青藤碱对脂多糖诱导的神经细胞环氧化酶-2表达的影响

目的 :了解青藤碱 (sinomenine ,Sin)对PC 12细胞增殖和表达环氧化酶-2 (COX-2 )及合成前列腺素E₂(PGE₂ )的影响 ,探讨Sin对神经细胞的作用机制。方法 :采用NGF诱导培养PC-12细胞 ,用不同浓度的Sin(0,3×10^-6,30×10^-6,150×10^-6mol·L^-1)预处理后 ,加或不加脂多糖 (LPS) ,分别用³H-TdR法、竞争ELISA、半定量逆转录 聚合酶联反应 (RT-PCR)法、细胞酶联免疫法及Western blot法 ,检测PC-12细胞的增殖活性、细胞培养上清液中PGE₂ 的水平、细胞COX-2 mRNA及蛋白表达水平。同时提取各组细胞蛋白 ,测定其核转录因子NF κB活性。结果 :Sin对LPS诱导的PC-12细胞的COX-2 mRNA、蛋白表达具有不同程度的抑制作用 ,与Sin浓度呈明显正相关性,且对LPS激活的PC-12细胞的核转录因子NF-κB活性有明显的抑制作用。实验未观察到Sin对PC-12细胞增殖的影响。结论 :Sin可显著抑制LPS诱导的PC-12细胞COX-2的表达及其产物PGE₂ 的合成 ,其作用机制可能是通过Sin抑制PC-12细胞核转录因子NF-κB活性而实现。     

青藤碱抑制H2O2诱导乳鼠心肌细胞凋亡

目的:探讨青藤碱对H₂O₂诱导乳鼠心肌细胞凋亡的影响及其可能的作用机制。方法:在原代培养的SD大鼠乳鼠心肌细胞上建立H₂O₂损伤模型,观察不同剂量青藤碱(10,30,100μmol.L^-1)对心肌细胞凋亡率、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、乳酸脱氢酶(LDH)活性及NF-κB蛋白表达的影响。结果:H₂O₂组与空白对照组相比,心肌细胞凋亡率显著增加(P<0.01),并随着时间的延长其凋亡率不断增高;青藤碱明显抑制H₂O₂所诱导各个时间段的心肌细胞凋亡率(P<0.01),降低MDA含量,增加SOD,LDH活性,抑制NF-κB蛋白表达。结论:青藤碱对H₂O₂诱导的乳鼠心肌细胞凋亡有抑制作用,其作用机制可能与其抗脂质氧化、抑制心肌细胞表达NF-κB有关。     

青藤碱对大鼠佐剂性关节炎治疗作用及机制的研究

目的:观察青藤碱对佐剂性关节炎的治疗作用和对继发性炎症区域一氧化氮(NO)及相关炎症介质的影响,进一步探讨其治疗类风湿性关节炎的可能作用机制。方法:用Freund’s完全佐剂诱导大鼠佐剂性关节炎,设立正常组、模型组、泼尼松组、青藤碱低、中、高剂量组(30,60,120 mg.kg^-1),记录各组大鼠足跖肿胀度和关节炎指数,检测大鼠关节浸液内NO,前列腺素E₂(PGE₂)及细胞因子白细胞介素1(IL-1),肿瘤坏死因子(TNF)水平。结果:与正常组比较,模型组大鼠关节浸液内NO,PGE₂及IL-1,TNF水平显著升高(P<0.05);与模型组相比,青藤碱组大鼠佐剂性关节炎继发病变显著受抑制,佐剂性关节炎大鼠关节浸液内NO,PGE₂及细胞因子IL-1,TNF水平显著降低(P<0.05)。结论:青藤碱对类风湿性关节炎的治疗作用与其抑制炎症局部区域细胞因子活性和降低炎症介质含量有关,其中青藤碱使NO水平下降可能是抗类风湿性关节炎的重要作用机制。     

