姜黄素对机械牵张引发的Ⅱ型肺泡上皮细胞ezrin/Akt通路及炎性因子的影响

目的探讨姜黄素对机械牵张引发的Ⅱ型肺泡上皮细胞(AT-Ⅱ细胞)ezrin/Akt通路及炎性因子的影响及其作用机制。方法原代培养大鼠AT-Ⅱ细胞,采用Western blot及ELISA方法检测AT-Ⅱ细胞在20%机械牵张力刺激4、8、16、24、48 h时p-ezrin、ezrin、p-Akt和Akt蛋白表达及炎性因子TNF-α、IL-1β及IL-6分泌的动态变化;并且进一步分析不同浓度姜黄素(10、20、30μmol/L)对ezrin/Akt通路蛋白及炎性因子水平的影响。结果机械牵张干预后,AT-Ⅱ细胞p-ezrin、p-Akt蛋白表达水平明显增高,且在4～16 h范围内表达水平持续增高,而ezrin、Akt蛋白表达无明显变化;AT-Ⅱ细胞TNF-α、IL-1β及IL-6分泌量在机械牵张刺激8 h内无明显变化,之后随刺激时间延长明显升高。在机械牵张刺激下,不同剂量姜黄素能够不同程度抑制p-ezrin、p-Akt蛋白表达及减少TNF-α、IL-1β及IL-6分泌。结论姜黄素对机械所致肺损伤有保护作用,其作用可能与抑制ezrin/Akt信号传导、调节炎性因子释放有关。     

白三烯受体拮抗剂对哮喘小鼠肺组织HMGB1表达的影响

目的 探讨白三烯受体拮抗剂对哮喘小鼠肺组织高迁移率族蛋白B1(HMGB1)表达的影响.方法 72只SPF级Balb/c小鼠随机分为哮喘组(A组)、孟鲁司特治疗组(B组)和生理盐水对照组(C组),每组24只.建守哮喘模型.于第1、2、3、4周4个时段,HE染色观察肺组织病理改变,RT-PCR法检测肺组织HMGB1 mRNA表达,免疫组化标记分析HMGB1在肺组织的分布.结果 HE染色B组肺组织炎症较A组明显减轻.第2周时A、B组肺组织HMGB1的表达明显高于C组[(1.58±0.47)、(1.42士0.30)vs.(0.65±0.15)](P<0.01);B组肺组织HMGB1的表达随治疗时间的延长而减少.结论 白三烯受体拮抗剂能抑制哮喘小鼠气道炎症,减少HMGB1的生成.     

连翘苷调控细胞因子信号转导抑制因子1对肺炎链球菌诱导的肺泡上皮细胞凋亡和氧化应激的影响

目的 研究连翘苷调控细胞因子信号转导抑制因子1(SOCS1)对肺炎链球菌诱导的肺泡上皮细胞凋亡和氧化应激影响。方法 将人Ⅱ型肺泡上皮细胞分为对照组、模型组(肺炎链球菌感染)、实验低、中、高剂量组(在模型组基础上分别给予5、10、20μmol·L^-1连翘苷)、实验高剂量+si-NC组(肺炎链球菌感染,转染siRNA NC、20μmol·L^-1连翘苷处理)、实验高剂量+si-SOCS1组(肺炎链球菌感染,转染siRNA SOCS1、20μmol·L^-1连翘苷处理)。用蛋白质印迹(Western blot)法检测SOCS1蛋白表达;用细胞计数试剂盒-8(CCK-8)检测细胞增殖;用碘化丙啶(PI)单染法检测细胞周期;用Annexin V-PI双染法检测细胞凋亡;用可见分光光度法检测丙二醛(MDA);用氮蓝四唑(NBT)法检测超氧化物歧化酶(SOD)。结果 对照组、模型组、实验高剂量组、实验高剂量+si-NC组和实验高剂量+si-SOCS1组肺泡上皮细胞SOCS1蛋白表达水平分别为1、0.54±0.04、0.96±0.04、0....     

