机动车尾气诱发大鼠慢性阻塞性肺疾病并上调气道上皮细胞COX-2/PGE2/EP受体信号通路

目的：探究机动车尾气(MVE)长期暴露引起大鼠慢性阻塞性肺疾病(COPD)发生时,气道上皮细胞中环加氧酶2(COX-2)/前列腺素E₂(PGE₂)/E-前列腺素类激素(EP)受体信号通路成员的表达变化。方法：(1)动物实验：健康雄性SD纯系大鼠(SPF级)16只,随机分为2组：MVE暴露组(n=8)和空白对照(CTL)组(n=8)。采用MVE暴露6个月的方法建立COPD大鼠模型。造模结束后,使用Buxco小动物有创肺功能仪检测大鼠肺功能;肺组织切片行HE染色并评估肺组织病理变化;ELISA法检测大鼠支气管肺泡灌洗液(BALF)中炎症因子白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和PGE₂的水平,评估大鼠肺部炎症情况;采用免疫荧光及Western blot法检测肺组织COX-2及EP受体蛋白水平;提取肺组织核蛋白,Western blot检测MVE对肺组织NF-κB核转位的影响。(2)细胞实验：采用MVE细颗粒物(PM2.5)标准品刺激人正常支气管上皮细胞BEAS-2B。ELISA法检测细胞培养液中PGE     

Multi-allergen Challenge Stimulates Steriod-Resistant Airway Inflammation via NF-κB-Mediated IL-8 Expression

Neutrophilic airway inflammation associated with multiple allergens has been related to steroid-resistant asthma. However, most animal models use only one allergen, which cannot simulate asthma closely as seen in patients. To determine the mechanism of inflammatory process involved in this severe condition, BALB/c mice were repetitively challenged with the pooled extract of dust mite, ragweed, and Aspergillus species (DRA). We found that DRA increased interleukin (IL)-10 and TGF-β levels and neutrophil recruitment in bronchial alveolar lavage fluid. We also found that although dexamethasone suppressed the release of these two cytokines, mast cells recruitment, and mucus hypersecretion, it actually increased neutrophil infiltration and the level of keratinocyte-derived chemokine (mKC), a functional homolog of human IL-8. Treatment of human lung alveolar A549 cells with Der p1, an extract of house dust mite Dermatophagoides pteronyssinus, increased the expression of IL-8 and activity of NF-κB. The elevated IL-8 level was suppressed by BAY11-7082, a selective NF-κB inhibitor, but not by dexamethasone. These results suggest that increased IL-8 (mKC) levels may be involved in steroid-resistant neutrophilic airway inflammation through an NF-κB-dependent pathway.     

葫芦素E通过抑制MAPKs/NF-κB通路减轻哮喘小鼠气道炎症

目的:探讨葫芦素E对哮喘小鼠气道炎症及MAPKs和NF-κB信号通路的影响。方法:将40只健康小鼠随机分成对照组、模型组、葫芦素E低剂量组、葫芦素E高剂量组和地塞米松组。用卵清蛋白致敏法制备哮喘模型,观察各组支气管肺泡灌洗液(BALF)中炎症细胞分类计数、肺组织炎症细胞浸润以及BALF中白细胞介素4(IL-4)、IL-5、IL-13及干扰素γ(IFN-γ)含量的变化;测定肺组织中磷酸化JNK(p-JNK)、磷酸化ERK1/2(p-ERK1/2)、磷酸化p38 MAPK(p-p38 MAPK)和磷酸化NF-κB p65(p-p65)的含量。结果:与正常组比,模型组BALF中炎症细胞数量明显增加,并且MAPKs和NF-κB信号通路相关蛋白活性显著增强。我们发现高剂量葫芦素E可减轻哮喘小鼠气道炎症反应,并明显抑制MAPKs和NF-κB信号通路相关蛋白活性。病理组织学结果显示,模型组小鼠肺组织内有杯状细胞及支气管黏膜上皮细胞增生,肺泡内有炎症细胞浸润,管腔狭窄;各剂量葫芦素E处理组病理改变均较模型组显著减轻。结论:葫芦素E可以减轻哮喘小鼠气道炎症反应,其机制可能与抑制MAPKs和NF-κB信号通路有关。     