固相萃取-薄层扫描法测定血清中青藤碱浓度

 目的:研究血清中青藤碱的液-固净化技术和在家兔体内的动力学特征。方法:自制硅胶萃取柱及萃取仪，将血清中青藤碱净化后，点样于硅胶G-硅胶GF₂₅₄ ( 2 : I )薄层板上，以氯仿-甲醇(19:2)为展开剂，在岛津CS-930上，用双波长(λ_s275 nm,λ_R 320 nm )锯齿法扫描测定。结果:血清中青藤碱的固相提取回收率为96.95%±s 7.59%,用薄层扫描法测绘了2只家兔静注青藤碱后的药物浓度-时间曲线。结论:自制萃取柱净化血清样品简便、有效;青藤碱的薄层扫描测定法灵敏、准确。     

青藤碱对人肝癌细胞HepG2增殖的抑制作用

目的:研究青藤碱对人肝癌HepG2细胞增殖的影响及其机制。方法:以不同浓度青藤碱处理体外培养的A549细胞,采用MTT法检测24h、48h、72h后HepG2细胞的增殖状态;流式细胞仪测定青藤碱处理过的细胞凋亡、细胞周期分布及凋亡率,相对定量RT-PCR测定Cox-2mRNA的表达、Western blot检测细胞凋亡相关基因Cox-2的表达。结果:青藤碱能显著抑制人肝癌HepG2细胞增殖,并具有时间和剂量依赖性。青藤碱处理组细胞早期凋亡率较对照组升高,呈时间剂量依赖性。相对RT-PCR结果显示其能下调HepG2细胞Cox-2mRNA的表达。Western blot检测Cox-2蛋白表达明显抑制。结论:青藤碱在体外能有效抑制肝癌细胞增殖。     

微透析法进行盐酸青藤碱肌注给药的药代动力学研究

目的 :微透析法进行盐酸青藤碱肌注给药的药代动力学研究。 方法 :微透析探针植入大鼠的右心室,实时、活体采集肌注盐酸青藤碱后的透析液样品,HPLC检测样品浓度,微透析样品浓度经校正后进行房室和非房室模型拟合。 结果 :盐酸青藤碱肌注后C_max为3.127 mg·L^-1,T_max为101 min,MRT为191 min,结果表盐酸青藤碱肌注后血药浓度呈一级速率、一室模型。 结论 :微透析法能较好的反映盐酸青藤碱肌注给药的药动学特征,微透析采样手段可作为药动学研究的理想手段。     

盐酸青藤碱缓释胶囊的家犬体内药动学和相对生物利用度研究

 目的研究口服盐酸青藤碱缓释胶囊在家犬体内药动学和相对生物利用度。方法以随机分组自身对照、间隔1周交叉试验设计法,用高效液相色谱法测定盐酸青藤碱的浓度,在6条健康家犬身上比较单剂量po 120 mg盐酸青藤碱缓释胶囊和正清风痛宁缓释片后的药动学参数和相对生物利用度。结果盐酸青藤碱缓释胶囊和正清风痛宁缓释片的药-时曲线相似,其t_1/2分别为(25.19±10.94)和(20.48±6.55)h,t_max分别为(8.67±1.63)和(5.7±1.51)h,ρ_max分别为(1 012.23±510.82)和(1 031.66±546.15)μg·L^-1,AUC_0-30分别为(13 797.04±4 915.68)和(15 507.17±6 263.07)μg·h·L^-1。结论盐酸青藤碱缓释胶囊和正清风痛宁缓释片具有等效性,盐酸青藤碱缓释胶囊t_max明显延后,t_1/2明显延长。     

盐酸青藤碱大鼠肠吸收实验研究

 目的研究盐酸青藤碱在大鼠不同肠段的吸收特性,为其剂型设计提供理论依据。方法以酚红为标示物,采用在体单灌流法进行肠吸收实验研究,高效液相色谱法测定灌流液中盐酸青藤碱和酚红的浓度,计算吸收性能参数。结果盐酸青藤碱在十二指肠、空肠、回肠、结肠的有效渗透系数(P_eff)分别为0.65±0.15,0.54±0.26,0.45±0.14,0.28±0.12(×10^-4cm·s^-1),表明该药在大鼠各肠段均有较好的吸收。结论盐酸青藤碱适于制成日服1次的缓控释制剂。     