TLR4/NF-κB通路在脂多糖诱导喉癌细胞释放HMGB1中的作用

目的探讨脂多糖(LPS)对喉癌细胞高迁移率族蛋白B1(HMGB1)释放的诱导作用。方法采用酶联免疫吸附试验、实时荧光定量PCR和Western blot检测LPS诱导后人喉癌细胞株Hep-2培养上清液中HMGB1含量、细胞HMGB1mRNA表达水平和细胞Toll样受体4(TLR4)、核因子-κB(NF-κB)蛋白表达水平的变化,观察TLR4单克隆抗体、NF-κB抑制剂二硫代氨基甲酸吡咯烷(PDTC)对HMGB1释放的影响。结果 LPS诱导12h和18h后,HMGB1mRNA表达水平和HMGB1含量均升高,且呈时间依赖性;LPS诱导24h后,TLR4及NF-κB p65蛋白明显升高。TLR4单克隆抗体和PDTC对LPS诱导的HMGB1释放有不完全的抑制作用。结论 LPS诱导喉癌细胞释放HMGB1;其机制可能与TLR4/NF-κB信号通路有关。     

HMGB1和MMP-9在食管鳞癌组织中的表达及临床意义

目的 探讨高迁移率族蛋白B1(HMGB1)和基质金属蛋白酶-9(MMP-9)在食管鳞癌组织中的表达及临床意义.方法 采用免疫组化EnVision法检测食管鳞癌组织(A组,87例)和癌旁正常黏膜组织(B组,40例)中HMGB1和MMP-9的表达.结果 A组HMGB1和MMP-9阳性表达率均高于B组(69.0％ vs.22.5％和62.1％ vs.12.5％)(P＜0.05).A组HMGB1和MMP-9阳性表达与肿瘤浸润深度、TNM分期有关(P＜0.05).A组HMGB1和MMP-9表达呈正相关(rs =0.295,P＜0.01).结论 联合检测食管鳞癌组织中HMGB1和MMP-9蛋白表达可能有助于评估食管鳞癌的恶性程度.     

高迁移率族蛋白B1及其与炎症反应相关性的研究进展

高迁移率族蛋白（High mobility group protein，HMG）因其在聚丙烯酰胺凝胶电泳中的高迁移能力而得名．是一大类富含电荷的低分子非组蛋白染色体核蛋白，HMGB1是其成员之一，山A区和B区及C端尾部3个结构域构成。B区的1-20号氨基酸是诱导TNF、IL-1、IL-6等细胞因子产生的部位。HMGB1因参与了DNA复制、转录和修饰，     

高迁移率族蛋白B1参与神经炎症反应及其抑制剂在神经精神疾病中的应用

高迁移率族蛋白B1(high mobility group box 1 protein,HMGB1)是一种高度保守的染色体结合蛋白,主要存在于真核细胞核中,参与基因的复制、转录和修复等过程,也可被免疫活性细胞和坏死细胞释放至细胞外,通过激活RAGE受体和Toll样受体2/4等引起炎症反应.大量研究表明,HMGB1炎症信...     

HMGB1的生物学作用

高迁移率族蛋白B1(HMGB1)是一组高度保守的非组蛋白核蛋白,在真核细胞中普遍存在.本文对HMGB1近几年的研究进展进行了综述.     

HMGB1介导的炎症通路在雷公藤致大鼠肝损伤中的作用研究

目的:研究高迁移率族蛋白B1(HMGB1)介导的炎症通路HMGB1-Toll样受体4(TLR4)/核转录因子κB(NF-κB)在雷公藤致大鼠肝损伤中的作用,为阐明雷公藤致肝损伤的作用机制提供参考。方法:将24只SD大鼠随机分为空白组(生理盐水,灌胃)、雷公藤组(以生药计16 g/kg,灌胃)和中和剂组(腹腔注射100 mg/kg甘草酸铵溶液3 h后再灌胃以生药计16 g/kg的雷公藤),每组8只,各组大鼠均连续给药3周。每周给药后均检测大鼠血清中天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)水平;给药结束后,采用酶联免疫吸附法检测大鼠血清中HMGB1、白细胞介素1β(IL-1β)、IL-2、肿瘤坏死因子α(TNF-α)水平,Western blot法检测大鼠肝组织中HMGB1、NF-κB p65、TLR4蛋白表达,苏木精-伊红染色后观察大鼠肝组织病理学改变。结果:给药3周后,雷公藤组大鼠血清中AST、ALT、HMGB1、IL-1β、IL-2、TNF-α水平以及肝组织中HMGB1、NF-κB p65、TLR4蛋白表达水平均显著高于空白组和中和剂组(P<0.05或P<0.01)。雷公藤组大鼠肝组织中央静脉周围肝细胞水肿,部分肝细胞浆内可见圆形空泡;中和剂组大鼠仅少量细胞内可见大小不一的空泡。结论:雷公藤致大鼠肝损伤的机制可能与其激活了HMGB1-TLR4/NF-κB炎症通路有关。     

