Epidermal growth factor protects the liver against alcohol-induced injury and sensitization to bacterial lipopolysaccharide

BACKGROUND: Whereas the role of proinflammatory cytokines in the pathogenesis of alcoholic liver disease has been at the forefront of investigation, a possible role for anti-inflammatory cytokines in this disease has received little attention. This study investigated (1) the hepatic protective effect of an anti-inflammatory cytokine, epidermal growth factor (EGF), against deleterious effects of alcohol and sensitization to bacterial lipopolysaccharide (LPS), and (2) the possible mechanisms that underlie such protection. METHODS: Male C57BL/6 mice were fed a Lieber-DeCarli liquid diet that contained alcohol or an isocaloric replacement for 6 weeks. The animals then were treated daily with human EGF for 7 days (5 microg/mouse), after which they were injected with either LPS (1 mg/kg of body weight) or vehicle and killed 8 hr later. Blood and liver were analyzed for plasma aminotransferase activity, liver histology, liver apoptotic nuclei, mRNA of several cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, and IL-10), apoptotic ligands (TRAIL), cytokine receptors (TNFRp55), pro- and antiapoptotic regulators/adaptors (Fas receptor, FasL, FADD, TRADD, RIP, Bak, Bax, Bcl-X, Bcl-2 and Bcl-w), and caspase-8. RESULTS: Alcohol increased plasma aminotransferase activity and sensitized the liver to the effects of LPS, such as polymorphonuclear infiltration, occurrence of necrotic foci and microabscesses, and increased apoptosis. These changes were associated with elevated mRNA expression of proapoptotic regulators/adaptors. EGF either counteracted or markedly blunted most of these effects. EGF did not affect liver mRNA expression of TNF-alpha, IL-1beta, IL-6, and IL-10, which suggested that these cytokines were not involved in EGF protective effect. EGF protection was mediated by down-regulation of apoptosis through suppression of proapoptotic gene expression. CONCLUSIONS: EGF protects the liver against both alcohol-induced liver damage and liver sensitization to bacterial LPS through down-regulation of apoptosis.     

支气管哮喘患者血浆白细胞介素-5、白细胞介素-10水平的变化

目的 探讨反映哮喘气道炎症的客观指标.方法 采用酶联免疫吸附法分别于急性期和缓解期对43例哮喘患者进行血浆白细胞介素-5(IL-5)、白细胞介素-10(IL-10)浓度测定,并与对照组比较.哮喘患者同期行肺通气功能检查,观察IL-5/IL-10与FEV1%、PEEF的关系.结果 与对照组比较,哮喘患者血浆IL-5浓度增高而IL-10浓度降低,差异均有显著性(P值均＜0.05).急性期哮喘患者血浆IL-5浓度比缓解期增高,而IL-10浓度低于缓解期,差异具有显著性(P值均＜0.05).急性期IL-/IL-10与FEV1%、PEEF呈负相关,相关系数分别为-0.6125和-0.5147(P值均＜0.05).结论 IL-5、IL-10水平变化参与了哮喘发病,其检测有助于哮喘气道炎症状态的评估.     

慢加急性乙型肝炎肝衰竭患者肝组织IFN-γ、TNF-α和IL-10的表达

目的研究乙型肝炎慢加急性肝衰竭（ACLF）患者肝内促炎细胞因子和抗炎细胞因子的表达特性。方法采用免疫组化法检测乙型肝炎ACLF患者、慢性乙型肝炎患者和正常人肝组织IFN-γ、TNF-α和IL-10的表达。结果在ACLF患者肝组织IFN-γ和TNF-α阳性细胞数均明显高于慢性乙型肝炎患者和正常对照组（P均〈0.001）;ACLF患者、慢性乙型肝炎患者和正常人肝组织IL-10的表达无统计学差异（P〉0.05）;肝内IFN-γ表达与TNF-α具有明显的相关性（r=0.886,P〈0.001）。结论乙型肝炎ACLF患者因肝内促炎细胞因子和抗炎细胞因子表达的失衡而发生了肝损伤。     