pGSK-3β和NF-κB在慢性支气管哮喘小鼠气道重塑中的表达水平及意义

目的探讨磷酸化糖原合成酶激酶3β(phosphor-Ser9 glycogen synthase kinase3β,pGSK-3β)和核因子kappa B(nuclear factor kappa B,NF-κB)的表达水平在慢性支气管哮喘气道重塑中的作用。方法 16只雄性BALB/c小鼠随机分为2组:支气管哮喘组与对照组,每组8只,通过卵蛋白致敏和雾化激发制备小鼠慢性支气管哮喘模型,对照组全部以生理盐水代替;2组小鼠分别在末次激发后24h内颈椎脱臼处死,取左肺通过HE染色,光学显微镜观察气道重塑情况;取右肺采用蛋白质印迹方法测定肺组织pGSK-3β、NF-κB、MMP-9的表达量。结果⑴支气管哮喘组出现管壁增厚,平滑肌增生、黏液分泌增加等气道重塑的特征性改变;⑵对照组pGSK-3β、NF-κB、MMP-9的表达水平均小于支气管哮喘组(P0.05),三者在慢性支气管哮喘小鼠中表达量增加。结论卵蛋白致敏反复雾化激发可导致气道重塑,GSK-3β/NF-κB信号转导途径在支气管哮喘气道重塑中起重要作用。     

Tetraspanin 1 inhibits TNFα-induced apoptosis via NF-κB signaling pathway in alveolar epithelial cells

Objective

Tetraspanin family plays an important role in the pathogenesis of cancer, but its role in lung fibrosis is unknown. To determine whether tetraspanin 1 (TSPAN1), a member of the family, may be involved in the pathogenesis of pulmonary fibrosis.

Methods

TNFα -stimulated human alveolar epithelial (A549) and alveolar epithelial type II cell (AT2) were treated in vitro. Murine pulmonary fibrosis model was generated by injection of bleomycin (BLM). The expression of TSPAN1 was examined in vivo using the bleomycin-induced lung fibrosis model and tissue sample of IPF patients. Then we transfected the cells with TSPAN1 siRNA or plasmid and detected the expression changes of related proteins and cell apoptosis.

Results

In our study, we found that TSPAN1 was markedly down-regulated in lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and in bleomycin-induced pulmonary fibrosis in mice. We also found that TSPAN1 was significantly down-regulated in A549 and primary (AT2) cells following exposure to TNFα. Meanwhile, TSPAN1 inhibited p-IκBα, which attenuated nuclear NF-κB translocation and activation and inhibited apoptosis. We demonstrated that TSPAN1 reduced Bax translocation and caspase-3 activation, inhibited the apoptosis by regulating the NF-κB pathway in response to TNFα.

Conclusions

We conclude that TSPAN1 mediated apoptosis resistance of alveolar epithelial cells by regulating the NF-κB pathway. TSPAN1 may be a potential therapeutic target for pulmonary fibrosis or acute lung injury.

     

XAV939, a Wnt/β-catenin pathway modulator,has inhibitory effects on LPS-induced inflammatory response

Aim: In this study, we report the anti-inflammatory activity of XAV939, a modulator of the Wnt/β-catenin pathway.

Methods: WNT/β-catenin pathway and NF-κB signaling pathway were examined in LPS-stimulated human bronchial epithelial cells and effects of XAV939 on these pathways were analyzed. The effect of XAV939 was confirmed in human umbilical vein endothelial cells.

Results: LPS-induced expressions of pro-inflammatory genes IL-6, IL-8, TNF-α, IL-1β, MCP-1, MMP-9, iNOS and COX-2 were significantly and dose-dependently suppressed by XAV939. LPS-induced NF-κB signaling, such as IκB phosphorylation and degradation as well as nuclear translocation of NF-κB, was also suppressed by XAV939. Target DNA binding of NF-κB was significantly and dose-dependently suppressed by XAV939 during LPS-induced inflammatory response. The suppressive effects of XAV939 on NF-κB signaling, target DNA binding of NF-κB and pro-inflammatory gene expression were all rescued by over expression of β-catenin, which shows that the anti-inflammatory effect of XAV939 is mediated by the modulation of β-catenin, a central component of the WNT/β-catenin pathway.

Conclusion: The findings of this study showed that XAV939 exerts anti-inflammatory effects through the modulation of the Wnt/β-catenin pathway.     