Metformin inhibits HMGB1 release in LPS-treated RAW 264.7 cells and increases survival rate of endotoxaemic mice

BACKGROUND AND PURPOSE

Recently, metformin, a well-known anti-diabetic drug, has been shown to possess anti-inflammatory activities. This study investigated the effect of metformin on the expression of pro-inflammatory cytokines including high mobility group box 1 (HMGB1) in lipopolysaccharide (LPS)-treated animals and cells.

EXPERIMENTAL APPROACH

We investigated whether metformin inhibits the release of HMGB1 in LPS-treated RAW 264.7 cells and increases survival rate in endotoxaemic mice (lethal endotoxaemia was induced by an i.p. injection of LPS). This was achieved by a range of techniques including Western blotting, enzyme-linked immunosorbent assay, specific pharmacological inhibitors, knock out of α₁-subunit of AMP-activated protein kinase (AMPK) and recombinant HMGB1.

KEY RESULTS

Both pre- and post-treatment with metformin significantly improved survival of animals during lethal endotoxaemia (survival rate was monitored up to 2 weeks), decreased serum levels of tumour necrosis factor-alpha (TNF-α), interleukin-1β, HMGB1 expression and myeloperoxidase activity in lungs. However, metformin failed to improve survival in endotoxaemic animals that had additionally been treated with recombinant HMGB1. In an in vitro study, metformin dose-dependently inhibited production of pro-inflammatory cytokines and HMGB1 release. Metformin activated AMPK by its phosphorylation. Compound C (pharmacological inhibitor of AMPK) and siAMPKα1 reversed the anti-inflammatory effect of metformin in LPS-treated cells.

CONCLUSIONS AND IMPLICATIONS

Our data indicate that metformin significantly attenuates the pro-inflammatory response induced by LPS both in vivo and in vitro. Metformin improved survival in a mouse model of lethal endotoxaemia by inhibiting HMGB1 release. AMPK activation was implicated as one of the mechanisms contributing to this inhibition of HMGB1 secretion.     

Astragaloside IV attenuates inflammatory reaction via activating immune function of regulatory T-cells inhibited by HMGB1 in mice

Context: High-mobility group box 1 (HMGB1) protein is a highly abundant protein that can promote the pathogenesis of inflammatory. Some experiments have demonstrated a vital role for HMGB1 to modulate the immune function of regulatory T-cells (Tregs). Astragaloside IV (AST IV), an extract from Astragalus membranaceus Moench (Leguminosae), has been shown to exert potent cardioprotective and anti-inflammatory effects. It is still unclear whether AST IV has a latent effect on the proinflammatory ability of HMGB1 with subsequent activation of Tregs in vivo.

Objective: This research explores the antagonism of different doses of AST IV on the immunologic function of Tregs mediated by HMGB1.

Materials and methods: Mouse models (BALB/c) were constructed by which normal saline or AST IV was administered i.p. at 2, 4 and 6 days after the administration i.p. of 20?μg recombinate HMGB1. Spleen was used to procure Treg and CD4?⁺?CD25^? T-cells which were co-cultured with Treg. Cell phenotypes of Tregs(Foxp3) were examined, and the cytokine levels in supernatants and the proliferation of T-cells were assayed. Gene expression was measured by RT-PCR.

