Anti-inflammatory effect of Citrus Unshiu peel in LPS-stimulated RAW 264.7 macrophage cells

Citrus Unshiu peel (CUP) has been traditionally used in East Asia as a drug for the treatment of vomiting and dyspepsia. However, its effects on inflammation remain unknown. In this study, we investigated the effects of CUP on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The research focused on determining whether CUP could inhibit the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and the activation of nuclear factor (NF)-κB, mitogen-activated protein kinases (MAPKs), as well as the secretion of nitric oxide (NO), prostaglandin (PG) E(2), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in LPS-stimulated RAW 264.7 cells. We found that CUP represses LPS-induced iNOS and COX-2 gene expression as well as NO, PGE(2), TNF-α and IL-6 production. Additionally, CUP inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK) MAPK, and suppressed IκBα degradation and nuclear translocation of NF-κB. Collectively, our results indicate that CUP inhibits the production of various inflammatory mediators via blockade of MAPK phosphorylation pursuant to the inhibition of IκBα degradation and the nuclear translocation of NF-κB. These findings are the first to clarify the mechanism underlying the anti-inflammatory effect exerted by CUP in RAW 264.7 macrophage cells stimulated by inflammatory agents.     

Heme oxygenase-1 mediates the anti-inflammatory effect of mushroom Phellinus linteus in LPS-stimulated RAW264.7 macrophages

This work aimed to elucidate the anti-inflammatory mechanism of the n-BuOH subfraction (PL) prepared from fruiting bodies of Phellinus linteus. PL induced heme oxygenase-1 (HO-1) of the RAW264.7 macrophages in concentration- and time-dependent manner. It suppressed induction of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO) through down-regulation of iNOS promoter activity in lipopolysaccharide (LPS)-stimulated macrophages. Zn(II) protoporphyrin IX (ZnPP), a specific inhibitor of HO-1, partly blocked suppression by PL on iNOS promoter activity and NO production, which were elevated in LPS-stimulated macrophages. LPS was able to enhance NO production via reactive oxygen species (ROS) generation, c-Jun NH(2)-terminal kinase (JNK) and c-Jun induction. ZnPP prevented PL from down-regulating ROS generation and JNK activation in LPS-stimulated macrophages. Taken together, PL shows its anti-inflammatory activity via mediation of HO-1 in an in vitro inflammation model.     

痹祺胶囊提取物对RAW264.7细胞模型的抗炎作用

目的通过比较研究痹祺胶囊(马钱子、党参、丹参、白术、茯苓、川芎、三七、地龙、甘草、牛膝)不同溶剂提取物的抗炎作用,从而确定最佳提取溶剂。方法 3种溶剂提取痹祺胶囊,水、石油醚、正丁醇的提取物与脂多糖(LPS)刺激小鼠单核/巨噬细胞RAW264.7共孵,用酶联免疫吸附法(ELISA)、Griess Reagent法检测细胞分泌的肿瘤坏死因子(TNF-α)和一氧化氮(NO)的水平;以实时定量逆转录-聚合酶链式反应(Real-time RT-PCR)检测细胞炎症相关指标白介素-6(IL-6)、环氧合酶-2(COX-2)、诱导型一氧化氮合酶(iNOS)、TNF-α的mRNA表达水平,比较3种提取物的抗炎作用。结果 3种提取物均能显著抑制LPS刺激的RAW264.7细胞分泌NO和TNF-α,其中水提取物的抑制作用最强,且剂量依赖性最显著。在基因表达水平,只有水提取物对LPS刺激的RAW264.7细胞的IL-6、COX-2、iNOS的mRNA表达有显著抑制作用。结论体外细胞药理学实验结果表明,痹祺胶囊内容物以水、石油醚、正丁醇3种溶剂体系提取得到提取物均具有抗炎作用,但水提取物抗炎作用更显著。     

