瘤果黑种草子总皂苷的抗炎和免疫调节作用

目的 考察瘤果黑种草子总皂苷(TSSN)的抗炎和免疫调节作用。方法 建立脂多糖(LPS)诱导的小鼠单核巨噬细胞白血病细胞RAW264.7炎症模型，采用Griess法检测NO分泌水平，ELISA法检测细胞上清TNF-α、IL-6、IL-1β含量，采用qRT-PCR方法考察TSSN对NF-κB信号通路相关靶点mRNA表达水平的影响；酶动力学实验检测总皂苷对环氧酶-2(COX-2)以及5-脂氧酶(5-LO)的抑制活性；通过MTS法及ELISA法检测TSSN对静息期淋巴细胞的影响，并以TSSN对刀豆蛋白(ConA)、LPS分别诱导的小鼠T、B淋巴细胞增殖的影响，以及对T淋巴细胞分泌细胞因子TNF-α、IL-2、IL-6、IFN-γ的影响来考察TSSN的免疫调节作用。结果 在LPS诱导的RAW 264.7炎症模型中，TSSN能显著降低LPS诱导的RAW264.7分泌炎症介质NO和促炎症因子TNF-α、IL-6、IL-1β水平，显著抑制TNF-α、IL-6、IL-1β m RNA表达水平，抑制iNOS、NF-κB p65 mRNA表达水平；在12.5～200μg·ml^-1剂量...     

三百棒通过调控TLR4/NF-κB信号通路对脂多糖诱导的RAW264.7细胞极化的影响

目的：探讨三百棒醇提物(TAAE)对脂多糖(LPS)诱导的小鼠单核-巨噬细胞RAW264.7极化趋向以及其对炎症反应的影响。方法：用LPS诱导RAW264.7细胞建立炎症模型。CCK-8法检测细胞活力;ELISA检测细胞因子(TNF-α、IL-1β、IL-6和IL-10)的水平;Griess法检测细胞培养上清液中一氧化氮(NO)的分泌情况;荧光染色双标法观察巨噬细胞的极化状态;免疫荧光染色检测NF-κB定位及表达;Transwell检测TAAE对RAW264.7细胞迁移和趋化性的影响;RT-qPCR检测TNF-α、IL-1β、IL-6、IL-10、Arg-1、NF-κB和TLR4的mRNA水平;Western blot检测iNOS、COX-2、TLR4、NF-κB、p-NF-κB、IκBα和p-IκBα蛋白水平。使用TLR4通路抑制剂TAK-242进一步验证TAAE对TLR4/NF-κB信号通路的影响。结果：与模型组相比较,TAAE降低LPS诱导的炎症模型RAW264.7细胞中M1型促炎因子(TNF-α、IL-1β和IL-6)及NO水平,促使M2型抑炎因子(IL-10和Arg-1)分泌...     

川续断皂苷Ⅵ对M1/M2型巨噬细胞极化的调节作用及机制

目的：研究川续断皂苷Ⅵ对M1/M2型巨噬细胞极化的调节作用及机制。方法：MTT法检测川续断皂苷Ⅵ对RAW264.7细胞活力的影响。ELISA检测脂多糖（LPS）诱导状态下RAW264.7细胞上清中TNF-α和IL-6的分泌量；Griess法检测LPS诱导状态下RAW264.7细胞上清中一氧化氮（NO）含量；荧光定量PCR检测TNF-α、IL-6、精氨酸酶-1(Arg-1)、血红素加氧酶1(HO-1)和细胞因子信号转导抑制蛋白-2(SOCS2)基因表达水平。Western blot检测一氧化氮合酶（iNOS）和p-p65蛋白表达。结果：在LPS诱导的RAW264.7细胞中，川续断皂苷Ⅵ抑制TNF-α、IL-6、iNOS和p-p65蛋白或基因表达水平，同时增加HO-1基因表达。川续断皂苷Ⅵ能抑制LPS诱导下RAW264.7细胞分泌的NO。川续断皂苷Ⅵ增加IL-4诱导下M2型巨噬细胞标志物Arg1和SOCS2基因表达。结论：川续断皂苷Ⅵ可以抑制RAW264.7巨噬细胞向M1型极化，同时促进其向M2型极化，可通过调节M1/M2型巨噬细胞极化发挥其抗炎免疫调节作用。     