邻甲氧基桂皮醛对IL-1刺激脑微血管内皮细胞COX活性及PGE2释放的影响

目的：观察桂枝汤有效部位A分离的邻甲氧基桂皮醛对发热相关的环氧酶（COX）和前列腺素E₂（PGE₂）的影响。方法：建立大鼠脑微血管内皮细胞（rCMEC）培养,进行Ⅷ因子相关抗原鉴定。待细胞生长至融合状态后加入不同含量的邻甲氧基桂皮醛（12.5,25,50,100,200 μg·mL^-1）孵育3 h,之后以30 ng·mL^-1的IL-1刺激12 h。ELISA方法检测细胞培养液中PGE2的含量及细胞内COX-1和COX-2的活性。结果：Ⅷ因子抗体免疫组化染色可见90％以上的培养细胞呈阳性,确认为rCMEC。暴露于30 ng·mL^-1 IL-1后,rCMEC内COX-2活性及释放的PGE₂量显著增加,COX-1活性变化无统计学差异。加入不同含量的邻甲氧基桂皮醛后,随含量增加可下调COX-1,COX-2活性及PGE₂量,且呈剂量依赖关系；至含量为200 μg·mL^-1时,COX-2活性及释放的PGE₂与IL-1单独作用组相比均有显著性差异,COX-1活性虽有所降低,但无统计学上的显著差异。结论：邻甲氧基桂皮醛能下调IL-1刺激rCMEC释放升高的PGE₂,作用机制可能与抑制COX-2活性有关。     

NF-κB在马钱子碱抑制非小细胞肺癌细胞环氧化酶2生成中的作用研究

目的:探讨马钱子碱抑制环氧化酶2(COX-2),从而诱导非小细胞肺癌细胞凋亡的分子机制。方法:构建COX-2启动子若干转录因子缺失突变体与含COX-2 mRNA 3’-UTR的报告质粒,与Renillia共转染至非小细胞肺癌A549细胞,测报告基因luciferase活性研究COX-2启动子受马钱子碱抑制的最小顺式作用元件;采用蛋白质免疫印迹法研究马钱子碱对IκBα磷酸化与p65进核的影响。结果:马钱子碱显著性抑制LPS诱导的COX-2启动子的激活,而对COX-2 mRNA转录后调控影响不大,COX-2启动子-262位附近NF-κB是马钱子碱抑制COX-2启动子活性的重要顺式作用元件。马钱子碱能抑制IκBα的磷酸化,并能抑制p65的进核。结论:马钱子碱抑制NF-κB的激活,进而从转录水平COX-2的基因表达,促进A549细胞凋亡。     

四君子汤对脾胃虚弱证型消化性溃疡患者肠道微生态变化及COX-1、COX-2、PGE2表达的影响

目的探讨中药方剂四君子汤对脾胃虚弱症型消化性溃疡患者肠道菌群变化、环氧化酶(COX-1、COX-2)及前列腺素E2表达水平的影响。方法从2017年1月-2018年12月收治的消化性溃疡患者中按年龄组、性别、溃疡类型1∶1匹配后,随机分配于试验组与对照组(试验组与对照组各240名患者)。其中,对照组患者给予思密达治疗,试验组使用思密达协同四君子汤,分别治疗8周。比较治疗前后2组患者的治疗效果、肠道微生态变化、环氧化酶及前列腺素E2水平表达情况。结果治疗后,试验组的阳性与阴性杆菌数减少,阳性球菌数增多,且阳性球菌与阴性球菌数量均高于对照组。而对照组的阳性、阴性杆菌与阴性球菌均显著减少(P<0.05);2组的COX-1、COX-2、PGE2表达水平均增高(P<0.05),且中药组显著高于对照组(P<0.05)。结论在思密达基础上加用四君子汤治疗脾胃虚弱症型消化性溃疡可有效调节其胃肠道菌群平衡、增强对胃黏膜的覆盖保护作用。     

