethyl-tetrazole-1-carboxylic acid dimethylamide (LY2183240), a selective anandamide uptake inhibitor, or 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl) methanone (AM630), selective cannabinoid CB₂ receptor antagonist. However, the cannabinoid CB₁ receptor antagonist, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), abolished anandamide effects on kinin B₁ receptor sensitization. The present results provide strong pharmacological evidence indicating that endocannabinoid anandamide inhibits kinin B₁ receptor up-regulation through cannabinoid CB₁ receptor stimulation in human umbilical vein.     

Evidence for the participation of kinins in Freund's adjuvant-induced inflammatory and nociceptive responses in kinin B1 and B2 receptor knockout mice

Experiments were designed to investigate the role of kinin B₁ and B₂ receptors in Freund's adjuvant (CFA)-induced inflammation and nociception responses by the use of B₁ and B₂ null mutant mice. Intradermal (i.d.) injection of CFA produced time-dependent and marked hyperalgesic responses in both ipsilateral and contralateral paws of wild-type mice. Gene disruption of the kinin B₂ receptor did not interfere with CFA-induced hyperalgesia, but ablation of the gene of the B₁ receptor reduced the hyperalgesia in both ipsilateral (48±13%, at 12 h) and contralateral (91±22%, at 12 h) paws. Treatment of wild-type mice with the selective B₁ antagonist des-Arg⁹-[Leu⁸]-BK (150 nmol/kg, s.c.) reduced CFA-evoked thermal hyperalgesia, to an extent which was similar to that observed in mice lacking kinin B₁ receptor. I.d. injection of CFA produced a time-related and long-lasting (up to 72 h) increase in paw volume in wild-type mice. A similar effect was observed in B₁ knockout mice. In mice lacking B₂ receptor, the earlier stage of the CFA-induced paw oedema (6 h) was significantly greater compared with the wild-type animals, an effect which was almost completely reversed (76±5%) by des-Arg⁹-[Leu⁸]-BK. This data demonstrates that kinin B₁ receptor, but not B₂ receptor, exerts a critical role in controlling the persistent inflammatory hyperalgesia induced by CFA in mice, while B₂ receptor appears to have only a minor role in the amplification of the earlier stage of CFA-induced paw oedema formation. The results of the present study, taken together with those of previous studies, suggest that B₁ receptor antagonists represent a potential target for the development of new drugs to treat persistent inflammatory pain.     

HPLC法测定四维钙片中维生素C、B1、B2的含量

目的：建立高效液相色谱法测定四维钙片中维生素C、维生素B1和维生素B2含量的方法.方法：采用SHISEIDOC18（250 mm×4.6 mm,5μm）色谱柱,以0.05％磷酸溶液（含0.005 mol·L-1己烷磺酸钠）-乙腈（88：12）为流动相,流速：1.0ml· min-1,检测波长：244 nm,柱温：30℃,进样量：20μm.结果：维生素C、维生素B1和维生素B2的线性范围分别为7.52～90.24 μg· ml-1（r=1.000 0）,1.32 ～15.82 μg·ml-1（r=1.000 0）和1.27～15.24μg·ml-1 （r=1.000 0）;平均回收率分别为101.63％、99.77％和100.25％,RSD分别为1.51％、1.48％和1.84％（n=9）.结论：本法操作简便,结果准确、可靠,可用于四维钙片中维生素C、B1、B2的含量测定.     

白头翁皂苷B4对咪喹莫特诱导的小鼠银屑病的治疗作用及其机制研究

目的 考察白头翁皂苷B₄对咪喹莫特诱导的银屑病模型小鼠的炎症及免疫相关指标的影响,探讨其对小鼠银屑病的治疗作用及其机制。方法 将56只BALB/c小鼠随机分成对照组,模型组,阿维A胶囊组（6 mg/kg）,白头翁皂苷B₄低、中、高剂量（5、10、20 mg/kg）组,白头翁皂苷B₄乳膏组（20 mg/kg）,每组各8只。通过局部涂抹5%咪喹莫特乳膏诱导小鼠银屑病模型,各组小鼠口服或涂抹相应药物,14 d后通过银屑病皮损面积及严重程度评分（PASI）评价小鼠皮损程度;HE染色观察皮肤组织病理学形态;血常规检测血液中免疫细胞的变化;酶联免疫吸附试剂（ELISA）检测小鼠皮肤中白细胞介素（IL）-6、IL-12、IL-17、IL-1β的水平。结果 与模型组相比,阿维A胶囊组,白头翁皂苷B₄ 5、10、20 mg/kg组,白头翁皂苷B₄乳膏组小鼠皮损组织棘层厚度减小,真皮层炎症细胞浸润减少,PASI评分、血液中的免疫细胞及皮肤组织中IL-6、IL-12、IL-17、IL-1β的表达水平均显著降低。结论 白头翁皂苷B₄对5%咪喹莫特诱导小鼠银屑病皮损具有明显的治疗作用,其机制可能是通过抑制IL-6、IL-12、IL-17、IL-1β等细胞因子表达发挥治疗作用。     