Results: (1) The expression levels of Foxp3 in Treg on post-stimulus days (PSD) 1–7 were significantly decreased in the HMGB1 group in comparison to those in the control group mice (p?<?0.01). The Foxp3 expression was markedly increased in a dose-dependent manner in the AST group as compared with those in the HMGB1 group (p?<?0.0 1–0.05). The same results were found in the contents of cytokines (IL-10 and TGF-β) released into supernatants by Treg. (2) When CD4?⁺?CD25^? T-cells were co-cultured with Treg stimulated by HMGB1, the cell proliferation and the levels of cytokines (IL-2 and IFN-γ) in supernatant were markedly increased as compared with those in the HMGB1 group. The level of IL-4 was markedly decreased as compared with that in the HMGB1 group. The same results were found when CD4?⁺?CD25^? T-cells were co-cultured with Treg in the NS group. Compared with those in the NS group, the contrary results were shown in a dose-dependent manner in the AST group.

Discussion and conclusion: These results showed that AST IV has a therapeutic effect on inflammation promoted by HMGB1, and it should be studied as a new drug for the treatment of sepsis.     

血清高迁移率蛋白B1水平与脓毒症病死率的关系及其机制

目的 探讨高迁移率蛋白B1（HMGB1）与脓毒症患者病死率的关系及其可能机制.方法 测定48例脓毒症患者血清HMGB1、超氧化物歧化酶（SOD）和丙二醛（MDA）的变化并记录患者转归.结果 发病后,死亡组和存活组患者血清HMGB1水平均逐渐上升,72 h达高峰.死亡组患者血清HMGB1水平明显高于存活组（12和24 h除外）（t=6.07、6.20、24.43,均P＜0.05）.12、24、48和72 h死亡组患者血清SOD活力均明显低于存活组（t=10.24、20.61、11.67、33.33,均P＜0.05）,所测时间点死亡组患者血清MDA水平均明显高于存活组（t=26.06、22.17、23.86、9.49、5.95,均P＜0.05）.HMGB1水平与MDA水平成显著正相关.结论 血清HMGB1水平升高可能是脓毒症患者病死率增加的重要原因,氧化抗氧化系统失衡可能是HMGB1升高的原因之一.     

Syringic acid suppresses ferroptosis of skeletal muscle cells to alleviate lower limb ischemia/reperfusion injury in mice via the HMGB1 pathway

Ischemia/reperfusion (I/R) of skeletal muscle in the lower limbs is an important factor affecting the outcome of lower limbs ischemia patients, with no effective preventive or therapeutic approaches available. The study was to investigate the effect of syringic acid (SA) on I/R skeletal muscle in the lower limbs injury. Mice femoral artery I/R models and C2C12 cell hypoxia/reoxygenation (H/R) models was establish, tissue damage, inflammatory status, and high mobility group box 1 (HMGB1) pathway were evaluated using histological analysis, enzyme-linked immunosorbent assay, and western blotting. Further, the study detected the effect of SA on cell apoptosis, lipid peroxidation, Fe²⁺ level, and ferroptosis-related proteins expression. Finally, the effect of HMGB1 expression on SA in H/R stimulation was studied. SA alleviated pathological damage and reduced levels of IL-1β, IL-6, and TNF-α in muscle tissues from femoral artery I/R mouse models. SA upregulated Bcl-2 and SOD as well as downregulated Bax, MDA, TBARS content, and Fe²⁺ level in H/R-induced cells. SA inhibited HMGB1 expression and promoted Nrf2, HO-1, GPX4, and SLC7A11 expressions in the injured tissues and cells. Such effects of SA on H/R-induced cells were rescued by HMGB1 overexpression. SA suppressed ferroptosis of skeletal muscle cells to alleviate lower limb I/R injury in mice by blocking the HMGB1 pathway, providing new insights for the treatment of lower limb ischemia–reperfusion injury.     