纤维化大鼠肝星状细胞IL-10/IL-10R的表达及IL-10干预的影响

目的研究实验性肝纤维化大鼠肝星状细胞白介素10(IL-10)/白介素10受体(IL-10R)表达及IL-10干预对其表达的影响。方法60只清洁级SD大鼠,随机分为正常对照(A组)、肝纤维化(B组)和IL-10干预组(C组)。B组和C组以CCl4腹腔内注射构建肝纤维化模型,C组并予IL-10腹腔内注射干预造模。于第7周和第11周分别随机选取一批大鼠,分离肝星状细胞,抽提总RNA,以半定量RT-PCR法检测各组HSC中IL-10和IL-10R mRNA表达情况。结果病理组织学证实肝纤维化模型构建成功,原代HSC分离培养成功,IL-10干预可以减轻其肝纤维化程度。肝纤维化时IL-10和IL-10R的mRNA表达均较正常组有所增强,但随着肝纤维化进展,IL-10的表达逐渐减弱。IL-10干预可以使二者的表达上调,以第11周时变化最为显著。结论大鼠肝HSC可表达IL-10及IL-10R,IL-10可作用于HSC而发挥抗肝纤维化作用。     

白细胞介素-10基因多态性与自身免疫性肝病相关性研究

目的研究白细胞介素10(IL-10)基因启动子区域-592、-819和-1082位单核昔酸多态性与自身免疫性肝病发病之间的关系。方法分别采用聚合酶链反应限制性片段长度多态性分析法(PCRRFLP)和聚合酶链反应一序列特异性引物扩增法(PCRSSP)检测54例自身免疫性肝炎(AIH)，77例原发性胆汁性肝硬化(PBC)患者及160例健康献血员外周血单个核细胞基因组DNA IL-10基因启动子区域3个多态位点-592，-819、-1082的基因多态性，并进行相关性分析。结果54例AIH和77例PBC患者与160例健康对照组的IL-10启动子-1082、-819和592基因型分布的统计学分析表明，差异无显著性。结论IL-10启动子基因多态性与自身免疫性肝病发病之间无显著关联性。     

血清IL-6和IL-8在还原型谷胱甘肽治疗肝硬化患者前后的变化

目的 探讨肝硬化患者血清白细胞介素6（IL-6）、白细胞介素8（IL-8）水平变化的临床意义。方法 采用ELISA法测定肝硬化患者治疗前后血清IL-6和IL-8水平。结果 36例肝硬化患者血清IL-6、IL-8水平较正常对照组显著增高。采用还原型谷胱甘肽治疗一个月后肝功能恢复者血清IL-6、IL-8水平较治疗前显著降低，而肝功能未恢复者血清IL-6、IL-8水平较治疗前降低不明显。结论 肝功能损伤可能是白细胞介素活性增加的重要原因，细胞因子的水平与肝硬化的形成及预后有关，还原型谷胱甘肽具有一定的治疗作用。     

乳凝集素调节EEI-10细胞IL-2,IL-4及IFN-γ的分泌表达

目的:探讨乳凝集素调节肠相关淋巴组织淋巴细胞分泌表达细胞因子的作用.方法:应用基因重组技术从MCF-7乳腺癌细胞中提取总RNA,通过逆转录PCR方法得到目的基因(乳凝集素)片段,利用酶切,连接等技术,将目的基因构建至PET28载体内,转化入DH5a细胞后,鉴定阳性质粒;将含有目的基因的阳性质粒转化入表达细胞BL-21,IPTG诱导表达目的蛋白乳凝集素,应用特异性镍鏊合的亲和层析柱得到纯化的目的蛋白.利用H3-Tdr法明确乳凝集素剂量与人类肠上皮内淋巴细胞株EEI-10细胞增殖之间的关系.给予适当剂量乳凝集素作用于EEI-10细胞后,利用ELISA方法检测EEI-10分泌IL-2,IL-4及IFN-γ浓度,应用RT-PCR方法检测IL-2,IL-4及IFN-γmRNA表达.结果:酶切阳性的质粒经测序证实与基因文库一致.所得纯化的目的蛋白进行SDS-PAGE电泳和特异性抗体鉴定,明确为目的蛋白乳凝集素.利用H3-Tdr法检测乳凝集素剂量与细胞增殖之间的关系,结果显示乳凝集素作用剂量为250mg/L时淋巴细胞增殖显著.给予乳凝集素处理后细胞培养上清中IL-2(P=0.0394)和IFN-γ(P=0.0082)的含量高于未处理细胞组,而IL-4没有显著的增高;同时处理后IL-2mRNA表达量升高,IL-4mRNA表达量无显著性改变,IFN-γmRNA表达量明显增高并高于PHA刺激组.结论:乳凝集素具有上调EEI-10细胞分泌和表达IL-2和IFN-γ的作用.     