粉防己碱对脂多糖诱导下RAW264.7细胞炎症模型细胞因子的作用

目的观察粉防己碱(tetrandrine,Tet)对脂多糖(LPS)诱导RAW264.7细胞炎症模型促炎细胞因子和抗炎细胞因子的影响。方法 LPS(1μg/mL)刺激生长良好的RAW264.7细胞,建立细胞炎症模型。四甲基偶氮唑盐(MTT)法检测不同浓度Tet对RAW264.7细胞增殖的影响,激光共聚焦显微镜观察Tet对细胞核因子-κB(NF-κB)核转运的作用,酶联免疫吸附试验(ELISA)检测细胞上清中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)的变化。结果当Tet浓度<1μmol/L时,单独或与1μg/mL LPS共培养,均对RAW264.7细胞生长无影响;而当Tet浓度>10μmol/L时,单独或与LPS共培养均表现出明显的细胞生长抑制效应;Tet可使IL-6、TNF-α的释放受到抑制,相反可促进IL-10的表达。激光共聚焦显微镜观察Tet大剂量组细胞核红染的阳性细胞显著减少,以阴性细胞(红染p65亚基主要集中在胞浆部位,使胞浆染色较深,胞核染色较浅,整个细胞成空泡状)为主。结论 Tet可以抑制LPS所致的RAW264.7细胞炎症反应,其抗炎作用与抑制NF-κB活化,进一步减少炎性细胞因子IL-6、TNF-α的产生,并促进抗炎细胞因子IL-10的表达等有关。     

Anti-inflammatory effects of ethyl acetate fraction from Melilotus suaveolens Ledeb on LPS-stimulated RAW 264.7 cells

Aim of the study

This paper aimed to elucidate the anti-inflammatory effects of EtOAc fraction prepared from Melilotus suaveolens Ledeb ethanol extract with a cellular model of LPS-stimulated RAW 264.7 cell.

Materials and methods

Some key pro-inflammatory cytokines and mediators including IL-1β, IL-6, NO, iNOS, COX-2 and TNF-α, two important anti-inflammatory cytokines and mediators IL-10 and HO-1, I-κB and NF-κB were studied by sandwich ELISA, real-time PCR, western blot analysis and immunocytochemistry. At last a HPLC fingerprint was taken to evaluate the fraction.

Results

The EtOAc fraction could significantly inhibit the production of IL-1β, IL-6, NO, TNF-α, COX-2 in LPS-stimulated cell than that of single LPS-stimulated cell (p < 0.01 or p < 0.05), and the extract could increase the production of IL-10 and HO-1 than that of single LPS intervention cell (p < 0.01 or p < 0.05). Meanwhile, the extract also could inhibit the production of NF-κB compared to single LPS-stimulated cell. All the results showed that the extract had a good anti-inflammatory effect on LPS-stimulated RAW264.7 cell.

Conclusions

Taken together, the anti-inflammatory actions of M. suaveolens Ledeb EtOAc fraction might be due to the down-regulation of IL-1β, IL-6, NO, TNF-α and COX-2 via the suppression of NF-κB activation, and another pathway was up regulating the production of IL-10 and HO-1. Meanwhile, the EtOAc fraction might be further studied to isolate the active anti-inflammatory ingredients besides coumarin.     

雪荔方总黄酮对LPS致RAW264.7细胞炎症的保护作用

目的观察雪荔方总黄酮(紫花地丁、六月雪、车前草、荔枝草,TFXL)对脂多糖(LPS)致RAW264.7巨噬细胞的抗炎作用。方法噻唑蓝(MTT)法检测不同浓度的雪荔方总黄酮对RAW264.7细胞活性的影响;NO试剂盒法检测雪荔方总黄酮对LPS致RAW264.7细胞的NO释放量;酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)和白介素-10(IL-10)的分泌;逆转录聚合酶链反应法(RT-PCR)检测诱导型一氧化氮合酶(iNOS)、TNF-α、IL6和IL10 mRNA的表达;免疫印迹法(Western blot)检测细胞IκB-α、p65蛋白表达。结果与LPS模型组相比,雪荔方总黄酮能显著降低NO、TNF-α和IL-6分泌,增加IL-10分泌;抑制iNOS、TNF-α、IL6mRNA表达,促进IL10 mRNA表达;抑制IκB-α、p65的磷酸化,其高剂量组能抑制IκB-α的降解。结论本研究初步证实了雪荔方总黄酮对LPS致RAW264.7巨噬细胞炎症的保护作用,其作用机制可能与NF-κB信号通路有关。     

Wogonoside displays anti-inflammatory effects through modulating inflammatory mediator expression using RAW264.7 cells

Ethnopharmacological relevance

The root of Scutellaria baicalensis Georgi, also called Huangqin in China, is an herbal-based nutraceutical which is usually used in Chinese medicated diet (CMD). As an abundant ingredient in Huangqin, wogonoside is a flavonoid glycoside. The present work investigated the anti-inflammatory activities of wogonoside in lipopolysaccharides (LPS)-induced RAW264.7 cells.

