OBJECTIVES:The knowledge that agmatine is found in the human body has existed for several years; however, its role in sepsis has not yet been studied. In the present study, we investigate the role of agmatine in the progression and treatment of sepsis.DESIGN:Clinical/laboratory investigations.SETTING:Medical centers/University-based research laboratory.SUBJECTS:Elective ICU patients with severe sepsis and healthy volunteers; C57BL/6 mice weighing 18-22 g.INTERVENTIONS:Serum agmatine level and its associations with inflammatory markers were assessed in patients with sepsis. Agmatine was administered intraperitoneally to mice before a lipopolysaccharide challenge. Human peripheral blood mononuclear cells and murine macrophages were pretreated with agmatine followed by lipopolysaccharide stimulation.MEASUREMENTS AND MAIN RESULTS:Serum agmatine levels were significantly decreased in patients with sepsis and lipopolysaccharide-induced mice, and correlated with Acute Physiology and Chronic Health Evaluation II score, procalcitonin, tumor necrosis factor-α, and interleukin-6 levels. In a therapeutic experiment, exogenous agmatine attenuated the cytokine production of peripheral blood mononuclear cells from patients with sepsis and healthy controls. Agmatine also exerted a significant beneficial effect in the inflammatory response and organ damage and reduced the death rate in lipopolysaccharide-induced mice. Imidazoline I2 receptor agonist 2-benzofuran-2-yl blocked the pharmacological action of agmatine; whereas, other imidazoline receptor ligands did not. Furthermore, agmatine significantly impaired the inflammatory response by inactivating nuclear factor-κB, but not protein 38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and inducible nitric oxide synthase signaling in macrophages. Activation of imidazoline I2 receptor or knockdown of ribosomal S6 kinase 2 counteracted the effects of agmatine on phosphorylation and degradation of inhibitor of nuclear factor-κBα.CONCLUSIONS:Endogenous agmatine metabolism correlated with the progression of sepsis. Supplemental exogenous agmatine could ameliorate the lipopolysaccharide-induced systemic inflammatory responses and multiple organ injuries through the imidazoline I2 receptor-ribosomal S6 kinase 2-nuclear factor-κB pathway. Agmatine could be used as both a clinical biomarker and a promising pharmaconutrient in patients with severe sepsis.

OBJECTIVE To explore the risk factors of multiple organ dysfunction syndrome (MODS) in severe trauma patients, put forward a new warning scoring system of MODS, and to provide a more accurate scoring method for doctors to judge the clinical condition and prognosis of patients. METHODS Clinical data of 342 patients with severe trauma admitted to intensive care unit (ICU) of the Affiliated Hospital of Zunyi Medical College and Daping Hospital of the Third Military Medical University from January 1st, 2015 to December 31st, 2016 were retrospectively analyzed. The patients were divided into MODS groups (n = 251) and non-MODS group (n = 91) according to clinical outcomes. The clinical data of patients, including gender, age, heart rate (HR) and blood pressure within 24 hours after admission to the hospital, indicators of blood routine and blood biochemistry, severity of disease, severity of trauma, whether received the emergency intubation or surgery within 24 hours or not, whether developed sepsis or acute respiratory distress syndrome (ARDS) during hospitalization, were recorded, and univariate analysis was conducted. The indicators with statistical significance found by univariate analysis were enrolled in multivariate Logistic regression analysis, and the risk factors for MODS in patients with severe trauma were screened and assigned, and the final total score was MODS warning score. Receiver operating characteristic (ROC) curve was plotted to evaluate MODS warning score for predicting the occurrence of MODS in patients with severe trauma. RESULTS Compared with non-MODS group, HR, Na+, serum creatinine (SCr), activated partial thromboplastin time (APTT), injury severity score (ISS), new injury severity score (NISS), acute physiology and chronic health evaluation II (APACHE II) score and sequential organ failure assessment (SOFA) score in MODS group were significantly increased, pH value, red blood cell (RBC), platelet (PLT), albumin (Alb) and Glasgow coma score (GCS) were remarkably decreased, and multiple injury, the patients with shock at admission, blood transfusion, central venous catheter, emergency intubation and infection were also increased, and more patients suffered from sepsis and ARDS. Multivariate Logistic regression analysis showed that the number of injured places equal or more than 2, shock at admission, APACHE II score ≥ 15, SOFA score ≥ 4 and APTT > 40 s were risk factors for MODS in patients with severe trauma, with total MODS warning score of 7.5. ROC curve analysis showed that the area under ROC curve (AUC) of MODS warning score for predicting MODS in patients with severe trauma was 0.822, which was significantly higher than that of APACHE II score (AUC = 0.698, P < 0.001), APTT (AUC = 0.693, P < 0.001) and SOFA score (AUC = 0.770, P = 0.025). When the cut-off value of MODS warning score was 2.5, the sensitivity was 61.35%, the specificity was 90.11%, and Youden index was 0.515. CONCLUSIONS MODS warning score is composed of five factors, including the number of injured places, shock at admission, APACHE II score, SOFA score and APTT, which could be regarded as early warning score system for predicting MODS in patients with severe trauma. MODS warning score can be more comprehensive and timely to assess the possibility of MODS and prognosis of patients with severe trauma, and the prediction result is better than the single use of APTT, APACHE II or SOFA score.