白头翁皂苷B4栓剂改善大鼠痛风药效学研究

目的 研究白头翁皂苷B₄栓剂抗痛风作用。方法 应用混合脂肪酸甘油酯制备白头翁皂苷B₄栓剂;使用尿酸钠诱导大鼠急性痛风性关节炎模型,观察白头翁皂苷 B₄栓剂（9、18、36 mg·kg^-1,直肠给药）对大鼠踝关节肿胀度的影响,试剂盒法检测血清炎症因子单核细胞趋化蛋白-1（MCP-1）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）水平,HE染色后观察踝关节组织病理学变化;使用氧嗪酸钾诱导大鼠急性高尿酸血症,给予别嘌醇（阳性药,22 mg·kg^-1,ip给药）和白头翁皂苷 B₄栓剂低 、中 、高剂量（9、18、36 mg·kg^-1）,试剂盒法检测大鼠血清中尿酸、黄嘌呤氧化酶（XOD）、尿素氮（BUN）水平,检测肝脏组织中尿酸、BUN、肌酐（CRE）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶（CAT）水平,HE染色后检测肾脏组织病理学变化;使用角叉菜胶诱导大鼠足肿胀建立炎症动物模型,足容积测量仪观察白头翁皂苷 B₄栓剂对肿胀率的影响;使用甲醛诱导小鼠致痛模型,观察白头翁皂苷 B₄栓剂（13、26、52 mg·kg^-1）对舔足次数的影响。结果 与模型组比较,白头翁皂苷 B₄栓剂能够显著抑制尿酸钠诱导急性痛风性关节炎大鼠的关节肿胀（P＜0.05、0.01、0.001）,降低血清炎症因子 MCP-1、TNF-α、IL-6、IL-1β水平（P＜0.05、0.01、0.001）;降低氧嗪酸钾诱导的高尿酸血症大鼠血清尿酸、XOD、BUN水平,降低肝脏中尿酸、BUN、CRE和MDA水平,升高肝脏中GSH-Px、CAT水平（P＜0.05、0.01、0.001）,改善肾脏组织病理改变;显著抑制角叉菜胶诱导大鼠足肿胀（P＜0.05）;显著减少甲醛诱导小鼠致痛模型的舔足次数（P＜0.01）。结论 白头翁皂苷B₄栓剂具有抗痛风性关节炎、抗急性高尿酸血症及抗炎镇痛作用。     

HPLC同时测定斑蝥酸钠维生素B6注射液中斑蝥酸钠和维生素B6的含量

目的 建立斑蝥酸钠维生素B₆注射液中斑蝥酸钠和维生素B₆含量的同时测定方法。方法 采用HPLC,色谱柱为Syncronis C₁₈（250 mm×4.6 mm,5 μm）,流动相为0.02 mol·L^-1磷酸二氢钾溶液（磷酸调pH值至2.6）-乙腈,梯度洗脱,流速为1.0 mL·min^-1,检测波长为193,290 nm,波长切换法,柱温：30℃。结果 斑蝥酸钠回归方程为Y=1.926X+0.010（r=0.999 7）,在0.064 6~0.322 8 mg·mL^-1内斑蝥酸钠与峰面积的线性关系良好,平均回收率为98.9%,RSD为1.2%（n=9）。维生素B₆回归方程为Y=24.153X+0.670（r=0.999 9）,在0.535 9~2.679 7 mg·mL^-1内维生素B₆与峰面积的线性关系良好,平均回收率为100.2%,RSD为1.0%（n=9）;仪器精密度、稳定性和重复性试验的RSD均<2.0%。结论 所建立的方法结果准确、重复性好,可用于斑蝥酸钠维生素B₆注射液的质量控制。     

The effects of CCK-4 on dopamine D1 agonist-induced grooming are blocked by a CCKA receptor antagonist: Evidence for a novel CCK receptor subtype?

