Evidence that genetic deletion of the TNF receptor p60 or p80 inhibits Fas mediated apoptosis in macrophages

Almost 19 members of the tumor necrosis factor (TNF) superfamily have been identified that interact with 29 different receptors. Whether these receptors communicate with each other is not understood. Recently, we have shown that receptor activator of NF-kappaB ligand signaling is modulated by genetic deletion of the TNF receptor. In the current report, we investigated the possibility of a cross-talk between Fas and TNF-alpha signaling pathway in macrophage cell lines derived from wild-type (WT) mice and from mice with genetic deletion of the type 1 TNF receptor (p60(-/-)), the type 2 TNF receptor (p80(-/-)), or both receptors (p60(-/-)p80(-/-)). We found that the macrophages expressing TNF receptors were highly sensitive to apoptosis induced by anti-Fas. The genetic deletion of TNF receptors, however, made the cells resistance to anti-Fas-induced apoptosis. Anti-Fas induced activation of caspase-3 and PARP cleavage in WT cells but not in TNF receptor-deleted cells. This difference was found to be independent of the expression of Fas, Fas-associated protein with death domain (FADD) or TNF receptor-associated death domain (TRADD). We found that anti-Fas induced recruitment of TNFR1 into Fas-complex. We also found that TRADD, which mediates TNF signaling, was constitutively bound to Fas receptor in TNF receptor-deleted cells but not in wild-type cells. Transient transfection of TNFR1 in TNFR1-deleted cells sensitized them to anti-Fas-induced apoptosis. Overall our results demonstrate that Fas signaling is modulated by the TNF receptors and thus provide the evidence of cross-talk between the receptors of two cytokines.     

罗格列酮对重症急性胰腺炎肺损伤保护作用的实验研究

【摘要】目的探讨罗格列酮对重症急性胰腺炎（SAP）肺损伤的保护作用。方法72只SD大鼠随机均分为假手术组（SO组）、SAP组及罗格列酮处理组（干预组）。分别观察各组大鼠血浆淀粉酶（AMY）、肿瘤坏死因子（TNF）-α、动脉血氧分压[p（O2）]、肺组织髓过氧化物酶（MPO）、肺湿/干比值的化及肺组织病理改变,免疫组化法及半定量逆转录聚合酶链反应（RT-PCR）法检测肺组织核因子（NF）-κB、TNF-αmRNA及细胞间黏附分子1（ICAM-1）mRNA的表达。结果SAP组AMY、TNF-α、MPO及肺组织NF-κB、TNF-αmRNA及ICAM-1mRNA表达较SO组显著增加(P<0.05）,干预组6h、12h肺组织湿/干比值较SAP组降低（P<0.05）,而动脉p（O2）较SAP组明显升高（P<0.05）,干预组各时点肺组织病理损伤较SAP组均明显减轻。结论罗格列酮可能通过抑制肺组织NF-κB的活性,减少肺组织TNF-α、ICAM-1的产生从而保护SAP合并肺损伤。     

Celastrol regulates innate immunity response via NF-κB and Hsp70 in human retinal pigment epithelial cells

Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA.Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB.     

罗浮山百草油抗炎作用机制及活性组分筛选研究

目的：研究罗浮山百草油体内及体外抗炎作用的药效及作用机制并初步筛选其活性组分。方法：分别使用二甲苯致小鼠耳肿胀法及脂多糖（lipopolysaccharide,LPS）诱导小鼠单核巨噬细胞白血病细胞RAW264.7炎性模型检测罗浮山百草油的体内及体外抗炎作用,使用RT-PCR法及ELISA法检测细胞内炎性因子变化;通过脂多糖LPS诱导人急性单核细胞白血病细胞THP1炎性模型,使用Western blot法检测核因子-κB p65（nuclear factor κB p65,NF-κB p65）及p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38 MAPK）的磷酸化水平。将罗浮山百草油分为13个组分,使用LPS诱导RAW264.7细胞炎性模型初步筛选其活性组分。结果：罗浮山百草油可剂量依赖性地抑制二甲苯诱导的小鼠耳肿胀及LPS诱导RAW264.7细胞一氧化氮（nitric oxide,NO）的产生,抑制细胞内白细胞介素1β（interleukin-1β,IL-1β）、白细胞介素6（interleukin-6,IL-6）、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS） mRNA和IL-1β、IL-6蛋白的表达;罗浮山百草油可抑制LPS诱导THP1细胞NF-κB p65及p38 MAPK的磷酸化。活性组分初步筛选发现,13个组分中,百草精（68种药材的茶油提取物）、丁香罗勒油、肉桂油以及八角茴香油具有较强的抗炎活性,是罗浮山百草油抗炎活性的主要贡献者。结论：本研究证实了罗浮山百草油抗炎活性的有效性,并筛选出了相应活性组分,可为临床应用及质量控制提供依据。     