Mechanism of pro-apoptotic action of prosthetic restorations on oral mucosa cells

PurposeThe aim of this study was to check the pro-apoptotic mechanism of prosthetic reconstruction on epithelial cells of the oral mucosa.Patients and methodsThe research was carried out on the saliva of healthy patients using prostheses. The sample swabs were stained using the May-Grünwald-Giemsa method and processed by immunohistochemistry for nuclear factor kappa B (NF-κB; p65) and caspase-3. Western blots were used to detect caspase-3, NF-κB, p53 and COX-2 expression.ResultsWe found an increased expression of caspase-3, NF-κB and p53 in the oral epithelial cells of patients using prosthetic restorations compared to the subjects from the control group. No differences in COX-2 expression were found between the groups. The strongest immunoreactivity and expression of caspase-3, NF-κB and p53 were observed in patients using full prosthesis for less than two years.ConclusionsThe results of the conducted research indicate that prosthetic restorations may affect the process of apoptosis of oral mucosa epithelial cells. Lack of difference in expression of COX-2 in the saliva of the studied patients suggests that apoptosis is not caused by inflammatory factors.     

Involvement of epidermal growth factor receptor-linked signaling responses in Pseudomonas fluorescens-infected alveolar epithelial cells

Pseudomonas fluorescens is an opportunistic indoor pathogen that can cause severe airway proinflammatory responses. Pulmonary epithelium, like other mucosal epithelial linings of the body, constitutes the first line of defense against airway microbial pathogens. Mucosal epithelial cells can be a sentinel of pathogenic bacteria via stimulation of specific cell surface receptors, including the epidermal growth factor receptor (EGFR) and Toll-like receptor (TLR). This study addressed the involvement of EGFR in airway epithelial pathogenesis by P. fluorescens. Human A549 pneumocytes showed prolonged production of proinflammatory interleukin-8 (IL-8) in response to infection with P. fluorescens, which was via the nuclear factor-kappa B (NF-κB) signaling pathway. Production of proinflammatory cytokine IL-8 was not mediated by P. fluorescens lipopolysaccharide, a representative TLR4 agonist, but was mediated through EGFR-linked signals activated by the opportunistic bacteria. Moreover, EGFR signals were involved in NF-κB signal-mediated production of proinflammatory cytokines. Along with persistent NF-κB activation, P. fluorescens enhanced the EGFR phosphorylation and subsequent activation of downstream mediators, including protein kinase B or extracellular-signal-regulated kinases 1/2. Blocking of EGFR-linked signals increased epithelial susceptibility to pathogen-induced epithelial cell death, suggesting protective roles of EGFR signals. Thus, airway epithelial exposure to P. fluorescens can trigger antiapoptotic responses via EGFR and proinflammatory responses via TLR4-independent NF-κB signaling pathway in human pneumocytes.     

薯蓣皂苷减轻卵清蛋白诱导的过敏性哮喘小鼠的气道炎症

目的探讨薯蓣皂苷对卵清蛋白(ovalbumin,OVA)诱导的过敏性哮喘小鼠中过敏性支气管炎的影响及机制。方法24只小鼠随机分为对照组、OVA组、OVA+30 mg/kg薯蓣皂苷组和OVA+60 mg/kg薯蓣皂苷组,每组纳入6只小鼠。全自动生化仪检测各组支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中嗜中性粒细胞、嗜酸性粒细胞和单核细胞的数量;ELISA法检测BALF中促炎因子IL-1β、IL-4、IL-5和TNF-α的含量;PAS染色法观察肺组织黏液分泌情况并对黏液分泌程度进行评分;免疫组化法观察肺组织p-NF-κB p65的表达和分布情况;Western blotting法检测肺组织中p-IκB和NF-κB p65的蛋白表达水平。结果薯蓣皂苷可降低过敏性哮喘小鼠BALF中炎性细胞数量,同时降低BALF中促炎因子IL-1β、IL-4、IL-5和TNF-α的水平以及肺部黏液的分泌和NF-κB的活化水平。结论薯蓣皂苷能减轻过敏性哮喘小鼠的气道炎症,且其抗炎作用与抑制NF-κB活化有关。     