Materials and methods

RAW264.7 cells were used. The inhibition of wogonoside against nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells were measured. Additionally, the effects of wogonoside on mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), TNF-α and IL-6 were also investigated.

Results and discussion

Wogonoside not only dose-dependently decreased the production of inflammatory mediators including NO and PGE₂ but also inhibited the release of pro-inflammatory cytokines including TNF-α and IL-6 in LPS-induced RAW264.7 cells. Furthermore, wogonoside possessed significantly in vitro inhibitory effects on the gene expression of iNOS, COX2, TNF-α and IL-6.

Conclusion

These results suggest that wogonoside may be used as a functional food component for prevention and treatment of inflammation.     

身痛逐瘀胶囊对LPS诱导RAW264.7细胞释放NO抑制作用的研究

目的:观察身痛逐瘀胶囊对LPS诱导RAW264.7细胞释放NO的抑制作用。方法:以脂多糖诱导RAW264.7细胞,观察不同浓度的身痛逐瘀胶囊(1、5、10、20 mg/ml)处理RAW264.7细胞,MTT法检测细胞活性;同时检测LPS诱导RAW264.7细胞释放NO量以及身痛逐瘀胶囊和12味单味中药水提物干预后RAW264.7细胞释放NO量。结果:身痛逐瘀胶囊作用RAW264.7细胞的安全范围<20mg/ml,与LPS组相比,身痛逐瘀胶囊(1、5、10mg/ml)能有效抑制NO释放,12味单味中药水提物无效。结论:身痛逐瘀胶囊对LPS诱导RAW264.7细胞产生的炎症具有保护作用,其保护机制与抑制NO的释放有关。     

The synergistic anti-inflammatory effect of the combination of sodium ferulate and oxymatrine and its modulation on inflammation-associated mediators in RAW 264.7 cells

Ethnopharmacological relevance

The combination of Radix Angelicae sinensis (Oliv.) Diels and Radix Sophora flavescens Ait. was extensively used in traditional Chinese medicine to treat inflammatory diseases, such as acne, heart disease, and hepatitis. Sodium ferulate (SF) and oxymatrine (OMT) were effective component of Radix Angelicae sinensis (Oliv.) Diels and Radix Sophora flavescens Ait., respectively.

Aim of the study

In this study, we investigated the synergistic anti-inflammatory effect of the combination of SF and OMT, and its modulation on inflammation-associated mediators in RAW 264.7 cells.

Materials and methods

In vivo, the anti-inflammatory effects of the combination of SF and OMT were evaluated with the xylene-induced mouse ear edema model and the carrageenan-induced rat paw edema model. In vitro, chemokines and cytokines mRNA expressions in lipopolysaccharide (LPS)-activated RAW 264.7 cells were determined by real-time PCR (RT-PCR) microarray analysis. The levels of interleukin-11 (IL-11), C-reactive protein (CRP) and interferon-γ (INF-γ) in the supernatant of LPS-stimulated RAW 264.7 cells were measured by enzyme-linked immune-sorbent assay (ELISA).

Results

The combination of SF and OMT could significantly inhibit the edema in the xylene-induced mouse ear edema and carrageenan-induced rat paw edema, but no effect was found when each drug was used alone according to above doses. The combination exhibited a better effect in down-regulating mRNA expressions of inflammation-associated mediators in LPS-stimulated RAW 264.7 cells than SF or OMT alone. The ELISA results showed that the combination synergistically inhibited LPS-induced IL-11, CRP and INF-γ production in a dose-dependent manner.

Conclusion

The combination of SF and OMT showed synergistic anti-inflammatory effect, and the activity was probably related to its modulation on inflammation-associated mediators, especially IL-11, CRP and INF-γ.     