Inhibitory effects of casticin on migration of eosinophil and expression of chemokines and adhesion molecules in A549 lung epithelial cells via NF-κB inactivation

Ethnopharmacological relevance

The fruits of Vitex rotundifolia L. have long been used for the treatment of inflammation of the respiratory tract in East Asia.

Aim

To determine if casticin, one of the constituents of Vitex rotundifolia L., has anti-allergic and anti-inflammatory effects in asthma.

Materials and methods

The in vitro anti-inflammatory activity of casticin was studied in A549 human type II-like epithelial lung cells using an eotaxin inhibition assay. Additionally, its effects on eotaxin, regulated on activation normal T cell expressed and secreted (RANTES), vascular cell adhesion molecule (VCAM)-1, and inter-cellular adhesion molecule (ICAM)-1 expression were investigated by real time-polymerase chain reaction (real time-PCR). The inhibition of nuclear factor κB (NF-κB) activity in the presence of casticin was determined by analyzing confocal microscopy images of fluorescence immunocytochemical analysis while the suppression of inhibitory κB (IκB)-α phosphorylation was studied using Western blot analysis. Finally, the inhibitory effect of casticin on eosinophil migration toward prestimulated A549 cell media was measured using the human eosinophilic leukemia cell line.

Results and discussion

Casticin significantly suppressed eotaxin production in cytokine activated A549 lung epithelial cells. Casticin also suppressed the mRNA expression levels of eotaxin, RANTES, VCAM-1, and ICAM-1, which subsequently contributed to the inhibition of eosinophil migration. Furthermore, casticin inhibited IκB-α phosphorylation and nuclear translocation of p65 in A549 cells.

Conclusion

Casiticin inhibited the eosinophil migration and activity of chemokines and adhesion molecules involved in the inflammatory process of asthma by suppressing the NF-κB pathway. These results suggest that casticin has the potential for use in the treatment of allergic asthma.     

愈疡胶囊对细菌感染皮炎模型大鼠的抗炎作用及对TLR/NF-κB通路的影响

目的:研究愈疡胶囊对细菌感染皮炎模型大鼠的抗炎作用及对Toll样受体/核因子κB(TLR/NF-κB)通路的影响.方法:采用肉汤稀释法测定愈疡胶囊对金黄色葡萄球菌的最低抑菌浓度(MIC)和最低杀菌浓度(MBC).将50只大鼠随机分为模型组、阳性对照组(阿莫西林克拉维酸钾,以阿莫西林计120 mg/kg)和愈疡胶囊高、中...     

The possible mechanisms of protocatechuic acid-induced central analgesia

It is aimed to investigate the central antinociceptive effect of protocatechuic acid and the involvement of stimulation of opioidergic, serotonin 5-HT_2A/2C, α2-adrenergic and muscarinic receptors in protocatechuic acid-induced central analgesia in mice. Time-dependent antinociceptive effects of protocatechuic acid at the oral doses of 75, 150 and 300?mg/kg were tested in hot-plate (integrated supraspinal response) and tail-immersion (spinal reflex) tests in mice. To investigate the mechanisms of action; the mice administered 300?mg/kg protocatechuic acid (p.o.) were pre-treated with non-specific opioid antagonist naloxone (5?mg/kg, i.p.), serotonin 5-HT_2A/2C receptor antagonist ketanserin (1?mg/kg, i.p.), α2-adrenoceptor antagonist yohimbine (1?mg/kg, i.p.) and non-specific muscarinic antagonist atropine (5?mg/kg, i.p.), respectively. The antinociceptive effect of protocatechuic acid was observed at the doses of 75, 150 and 300?mg/kg in tail-immersion test, at the doses of 150 and 300?mg/kg in hot-plate test at different time interval. The enhancement in the latency of protocatechuic acid-induced response to thermal stimuli was antagonized by yohimbine, naloxone and atropine in tail-immersion test, while it was antagonized only by yohimbine and naloxone pretreatments in hot-plate test. These results indicated that protocatechuic acid has the central antinociceptive action that is probably organized by spinal mediated cholinergic and opiodiergic, also spinal and supraspinal mediated noradrenergic modulation. However, further studies are required to understand how protocatechuic acid organizes the interactions of these modulatory systems. As a whole, these findings reinforce that protocatechuic acid is a potential agent that might be used for pain relief. Additionally, the clarification of the effect and mechanisms of action of protocatechuic acid will contribute to new therapeutic approaches and provide guidance for new drug development studies.     