Cannabinoid receptor 2 promotes the intracellular degradation of HMGB1 via the autophagy-lysosome pathway in macrophage

High mobility group box 1 (HMGB1) is a late phase inflammatory mediator in many inflammatory diseases. Extracellular HMGB1 could bind to many membrane receptors to activate downstream signaling molecules and promote inflammation resulting in cell and tissue damage. In our previous work, we found cannabinoid receptor Ⅱ(CB2R) inhibited the expression of HMGB1 in lipopolysaccharide (LPS)-induced septic models in vivo and in vitro, but the underlying mechanism is still unclear. The present study was aimed to explore the possible pathway through which CB2R suppressed HMGB1. Here, we found that the specific agonist of CB2R, GW405833 (GW) could induce intracellular HMGB1 degradation without influencing HMGB1 mRNA in peritoneal macrophages. Then we observed that autophagy inhibitor 3-methyladenine (3-MA) but not proteasome inhibitor MG-132 (MG) could block GW-induced HMGB1 degradation, which indicated that the autophagy-lysosome but not the ubiquitination pathway was involved in this process. Further study showed that GW could promote the integrity of autophagy flux in macrophages in terms of increased level of LC3Ⅱand decreased expression of p62 protein. It also observed that inhibition of autophagy blocked GW-induced nuclear translocation of HMGB1 in macrophages. GW could up-regulate expression of Cathepsin B (CTSB), and inhibition of CTSB blocked GW-induced HMGB1 degradation. In summary, all the data showed that activation of CB2R could promote the intracellular degradation of HMGB1 via the autophagy-lysosome pathway in macrophage.     

Lung epithelial binding peptide-linked high mobility group box-1 A box for lung epithelial cell-specific delivery of DNA

High mobility group box-1 A box (HMGB1A) is an anti-inflammatory peptide originating from HMGB1. A previous report demonstrated that recombinant HMGB1A could deliver DNA into cells. Lung epithelial-specific gene delivery is required for the gene therapy of various lung diseases such as acute lung injury. In this study, a lung epithelial-specific DNA carrier was produced by linking the lung epithelial binding peptide (LEBP) to HMGB1A. An LEBP-linked HMGB1A (LEBP-HMGB1A) expression vector, pET21a-LEBP-HMGB1A, was constructed. LEBP-HMGB1A was expressed in BL21 strain and purified by consecutive applications of nickel affinity chromatography and cationic exchange chromatography. In a gel retardation assay, LEBP-HMGB1A completely retarded DNA at a 5:1 weight ratio (peptide:DNA). LEBP-HMGB1A/DNA complexes were prepared at various weight ratios, to which a fixed amount of polyethylenimine (2?kDa, PEI2k) was added to increase the proton buffering effect of the complex. LEBP-HMGB1A had the highest transfection efficiency to L2 lung epithelial cells at a 20:1 weight ratio (peptide:DNA). At this ratio, LEBP-HMGB1A had a higher transfection efficiency than poly-L-lysine (PLL) as well as HMGB1A without LEBP. A cytotoxicity assay showed that LEBP-HMGB1A was not toxic to L2 cells. Therefore, LEBP-HMGB1A may be useful in developing gene therapies for lung diseases.     

HMGA1和HMGA2在胰腺癌中的表达及临床意义

目的 探讨高迁移率族蛋白A1(high mobility group protein A1,HMGA1)、高迁移率族蛋白A2(high mobility group protein A2,HMGA2)在胰腺癌组织中的表达及其临床意义.方法 采用免疫组化SP法检测60例胰腺癌组织和 30 例正常胰腺组织中的HMGA1、HMGA2表达,并分析两者与胰腺癌临床病理参数间的相互关系.结果 在胰腺癌组织中HMGA1和HMGA2的表达均显著高于正常胰腺组织(P<0.01);HMGA1的表达强度与胰腺癌的TNM分期、组织学分级、淋巴结转移情况有关(P<0.05);而HMGA 2的表达强度仅与胰腺癌的组织学分级及TNM分期有关(P<0.05);HMGA1和HMGA2的表达呈正相关关系(r=0.428,P=0.001).结论 HMGA1、HMGA2在胰腺癌组织中呈高表达,二者在胰腺癌的发生、发展过程中具有协同作用,共同促进胰腺癌的侵袭和转移,HMGA1及HMGA2联合检测可作为胰腺癌早期诊断和判断预后的辅助指标.