High-mobility group box 1 inhibition protects against hepatic ischemia-reperfusion injury

Introduction. Hepatic ischemia followed by reperfusion (I/R) occurs in the settings of trauma, transplantation, and elective liver resections. The mechanisms that account for local organ damage are only partially understood. High-mobility group box 1 (HMGB1) has been identified as a late mediator of lethality in sepsis and is released by damaged tissues where it could also act as a mediator of inflammation and organ injury. We hypothesized that HMGB1 would contribute to organ injury following hepatic I/R. Methods. HMGB1 protein expression and hepatocellular secretion were determined in rat hepatocytes subjected to hypoxia (1% O₂) versus normoxic cells. The role of HMGB1 in mediating hepatic I/R injury was examined in C57Bl/6 mice that underwent 90 min of partial I/R injury. These mice were pretreated with neutralizing HMGB1 antibody (600 μg) or control normal saline 1 h prior to ischemia. Results. Basal HMGB1 expression was observed in normoxic hepatocytes and was dramatically up regulated 12-24 h after hypoxia. This corresponded to increased HMGB1 secretion 18-24 h after hypoxia. In mice undergoing partial warm liver I/R injury, HMGB1 protein expression is increased as early as 1 h after reperfusion and then increases in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after warm ischemia compared to control animals.

TABLE—ABSTRACT 81.

Group	ALT-1 h	ALT-6 h	ALT-24h
Sham	67 ± 24	57 ± 8	51 ± 2
Control	3101 ± 1167	8140 ± 1656	905 ± 65
Anti-HMGB1	662 ± 120∗	2382 ± 687∗	265 ± 16∗

Note. Data are mean ± SEM, n = 3-4 per group;

∗

indicates P < 0.05 versus control.

Full-size table

     

High-mobility group box 1 expressions in hypoxia-induced damaged mouse islets

Introduction

Discovering a new, accurate, and useful damage marker for isolated islets is critical for avoiding the transplantation of nontherapeutic preparations. Recently, we have reported that islets that contained uniquely high levels of high-mobility group box 1 (HMGB1) protein and cytokine induced damaged islets released HMGB1 in a mouse model. Islets are frequently exposed to hypoxic conditions during organ procurement, organ transportation, islet isolation, and islet storage before transplantation. In the present study, we analyzed HMGB1 expressions in hypoxia-induced damaged mouse islets.

Methods

Damaged mouse islets were generated by hypoxic conditions (1% O2, 5% CO₂, and 94% N₂). HMGB1 expressions and production levels were assessed by quantitative real-time polymerase chain reaction (PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA) studies. In vivo islet function was analyzed using transplantation assay using streptozotocin-induced diabetic mice.

Results

HMGB1 was mainly stained in the nucleus in the intact islets; however, HMGB1 was present in not only the nucleus, but also the cytoplasm in hypoxia-induced damaged islets. HMGB1 messenger RNA (mRNA) levels were up-regulated in the hypoxia-induced damaged islets, suggesting that HMGB1 was intentionally generated during hypoxia. HMGB1 protein levels in the islets were gradually decreased with time under hypoxic conditions. The amount of released HMGB1 levels and the amount of released HMGB1 levels per hour were significantly increased in damaged (noncurable) islets.

Conclusions

When islets were damaged by hypoxic condition, HMGB1 was synthesized and released from hypoxia-induced damaged islets. The amount of released HMGB1 and/or the amount of released HMGB1 per hour might be a useful marker for detecting damaged islets and might be used for islet potency assay.     

胆红素浓度对肝星状细胞活化增殖和凋亡的影响

目的 探讨低浓度胆红素( bilirubin)对活化态肝星状细胞(hepatic stellate cells,HSCs)增殖、活化、凋亡的影响.方法 从肝脏分离纯化培养HSCs,DCFH-DA法检测不同浓度胆红素对HSCs中活性氧的影响.通过CCK-8比色法检测胆红素对HSCs增殖的影响.Western blot检测HSCsα-肌动蛋白(d-SMA)的表达.流式细胞仪检测胆红素对HSCs凋亡的影响.PCR检测胆红素对HSCs纤维化相关基因表达的影响.结果 分离并培养HSCs,低浓度胆红素浓度(0、1、10、20 mg/L)抑制HSCs中活性氧的产生,明显抑制HSCs的增殖(0.624±0.092,0.536±0.127,0.407±0.033,0.399±0.022,F=13.454,P＜0.05)及α-SMA的表达(339 ±2,336±10,246±7,242±5,3.7±0.3,F=191.107,P＜0.05),促进HSCs的凋亡(2.69±0.07％,2.95±0.10％,4.41±0.22％,4.91±0.86％,F=34.731,P＜0.05),改变HSCs纤维化相关基因的表达,TIMP-1 mRNA/MMP-2 mRNA的比值呈降低趋势(54±2,65±2,47 ±2,44±2,F=73.400,P＜0.05).结论 低浓度胆红素具有抑制HSCs活化增殖,促进凋亡的潜能.胆红素的作用可能与其抗氧化功能有关.     