草木犀石油醚提取物对小鼠巨噬细胞促炎介质的影响

目的 研究草木犀石油醚提取物在体外的抗炎作用.方法 采用小鼠巨噬细胞系RAW 264.7建立炎症细胞模型,加入10 μg/L的LPS培养液和不同浓度的草木犀石油醚提取物进行干预.ELISA法检测上清液中TNF-α, IL-1β, IL-6和NO的分泌量;实时荧光定量RT-PCR检测TNF-α, iNOS 和 COX-2的 mRNA表达;Western 印迹法检测COX-2 蛋白的表达.结果 草木犀提取物干预后细胞所分泌的炎性介质(TNF-α, IL-1β, IL-6和NO)与模型组相比均显著降低(P＜0.01),并存在剂量依赖关系;RT-PCR结果显示干预后细胞TNF-α,iNOS和COX-2的mRNA表达水平显著降低(P＜0.01),也存在剂量依赖关系;Western印迹结果显示草木犀石油醚提取物及地塞米松干预后COX-2蛋白水平明显降低(P＜0.01).结论 草木犀的石油醚提取物通过下调LPS诱导的巨噬细胞表达炎性介质而发挥其体外抗炎作用, 且其下调作用呈剂量依赖性.     

醛肽类蛋白酶体抑制剂对LPS诱导小鼠巨噬细胞炎症介质表达的影响

目的： 探讨醛肽类蛋白酶体抑制剂MG132对脂多糖（LPS）诱导小鼠巨噬细胞Raw264.7 核转录因子-κB（NF-κB)活化、炎性因子一氧化氮(NO)和肿瘤坏死因子α(TNF-α)分泌以及诱导型一氧化氮合酶(iNOS)表达的影响。方法： 利用报告基因检测法分析转染细胞（pNiFty-SEAP/HEK293）中NF-κB的活性;采用DAF-2DA荧光探针法检测Raw264.7细胞内NO的产生;蛋白免疫印迹法探讨细胞中iNOS和IκB-α蛋白表达情况;酶联免疫吸附法测定Raw264.7细胞上清中TNF-α的含量。结果： 预先加入MG132能够显著抑制LPS诱导的TNF-α分泌,其抑制率由5 μmol/L时的36.7%升高到10 μmol/L时的60.4%。反映NO含量的荧光强度值随着MG132给药浓度的增加而降低,其抑制率从2 μmol/L时的29.5%达到10 μmol/L的55.9%。预刺激后MG132可使胞浆中iNOS蛋白表达减少,IκB-α蛋白却明显增加,NF-κB的活性随着给药浓度的升高而不断降低。结论: MG132能够抑制LPS诱导的TNF-α和NO的产生,减少iNOS表达,具有抗炎作用。其作用机制可能与IκB降解受阻,导致NF-κB活性降低有关。     

黄连乙醇提取物与盐酸小檗碱体外抗炎对比研究

目的:比较黄连乙醇提取物与盐酸小檗碱体外抗炎活性,探索体外抗炎机制。方法:通过脂多糖体外刺激小鼠单核巨噬细胞建立细胞炎症模型,给药干预后,LPS 长时间刺激RAW264.7 细胞,MTT 比色法分析黄连乙醇提取物及盐酸小檗碱对RAW264.7 细胞生长活性的影响。酶联免疫吸附法检测细胞上清液中IL-β、IL-6、TNF-α、NO、前列腺素E2 (PGE2)含量。实时荧光定量RT-PCR 法检测iNos、HO-1、TNF-αmRNA 表达。结果:在5 ~80 mg/ L 范围内,黄连乙醇提取物及盐酸小檗碱对RAW264.7 细胞无抑制作用;各浓度给药组IL-6、IL-1β、TNF-α、NO、前列腺素E2 (PGE2 )含量与LPS 刺激模型组比较均有显著性(P<0.01),且浓度与剂量无效应相关。实时荧光定量RT-PCR 结果显示,各浓度给药组均明显降低iNos、HO-1、TNF-αmRNA 表达(P<0.05,P<0.05,P<0.01),且与浓度不呈效应关系。结论:黄连乙醇提取物具有体外抗炎作用,抗炎活性优于盐酸小檗碱,其作用机制可能与抑制TNF-α、NO 等炎症因子的活化,进而影响花生四烯酸(AA)代谢有关。     