木犀草素对LPS诱导的RAW264.7细胞COX-2及mPGES-1表达的影响

目的:探讨木犀草素对脂多糖(LPS)诱导RAW264.7细胞COX-2及mPGES-1表达的影响。方法:酶免疫测定法(EIA)检测木犀草素对PGE2生成的影响,逆转录聚合酶链反应(RT-PCR)检测COX-2及mPGES-1 mRNA的表达。免疫印迹法(Western blotting)检测COX-2及mPGES-1蛋白的表达。结果:木犀草素抑制LPS诱导的RAW264.7细胞PGE2的生成,同时下调LPS诱导的RAW264.7细胞COX-2及mPGES-1 mRNA和蛋白的表达。结论:木犀草素可以抑制PGE合成途径中两个诱导酶COX-2和mPGES-1的表达。     

青藤碱对脑缺血再灌注损伤大鼠前列腺素E2含量和血脑屏障通透性的影响

目的探讨青藤碱(Sin)对缺血再灌注损伤大鼠前列腺素E2(PGE2)含量和血脑屏障通透性的影响。方法将大鼠随机分为假手术组、缺血再灌注组、Sin低剂量治疗组和Sin高剂量治疗组,线栓法建立局灶性脑缺血再灌注模型。Sin低(30mg/kg)、高剂量(60mg/kg)治疗组于术前30min分别给予大鼠腹腔注射。用放免法检测缺血90min再灌注24h大鼠额顶部皮质PGE2含量、脑含水量和伊文斯蓝(EB)含量变化。结果与假手术组比较,缺血再灌注组额顶部皮质PGE2含量明显升高;与缺血再灌注组比较,Sin高、低剂量治疗组PGE2含量明显降低,高、低剂量组之间差异有统计学意义。与假手术组比较,缺血再灌注组脑含水量和EB含量明显升高;与缺血再灌注组比较,Sin高、低剂量治疗组脑含水量和EB含量明显降低,高、低剂量组之间差异有统计学意义。结论Sin对缺血再灌注脑损伤具有保护作用,其机制可能与减少再灌注损伤后PGE2含量、减轻血脑屏障通透性有关。     

Effects of taraxasterol on iNOS and COX-2 expression in LPS-induced RAW 264.7 macrophages

Ethnopharmacological relevance

Taraxasterol was isolated from the Chinese medicinal herb Taraxacum officinale which has been frequently used as a remedy for inflammatory diseases. Our previous study has shown that taraxasterol inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in RAW 264.7 macrophages. To elucidate the underlying mechanism responsible for these effects, in the present study, we investigated the effects of taraxasterol on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, and mitogen-activated protein kinases (MAPKs) signaling pathway in LPS-induced RAW 264.7 macrophages.

Materials and methods

RAW 264.7 cells were pretreated with 2.5, 5 and 12.5 μg/ml of taraxasterol 1 h prior to treatment with 1 μg/ml of LPS. The mRNA expression levels of iNOS and COX-2 were examined by RT-PCR. The protein expression levels of iNOS and COX-2, and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) MAPKs were measured by Western blot.

Results

The mRNA and protein expression levels of iNOS and COX-2 were inhibited by taraxasterol in a concentration-dependent manner. Further studies revealed that taraxasterol suppressed the phosphorylation of ERK1/2 and p38 in LPS-induced RAW 264.7 macrophages.

Conclusions

These results indicate that taraxasterol inhibits iNOS and COX-2 expression by blocking ERK1/2 and p38 MAPKs signaling pathway.     