白藜芦醇调节肝星状细胞活性的实验研究

目的 探讨白藜芦醇（resveratrol,Res）对活化态肝星状细胞（HSCs）增殖、活化的影响及其机制。方法 从肝脏分离纯化培养HSCs,DCFH-DA法检测不同浓度Res对HSCs中活性氧的影响,CCK-8比色法检测Res对HSCs增殖的影响,Western blotting检测HSCsα-肌动蛋白（α-SMA）的表达,PCR检测Res对HSCs活性相关基因表达的影响。结果 Res抑制HSCs中活性氧的产生,明显抑制HSCs的活化与增殖（P＜0.05）,改变了HSCs活性相关基因（大鼠生肌调节因子MyoD、胶原蛋白Ⅲ及胶原蛋白I）的表达（P＜0.05）。结论 Res能够抑制HSCs的活化增殖,Res对HSCs的作用可能与抗氧化及抑制MyoD的表达有关。     

肝星状细胞在肝脏疾病中的作用

肝星状细胞（HSC）是肝脏内重要的非实质细胞之一,可分泌、释放多种胶原纤维和细胞骨架蛋白参与肝脏疾病的病理生理过程。正常状态下,HSC通过调节细胞外基质蛋白的合成和降解维持肝脏正常的组织结构;肝脏损伤时,HSC被激活,活化的HSC导致细胞外基质的增加是肝纤维化形成并最终导致肝硬化、肝衰竭的主要原因。因此,深入研究HSC在肝脏疾病发生与发展中的作用和机制,并研究与HSC相关的治疗策略,对于提高患者生存率具有一定意义。     

Liver cirrhosis and hepatic stellate cells

The cirrhosis represents the final stage of several chronic hepatic diseases and it is characterized by the presence of fibrosis and morphologic conversion from the normal hepatic architecture into structurally abnormal nodules. In the evolution of the disease there is loss of the normal vascular relationship and portal hypertension. There are also regenerative hepatocellular alterations that become more prominent with the progression of the disease. The liver transplantation continues to be the only therapeutic option in cases of disease in terminal phase. The hepatic stellate cells (HSC) are perisinusoidal cells that store vitamin A and produce growth factors, citocins, prostaglandins and other bioactive substances. They can suffer an activation process that convert them to cells with a phenotype similar to myofibroblasts. When activated, they present increased capacity of proliferation, mobility, contractility and synthesis of collagen and other components of extracellular matrix. They possess cytoplasmic processes adhered to sinusoids and can affect the sinusoidal blood flow. HSC are important in pathogenesis of fibrosis and portal hypertension.     

脂肪特异性蛋白27基因抑制大鼠肝星状细胞增殖活化

目的 探讨脂肪特异性蛋白27(Fsp27)基因对活化态肝星状细胞(HSCs)增殖、活化的影响及其对纤维化相关基因的调节作用.方法 提取HSCs并培养.用实时定量PCR技术检测原代HSCs及活化HSCs中的Fsp27基因表达.构建携带Fsp27目的基因的慢病毒,转染活化的HSCs并继续培养72 h.通过CCK-8比色法检测Fsp27基因对HSCs增殖的影响.Western blot检测HSCsα-肌动蛋白(α-SMA)的表达,了解HSCs的活化状态.实时定量PCR技术研究Fsp27基因对HSCs纤维化相关基因表达的影响.结果 成功分离原代HSCs,Fsp27基因在原代HSCs及活化HSCs中表达差异显著(P＜0.01).活化HSCs成功转染携带Fsp27目的基因的慢病毒后继续培养72 h,与对照组比较,HSCs的活化与增殖受到明显抑制(P＜0.05);Fsp27基因促进MMP-2的表达(P＜0.05),降低了TIMP-1及TGF-β1的表达(P＜0.05).结论 Fsp27基因具有抑制HSCs活化增殖的潜能,Fsp27基因可调节纤维化相关基因的表达.Fsp27基因的作用可能与维持HSCs静息状态细胞表型有关.     