GW4064 对LPS 诱导的RAW264.7 细胞中炎症反应的影响

目的:研究FXR激动剂GW4064对脂多糖(LPS)诱导的RAW264.7巨噬细胞炎症因子TNF-α、MCP-1和NO的分泌及IL-1β、TNF-α的表达,以及诱导型一氧化氮合酶(iNOS)表达的影响。方法:通过流式高通量多因子检测技术和Griess法检测炎症因子TNF-α、MCP-1和NO分泌的影响;采用qPCR技术检测炎症因子IL-1β、TNF-α和iNOS的mRNA的表达;Western blot检测iNOS及NF-κB蛋白的表达的水平。结果:GW4064能抑制LPS诱导的细胞促炎因子IL-1β、TNF-α和iNOS的mRNA的表达(P0.05),同时能不同程度的抑制细胞上清中TNF-α,MCP-1中的分泌量(P0.05);并且呈剂量依赖性的抑制NO的释放(P0.05);除此之外,GW4064(10、20μmol/L)可显著抑制LPS诱导的RAW264.7巨噬细胞iNOS蛋白的产生(P0.01),经过GW4064处理后,LPS诱导的RAW264.7巨噬细胞NF-κB的活性也显著下调(P0.01)。结论:FXR激动剂GW4064在巨噬细胞中具有一定的抗炎作用,抑制iNOS的表达及下调NF-κB的活性是其发挥抗炎作用的机制之一。     

厚朴酚对骨关节炎的抗炎和软骨保护作用

目的探讨厚朴酚的体外抗炎活性和对碘乙酸单钠(MIA)诱导的骨关节炎(OA)的影响。方法脂多糖(LPS)处理的RAW264.7细胞中体外评价厚朴酚的抗炎作用,设立LPS组、厚朴酚不同浓度组(5、10、20μmol/L),以正常培养的细胞作为对照组,Griess试剂测量培养基中NO的累积;Western blot检测细胞iNOS、COX-2蛋白的表达。MIA诱导OA的SD大鼠模型,分为MIA组、厚朴酚不同浓度组(5、10、20 mg/kg),以正常大鼠作为对照组,取大鼠膝关节组织样本进行组织学分析,实时PCR分析大鼠IL-1β、TNF-α、IL-6、MMP-2、MMP-9和COX-2的mRNA表达;ELISA检测培养基上清液中PGE 2、IL-6及各组大鼠血清IL-1β、IL-6和TNF-α含量。结果厚朴酚抑制LPS处理的RAW264.7细胞中的NO、PGE2和IL-6的产生(P0.05)。此外,厚朴酚抑制MIA诱导OA大鼠模型的IL-1β、TNF-α和IL-6的升高及MMP-2、MMP-9和COX-2的合成(P0.05)。结论厚朴酚在OA大鼠模型中发挥有效的抗炎活性并保护软骨。     