The neuropeptide cholecystokinin (CCK) has been shown to interact with dopamine in various ways, including attenuation of dopamine D₁ receptor-mediated vacuous chewing and grooming. While we have demonstrated a clear role for the CCK_A receptor in the attenuation of dopamine D₁ agonist-induced vacuous chewing, studies of grooming yielded anomolous results. We examined the effects of selective CCK receptor antagonists on the attenuation of SKF 38393-induced grooming by the CCK_B agonist CCK-4. Administration of SKF 38393 (5 mg/kg s.c.) to male Sprague-Dawley rats resulted in a significant increase in grooming which was reduced to control levels by CCK-4 (20 mg/kg i.p.). Pretreatment with either the CCK_A receptor antagonist devazepide or the CCK_B receptor antagonist L-365,260 significantly attenuated this effect over a range of doses (20, 100, 500 μg/kg i.p.). The suppression of dopamine D₁ agonist-induced grooming by CCK-4 does not appear to reflect a non-specific effect of anxiogenesis, as it was unaffected by the anxiolytic diazepam. The CCK receptor antagonists alone were without behavioural effect. Taken together with previous studies in models of anxiety and analgesia, our findings lend further support to the hypothesis that CCK-4 may act at a novel receptor subtype.     

白头翁皂苷B4栓剂治疗大鼠痔疮的药效学研究

目的 探究白头翁皂苷B₄栓剂对冰醋酸、巴豆油及细菌所致痔疮模型大鼠的治疗作用。方法 应用混合脂肪酸甘油酯36型号和38型号制备含白头翁皂苷B₄ 5、10、20 mg·粒^-1的栓剂。分别用冰醋酸、巴豆油、细菌建立急性痔疮大鼠模型,每种模型制备完成后大鼠均随机分为模型组、空白栓剂组、马应龙麝香痔疮膏（阳性药,1 g·kg^-1）组和白头翁皂苷B₄栓剂（5、10、20 mg·粒^-1）组,每组10只,对照组未经任何处理。白头翁皂苷B₄栓剂各剂量组每天给药2次,给药3 d,共6次;马应龙麝香痔疮膏每天1次,给药3 d,共3次。3 d后取大鼠肛门组织,HE染色后比较各组大鼠肛周组织病理形态学变化;血常规检测仪检测血液中白细胞（WBC）、中性粒细胞（NEUT）、淋巴细胞（LYMPH）水平;ELISA法检测大鼠血清和肛门组织中白细胞介素-6（IL-6）、IL-1β、肿瘤坏死因子-α（TNF-α）水平。结果 与模型组比较,白头翁皂苷B₄栓剂明显改善大鼠肛周组织充血肿胀及肛周组织病变,显著降低血液中WBC、LYMPH和NEUT水平,显著抑制血清和组织中IL-6、IL-1β和TNF-α水平（P＜0.05、0.01、0.001）。结论 白头翁皂苷B₄栓剂能有效改善急性痔疮模型大鼠的组织病变,调节免疫细胞,抑制炎症因子反应,对急性痔疮具有治疗作用。     

Leukotriene B4 receptor antagonists as therapeutics for inflammatory disease: preclinical and clinical developments

Leukotriene B₄ (LTB₄) is a lipid inflammatory mediator derived from membrane phospholipids by the sequential actions of cytosolic phospholipase A2 (PLA2), 5-lipoxygenase (5-LO) and leukotriene A₄ (LTA₄) hydrolase. Several inflammatory diseases, including asthma, chronic obstructive pulmonary disease, arthritis and inflammatory bowel disease, have been associated with elevated levels of LTB₄. As a result, pharmacological strategies to modulate the synthesis of LTB₄ (inhibition of PLA2, 5-LO or LTA₄ hydrolase) or the effects of LTB₄ itself (antagonism of LTB₄ receptors) are being developed by several companies. Two G-protein-coupled receptors mediate the effects of LTB₄, namely BLT1 and BLT2. The pharmacology, expression and function of these two receptors were last reviewed by Tager and Luster in 2004. Since then, there has been an increased understanding of the function of these receptors, in particular for the lesser understood of the two receptors, BLT2. Furthermore, since last reviewed in 1996, there have been several clinical developments in the use of BLT receptor antagonists for inflammatory diseases. This review summarizes the latest preclinical and clinical developments in BLT antagonism for inflammatory diseases and discusses potential future developments.     