Screening of ubiquitous plant constituents for COX-2 inhibition with a scintillation proximity based assay

A rapid semi-homogeneous cyclooxygenase-2 (COX-2) enzymatic assay using scintillation proximity assay (SPA) technology was developed, and 49 ubiquitous plant secondary metabolites were screened for inhibition of COX-2-catalyzed prostaglandin E(2) (PGE(2)) biosynthesis. Assay conditions were optimized with respect to reaction time, amount of antibody, radiolabeled PGE(2), and SPA beads, and the kinetic parameter, K(m), was estimated. The assay was validated with two natural triterpenoids, ursolic and oleanolic acid, known to inhibit COX-2, as well as with four synthetic COX inhibitors, NS-398, rofecoxib, indomethacin, and aspirin. Plant metabolites of different biosynthetic origin representing several substance classes, including alkaloids, anthraquinones, flavonoids, phenylpropanes, steroids, and terpenes, were screened for inhibition of COX-2-catalyzed PGE(2) production. Of these 49 plant metabolites, eugenol, pyrogallol, and cinnamaldehyde (with IC(50) values of 129, 144, and 245 microM, respectively) were found to inhibit COX-2. This study showed that a COX-2-catalyzed PGE(2) assay using SPA is suitable for screening natural compounds with respect to COX-2 inhibition.     

Plant‐derived alkaloid sinomenine potentiates glucocorticoid pharmacodynamics in mitogen‐activated human peripheral blood mononuclear cells by regulating the translocation of glucocorticoid receptor

Sinomenine has been used as an antirheumatic drug in China. Glucocorticoid combined with sinomenine could be an alternative therapeutic approach. In this study, we evaluated the sinomenine potential effect on glucocorticoid pharmacodynamics in vitro using a human peripheral blood mononuclear cell (PBMC) culture system. We also disclosed the possible action mechanism of sinomenine with a focus on P‐glycoprotein function and glucocorticoid receptor (GR) translocation into nucleus. The median (range) of methylprednisolone IC₅₀ values against the PBMC proliferation was 3.18 (0.45–6.81) ng/mL, whereas the median (range) IC₅₀ values of methylprednisolone combined with 0.03, 0.3, 3, and 30 μM sinomenine were 1.85 (0.05–5.15), 0.83 (0.10–3.90), 0.56(0.09–1.62), and 0.59(0.05–1.30) ng/mL, respectively. Sinomenine significantly decreased the IC₅₀ values of methylprednisolone and enhanced the immunosuppressive effect of methylprednisolone (p < 0.05). Sinomenine alone regulated the GR translocation in both Jurkat T cells and normal human PBMCs, and the combination of sinomenine and methylprednisolone showed stronger GR‐modulatory activity than methylprednisolone alone. Thus, the additive effect of sinomenine to promote the methylprednisolone immunosuppressive efficacy was suggested to be related to nuclear GR‐translocation. However, sinomenine did not significantly inhibit the P‐glycoprotein function in the activated PBMCs, suggesting that sinomenine's additive effect seemed to be unrelated with the P‐glycoprotein inhibition.     

San-Huang-Xie-Xin-Tang reduces lipopolysaccharides-induced hypotension and inflammatory mediators

San-Huang-Xie-Xin-Tang (SHXT) is a traditional Chinese medicinal formula containing Coptidis rhizoma, Scutellariae radix and Rhei rhizoma. The present study aimed to determine the preventive effects of standardized SHXT on lipopolysaccharides (LPS)-induced arterial hypotension, protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), cytokines formation and prostaglandin E2 (PGE2) production. LPS-induced activation of iNOS has been recognized to increase cytokines and nitric oxide, some of them play predominant roles in sepsis. Intravenous injection of LPS (10 mg/kg) caused a marked decrease of the mean arterial pressure in normotensive rats. However, the LPS-induced arterial hypotension was inhibited by SHXT (0.01 and 0.03 g/kg), when it was given 30 min before LPS. Moreover, plasma level of cytokines and PGE2 were lowered by SHXT. In RAW 264.7 cells, SHXT (20-200 microg/ml) dose-dependently inhibited LPS (1 microg/ml)-induced iNOS and COX-2 expression, and it also significantly decreased LPS-induced cytokines in a dose-dependent manner. In conclusion, our data suggest that SHXT prevented LPS-induced arterial hypotension, which might be mediated through its inhibition activities on the expression of iNOS and COX-2, cytokines formation and PGE2 production. Therefore, its protection activity against LPS-induced arterial hypotension and inflammatory mediators release might be beneficial in the treatment of endotoxin shock and/or associated inflammation.