没食子酸通过拮抗脂多糖诱导的TLR4/NF-κB活化抑制RAW264.7巨噬细胞炎性反应

目的观察没食子酸对脂多糖(LPS)诱导RAW264.7巨噬细胞Toll样受体4/核因子κB(TLR4/NF-κB)通路的影响。方法将巨噬细胞分为空白对照组、LPS组、LPS联合没食子酸组、LPS联合NF-κB抑制剂吡咯二硫代甲酸(PDTC)组和LPS联合地塞米松(DM)组。处理后的细胞培养24 h,ELISA检测肿瘤坏死因子α(TNF-α)、白细胞介素1(IL-1)、IL-6水平,实时定量PCR检测TLR4、NF-κB mNRA水平,Western blot法检测p65、p-p65、TLR4、磷酸化的NF-κB抑制蛋白α(IκBα)的蛋白表达水平。结果 LPS诱导RAW264.7巨噬细胞后TNF-α、IL-1、IL-6水平升高,没食子酸可降低LPS诱导引起的TNF-α、IL-1和IL-6表达水平升高。LPS刺激后TLR4 mRNA及蛋白表达增加,NF-κB活化,没食子酸可拮抗以上作用,阻止NF-κB活化。结论没食子酸可通过TLR4/NF-κB通路抑制LPS诱导的RAW264.7巨噬细胞炎性反应。     

黄连乙醇提取物与盐酸小檗碱体外抗炎对比研究北大核心CSCD

目的:比较黄连乙醇提取物与盐酸小檗碱体外抗炎活性,探索体外抗炎机制。方法:通过脂多糖体外刺激小鼠单核巨噬细胞建立细胞炎症模型,给药干预后,LPS长时间刺激RAW264.7细胞,MTT比色法分析黄连乙醇提取物及盐酸小檗碱对RAW264.7细胞生长活性的影响。酶联免疫吸附法检测细胞上清液中IL-1β、IL-6、TNF-α、NO、前列腺素E2(PGE2)含量。实时荧光定量RT-PCR法检测i Nos、HO-1、TNF-αmRNA表达。结果:在5~80 mg/L范围内,黄连乙醇提取物及盐酸小檗碱对RAW264.7细胞无抑制作用;各浓度给药组IL-6、IL-1β、TNF-α、NO、前列腺素E2(PGE2)含量与LPS刺激模型组比较均有显著性(P<0.01),且浓度与剂量无效应相关。实时荧光定量RT-PCR结果显示,各浓度给药组均明显降低i Nos、HO-1、TNF-αmRNA表达(P<0.05,P<0.05,P<0.01),且与浓度不呈效应关系。结论:黄连乙醇提取物具有体外抗炎作用,抗炎活性优于盐酸小檗碱,其作用机制可能与抑制TNF-α、NO等炎症因子的活化,进而影响花生四烯酸(AA)代谢有关。     

Anti-Inflammatory Mechanism of a Folk Herbal Medicine,Duchesnea indica (Andr) Focke at RAW264.7 Cell Line

This study is to explore the anti-inflammatory mechanism of the ethanol extract of Duchesnea indica (Andr) Focke. An inflammatory cellular model was established by addition of lipopolysaccharide (LPS) on RAW264.7 cell line. The cellular secretion of TNF-α, IL-1β, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-α, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, and activation of NF-κB were assayed by ELISA, the Griess method, real-time quantitative PCR, and Western blot and immunocytochemistry method, respectively. The ethanol extract of D. indica not only reduced production of pro-inflammatory cytokines and mediators and blocked NF-κB activation, but also slightly promoted release of the anti-inflammatory mediator HO-1 and suppressed IL-10 secretion. In conclusion, the anti-inflammatory effects of the extract of D. indica are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-κB activation. The extract of D. indica can also slightly promote HO-1 production to reduce inflammation.     

Upregulation of heme oxygenase-1 via PI3K/Akt and Nrf-2 signaling pathways mediates the anti-inflammatory activity of Schisandrin in Porphyromonas gingivalis LPS-stimulated macrophages

