Inhibition of interleukin-12 production in lipopolysaccharide-activated macrophages by curcumin

Pharmacological control of interleukin-12 production may be a key therapeutic strategy for modulating immunological diseases dominated by type-1 cytokine responses. In this study we investigated the effects of curcumin (1,7-bis[4-hydroxy-3-methoxyphenyl]-1, 6-heptadiene-3,5-dione) on the production of interleukin-12 from mouse macrophages stimulated with lipopolysaccharide. Curcumin potently inhibited the production of interleukin-12 in a dose-dependent manner. The effect of curcumin on interleukin-12 p40 promoter activation was analyzed by transfecting RAW264.7 monocytic cells with p40 promoter/reporter constructs. The repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor kappaB (p40-kappaB). Furthermore, activation of macrophages by lipopolysaccharide resulted in markedly enhanced binding activity to the kappaB site, which significantly decreased upon addition of curcumin. These results suggest that curcumin-induced inhibition of interleukin-12 production in macrophages may explain some of the biological effects of curcumin including its anti-inflammatory activity.     

Enhancement of interleukin-1 production in mouse peritoneal macrophages by methionine-enkephalin

Methionine-enkephalin, an endogenous opioid, has been reported to have some effects on immune responses. By thymocyte proliferation method, we herein report that methionine-enkephalin over a wide range of concentrations (1 pmol-0.1 mumol/L) significantly increases both extracellular interleukin-1 release and intracellular interleukin-1 production from peritoneal macrophages induced by lipopolysaccharide in mice. Naloxone, having no effect per se on interleukin-1 production, does not block the enhancing effect of the neuropeptide. Interleukin-1 production was also elevated following ip methionine-enkephalin into mice. The results suggest that methionine-enkephalin mediates the enhancement of interleukin-1 synthesis and release as well, and that the effect is not mediated through classical opioid receptors. The results also provide the further links between immune and nervous systems.     

Involvement of nuclear factor-kappaB in the inhibition of interleukin-12 production from mouse macrophages by baicalein, a flavonoid in Scutellaria baicalensis

Pharmacological inhibition of interleukin-12 (IL-12) production may be a therapeutic strategy for preventing development and progression of disease in experimental models of autoimmunity. In this study we investigated the effects of baicalein, a flavonoid present in the root of Scutellaria baicalensis, on the production of IL-12 from mouse macrophages stimulated with lipopolysaccharide (LPS). Baicalein potently inhibited the LPS-induced IL-12 production from both primary macrophages and RAW264.7 monocytic cell-line in a dose-dependent manner (the IC50 values were 43.7 and 17.4 microM, respectively). The effect of baicalein on IL-12 gene promoter activation was analyzed by transfecting RAW264.7 cells with IL-12 gene promoter/luciferase constructs. The repressive effect mapped to a region in the IL-12 gene promoter containing a binding site for NF-kappaB. Furthermore, activation of macrophages by LPS resulted in markedly enhanced binding activity to the NF-kappaB site, which significantly decreased upon addition of baicalein, indicating that baicalein inhibited IL-12 production in LPS-activated macrophages via inhibition of NF-kappaB binding activity.     

贯叶连翘提取物中金丝桃苷的HPLC测定

建立了HPLC法测定贯叶连翘提取物中金丝桃苷的含量。采用C18柱，以甲醇-0．025mol／L磷酸溶液（50：50）为流动相，检测波长363nm。金丝桃苷在0．25～2．5μg／ml范围内线性关系良好。平均同收率为101．4％，RSD为0．66％。     

贯叶连翘对PC12细胞缺血性损伤的保护作用

目的:研究贯叶连翘对PC12细胞缺血性损伤的保护作用。方法:在离体培养的PC12细胞,用NaCN加缺糖造成拟缺血损伤模型,噻唑蓝(MTT)、LDH活力测定、细胞内肌酸激酶(CK)活力测定、流式细胞仪测定细胞凋亡率和线粒体跨膜电位。结果:在10-7~10-5mol.L-1范围内,贯叶连翘浓度依赖地增加MTT比色值,降低缺血性损伤所致培养介质内LDH的释放,增加细胞内CK活力。贯叶连翘还可剂量相关性地降低PC12细胞的凋亡百分率和稳定细胞线粒体跨膜电位。结论:贯叶连翘对PC12细胞缺血性损伤具有保护作用,其机制可能与增加细胞内CK活力和稳定细胞线粒体跨膜电位而抑制缺血性损伤诱导的PC12细胞的凋亡有关。     