Effects of interleukin-10 on activation and apoptosis of hepatic stellate cells in fibrotic rat liver

AIM:To study the effects of interleukin-10(IL-10)on the expression of α-smooth muscle actin(α-SMA),nuclear factor-κB(NF-κB)and Fas/Fas ligand(FasL)inhepatic stellate cells of experimental rats with hepaticfibrosis.METHODS:Sixty clean SD rats were randomly dividedinto control group(group N),liver fibrotic group(groupC)and IL-10 treatment group(group I).Control groupreceived intraperitoneal injection of saline(2ml·kg~(-1)),twicea week.Fibrotic group was injected intraperitoneallywith 50% carbon tetrachloride(CCl_4)(2 ml·kg~(-1)),twicea week.IL-10 treatment group was given IL-10 at adose of 4 μg·kg~(-1)20 minutes before CCl_4 administrationfrom the third week.Hepatic stellate cells(HSCs)wereisolated from these rats at the seventh and eleventhweeks during the course of liver fibrosis,respectively.The expression of α-SMA and NF-κB in HSCs wasmeasured by S-P immunohistochemistry.The expressionof Fas and FasL mRNA was measured by RT-PCR.Furthermore,liver tissues were harvested from threegroups at the same time.RESULTS:The CCl_4- induced experimental rat hepaticfibrosis model was established successfully.The purityof extracted hepatic stellate cells was about 95% andthe yield of hepatic stellate cells was 1.2-2.3×10~6/g livertissue averagely.The positive expression of α-SMA andNF-κB was 36.5% and 28.5% respectively in group N.The positive levels of α-SMA and NF-κB were increasedsignificantly in group C compared to group N(P<0.01).The positive signals decreased significantly(P<0.05)ingroup I.In the 11~(th)week,the HSCs of group I becameround with visible pyknotic nuclei.The expression ofNF-μB in group C was significantly increased in a time-dependentmanner(P<0.01),but there was no difference in the α-SMA expression(P>0.05).The mRNA of Fasand FasL in group C was significantly increased in a time-dependent manner compared to that in control group.After treated with IL-10,the expression level of Fas andFasL was higher in group I than in group C.CONCLUSION:The positive expression of α-SMA andNF-κB in hepatic stellate cells is decreased by ectogenicIL-10 in liver fibrosis induced by CCl_4.The expression ofFas and FasL is increased in the course of liver fibrosis,and is further increased by IL-10.IL-10 could inhibitthe activation of HSCs and cause apoptosis of activatedHSCs.     

hIL-10基因修饰的L02肝细胞的克隆培养和最高表达株的筛选

目的:确认hIL-10基因修饰的L02肝细胞的克隆培养可实现hIL-10在L02肝细胞中的高效表达．方法:通过构建真核质粒表达载体pchIL-10, 并纯化后转染L02肝细胞．通过G418的压力选择获得hIL-10高表达的克隆株,并以ELISA测定其表达水平．结果:经过测序和酶切验证,真核质粒表达载体pchIL-10构建成功．电泳显示一长约540 bp 条带．hIL-10基因转导可在L02肝细胞中实现 hIL-10的高效表达．最高表达株表达量为每小时69．875 ng/106细胞．结论:hIL-10基因修饰的L02肝细胞的克隆培养可实现hIL-10的高效表达,为抗肝纤维化、肝硬化提供有效途径．     

白细胞介素-10与肝脏疾病

肝脏疾病与各种因素导致的肝脏急、慢性损伤关系密切,而炎症反应和机体的免疫调节机制在各种肝病的发生和发展中具有举足轻重的作用.白细胞介素-10是一种重要的炎症负性调控因子,参与多种疾病的病理生理调节过程,对各种肝脏疾病均有一定的影响.     