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is thought to induce periodontitis. In this study, we isolated Schisandrin from the dried fruits of Schisandra chinensis and examined the anti-inflammatory effect of Schisandrin in macrophages stimulated with LPS from P. gingivalis. First, Schisandrin inhibited LPS-induced pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. And Schisandrin suppressed the nuclear translocation and activity of NF-κB and phosphorylation of IκBα in LPS-stimulated RAW 264.7 cells. Next, the presence of a selective inhibitor of HO-1 (SnPP) and a siRNA specific for HO-1 inhibited Schisandrin-mediated anti-inflammatory activity. Furthermore, Schisandrin induced HO-1 expression of RAW 264.7 cells through Nrf-2, PI3K/Akt, and ERK activation. Therefore, these results suggest that the anti-inflammatory effects of Schisandrin on P. gingivalis LPS-stimulated RAW 264.7 cells may be due to a reduction of NF-κB activity and induction of the expression of HO-1, leading to TNF-α, IL-1β, and IL-6 down-regulation.     

Dichloromethane Fraction of Laminaria japonica Ethanolic Extract Inhibits Lipopolysaccharide-Induced Nitric Oxide Synthase and Cyclooxygenase-2 Expression in RAW 264.7 Cells via NF-κB Pathway

Strong anti-inflammatory activity has been found in Laminaria japonica dichloromethane fraction (LDF); however, the molecular mechanisms underlying its anti-inflammatory activity are not reported. Our results indicated that LDF inhibited LPS-induced nitric oxide and prostaglandin E(2) production in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW 264.7 cells. Also, levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were remarkably reduced by LDF in LPS-treated RAW 264.7 cells. LDF greatly inhibited promoter activity of nuclear factor-κB (NF-κB) and translocation of NF-κB subunits by prevention of the degradation of inhibitor κB-α in LPS-treated RAW 264.7 cells (p?相似文献   

Pyranocoumarins Isolated from Peucedanum praeruptorum Dunn Suppress Lipopolysaccharide-Induced Inflammatory Response in Murine Macrophages Through Inhibition of NF-κB and STAT3 Activation

Praeruptorin C, D, and E (PC, PD, and PE) are three pyranocoumarins isolated from the dried root of Peucedanum praeruptorum Dunn of Umbelliferae. In the present study, we investigated the anti-inflammatory effect of these compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Pyranocoumarins significantly inhibited LPS-induced production of nitric oxide, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The mRNA and protein expressions of inducible nitric oxide synthase, IL-6, and TNF-α were also suppressed by these compounds. Both PD and PE exhibited greater anti-inflammatory activities than PC. Further study showed that pyranocoumarins suppressed the cytoplasmic loss of inhibitor κB-α protein and inhibited the translocation of NF-κB from cytoplasm to nucleus. In addition, pyranocoumarins suppressed LPS-induced STAT3 tyrosine phosphorylation. Taken together, the results suggest that pyranocoumarins may exert anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages through the inhibition of NF-κB and STAT3 activation.     

Inhibition of LPS-induced inflammatory biomarkers by ethyl acetate fraction of Patrinia scabiosaefolia through suppression of NF-κB activation in RAW 264.7 cells

Patrinia scabiosaefolia (PS) has been used for curing various types of inflammatory-related disorders. However, the precise mechanism of the anti-inflammatory activity of PS remains unclear. Here, we investigated the anti-inflammatory effects of several fractions isolated from the PS in RAW 264.7 macrophages. The results indicated that the ethyl acetate fraction of PS (EAPS) concentration highly suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) and IL-6 productions without a cytotoxic effect on RAW 264.7 cells. EAPS inhibited the expressions of LPS-induced iNOS and COX-2 protein and their mRNA in a dose-dependent manner. Particularly, EAPS suppressed the level of nuclear factor-κB (NF-κB) activity, which was linked with the suppression of LPS-induced phosphorylation of p65 at serine 276 and p65 translocation into nuclei, but not MAPK signaling. In addition, treatment with EAPS inhibited the production of TNF-α in LPS-injected mice and suppressed the production of IL-6 and TNF-α in LPS-stimulated splenocytes from BALB/c mice. Therefore, we demonstrate here that Patrinia scabiosaefolia potentially inhibits the biomarkers related to inflammation through the blocking of NF-κB p65 activation, and it may be a potential therapeutic candidate for the treatment of inflammatory diseases.     