Effects of Hypericum perforatum and paroxetine in the mouse defense test battery

Since (a) Hypericum perforatum shows anxiolytic-like effect in some animal models, (b) antidepressant drugs (AD) have been used as the main drug treatment for panic disorder (PD), (c) AD are also effective in generalized anxiety disorder (GAD), and (d) H. perforatum exhibits antidepressant activity, it was hypothesized that H. perforatum might possess an antipanic-like and/or anxiolytic-like effect. Previous studies with the mouse defense test battery (MDTB) have suggested that this model may be useful for the investigation of anxiolytic-like and antipanic-like compounds. Thus, the aim of the present study was to evaluate the effect of H. perforatum extract in the MDTB. The effect of acute, subchronic (7 days), and chronic (21 days) H. perforatum (150 and 300 mg/kg) extract administration was evaluated in mice submitted to the MDTB. Paroxetine (5 mg/kg), a selective serotonin re-uptake inhibitor with anxiolytic and antipanic effect, was used as a positive control. The results showed that 21 days of repeated administration of H. perforatum 300 mg/kg and paroxetine 5 mg/kg reduced flight reactions (number of avoidances, avoidance distance, and overall flight speed) to the presence of the predator. While the effect of paroxetine confirms that MDTB is useful for the detection of antipanic-like drugs, the effect of H. perforatum suggests a putative antipanic-like effect for this extract. Moreover, after 21 days of repeated administration, paroxetine increased the number of approaches/withdrawals and reduced the number of upright postures, suggesting a partial anxiolytic-like effect, while H. perforatum only reduced the number of upright postures. The present results suggest anxiolytic-like and antipanic-like effects of H. perforatum extract. However, it should be emphasized that the risk assessment (the main index of anxiety) was not affected by the extract, while the attack reactions were only weakly modified.     

紫外分光光度法测定贯叶金丝桃胶囊中金丝桃素的含量

目的：建立制剂贯叶金丝桃胶囊中金丝桃素的含量测定方法。方法：采用紫外分光光度法，在590nm波长处检测。结果：金丝桃素在3.29—16.49mg.L^-1(r＝0．9999)范围内吸收值与其浓度呈良好的线性关系，方法平均回收率99．54％，RSD为0.44％(n＝6)。结论：该方法简便、快速、准确，可作为本制剂的质量控制方法。     

Stimulation of interleukin-12 production in mouse macrophages via activation of p38 mitogen-activated protein kinase by alpha2-adrenoceptor agonists

Interleukin-12 is a cytokine primarily produced by monocytes and macrophages. It plays an essential role in the development of cell-mediated immunity and stimulates T helper type 1 (Th1) immune responses. This study was designed to determine if alpha(2)-adrenoceptor agonists are involved in the induction of interleukin-12 production by macrophages. alpha(2)-adrenoceptor agonists such as clonidine, guanfacine, and oxymetazoline significantly induced interleukin-12 secretion and interleukin-12 mRNA expression by macrophages in a concentration-dependent manner. Moreover, stimulation of alpha(2)-adrenoceptor by their agonists triggered the activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Inhibitors of p38 MAPK prevented the stimulatory effects of alpha(2)-adrenoceptor agonists on IL-12 production. Yohimbine and 2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)4,5-dihydro-1H-imidazole (RX821002), alpha(2)-adrenoceptor antagonists, significantly blocked agonist-induced interleukin-12 production and p38 MAPK activation, indicating that the effects of the agonists were mediated through alpha(2)-adrenoceptor. In addition, protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and chelerythrine, significantly inhibited guanfacine-induced interleukin-12 production and p38 MAPK in a concentration-dependent manner. These findings show that alpha(2)-adrenoceptor agonists induce interleukin-12 production in mouse macrophages via a PKC/p38 MAPK signaling pathway and suggest that the effect of alpha(2)-adrenoceptor agonists on interleukin-12 secretion may be a new and novel means of augmenting cell-mediated immune responses.     

Involvement of p38 mitogen-activated protein kinase in the induction of interleukin-12 p40 production in mouse macrophages by berberine,a benzodioxoloquinolizine alkaloid

Interleukin (IL)-12 plays a pivotal role in the development of T helper type 1 (Th1)-immune response, which may have therapeutic effects on diseases associated with pathologic Th2 responses such as allergic disorders and asthma. In this study, we investigated the effects of berberine, a benzodioxoloquinolizine alkaloid with anti-microbial and anti-tumor activities, on the production of IL-12 p40, an inducible subunit of IL-12, in mouse macrophages. Berberine-induced IL-12 p40 production and activation of p38 mitogen-activated protein kinase (MAPK) in dose-dependent manners, which were significantly inhibited by p38 MAPK inhibitors and yohimbine, indicating that p38 MAPK and alpha(2)-adrenergic receptor were involved in the induction of IL-12 p40 production in mouse macrophages by berberine. Furthermore, berberine significantly enhanced IL-12 p40 production in mouse macrophages when combined with lipopolysaccharide, a well-known inducer of IL-12 production. These findings may explain some of the known biological effects of berberine and suggests berberine as an immunotherapeutic compound for induction of IL-12, which is potentially applicable for tumors, infectious disease, and airway inflammation.     