高迁移率族蛋白1和急性肺损伤

背景 高迁移率族蛋白1(high mobility group box protein 1,HMGB1)是一种非组蛋白染色体结合蛋白,进化上高度保守.HMGB1广泛存在于细胞核内,在核内作为DNA分子伴侣,被释放至细胞外将发挥促炎作用.急性肺损伤(acute lung injury,ALI)和急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是全身性炎症的肺部表现,特征为血管通透性增高致组织水肿、器官功能障碍.越来越多的研究证实细胞外的HMGB1能够通过促进NF-κB核转位和显著增加促炎因子释放引起ALI和致死性的炎症反应.目的 阐述HMGB1和ALI之间的关系以及HMGB1在治疗ALI中的研究现况,为临床治疗ALI提供新的靶点.内容 重点回顾有关HMGB1在ALI发病中的作用、HMGB1抑制剂在改善ALI中作用的研究.趋向 HMGB1抑制剂可以改善ALI结局,在治疗ALI中具有广阔前景.     

高迁移率族蛋白B1对体外人肺动脉血管平滑肌细胞增殖、迁移和凋亡的影响

目的 评价高迁移率族蛋白B1(HMGB1)对体外培养人肺动脉血管平滑肌细胞(hPASMC)增殖、迁移和凋亡的影响.方法 体外培养hPASMC,调整细胞密度(2× 105个/ml)后接种到96孔板(100 μl/孔,2× 105个/ml)、6孔板(1ml/孔,2× 106个/ml)和改良24孔Boyden趋化小室(100μg/孔,5×103个/ml),采用随机数字表法,将其分为5组:对照组(C组)和不同浓度HMGB1组(H1组～H4组),分别在DMEM和含HMGB1 1、10、100、1000 ng/ml的DMEM培养液孵育.孵育24和48 h时,采用MTT法检测细胞增殖率,Boyden小室法检测透膜细胞数,TUNEL法检测hPASMC凋亡情况.结果 与C组比较,H1组～H4组细胞增殖率升高,透膜细胞数增多(P＜ 0.05);与H1组比较,H2组～H4组细胞增殖率升高,H3组和H4组透膜细胞数增多(P＜0.05);与H2组比较,H3组和H4组细胞增殖率升高,透膜细胞数增多(P＜ 0.05);H3组和H4组间各指标比较差异无统计学意义(P＞0.05);与孵育24h时比较,各组孵育48 h时细胞增殖率升高(P＜0.05).各组细胞凋亡率比较差异无统计学意义(P＞ 0.05).结论 HMGB1可促进hPASMC的增殖和透膜迁移,可能参与肺损伤肺血管重构的发生.     

肝星状细胞对同种异体胰岛移植物的保护作用

目的 研究小鼠肝星状细胞对同种异体胰岛移植物的保护作用.方法 将糖尿病小鼠随机分为三组,分别为糖尿病组、单纯胰岛移植组及与肝星状细胞共同移植组.单纯移植组于肾被膜下移植入同种异体胰岛300个;共同移植组移植入肝星状细胞(3×105个)与同种异体胰岛(300个)混合物.分别于移植术后监测受体小鼠血糖值及正常血糖维持时间;血糖正常一周后移植组及糖尿病组小鼠分别采血检测血清中TGF-β,TNF-α,IL-1β,IFN-γ的含量,同时取出移植物进行免疫组织化学检测.结果 术后共同移植组受体正常血糖维持时间为(23.75±8.96)d,单纯胰岛移植组受体正常血糖维持时间为(11.9±6.92)d,差异有统计学意义(P<0.05);三组受体血清中TNF-α、IL-1β、IFN-γ含量差异无统计学意义(P>0.05),共同移植组受体血清中TGF-β含量为(2292.31±5.87)pg/ml,单纯移植组为(1246.55±38.91)pg/ml,两组比较差异有统计学意义(P<0.05);病理学结果显示共同移植组胰岛素表达量大,并且在移植物周围有生物包膜形成.结论 在同种异体移植模型中,肝星状细胞可能通过高分泌TGF-β、局部形成包囊等方式保护胰岛移植物并延长其存活时间.     

