Effects of schisandrin on transcriptional factors in lipopolysaccharide-pretreated macrophages

Schisandrin is the main active ingredient isolated from Schisandra chinensis Baill. Recent studies have demonstrated that schisandrin exhibits anti-inflammatory effects in vivo and in vitro. In this study, we examined whether the order of lipopolysaccharide (LPS) treatment affects the mechanism of schisandrin anti-inflammatory activity. We found that the antiinflammatory mechanisms are not the same depending on whether macrophages were treated with schisandrin before or after LPS. The main difference is that inhibitor kappaBα (IκBα) degradation was not inhibited when macrophages were pretreated by LPS before schisandrin and was weakly inhibited when macrophages were pretreated by schisandrin before LPS.     

Anti-inflammatory activity of lansais from endophytic Streptomyces sp. SUC1 in LPS-induced RAW 264.7 cells

This research was undertaken to find the in vitro inflammatory action of lansai A–D produced by Streptomyces sp. SUC1. We investigated the effects of lansai C not only on the formation of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumour necrosis factor (TNF-α), interleukin (IL)-1α, IL-6 and IL-10, but also on inducible NO synthase and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells. The data obtained were consistent with the modulation of inducible nitric oxide synthase enzyme expression. A similar fashion was also observed when LPS-induced PGE₂ release and COX-2 expression were tested. The significant inhibitory effects were shown in concentration-dependent manners. In addition, lansai C also mildly but significantly reduced the formation of TNF-α. These findings support the application of lansai C as anti-inflammatory agent.     

八肽胆囊收缩素抑制LPS诱导的RAW264.7细胞AP-1活性及IL-6表达

目的研究八肽胆囊收缩素(CCK-8)对LPS诱导RAW264.7细胞IL-6表达的影响及相关机制。方法用ELISA及RT-PCR法检测RAW264.7细胞IL-6蛋白及mR-NA表达;用EMSA方法检测RAW264.7细胞AP-1 DNA结合活性。结果①LPS可时间依赖性的诱导RAW264.7细胞IL-6蛋白及mRNA表达;②10-10 mol.L-1 CCK-8对LPS诱导的RAW264.7细胞IL-6表达无明显影响;10-8、10-6 mol.L-1 CCK-8浓度依赖性地抑制了LPS诱导的RAW264.7细胞IL-6表达;③10-10 mol.L-1 CCK-8未影响LPS诱导的AP-1活性,10-8、10-6 mol.L-1 CCK-8浓度依赖性地抑制了LPS诱导的AP-1活性。结论 CCK-8通过抑制AP-1 DNA结合活性而抑制了LPS诱导的RAW264.7细胞IL-6表达,这可能是CCK-8发挥抗炎作用的信号转导机制之一。     

熵权法结合星点设计-效应面法优化祛湿清肺方提取工艺及体外抗炎活性评价

目的:优化祛湿清肺方提取工艺,并对祛湿清肺方提取液进行体外抗炎活性评价。方法:以绿原酸、虎杖苷、黄芩苷、汉黄芩苷、黄芩素、芦荟大黄素、汉黄芩素、大黄素成分含量及得膏率为指标,加水量及提取时间为考察因素,采用熵权法结合星点设计-效应面法对祛湿清肺方提取工艺进行优化。以脂多糖(LPS)诱导大鼠腹腔巨噬细胞(RAW_264.7)为炎症模型,酶联免疫吸附法测定白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、一氧化氮(NO)的含量,蛋白质印迹法检测磷酸化NF-κB抑制蛋白激酶(phosphorylated inhibitor of NF-κB kinase,p-IKK)、磷酸化NF-κB p65(phosphorylated NF-κB p65,p-NF-κB p65)、NF-κB抑制蛋白α(inhibitor of NF-κBα,IκBα)蛋白表达水平变化,评价祛湿清肺方提取液抗炎活性。结果:星点设计-效应面法优化所得的最佳提取工艺为加水量13倍,提取2次,每次提取时间105 min。祛湿清肺方提取液能降低IL-6、IL-1β、TNF-α、NO的含量,抑制p-IKK、p-NF-κB p65蛋白表达,促进IκBα蛋白表达,具有较好的体外抗炎活性。结论:熵权法结合星点设计-效应面法优选的祛湿清肺方提取工艺稳定可行,所得提取液具有较好的抗炎活性,为祛湿清肺方开发和现代化研究提供参考奠定基础。     

HMCO5, herbal extract, inhibits NF-kappaB expression in lipopolysaccharide treated macrophages and reduces atherosclerotic lesions in cholesterol fed mice

HMCO5 is a herbal extract which comprises of eight different herbs. We studied whether this extract has anti-atherosclerotic effects. In lipopolysaccharide (LPS) stimulated RAW264.7 cells, HMCO5 inhibited NF-kappaB activation as well as iNOS promoter activity, inhibited the secretion of TNF-alpha and IL-1beta, and directly inhibited the intracellular accumulation of reactive oxygen species. ApoE knock-out mice fed a high-fat high-cholesterol diet with HMCO5 for 10 weeks showed a significant reduction in atherosclerotic lesions. A notable finding was the preservation of the smooth muscle cell layer in the media of aorta in the HMCO5 co-treated mice. HMCO5 treated mice did not show significant decrease in serum level of cholesterol. These results suggest that HMCO5 has anti-atherosclerotic effects which in part may be attributable to the inhibition of production of NF-kappaB dependent pro-inflammatory cytokines.     