The inhibition of lipopolysaccharide-induced tumor necrosis factor-α and nitric oxide production by Clostridium perfringens α-toxin and its relation to α-toxin-induced intracellular ceramide generation

The effect of Clostridium perfringens α-toxin on production of tumor necrosis factor (TNF)-α and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The pretreatment of wild type α-toxin, but not the inactive mutant, significantly decreased LPS-induced TNF-α and NO production. α-Toxin inhibited the expression of TNF-α and an inducible type of NO synthase protein and mRNA. Furthermore, it inhibited the phosphorylation of IκB-α and p65 NF-κB subunit, and the NF-κB luciferase reporter gene activity in LPS-stimulated cells. The pretreatment of α-toxin increased the level of intracellular ceramide. Taken together, Clostridium perfringens α-toxin pretreatment was suggested to inhibit LPS-induced TNF-α and NO production through the inhibition of NF-κB activation. The relationship between α-toxin-induced intracellular ceramide generation and the NF-κB inhibition is discussed.     

β-sitosteryl-3-O-β-glucopyranoside isolated from the bark of Sorbus commixta ameliorates pro-inflammatory mediators in RAW 264.7 macrophages

The bark of Sorbus commixta has been used in Asian traditional medicine for treatment of cough, asthma, bronchial disorders, gastritis and dropsy. However, the anti-inflammatory effect of β-sitosteryl-3- O -β-glucopyranoside, a major compound of the bark of S. commixta, is poorly understood. In this study, we investigated the anti-inflammatory effect and the underlying molecular mechanisms of β-sitosteryl-3-O-β-glucopyranoside in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Prostaglandin E₂ (PGE₂) and cytokines released from cells were measured using EIA assay kit. The expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, Tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) was measured by real-time polymerase chain reaction (RT-PCR) and/or Western blot analysis. β-sitosteryl-3-O-β-glucopyranoside inhibited the production of nitric oxide (NO) and PGE₂ along with the expression of iNOS and COX-2 in LPS-stimulated RAW264.7 cells. In addition, β-sitosteryl-3-O-β-glucopyranoside reduced the release of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6. Moreover, β-sitosteryl-3-O-β-glucopyranoside inhibited the NF-κB activation induced by LPS, which was associated with the abrogation of IκBα degradation and subsequent decreases in nuclear p65 levels. The result suggested that the β-sitosteryl-3-O-β-glucopyranoside inhibited NO and pro-inflammatory productions by down-regulating the gene expression of pro-inflammatory mediators via the negative regulation of the NF-кB pathway in LPS-stimulated RAW 264.7 cells.     

Inhibition of LPS-induced inflammatory biomarkers by ethyl acetate fraction of Patrinia scabiosaefolia through suppression of NF-κB activation in RAW 264.7 cells

Patrinia scabiosaefolia (PS) has been used for curing various types of inflammatory-related disorders. However, the precise mechanism of the anti-inflammatory activity of PS remains unclear. Here, we investigated the anti-inflammatory effects of several fractions isolated from the PS in RAW 264.7 macrophages. The results indicated that the ethyl acetate fraction of PS (EAPS) concentration highly suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) and IL-6 productions without a cytotoxic effect on RAW 264.7 cells. EAPS inhibited the expressions of LPS-induced iNOS and COX-2 protein and their mRNA in a dose-dependent manner. Particularly, EAPS suppressed the level of nuclear factor-κB (NF-κB) activity, which was linked with the suppression of LPS-induced phosphorylation of p65 at serine 276 and p65 translocation into nuclei, but not MAPK signaling. In addition, treatment with EAPS inhibited the production of TNF-α in LPS-injected mice and suppressed the production of IL-6 and TNF-α in LPS-stimulated splenocytes from BALB/c mice. Therefore, we demonstrate here that Patrinia scabiosaefolia potentially inhibits the biomarkers related to inflammation through the blocking of NF-κB p65 activation, and it may be a potential therapeutic candidate for the treatment of inflammatory diseases.