高迁移率族蛋白-1在急性胰腺炎中的表达及意义

目的 观察急性胰腺炎大鼠高迁移率族蛋白-1(HMGBl)表达水平的变化,探讨其在急性胰腺炎发病过程中的作用和意义.方法 分别以2%及3.8%的牛磺胆酸钠逆行注入大鼠胰胆管,制作大鼠轻型和重症急性胰腺炎模型.将84只大鼠随机分成3组:假手术组(SO组n=28),轻型胰腺炎组(MAP组n=28),重症胰腺炎组(SAP组n=28).对胰腺损伤进行病理评分,免疫组织化学方法观察胰腺中HMGB1的表达情况,ELISA法测定血清中HMGB1水平.结果 MAP和SAP组胰腺组织HMGB1的表达在建模后12h明显升高,于24h达高峰,至48h明显下降,各时段胰腺组织HMGB1的表达与SO组比较差异具有显著统计学意义(P＜0.05).建模后12、24、48、72 h SAP组胰腺HMGBl免疫组化IOD均值明显高于MAP组(P＜0.05),胰腺HMGBl表达程度与胰腺病理损伤程度呈正相关.MAP和SAP组大鼠的血清HMGB1水平在建模后12h明显升高,于48h达高峰,至72h明显下降,各时段的血清HMGB1水平与SO组比较差异具有显著统计学意义(P＜0.05).SAP组的血清HMGB1水平于建模后24、48、72 h明显高于MAP组(P＜0.05),血清HMGB1水平与胰腺病理损伤程度呈正相关.结论 HMGB1可能作为晚期炎症介质参与了胰腺炎的局部及全身炎症反应,并可作为评价胰腺炎病变和炎症反应程度的有效指标.     

ICAM-1 expressed on hepatic stellate cells plays an important role in immune regulation

The authors have demonstrated a strong T-cell inhibitory activity of hepatic stellate cells (HSC), which may participate in the establishment of hepatic tolerance. The underlying mechanism is not completely understood. This study showed that intercellular adhesion molecule 1 (ICAM-1) was constitutively expressed on HSC, and up-regulated upon activation. ICAM-1 knockout mice was used to analyze the role of ICAM-1 expressed on HSC, and showed that deficiency in ICAM-1 expression partially reverses HSC immune inhibitory activity both in vitro and in vivo, but did not significantly affect their capacity to induce T-cell apoptosis.     

活化的肝星状细胞促进调节性T细胞的表达

目的 越来越多的研究证实,活化的肝星状细胞(hepatic stellate cells,HSCs)具有免疫抑制功能.本研究观察了HSCs对T细胞介导的适应性免疫应答的影响及其与调节性T细胞(regulatory T cells,Treg)的关系.方法 分离培养小鼠HSCs,运用混合淋巴细胞反应(mixed lymphocyte reaction,MLR),体外观察活化的HSCs对T细胞增殖和Treg细胞的影响.结果 活化的HSCs能导致树突状细胞(dendritic cells,DCs)介导的适应性免疫应答中T细胞的低反应性:抑制T细胞的增殖,同时诱导Treg细胞的表达.结论 活化的HSCs能通过促进Treg细胞表达而诱导适应性免疫应答中T细胞的活化无能,促进免疫耐受.     

胰岛素样生长因子结合蛋白7在活化肝星状细胞促肝纤维化中的作用

目的 探讨胰岛素样生长因子结合蛋白7(IGFBP7)对肝星状细胞-T6合成分泌纤维连接蛋白的调节作用及抗IGFBP7抗体诱导肝星状细胞-T6凋亡的作用.方法 体外培养活化的肝星状细胞-T6分为5组:(1)空白对照组;(2)IGFBP7 20μg/L组;(3)抗IGFBF7抗体0.25 mg/L组;(4)抗IGFBP7抗体0.50 mg/L组;(5)抗IGFBP7抗体1.00 mg/L组.采用Western blot及ELISA法检测肝星状细胞-T6经IGFBP7作用24 h后纤维连接蛋白的表达及合成分泌量的变化;MTT比色法检测抗IGFBP7抗体对肝星状细胞-T6作用14 h后增殖抑制的影响,同时用流式细胞仪测定抗IGFBP7抗体对肝星状细胞-T6凋亡发生率的影响.采用单因素方差分析,两样本均数比较采用t检验,多重比较采用SNK-q检验.结果 肝星状细胞-T6经IGFBP7作用后纤维连接蛋白的表达及合成分泌量较空白对照组显著增加(t=22.06,7.43,P<0.05).抗IGFBP7抗体对肝星状细胞-T6的增殖具有明显抑制作用(F=14.70,P<0.05).抗IGFBP7抗体能够上调肝星状细胞-T6的凋亡率(F=63.79,P<0.05).结论 IGFBP7能够使细胞外基质的重要组成成分纤维连接蛋白的合成与分泌增加,抗IGFBP7抗体可以抑制肝星状细胞-T6的增殖并诱导其发生凋亡.     