IL-10对实验性肝纤维化大鼠基质金属蛋白酶-2影响的研究

目的研究IL-10对实验性肝纤维化大鼠基质金属蛋白酶-2(MMP-2)的影响.方法建立大鼠肝纤维化模型并行IL-10干预实验,从正常大鼠(N组)和CCl4诱导肝纤维化大鼠(C组)及IL-10干预肝纤维化大鼠(E组)中取肝脏组织,采用S-P免疫组织化学方法检测分析不同组大鼠肝脏组织中MMP-2的表达状况.结果N组MMP-2阳性染色偶见于窦内皮细胞及肝细胞的胞浆;C组MMP-2阳性染色多见于汇管区新生的胆管细胞及纤维隔内条索状的成纤维细胞,26例标本中阳性13例,强阳性8例.第5周开始MMP-2有阳性表达,第9周阳性表达明显增强;E组MMP-2阳性染色明显减少,多见于汇管区的新生胆管细胞,27例标本中阳性10例,强阳性1例.Redit分析表明3组间MMP-2阳性表达有显著性差异(P＜0.01).结论MMP-2随着大鼠肝纤维化进展阳性表达升高,IL-10对CCl4诱导的大鼠肝纤维化具有保护作用,可明显抑制纤维化的生成和沉积.     

IL-6 and IL-10 Closely Correlate with Bacterial Bloodstream Infection

Background: Given the high mortality of bacterial bloodstream infections (BSI), blood culture results do not meet clinical needs timely due to being time-consuming and having low positive rate. Whether we can identify the severity and type of bacterial infections by cytokines is a controversial issue. Objective: To investigate the dynamic change of cytokines in BSI. Methods: 55 patients with Gram-positive (GP) BSI, 64 patients with Gram-negative (GN) BSI and 52 healthy controls were enrolled. We quantitatively detected the cytokines interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) by flow cytometry in the sera. The levels of procalcitonin, C-reactive protein, leukocytes and neutrophils were also detected simultaneously. Results: There were significantly up-regulated IL-6 and IL-10 expression in BSI patients, particularly in the GN-BSI, for instance Escherichia coli and Klebsiella pneumoniae infections; following the treatment, IL-6 and IL-10 decreased by 10-23 and 4-27 times, respectively. Additionally, IL-2, TNF-α and IFN-γ expression increased slightly in BSI patients and IFN-γ expression declined as GN-BSI progressed. Conclusion: IL-6 and IL-10 are closely associated with the severity and treatment efficacy of BSI, and can help to distinguish between GP-BSI and GN-BSI at an early stage.     

IL-10基因-592C/A多态性与儿童过敏性紫癜的关系

目的探讨白细胞介素10(IL-10)启动子区-592位点的单核苷酸多态性(SNP)与儿童过敏性紫癜(HSP)易感性之间的关系。方法收集100例HSP患儿和153名健康体检儿童全血标本,提取DNA,采用限制片断长度多态分析-聚合酶链反应方法(RFLP/PCR)对IL-10的基因启动子区592位点基因型进行分析,比较病例组与正常对照组的基因分布。结果 HSP患儿IL-10基因-592C/A位点等位基因频率和基因型频率与正常对照组差异无统计学意义(P0.05)。结论 IL-10基因-592C/A多态性与儿童HSP易感性无关。     

IL-10抗肝纤维化的实验研究及临床现状

肝纤维化是慢性肝损伤时的修复和疤痕形成过程,目前尚缺乏有效的治疗方法.白细胞介素-10(IL-10)为一种下调炎症反应的细胞因子,体内外试验证实其在肝纤维化过程中有重要作用.重组人IL-10已在临床试用.IL-10可能是治疗肝纤维化的一种有效方法,具有潜在的临床应用价值.     

Interleukin 10: the critical role of a pleiotropic cytokine in food allergy

Despite advances in research, the pathophysiology of food allergy has not yet been fully elucidated. IL-10 has both a pro- and anti-inflammatory effect on the development of food allergy and in order to understand its different immune-modulatory effects the factors that influence the inflammatory microenvironment need to be taken into account. Specific single nucleotide polymorphisms of the IL-10 gene seem to confer an increased risk of developing food allergy, but to date there is a substantial lack of genome- wide association studies regarding the genetic and epigenetic underpinnings of the disease. Special interest has been drawn to the development of allergen-specific regulatory CD4+CD25+ T-cells secreting IL-10 in the immunotherapy of allergic diseases. In addition, a distinct population of human tolerogenic dendritic cells (DC), DC-10 seems to hold great potential and could potentially serve as a therapeutic tool to improve the management of food allergy.     