TLR4/PI3K信号相关分子在气道上皮细胞诱导的哮喘气道平滑肌细胞迁移功能中的作用

目的 探讨TLR4、PI3K和NF-kB在气道上皮细胞诱导的哮喘气道平滑肌细胞(airway smooth muscle cells,ASMCs)迁移中的作用.方法 细胞消化法培养原代哮喘ASMCs,TNF-α刺激上皮细胞系RTE细胞收集细胞培养上清液,ELISA检测上清液中IL-8和RANTES的含量,以TLR4抗体、渥曼青霉素(Wortmannin)、二硫代氨基吡咯烷(PDTC)作为工具药,改良Boyden趋化小室检测其在上皮细胞诱导的哮喘ASMCs跨膜迁移中的作用.结果 各TNF-α组RTE培养上清液中IL-8和RANTE水平均显著增高(P＜0.01),20 ng/ml组较其他组显著增高(P＜0.01).各哮喘组ASMCs的跨膜迁移数较正常组均显著增加(P＜0.01),各处理组哮喘ASMCs的跨膜迁移数亦较哮喘组明显增加(P＜0.01),TNF-α+TLR4抗体组、TNF-α +Wortmannin组和TNF-α+Wortmannin+PDTC组哮喘ASMCs跨膜迁移数较TNF-α组明显减少(P＜0.01);TNF-α+Wortmannin +PDTC组ASMCs跨膜迁移数亦低于Wortmannin组(P＜0.05).结论 TLR4/PI3K信号相关分子参与哮喘气道上皮细胞诱导的ASMCs跨膜迁移,可能是哮喘气道重塑的机制之一.     

Immunomodulatory role of recombinant human erythropoietin in acute kidney injury induced by crush syndrome via inhibition of the TLR4/NF-κB signaling pathway in macrophages

Abstract

Objective: The present study aimed to investigate whether recombinant human erythropoietin (rHuEPO) plays an immunomodulatory function by regulating the TLR4/NF-κB signaling pathway.

Materials and methods: C57BL/6 mice were intraperitoneally injected with rHuEPO and, half an hour later, with 50% glycerol at the dose of 7.5?ml/kg to induce crush syndrome (CS)-acute kidney injury (AKI). The levels of TNF-α, IL-1β, IL-6, serum creatinine (Scr), and creatine kinase (CK) were measured. The kidney tissues were analyzed by HE staining, and macrophage infiltration was detected by immunohistochemistry. Double immunofluorescence staining, RT-qPCR, and western blotting were conducted to analyze TLR4/NF-κB p65 expression. Ferrous myoglobin was co-cultured with RAW264.7 cells to mimic crush injury and the production of proinflammatory cytokines. The expression levels of TLR4 and NF-κB p65 were measured.

Results: In vivo study results revealed that rHuEPO ameliorated renal function, tissue damage, production of proinflammatory cytokines, and macrophage infiltration in the kidneys. The protein and mRNA expression levels of genes involved in the TLR4/NF-κB signaling pathway in CS-induced AKI mice were upregulated (p?<?.05). Meanwhile, the expression levels of TLR4, NF-κB p65, and proinflammatory cytokines in RAW264.7 cells were downregulated in CS-AKI mice injected with rHuEPO (p?<?.05).

Conclusions: Our results demonstrated the immunomodulatory capacity of rHuEPO and confirmed that rHuEPO exerts protective effects against CS-induced AKI by regulating the TLR4/NF-κB signaling pathway in macrophages. Therefore, our findings highlight the therapeutic potential of rHuEPO in improving the prognosis of CS-AKI patients.     

不可分型流感嗜血杆菌通过p38 MAPK和NF-κB依赖的方式上调IL-8的表达

目的：探讨不可分型流感嗜血杆菌（NTHi）致肺泡上皮细胞炎症反应的分子机制。方法：A549肺泡上皮细胞株与NTHi（感染复数：10）共孵育15 min、30 min后收集细胞,用Western blotting检测p38丝裂原活化蛋白激酶（p38 MAPK）的磷酸化;4 h后用流式细胞仪检测胞内NF-κB p65亚单位的表达。预先用p38 MAPK抑制剂（SB203580）和NF-κB抑制剂（PDTC）与A549细胞共孵育1 h,然后加入NTHi,24 h后收集上清,以酶联免疫吸附法检测白介素8（IL-8）的水平。结果： NTHi能迅速地诱导p38 MAPK通路的磷酸化。 NTHi刺激4 h后A549细胞胞内NF-κB p65的表达较与未加细菌组明显增加（P<0.05）。NTHi刺激A549细胞24 h后上清中的IL-8较未加细菌组显著增加,差异显著（P<0.05）。与细菌攻击组比较,阻断p38 MAPK或NF-κB通路,能显著地降低A549细胞生成IL-8（P<0.05）。结论：NTHi 以p38 MAPK和NF-κB依赖的方式诱导肺泡上皮细胞的炎症反应。     

MiR-19a mediates the negative regulation of the NF-κB pathway in lipopolysaccharide-induced endometritis by targeting TBK1

Objective

In both humans and animals, endometritis is severe inflammation of the uterus, and it causes great economic losses in dairy cow production. MicroRNAs have been reported to play an important role in various inflammatory diseases. However, the regulatory mechanisms of miR-19a in endometritis remain unclear. Thus, the aims of this study are to investigate the role of miR-19a in a mouse model of lipopolysaccharide (LPS)-induced endometritis and elucidate the possible mechanisms in bovine endometrial epithelial cells (bEECs).