紫金龙乙醇组分对脂多糖诱导的RAW264.7细胞分泌炎症因子的影响

目的:研究紫金龙乙醇组分(AVEC)对脂多糖(LPS)诱导小鼠单核巨噬细胞RAW 264.7分泌炎症因子的影响。方法:用脂多糖(10μg·L-1)刺激生长良好的RAW 264.7细胞24 h建立体外细胞炎症模型,以MTT法测定不同浓度AVEC对RAW 264.7细胞的毒性作用,Griess试剂法检测一氧化氮(NO)含量,ELISA法检测细胞上清液中肿瘤坏死因子α(TNF-α)和白介素6(IL-6)含量。结果:AVEC在低于400 mg·L-1时对RAW 264.7细胞无毒性作用。与空白对照组相比,LPS可以明显诱导RAW 264.7细胞分泌炎症因子TNF-α,IL-6和NO(P<0.01);与模型组相比,100~400 mg·L-1的AVEC可明显下调LPS诱导的RAW 264.7细胞释放炎症因子TNF-α,IL-6和NO(P<0.05,P<0.01),并呈现良好的剂量依赖关系。结论:AVEC可以抑制脂多糖诱导的RAW 264.7细胞炎症反应,其抗炎作用可能与减少炎症因子TNF-α,IL-6和NO有关。     

葛根素预处理对LPS诱导RAW264.7细胞活化的影响

目的:观察葛根素预处理对脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7活化和分泌细胞因子的影响,探讨其抗炎机制。方法:取对数期生长良好的RAW264.7细胞,随机分为空白对照组、LPS组、葛根素预处理+LPS组。CCK-8法检测葛根素对RAW264.7的细胞毒性作用,Giemsa染色法观察细胞形态学变化,酶联免疫吸附试验(ELISA)检测细胞上清中肿瘤坏死因子-α(TNF-α)、巨噬细胞炎性蛋白-2(MIP-2)的变化,实时荧光定量PCR(qRT-PCR)动态测定NF-κB p65 mRNA的表达。结果:当葛根素的浓度为100,200,400μmol·L-1时,与1 mg·L-1LPS共培养均未显示出细胞毒性作用(P<0.05);与空白对照组相比,LPS组可明显改变RAW264.7细胞的形态(胞体增大,形态不规则,伪足大量伸出),葛根素100μmol·L-1组干预后可明显抑制LPS导致的细胞形态学变化,葛根素200,400μmol·L-1干预组的抑制效果更显著,但2组之间无明显差异;葛根素预处理可使细胞上清液中TNF-α,MIP-2以及细胞内NF-κB p65 mRNA的表达受到抑制(P<0.05),并随着葛根素浓度的增加,抑制效果逐渐增加(P<0.05),但未达到对照组水平。结论:葛根素是一种安全有效的天然抗炎药物,可显著下调炎症细胞因子(如TNF-α,MIP-2)的表达,其作用机制可能与下调NF-κB p65 mRNA的表达有关。     

Panax notoginseng attenuates LPS-induced pro-inflammatory mediators in RAW264.7 cells

Herbals or dietary supplements are not regulated as drugs by the United States Food and Drug Administration (USFDA) although many may have associated therapeutic effects and toxicities. Therefore, the immunomodulatory effects of the herbal extract Panax notoginseng on cultured macrophages (RAW264.7 cells) were investigated to address potential therapeutic or toxic effects. Cells were stimulated with LPS (1 microg/ml) and treated with notoginseng at 5, 25 and 50 microg/ml. Notoginseng inhibited the LPS-induced production of TNF-alpha and IL-6 by the cultured macrophages in a concentration-dependent manner. The expression of COX-2 and IL-1 beta mRNA was also attenuated by notoginseng. TNF-alpha production was inhibited in samples treated with notoginseng 24h before, or at the same time as LPS stimulation, but not in samples treated 8h after LPS stimulation. Notoginseng reduced expression of the accessory molecules CD40 and CD86 on the RAW264.7 cells while CD14 and TLR4 expression remained unaffected. Furthermore, Rb1 and Rg1 ginsenosides also inhibited macrophage production of TNF-alpha, but to a lesser extent than did the whole notoginseng extract. Collectively, these results indicate that notoginseng inhibits LPS-induced activation of RAW264.7 macrophages and demonstrates that notoginseng possesses anti-inflammatory and immunosuppressive properties in vitro.     