Plasma Interleukin-10: A Likely Predictive Marker for Hepatitis B Virus-Related Acute-on-Chronic Liver Failure

Background:

The pathogenesis of HBV-related acute-on-chronic liver failure (HBV-ACLF) is mainly based on a heightened immune-inflammatory reaction; however, the intimate underlying mechanism remains unclear.

Objectives:

The aim of the study was to explore potential key immune molecular targets that could serve as early predictive markers for HBV-ACLF.

Patients and Methods:

Twenty-seven patients with acute exacerbation of chronic hepatitis B (CHB) (defined by: alanine transaminase ≥ 20 ULN, total bilirubin ≥ 5 ULN, 40% < prothrombin time activity ≤ 60%) and without cirrhosis were divided into 18 cases which did not progress to HBV-ACLF (defined by: prothrombin time activity < 40% and development within four weeks of hepatic encephalopathy and/or ascites) and nine cases that developed HBV-ACLF. Nine healthy people defined the normal control group (NC). Interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, TNF-α and IFN-γ protein levels were assayed by Cytometric Bead Array (CBA) in blood plasma. The ELISA method was applied to confirm IL-10 detection using the CBA method.

Results:

IL-4, IL-12p70 and IFN-γ were undetectable; IL-1β, IL-6, IL-8, IL-10 and TNF-α levels were significantly higher than in NC. Moreover, cytokines reached the highest levels in acute exacerbation of CHB, with the exception of IL-2 and IL-8. When comparing the HBV-ACLF patients prior to and at the time of ACLF diagnosis, IL-10 was the only cytokine that exhibited a significant decrease (P = 0.008). IL-10 concentrations were positively correlated to ALT levels (r = 0.711, P < 0.001).

Conclusions:

The assessment of plasma IL-10 levels in chronic hepatitis B acute exacerbation may provide an early predictive marker for progression to HBV-ACLF.     

白细胞介素-10与减体积大鼠肝移植后肝再生的关系

目的 探讨白细胞介素-10(IL-10)与减体积大鼠肝移植术后移植肝再生的关系。方法 建立减体积大鼠肝移植模型,实验分为:肝切除组、全肝移植组和减体积肝移植组,分别于术后1、2、4、7d取肝组织,免疫组织化学检测各组IL-10的表达,流式细胞仪检测移植肝的增殖活性。结果 肝切除组、全肝移植组和减体积肝移植组肝细胞增生活跃,术后4d增殖高峰分别为26.3±0.9、35.8±2.2、32.4±1.8。IL-10与移植后肝再生呈负相关(r=-0.58,P<0.01)。结论 减体积肝移植和全肝移植术后肝脏具有同样的增殖活性,但增殖峰值较肝切除延迟。IL-10对移植肝肝再生具有明显的调控作用,同时受免疫系统产生的其它细胞因子和激素的影响。     

The role of interleukin-1 receptor antagonist in the prevention and treatment of disease

Abstract

?Interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) play key proinflammatory roles in a variety of human diseases, including rheumatoid arthritis (RA). IL-1 receptor antagonist (IL-1Ra) is a naturally occurring structural variant of IL-1 that competitively inhibits receptor binding of IL-1. Four forms of IL-1Ra have been described: secretory IL-1Ra (sIL-1Ra) and three intracellular molecules (icIL-1Ra1, 2, and 3). Excess amounts of IL-1Ra are necessary to inhibit the biological effects of IL-1. The endogenous production of IL-1Ra plays an anti-inflammatory role, but the level of production of IL-1Ra in inflamed tissues may not be adequate to block IL-1 effectively. An allelic polymorphism in the IL-1Ra gene is associated with a variety of human diseases, largely of epithelial or endothelial cell origin. The disease associated allele IL1RN*2 may lead to a decreased production of icIL-1Ra1 by these cells, predisposing the patient to an imbalance in the IL-1 system. The therapeutic administration of IL-1Ra was found to be safe and efficacious in the treatment of RA. Intraarticular delivery of the IL-1Ra cDNA by ex vivo gene therapy in patients with RA was effective in enhancing local IL-1Ra production. This unique form of therapy is under further evaluation.