Methods and results

Histological analysis showed that LPS induced severe pathological changes, suggesting that the endometritis mouse model was well established. The qPCR assay indicated that miR-19a expression in the uterine tissues of mice with endometritis and in bEECs with LPS stimulation was significantly reduced. The overexpression of miR-19a significantly decreased the expression of inflammatory cytokines (TNF-α, IL-6 and IL-1β) and the phosphorylation of NF-κB p65 and IκBα. Similar results were also obtained following the knockdown of TBK1. Furthermore, a dual luciferase reporter assay further validated that miR-19a inhibited TBK1 expression by binding directly to the 3′-UTR of TBK1.

Conclusion

We demonstrated that miR-19a has anti-inflammatory effects and mediates the negative regulation of the NF-κB Pathway in LPS-induced endometritis by targeting TBK1.

     

沉默NF-κB p65基因下调TNF-α诱导的肺泡上皮细胞的炎症反应

目的建立急性肺损伤体外细胞炎症模型,探讨NF-κB p65基因沉默减轻细胞炎症、保护肺结构细胞的可行性。方法以TNF-α(10 ng/ml)刺激肺泡Ⅱ型上皮细胞(A549),运用RNA干扰技术沉默NF-κB p65基因,采用RT-PCR及Westernblotting法检测沉默效率,ELISA法检测细胞培养上清中IL-1β、IL-8、IL-10等炎症因子浓度。结果 TNF-α刺激A549细胞可在基因水平上调NF-κB p65的表达,并增加NF-κB蛋白的核转位,上调细胞培养上清中IL-1β、IL-8、IL-10的浓度;预转染NF-κB p65 siRNA可在基因水平及蛋白水平有效沉默NF-κB p65表达,降低上述各炎症因子浓度(P<0.05)。结论急性肺损伤体外细胞炎症模型构建成功,RNA干扰技术能有效沉默该模型NF-κB p65基因,下调炎症反应水平,保护肺泡上皮细胞,为急性肺损伤免疫调控机制的研究和基因靶向治疗提供实验依据。     

Ketamine delays progression of oxidative and damaged cataract through regulating HMGB-1/NF-κB in lens epithelial cells

Objective: Lens epithelial cell (LEC) membrane damage is one of the pathogenesis of cataract. High mobility group box-1 (HMGB-1) and nuclear factor-κB (NF-κB) play vital roles in a variety of diseases, such as inflammation. Ketamine has numerous pharmacological effects that can inhibit inflammation. However, its role in cataract rats LECs has not yet been elucidated.

Materials and methods: LECs were isolated from SD rats and cultured in vitro. The cells were randomly divided into three groups, including the control group, cataract model group induced by H₂O₂, and ketamine group treated by 10?mM ketamine under H₂O₂ environment. LECs proliferation was assessed by MTT assay. LECs apoptosis was evaluated by Caspase-3 activity detection. NF-κB mRNA and protein expressions were tested by real-time PCR and Western blot. HMGB-1 expressions in cells and supernatant were detected by real-time PCR and ELISA. TNF-α and IL-1β secretions were detected by ELISA.

Results: In H₂O₂ model group, the LECs proliferation was significantly inhibited, the caspase-3 activity significantly increased, HMGB-1 mRNA and secretion significantly enhanced, NF-κB mRNA and protein levels significantly elevated, compared to the Control group (p?2O₂ model group compared to the Control group (p?2O₂ model group (p?p?Conclusions: Ketamine delays the progression of oxidative and damaged cataract by regulating HMGB-1/NF-κB expression, inhibiting TNF-α, IL-1β, and apoptosis, and promoting cell proliferation.     

川芎嗪通过AMPK/NF-κB和Nrf-2/HO-1途径减轻过敏性气道炎症和氧化应激的实验研究

目的 本研究旨在探讨川芎嗪对过敏性炎症反应和氧化应激的影响,并探讨其机制是否与AMPK/NF-κB和Nrf-2/HO-1信号通路有关.方法 用组织化学染色来观察小鼠肺组织病理学改变;小鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中炎症细胞数量用Diff-quik染色法检测;用...