Inhibition of superoxide anion production in macrophages by anti-inflammatory drugs.

The inhibition by anti-inflammatory drugs of the production of Superoxide anions (O₂^?) by isolated guinea pig macrophages was studied spectrophotometrically using NADH and lactate dehydrogenase. id₅₀ values were: 4 × 10^?7M (diclophenac sodium), 1 × 10^?6M (oxyphenbutazone), 1 × 10^?5M (indomethacin), 4 × 10^?5M (phenylbutazone), 7 × 10^?5M (mefenamic acid), 8 × 10^?5 M (flufenamic acid), 8 × 10^?5M (colchicine), 3 × 10^?4M (aspirin), 3 × 10^?4M (benzydamine), 10^?3M < (dexamethasone) and 10^?3M < (gold sodium thiomalate). They seemed to block the cell membrane-associated mechanism to produce Superoxide anions, since most of them did not abolish the generation of superoxide anions from the xanthine oxidase plus hypoxanthine system. Cytochalasin B, pyrogallol, ascorbate, NEM, l-epinephrine and chlorpromazine also inhibited, the production of Superoxide anion, but many non anti-inflammatory drugs were ineffective. This technique was evaluated as a screening method in vitro for nonsteroidal anti-inflammatory drugs.     

HPLC测定新疆贯叶金丝桃中金丝桃素的含量

目的　建立测定新疆贯叶金丝桃中金丝桃素含量的测定方法。方法　采用HPLC法 ,KromasilODS - 1色谱柱 (4 .6mm× 2 0 0mm ,5 μm) ,流动相为甲醇 -pH 6 .5磷酸盐缓冲液 (90∶10 ) ,流速 1ml·min- 1 ,柱温 4 0℃ ,检测波长 5 88nm。结果　金丝桃素和其他组分可达基线分离 ,线性范围为 0 .0 16 5～ 0 .0 82 5 μg(r=0 .9999) ,平均回收率为 10 1.1% ,RSD =3.7% (n =9)。 结论　本法操作简便快速 ,灵敏准确。新疆贯叶金丝桃药材中金丝桃素的含量略高于美国药典的要求。     

高效液相色谱—荧光检测法测定贯叶连翘中金丝桃素的含量

目的：建立高效液相色谱－荧光检测法测定贯叶翘中金丝桃素的含量。方法：色谱柱为18柱（5μm,4．6mmx200mm),流动相为甲醇－pH6．5的磷酸盐缓冲液（55：45）,流速为1．0mL.min^-1,柱温为30℃,荧光检测波长λEX＝580nm,λEM＝600nm。结果：该方法的线性范围为2．55－102ng(r=0.9999)。平均加样回收率为101．1％,RSD为1．4％（n =5)。最低检测限为80pg。结论：本方法快速,简单,灵敏度极高。     

贯叶连翘提取物中金丝桃素的初步HPLC测定

采用ＨＰＬＣ荧光检测法检测围产贯叶连翘提取物中的线桃素。色谱柱为μ－Ｂｏｎｄａｐａｋ Ｃ１８,流动相为０．０５ｍｏｌ／Ｌ,磷酸盐缓冲液（ＰＨ７．０）－甲醇（３：７）和水－甲醇（３：７）,荧光检测激发波长Ｅｘ４７０ｎｍ,发射波簪Ｅｍ５９０ｎｍ。     

贯叶连翘中金丝桃素的提取与含量测定方法概述

贯叶连翘作为传统中药材入药,其活性成分在治疗抑郁症、肝炎、爱滋病及抗肿瘤方面具有重要的药用价值而被广泛关注。本文收集贯叶连翘近年来国内研究开发动态,介绍了有关金丝桃素的提取与含量测定方法的研究进展。     

Retinoid-mediated inhibition of interleukin-12 production in mouse macrophages suppresses Th1 cytokine profile in CD4(+) T cells

Interleukin-12 (IL-12) plays a central role in the immune system by driving the immune response towards T helper 1 (Th1) type responses characterized by high IFN-gamma and low IL-4 production. In this study we investigated whether retinoid-mediated inhibition of interleukin-12 production in mouse macrophages could regulate cytokine profile of antigen (Ag)-primed CD4(+) Th cells. Pretreatment with retinoids (9-cis-RA, all-trans-RA, TTNPB) significantly inhibited IL-12 production by mouse macrophages stimulated with lipopolysaccharide (LPS) or heated-killed Listeria monocytogenes (HKL). Retinoid-pretreated macrophages reduced their ability to induce IFN-gamma and increased the ability to induce IL-4 in Ag-primed CD4(+) T cells. Addition of recombinant IL-12 to cultures of retinoid-pretreated macrophages and CD4(+) T cells restored IFN-gamma production in CD4(+) T cells. The in vivo administration of 9-cis-RA resulted in the inhibition of IL-12 production by macrophages stimulated in vitro with either LPS or HKL, leading to the inhibition of Th1 cytokine profile (decreased IFN-gamma and increased IL-4 production) in CD4(+) T cells. These findings may explain some known effects of retinoids including the inhibition of encephalitogenicity, and point to a possible therapeutic use of retinoids in the Th1-mediated immune diseases such as autoimmune diseases.     