Blockade of LTB4-induced chemotaxis by bioactive molecules interfering with the BLT2-Gαi interaction

BLT2, a low-affinity leukotriene B₄ (LTB₄) receptor, is a member of the G-protein coupled receptor (GPCR) family and is involved in the pathogenesis of inflammatory diseases such as asthma. Despite its clinical implications, however, no pharmacological inhibitors are available. In the present study, we screened for small molecules that interfere with the interaction between the third intracellular loop region of BLT2 (BLT2_iL3) and the Gα_i3 protein subunit (Gα_i3), using a high-throughput screening (HTS) assay with a library of 1040 FDA-approved drugs and bioactive compounds. We identified two small molecules—purpurin [1,2,4-trihydroxy-9,10-anthraquinone; IC₅₀ = 1.6 μM for BLT2] and chloranil [tetrachloro-1,4-benzoquinone; IC₅₀ = 0.42 μM for BLT2]—as specific BLT2-blocking agents. We found that blockade of the BLT2_iL3-Gα_i3 interaction by these small molecules inhibited the BLT2-downstream signaling cascade. For example, BLT2-signaling to phosphoinositide-3 kinase (PI3K)/Akt phosphorylation was completely abolished by these molecules. Furthermore, we observed that these small molecules blocked LTB₄-induced chemotaxis by inhibiting the BLT2-PI3K/Akt-downstream, Rac1-reactive oxygen species-dependent pathway. Taken together, our results show that purpurin and chloranil interfere with the interaction between BLT2_iL3 and Gα_i3 and thus block the biological functions of BLT2 (e.g., chemotaxis). The present findings suggest a potential application of purpurin and chloranil as pharmacological therapeutic agents against BLT2-associated inflammatory human diseases.     

白头翁皂苷B4治疗大鼠子宫肌瘤的作用机制研究

目的 基于大鼠体内实验以及网络药理学探讨白头翁皂苷B₄治疗子宫肌瘤的作用机制。方法 大鼠按体质量随机分为 6组：对照组、模型组、米非司酮（阳性药,1.25 mg·kg^-1）组和白头翁皂苷B₄低、中、高剂量（5、10、20 mg·kg^-1）组,每组 8只。采用雌激素负荷法制备子宫肌瘤模型,于造模成功后ip给药,对照组及模型组每天ip等量0.9%氯化钠溶液。通过对大鼠一般行为学的观察、子宫组织的明场以及病理HE染色切片观察、ELISA试剂盒检测血清与子宫组织中雌二醇、孕酮、雌激素受体的表达水平,明确白头翁皂苷 B₄对大鼠子宫肌瘤是否有治疗作用;通过网络药理学收集白头翁皂苷 B₄与子宫肌瘤的交互靶点,并对其进行蛋白质-蛋白质相互作用（PPI）网络信息的构建以及基因功能富集分析,并将靶点以及药物进行AutoDockVina分子对接分析,确定作用靶点;最终对组织进行相关靶点Bcl-2、Bax、Caspase-3蛋白水平的Western blotting检测进行验证。结果 体内实验结果显示,与模型组比较,白头翁皂苷B₄组大鼠子宫形态明显好转,子宫指数显著降低（P＜0.001）;HE染色显示,白头翁皂苷B₄组大鼠子宫平滑细胞排列整齐,形态正常,色泽均一,肌层炎症细胞浸润减少 ;血清及子宫组织中雌二醇、孕酮、雌激素受体的水平显著降低（P＜0.05、0.01、0.001）。通过Pubchem数据库筛选得到白头翁皂苷B₄相关作用靶点23个,与子宫肌瘤共同作用靶点8个,富集得到69条KEGG信号通路,主要包括细胞凋亡、脂质和动脉粥样硬化、IL-17、NF-κB、小细胞肺癌、MAPK等相关信号通路。AutoDock Vina结果显示白头翁皂苷 B₄与关键靶点 BCL2、BAX、Caspase3结合良好。与模型组比较,白头翁皂苷 B₄中、高剂量组 Bax、Caspase-3蛋白水平显著升高（P＜0.001）,Bcl-2蛋白水平显著降低（P＜0.001）。结论 白头翁皂苷B₄可能通过BCL2、BAX、Caspase-3等关键靶点诱导凋亡,同时降低雌二醇、孕酮、雌激素受体的表达,从而治疗子宫肌瘤。