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells,indicative of alternative macrophage activation

Ethnopharmacological relevance

The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages.

Aim of the study

In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity.

Materials and methods

Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/− LPS.

Results

Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of Echinacea pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, l-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8-bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression.

Conclusions

These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation.     

丹皮酚对脂多糖刺激的RAW264.7细胞炎症与自噬的影响

目的:以脂多糖(lipopolysaccharide,LPS)刺激的RAW264.7细胞,以自噬诱导剂雷帕霉素(rapamycin,Rap)促进自噬为主要模型,研究丹皮酚(paeonol,P)对RAW264.7自噬与免疫功能的影响。方法:使用LPS 1μg·ml-1刺激RAW264.7细胞6 h,丹皮酚200μmol/L处理12 h,Rap 100 ng·ml-1作用6 h,蛋白免疫印迹法(western blot,WB)检测自噬相关因子LC3B、Beclin、P62和炎症相关因子TN F-α、IL-1β的表达水平,激光共聚焦荧光共定位法(immunofluorescence,IF)检测LC3B、P62蛋白的荧光表达和定位;酶联免疫分析(enzyme-linked immunosorbent assay,ELISA)检测RAW264.7细胞TNF-α的分泌。结果:在脂多糖诱导RAW264.7炎症反应的细胞模型中,添加丹皮酚药物后LC3B、Beclin表达增多,P62表达减少,LC3B-l向LC3B-ll的转换效率增强;雷帕霉素抑制IL-1β、TNF-α的表达,与丹皮酚联用进一步抑制IL-1β、TNF-α的表达和分泌。结论:丹皮酚通过促进细胞自噬来抑制炎症反应,丹皮酚与雷帕霉素联用加强了对炎症的抑制作用。     

雷公藤红素对巨噬细胞焦亡的影响

目的探讨雷公藤红素对小鼠巨噬细胞RAW264.7焦亡的影响及相关机制。方法采用MTT法测定细胞增殖;吖啶橙(AO)/溴化乙啶(EB)、Hoechst/PI荧光染色观察细胞焦亡形态变化;ELISA法检测细胞上清液中白细胞介素-1β(IL-1β)分泌量;Western blotting法检测Caspase-1蛋白表达水平;Caspase-1活性检测试剂盒检测细胞内Caspase-1酶活性。结果经MTT法检测发现,雷公藤红素在50 nmol/L以下对巨噬细胞RAW264.7增殖活性无显著影响;AO/EB、Hoechst/PI染色显示雷公藤红素能够改善脂多糖(LPS)与三磷酸腺苷(ATP)诱导的细胞焦亡;ELISA结果显示,雷公藤红素能够抑制IL-1β的分泌(P0.05),且具有一定的浓度依赖性;Western blotting检测发现cleaved-Caspase-1蛋白表达量降低,同时雷公藤红素能抑制Caspase-1的酶活性。结论雷公藤红素抑制LPS与ATP诱导的细胞焦亡,可能与抑制IL-1β的分泌,抑制Caspase-1的活化有密切关系。     

槲皮素对LPS诱导小鼠RAW264.7细胞炎症的保护作用

目的研究槲皮素对脂多糖(LPS)诱导小鼠RAW264.7细胞炎症的保护作用。方法 CCK-8法检测细胞活力,Griess法检测NO释放,RT-PCR法检测TNF-α、IL-6、IL-1β、iNOS、COX-2、MCP-1、TLR2、TLR4、MyD88 mRNA表达,Western blot法检测iNOS、COX-2、TLR4、IκBα、p-IκBα、p-NF-κB p65蛋白表达。结果不同浓度(0～50μmol/L)槲皮素对细胞活力无明显影响(P0.05)。与LPS组比较,槲皮素组(25、50μmol/L)显著抑制NO释放(P0.05);槲皮素组(5、15、25μmol/L)显著降低TNF-α、IL-6、IL-1β、iNOS、COX-2、MCP-1、TLR4、MyD88 mRNA表达和iNOS、COX-2蛋白表达(P0.05,P0.01),并呈浓度依赖性;槲皮素组(25μmol/L)显著下调TLR4、p-IκBα、p-NF-κB p65蛋白表达(P0.05),显著上调IκBα蛋白表达(P0.05)。结论槲皮素可抑制LPS诱导小鼠RAW264.7细胞炎症,其机制可能与调节TLR4/NF-κB信号通路有关。     