泻心汤有效组分不同配伍对内毒素肺损伤大鼠NF-κB及IκB表达的影响

目的研究泻心汤有效组分及其配伍对内毒素肺损伤大鼠NF-κB及IκB表达的影响。方法大鼠灌胃给予黄芩总黄酮提取物(TFL)、大黄总游离蒽醌提取物(TFA)、大黄总结合蒽醌提取物(TCA)、TFL与TFA配伍高、低剂量(A高A低)、TFL与TCA配伍(B)及醋酸地塞米松(Dex)4d,末次给药后1h股静脉注射脂多糖(LPS)建立大鼠内毒素肺损伤模型,1、2、4h各组分别处死6只动物,收集肺组织,免疫印记(Western blot)检测核因子κB(NF-κB)、κB抑制因子(IκB)的蛋白表达。结果大黄总游离蒽醌、A高及Dex在1、2、4h均能够明显抑制NF-κB p65的核转位(P<0.01),所有给药组在2、4h均能够明显抑制胞质中的IκBα的降解(P<0.01)。结论泻心汤有效组分及其配伍对内毒素肺损伤大鼠保护作用与抑制NF-κB的核转位及IκB的降解有关。     

异丙酚对大鼠心肌缺血/再灌注损伤时核因子-κB信号途径的影响

目的观察异丙酚对大鼠在体心肌缺血/再灌注时核因子-κB(NF-κB)信号途径的影响,以探讨异丙酚的心肌保护作用机制。方法阻断大鼠左冠状动脉前降支30min,再灌注2h引起心肌缺血/再灌注损伤。60只SD大鼠随机分为假手术组(Sham)、缺血/再灌注组(I/R)和异丙酚3、6、12mg·kg-1·h-1组。分别于缺血前、缺血30min末、再灌注2h末记录心率、平均动脉压,并计算心率-血压指数。ELISA及放射免疫法分别测定血清、心肌中肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)的含量。分别用原位杂交法(ISH)和半定量RT-PCR法检测心肌组织中诱导型一氧化氮合酶(iNOS)、粘附分子1(ICAM-1)的mRNA的表达。免疫组化染色分析心肌组织中NF-κB的核移位,Western blot检测心肌组织NF-κB的表达量。结果缺血/再灌注后各组大鼠的平均动脉压、血压-心率指数均呈进行性下降(与Sham组相比P<0.05)。异丙酚12mg·kg-1·h-1组在缺血/再灌注后的平均动脉压和血压-心率指数明显高于I/R组(P<0.05)。与Sham组相比,I/R组NF-κB活化,明显从细胞质移位于细胞核,表达量也增加(P<0.05);血清、心肌中TNF-α、IL-1β含量明显升高(P<0.01),心肌中iNOSmR-NA、ICAM-1 mRNA表达增强(P<0.01)。而异丙酚6、12mg·kg-1·h-1组,NF-κB从细胞质向细胞核的移位被明显限制,NF-κB的表达量也明显低于I/R组(均P<0.05);血清、心肌中TNF-α、IL-1β含量明显较I/R组降低(P<0.05),心肌中iNOS mRNA、ICAM-1 mRNA表达减弱(与I/R组相比,P<0.05,P<0.01)。结论异丙酚能减轻缺血/再灌注损伤,同时明显抑制心肌缺血/再灌注时NF-κB的活化,阻断NF-κB相关信号通路的传导,下调TNF-α、IL-1β、ICAM-1和iNOS等炎症相关因子的表达。