The aryl hydrocarbon receptor (AhR) is an important immune regulator with a role in inflammatory response. However, the role of AhR in IL-10 production by inflammatory macrophages is currently unknown. In this study, we investigated LPS-induced IL-10 expression in macrophages from AhR-KO mice and AhR-overexpressing RAW264.7 cells. AhR was highly expressed after LPS stimulation through NF-κB pathway. Loss of AhR resulted in reduced IL-10 expression in LPS-induced macrophages. Moreover, the IL-10 expression was elevated in LPS-induced AhR-overexpressing RAW264.7 cells. Maximal IL-10 expression was dependent on an AhR non-genomic pathway closely related to Src and STAT3. Furthermore, AhR-associated Src activity was responsible for tyrosine phosphorylation of STAT3 and IL-10 expression by inflammatory macrophages. Adoptive transfer of AhR-expressing macrophages protected mice against LPS-induced peritonitis associated with high IL-10 production. In conclusion, we identified the AhR-Src-STAT3-IL-10 signaling pathway as a critical pathway in the immune regulation of inflammatory macrophages, It suggests that AhR may be a potential therapeutic target in immune response.

Evodiamine (EVO), an important alkaloidal component extracted from the fruit of Evodiae fructus, has been known to possess anti-tumor, anti-inflammatory, anti-oxidative, and other therapeutic capabilities. In the present study, the effects of EVO on zymosan-induced inflammation and its underlying mechanism were investigated both in vitro and in vivo. Our results showed that EVO effectively suppressed both protein and mRNA expression of interleukin-1β, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in vitro. Zymosan-induced DNA-binding activity of nuclear factor-kappa B (NF-κB) was attenuated by EVO, which was achieved through inhibitory effects on the phosphorylation of inhibitory κB α and p65 nuclear translocation, but there was very little association with mitogen-activated protein kinase activation. In vivo, treatment with EVO markedly decreased TNF-α and IL-6 levels in plasma. EVO also repressed inflammatory cytokine expression and ameliorated the abnormal state in both lung and intestine tissues by inactivation of NF-κB. Furthermore, EVO significantly reduced the mortality caused by zymosan. In summary, these results suggested that EVO could effectively suppress inflammatory responses in vitro and in vivo, and may be a potential therapeutic agent against inflammatory disorders.

芳香烃受体(aryl hydrocarbon receptor,AhR)是一种配体激活的转录因子,对环境中毒物和异物质代谢、机体免疫调节具有重要作用.炎性细胞因子是由多种细胞合成、分泌的一类小分子蛋白质,介导各阶段的炎症和免疫反应.不同配体活化的和未被活化的AhR能够通过不同方式调控多种炎性细胞因子的表达,深入研究调控的机理及信号通路有助于进一步阐明异物质代谢和炎症反应的机制.本文将重点论述AhR调控炎性细胞因子IL-6、TNF-α、IL-1β及炎性趋化因子的研究进展.

目的 探讨吴茱萸碱(evodiamine,EVO)对酵母多糖(zymosan,ZYM)诱导的小鼠腹膜炎中炎症反应及中性粒细胞(polymorphonuelear neutrophil,PMN)浸润的影响.方法 将54只成年雄性C57BL/6小鼠按随机数字表法分为正常对照组、模型组和EVO治疗组.模型组腹腔注射1 mg/mLZYM溶液(1 mL)建立急性腹膜炎小鼠模型,EVO治疗组则同时腹腔注射10 mg/kg EVO及1 mg/mLZYM溶液,正常对照组腹腔注射等量磷酸盐缓冲液.各组处理后2、6、12 h处死6只小鼠,收集眼眶血及腹腔灌洗液.采用酶联免疫吸附法检测血清及腹腔灌洗液中白介素6(interleukin-6,IL-6)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、角化细胞来源趋化因子(keratinocyte-derived chemokine,KC)及巨噬细胞炎性蛋白2(macrophage inflammatory protein 2,MIP-2)的含量;通过血细胞计数板计数腹腔灌洗液中混合细胞总数;利用流式细胞仪检测腹腔灌洗液中PMN比例及PMN中活性氧(reactive oxygen species,ROS)水平;采用Western blot检测6h时腹腔灌洗液混合细胞中核转录因子-kB(nuclear factorκB,NF-κB) p65入核情况.结果 建模后2、6、12 h,模型组血清及腹腔灌洗液中IL-6、TNF-α、KC及MIP-2含量均高于正常对照组(P＜0.05),EVO治疗组上述指标均较模型组明显降低(P＜0.05),其中,血清中IL-6、KC及MIP-2均于2h达高峰,随后逐渐下降,而TNF-α在2h逐渐上升,6h达到高峰,随后逐渐下降;腹腔灌洗液中TNF-α、KC及MIP-2均于2h达高峰,随后逐渐下降,而IL-6在2h逐渐上升,6h达到高峰,随后逐渐下降.制模后6h,EVO治疗组可显著降低腹腔混合细胞数与PMN数及其百分比(P＜0.05),且PMN中ROS显著降低(P＜0.05).另EVO治疗可显著降低腹腔混合细胞中细胞核的NF-κB p65表达(P＜0.05).结论 EVO可抑制ZYM诱导急性小鼠腹膜炎模型中血清及腹腔灌洗液中炎症因子及趋化因子的分泌,减轻腹腔PMN的浸润,并抑制PMN中ROS的产生,该作用可能是通过抑制NF-kB p65入核实现的.