金雀异黄素对巨噬细胞RAW 264.7 增殖、形态和细胞周期的影响

目的研究金雀异黄素对巨噬细胞RAW264．7增殖、细胞形态及细胞周期的影响,以探讨其对免疫系统的作用。方法以鼠源巨噬细胞系RAW264．7为细胞模型,将金雀异黄素用二甲基亚砜（DMSO）溶解,再用培养液稀释成所需终浓度为12．5、25、50、100kcmol&#183;L^-1的4组及溶剂对照组（DMSO体积含量比低于0．1％）。通过WST-1细胞增殖实验、倒置相差显微镜及流式细胞仪分别检测金雀异黄素对巨噬细胞的增殖、细胞形态及细胞周期的影响。结果金雀异黄素对巨噬细胞的影响呈时间和剂量依赖关系。50-100μmol&#183;L^-1,浓度的金雀异黄素作用巨噬细胞24h和48h后均能抑制细胞增殖,细胞增殖率分别为77％～81％、58％-73％（P〈0．01）;并能影响细胞形态改变,表现为细胞体积增大,并形成较长伪足。流式细胞结果显示．该浓度金雀异黄素可将巨噬细胞阻滞在S-G2／M期。结论金雀异黄素能抑制巨噬细胞细胞增殖,影响巨噬细胞形态,可能与干扰细胞周期有关。     

Anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium on LPS-stimulated Raw264.7 cells

Aim of the study

Lilium lancifolium is commonly used to treat bronchitis, pneumonia, etc. In this study, we investigated the anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium (LL extracts) in LPS-stimulated Raw264.7 cells.

Material and methods

Levels of NO, PGE₂ and pro-inflammatory cytokines (IL-6 and TNF-α) in the supernatant fraction were determined using sandwich ELISA. Expression of COX-2 and iNOS, phosphorylation of MAPK subgroups (ERK and JNK), and NF-κB activation in extracts were detected via Western blot and immunocytochemistry assays.

Results

The LL extract significantly inhibited NO, PGE₂, IL-6 and TNF-α production in LPS-stimulated cells, and suppressed iNOS and COX-2 expression. A mechanism-based study showed that phosphorylation of ERK1/2 and JNK and translocation of the NF-κB p65 subunit into nuclei were inhibited by the LL extract. Furthermore, interleukin-4 and interleukin-13 production in Con A-induced splenocytes was suppressed.

Conclusion

These results indicate that anti-inflammatory effects of methanol extracts from Lilium lancifolium are due to downregulation of iNOS and COX-2 via suppression of NF-κB activation and nuclear translocation as well as blocking of ERK and JNK signaling in LPS-stimulated Raw264.7 cells.     

白背叶根含药血清对脂多糖诱导下RAW264.7细胞分泌的影响

目的:观察不同浓度的白背叶根含药血清对脂多糖(lipopolysac-charide,LPS)诱导下RAW264.7细胞分泌的影响。方法:制作高、中、低浓度白背叶根含药血清,以CCK-8法检测不同浓度白背叶根含药血清对RAW264.7细胞活力的影响。以脂多糖刺激生长良好的RAW264.7细胞,建立细胞炎症模型,检测白背叶根含药血清对细胞上清液中炎症介质一氧化氮(NO)、前列腺素E2(PGE2)、白三烯B4(LTB4)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量的影响。结果:不同浓度的白背叶根含药血清均对RAW264.7细胞活力无影响;与模型组比较,10%、5%的白背叶根含药血清可显著抑制NO的释放(P<0.05或P<0.01),10%白背叶根含药血清可显著抑制LTB4的分泌(P<0.05),2.5%白背叶根含药血清可显著抑制IL-1β的分泌(P<0.05);但不同浓度的白背叶根含药血清对PGE2、TNF-α的分泌无显著影响。结论:白背叶根的抗炎机制可能是通过抑制炎症反应中NO,LTB4,IL-1β的释放而实现。