RP-HPLC测定贯叶连翘中金丝桃素、芦丁和槲皮素的含量

目的:采用高效液相法测定贯叶连翘中的金丝桃素、芦丁和槲皮素的含量方法.方法:色谱柱为Symmtry C18(4.6mm×250 mm,5μm),检测波长分别为588 nm,359 nm,流动相分别为甲醇-乙腈-1.0%磷酸二氢钠溶液(340:12:7)和甲醇-水(40:60).结果:线性范围分别在0.02704～0.1352μg和0.264～1.32μg,0.052～0.260μg内良好,回收率分别为98.39%(n=3)、98.56%(n=3)和98.85%(n=3).RSD分别为0.78%和1.01%和1.02%.结论:该法简便,精密度高,结果准确可靠.     

Hyperforin contributes to the hepatic CYP3A-inducing effect of Hypericum perforatum extract in the mouse.

This study in mice investigated whether hyperforin accounts for the inductive effects on cytochrome P4503A enzymes of St. John's wort extracts (SJW; Hypericum perforatum), one of the most popular herbal preparations because of its alleged activity in mild to moderate depression. A hydroalcoholic extract containing 4.5% hyperforin was given at a dose of 300 mg/kg, bis in die (b.i.d.), for 4 and 12 days. Hyperforin, its main phloroglucinol component, was given as dicyclohexylammonium (DCHA) salt (18.1 mg/kg, b.i.d.) on the basis of its content in the extract, to ensure comparable exposure to hyperforin. The extract increased hepatic erythromycin-N-demethylase (ERND) activity, which is cytochrome P450 enzyme (CYP) 3A-dependent, about 2.2-fold after 4 days of dosing, with only slightly greater effect after 12 days (2.8 times controls). Hyperforin too increased ERND activity within 4 days, much to the same extent as the extract (1.8 times the activity of controls), suggesting that it behaves qualitatively and quantitatively like the extract as regards induction of CYP3A activity. This effect was confirmed by Western blot analysis of hepatic CYP3A expression. Exposure to hyperforin at the end of the 4-day treatment was still similar to that with SJW extract, although it was variable and lower than after the first dose in both cases, further suggesting that hyperforin plays a key role in CYP3A induction by the SJW extract in the mouse. Standardization of the extracts based on the hyperforin content can be proposed for further evaluation of their potential action on first-pass metabolism and clearance of coadministered CYP3A substrates.     

一氧化氮和白细胞介素—10对小鼠肺泡巨噬细胞产生肿瘤坏死因？…

目的 观察一氧化氮和ＩＬ－１０对肺泡巨噬细胞炎症反应的调节作用,方法：小鼠肺泡汇噬细胞（ＡＭ）受脂多糖（ＬＰＳ）１０ｍｇ．Ｌ＾－１刺激同时,加入一氧化氮合酶抑制剂Ｓ－硫酸甲基异硫脲（ＳＭＴ）或一氧化氮供体Ｓ－亚硝基乙酰青霉胺（ＳＮＡＰ）,ＥＬＩＳＡ法测定上清液中ＴＮＦα,ＩＬ－１β,ＩＬ－６和ＩＬ－１０浓度,结果：ＡＭ受ＬＰＳ刺激后,ＴＮＦα,ＩＬ－１β和ＩＬ－６释放峰值分别在６,１２和２４小时,     

HPLC法测定药材贯叶金丝桃中金丝桃苷和槲皮素的含量

目的建立测定药材贯叶金丝桃中槲皮素和金丝桃苷含量的方法。方法采用高效液相色谱法(HPLC)。色谱柱:Hypersil CN(150mm×4.6mm,5μm);流动相:甲醇-4mL·L-1磷酸(30/70),流速1.0mL·min-1;检测波长(241,360nm);柱温30℃;进样量20μL。结果槲皮素和金丝桃苷浓度分别在0.02~10和0.03~8μg·mL-1质量范围内与峰面积积分分值呈良好线性关系。加样回收率分别为98.0%和98.1%。结论该方法专属性强,灵敏度高,重复性好,可作为该药材测定槲皮素和金丝桃苷质量控制的方法。