Raw264.7细胞蛋白和PD-L1抗体在小鼠黑色素瘤(B16)中的抑瘤效果评价

目的探讨小鼠来源白血病毒诱导的肿瘤细胞(Raw264.7)蛋白和程序性死亡-配体(PD-L1)在C57BL/6小鼠黑色素瘤(B16)中的抑瘤效果。方法采用6~8周龄清洁级C57BL/6小鼠30只(雌雄各半),平均分为3组。各组小鼠分别于皮下接种B16细胞(2×10⁵个/只),待小鼠肿瘤平均体积达到100 mm³时,给予治疗。group A:腹腔注射Raw264.7细胞蛋白(50μg/只),每周1次,共2次。group B:腹腔注射PD-L1抗体(600μg/只,本课题组生产纯化),每3天1次,共4次。group C:相同时间点给予等体积生理盐水对照。各组小鼠给药后,测量肿瘤大小,末次测量后,各组小鼠无菌取脾脏,应用流式细胞术分析CD3/4/8⁺T细胞群比例。结果各组荷瘤小鼠给予相应治疗后,组C小鼠肿瘤体积增长迅速,末次测量平均体积达到4004 mm³;组B小鼠增长次之,末次平均体积达到1637 mm³;组A小鼠体积增长最慢,末次平均体积仅为882 mm³(P<0.01)。对小鼠脾脏免疫细胞分析显示,组B小鼠中CD3⁺CD8⁺T cell比例明显上调,平均达到77.65%,而CD3⁺CD4⁺T比例明显下调,仅为11.86%(P<0.01);而组A和组C两组小鼠CD3⁺CD4⁺T和CD3⁺CD8⁺T没有明显区别(P>0.05)。结论制备的RAW264.7细胞灭活蛋白和PD-L1抗体对于B16荷瘤小鼠均有抑瘤作用,且前者效果更为明显。     

Effect of iRoot SP and mineral trioxide aggregate (MTA) on the viability and polarization of macrophages

ObjectiveThis study was performed to investigate the effect of iRoot SP and mineral trioxide aggregate (MTA) on the viability and polarization of macrophages.MethodsThe effect of iRoot SP and MTA on the viability of RAW 264.7 macrophages was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 1 and 2 days of culture. The gene expression levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), interleukin 12p40 (IL-12p40) were measured by quantitative real time polymerase chain reaction (qRT-PCR) after stimulation of the RAW 264.7 macrophages with iRoot SP and MTA. The expression levels of CD11c and CD206 in RAW 264.7 macrophages were examined by immunofluorescence and flow cytometry after stimulation with iRoot SP and MTA. The data were analyzed by one-way analysis of variance and the Tukey test.ResultsBoth iRoot SP and MTA were non-toxic to the RAW 264.7 macrophages. The use of iRoot SP and MTA increased the expression of IL-1β, TNF-α, IL-10, IL-12p40 on the first day of culture and could promote macrophage M1 and M2 polarization.ConclusionsMTA and iRoot SP have good biocompatibility with macrophages, and they induced both M1 and M2 polarization of the RAW 264.7 macrophages.     

应用UPLC-MS/MS探讨芫花酯甲对巨噬细胞分泌花生四烯酸类水平的影响

目的:研究细胞上清中6种炎症介质花生四烯酸(AA),白三烯B4(LTB4),前列腺素G2(PGG2),前列腺素F2α(PGF2α),15-脱氧-δ-12,14-PGJ2(15d-PGJ2),5,15-过氧化氢二十碳四烯酸(5,15-Di HETE)的变化,探讨芫花酯甲干预花生四烯酸代谢产生的致炎、诱导平滑肌收缩的作用机制。方法:选取RAW264.7作为体外实验模型,采用三重四极杆质谱,分析空白组及低、高质量浓度组芫花酯甲(0,1.62,6.48 mg·L-1)作用于RAW264.7细胞(5×104个/m L)48 h后细胞上清中6种炎症相关介质的相对含量变化,以探讨芫花酯甲的致炎机制。结果:与空白组相比,AA,PGF2α,PGG2,5,15-Di HETE的相对含量显著升高(P0.05);与空白组相比较,LTB4在高浓度芫花酯甲刺激时相对含量显著升高,但对于15d-PGJ2,高浓度的芫花酯甲刺激时相对含量呈现下降的趋势(P0.01)。结论:芫花酯甲增加了巨噬细胞上清液中炎症相关介质AA,5,15-Di HETE及促炎介质LTB4,PGG2,PGF2α水平,减低了抗炎介质15d-PGJ2的水平,提示芫花酯甲通过影响促炎介质和抗炎介质的双重途径,发挥致炎和诱导平滑肌收缩作用。     

荆三棱顺式茋类化合物的结构修饰及抗炎活性

 目的 对荆三棱顺式茋类化合物进行结构修饰及体外抗炎活性评价。方法 分别运用甲基化、乙酰化、去甲基化反应对荆三棱中新的顺式茋类化合物sciryagarol I(1)进行结构修饰。采用噻唑蓝(MTT)法考察化合物1及其结构修饰物3~5和荆三棱中另一新顺式茋类化合物sciryagarol II(2)对RAW264.7细胞活力的影响。以脂多糖(LPS)与Pam3csk4分别诱导RAW264.7细胞炎症模型,运用酶联免疫吸附法(ELISA )法检测化合物1~5对细胞释放炎性因子肿瘤坏死因子-ɑ与白细胞介素-6(IL-6)的影响。结果 通过¹H-NMR和HRESI-MS确定修饰物3~5的结构;化合物1,2 和5能显著抑制由LPS或Pam3csk4诱导的RAW264.7细胞释放的炎性因子肿瘤坏死因子-ɑ和白细胞介素-6。结论 修饰物3~5皆为新化合物,顺式茋类化合物的抗炎作用强度与其结构上的酚羟基数目有一定关系。