高迁移率族蛋白B1在烧伤小鼠大脑中表达的研究

目的:检测烫伤小鼠高迁移率族蛋白B1(HMGB1)在大脑中的表达.方法:应用BALB/c小鼠15%TBSAⅢ度烫伤(95 ℃热水浸烫8 s)和假伤(室温水,8 s)模型,并以正常小鼠作为对照(6只).在4、8、24和48h(每个时间点6只)处死动物.采用苏木精-伊红染色观察大脑形态学改变,免疫荧光方法检测caspase-3和HMGB1在大脑的表达,ELISA方法检测大脑组织HMGB1含量.结果:15%体表面积Ⅲ度烫伤8 h即导致大脑发生显著性病理改变,如炎性细胞浸润,烫伤24h大脑终纹出现caspase-3阳性细胞,而在假伤组未见特异性染色.免疫荧光染色发现,HMGB1位于假伤组小鼠神经细胞的胞核;而在烫伤后4h即释放至细胞浆.释放到脑组织中的细胞外HMGB1在烧伤后 24和48h,显著高于假伤组和对照组(P<0.01).结论:烫伤导致小鼠大脑HMGB1的分布和含量发生改变,可能参与了烧伤后脑损伤的病理过程.     

Cotransplanted hepatic stellate cells enhance vascularization of islet allografts

Islet transplantation is an alternative to whole pancreas transplantation in curative therapy of diabetics. The outcome of engraftment of islet, however, remains disappointing. Rapid and adequate islet revascularization is crucial for the survival and function of transplanted islets. In this study, hepatic stellate cells (HSC) were cotransplanted with islet allografts, achieving marked prolongation of islet allografts. This was associated with enhanced revascularization within islet grafts as determined by anti-CD31 antibody staining.     

辣椒素对肝星状细胞增殖凋亡的影响

目的 探讨辣椒素(capsaicin)对大鼠肝星状细胞(hepatic stellate cell,HSC) T6细胞系生长的影响及其机制.方法 用CCK-8(Cell Counting Kit-8)法检测终浓度50、100、150、200 μmol/L的辣椒素对大鼠肝星状细胞T6细胞株生长的影响;采用流式细胞仪检测辣椒素对大鼠肝星状细胞T6细胞凋亡率的影响;Western blotting法检测Bcl-2、Bax、细胞色素酶c(Cyt c)蛋白的表达.结果 辣椒素能显著抑制大鼠肝星状细胞T6细胞株增殖,呈时间浓度依赖性(P＜0.05).辣椒素作用24 h后T6细胞凋亡率与对照组相比明显升高(P＜0.05),与对照组相比,辣椒素作用组Bcl-2蛋白表达量明显下降,bax蛋白的表达明显上升,Cyt c蛋白的表达明显上升,Bcl-2/Bax灰度值明显下降(P＜0.05).结论 辣椒素抑制T6细胞增殖并诱导其凋亡,其机制可能是与下调Bcl-2蛋白的表达,上调Bax蛋白的表达,促进Cyt c蛋白的释放有关.     

缺氧诱导肝癌细胞释放高迁移率族蛋白B1及其机制

目的 探讨缺氧对肝癌细胞高迁移率族蛋白B1(HMGB1)释放的诱导作用及其机制.方法 采用正常肝细胞株QSG-7701和肝癌细胞株SMMC-7721,观察缺氧(1％O2)培养3～24h后对HMGB1胞外释放、HMGB1基因表达和HMGB1胞内分布的影响,使用丝裂原活化蛋白激酶(MAPK)通路抑制剂探讨缺氧诱导HMGB1释放的机制.观察28例肝细胞癌患者血清HMGB1水平的改变.结果 肝细胞癌患者血清HMGB1水平[(19.3±7.2) μg/L]明显高于健康对照者[(4.1±1.6) μg/L,P＜0.01].QSG-7701和SMMC-7721细胞经缺氧培养后,3h即见培养上清液中HMGB1含量明显升高,HMGB1 mRNA表达于6h后显示增强,且SMMC-7721株改变更为显著.SMMC-7721细胞经缺氧培养12 h后,核浆HMGB1分布发生明显改变,胞质HMGB1蛋白表达量升高.SB203580、SP600125和PD98059对缺氧诱导HMGB1释放均显示不同程度的抑制作用.结论 缺氧能诱导肝癌细胞表达、释放HMGB1,其诱导HMGB1释放机制与MAPK信号通路有关.