This study was performed to establish a murine macrophage RAW264.7 cell line stably overexpressing AhR for evaluating the regulatory effects of AhR in inflammation induced by LPS stimulation.A lentiviral vector containing AhR-FIag-EGFP gene was constructed and then packaged in HEK293T cells;RAW264.7 cells were infected with collected supernatant and screened by puromycin.The gene and protein expression of AhR were analyzed by qRT-PCR and Western blotting,respectively;the expression of inflammatory cytokines were detected by qRT-PCR and ELISA.Data showed that a RAW264.7 cell strain with stable expression of AhR-Flag-EGFP was successfully constructed.Fluorescent microscopy and flow cytometry demonstrated that the expression rates of EGFP reached to 100％;the qRT-PCR and Western blot showed that the gene and protein expression of AhR were significantly upregulated in RAW/AhR group compared to RAW/Vector group.The expression of IL-6 and IL-1β in LPS-induced RAW/AhR group were much lower than that in LPS-induced RAW/Vector group.In contrast,the level of IL-10 in LPS-induced RAW/AhR group was much higher than that in LPS-induced RAW/Vector group.In conclusion,a RAW264.7 cell line stably expressing AhR has constructed,and AhR negatively regulates the LPS-induced inflammatory response in macrophages,which lay a foundation for further study AhR function.

目的 探究吴茱萸碱(evodiamine,EVO)对酵母多糖(zymosan)诱导的急性肺损伤的保护效应及其作用机制.方法 24只6～8周龄雄性C57BL/6小鼠,按随机数字表法分为正常对照组、EVO对照组、模型组、EVO治疗组,每组6只.各组分别于处理作用12 h后处死小鼠,收集眼眶血及肺组织.酶联免疫吸附法(ELISA)检测血液及肺组织中白介素-6(interleukin-6,IL-6)、肿瘤坏死因子-0(tumor necrosis factor-α,TNF-α)水平及肺组织髓过氧化物酶(myeloperoxidase,MPO)活性及核转录因子NF-κBp65的DNA结合活性,TUNEL法检测肺组织细胞凋亡情况,HE染色观察肺组织病理改变并测量肺湿干质量比.结果 酵母多糖作用12h后,小鼠出现精神萎靡、活动减少等症状.EVO治疗组小鼠状态有所好转.EVO可明显改善酵母多糖所致的肺泡壁毛细血管扩张充血及炎性细胞浸润,降低肺湿干质量比,并显著下调酵母多糖刺激下血清和肺脏中IL-6和TNF-α的含量(P＜0.01),同时抑制肺组织中MPO含量以及NF-κB p65的DNA结合活性(P＜0.01).结论 EVO可缓解酵母多糖诱导的急性肺损伤.该作用可能是通过抑制NF-κB的活性以减少炎症介质释放实现.

Objective To investigate the role of Tmub1 protein in liver cell proliferation. Methods Using silenced lentivirus (LV)-Tmub1 vector, over-expression LV-Tmub1 vector, and corresponding empty vectors to transfect rat's liver cell line BRL-3A to obtain stable transfected cells, which bear the expression of Tmub1 protein as detected by Western blotting, cell proliferation with MTT, and cell cycle with flow cytometry. Results The Tumb1 protein expression in the hepatocytes transfected by LV-Tmub1 was increased significantly and that by LV-Tmub1-RNAi decreased significantly, with a statistically significant difference compared with normal hepatocytes (P<0.01). The proliferation was fastest in hepatocytes transfected by LV-Tmub1 and slowest in those by LV-Tmub1-RNAi, and the difference was statistically significant (P<0.01). The G2+S phase was longer in hepatocytes transfected by over-expression LV-Tmub1 vectors than in those by silenced LV-Tmub1 vectors, and the difference was statistically significant (P<0.01). Conclusion Tmub1 protein can promote the hepatocyte proliferation in BRL-3A cell line. DOI: 10.11855/j.issn.0577-7402.2016.01.03

目的 构建Tmub1基因的过表达慢病毒载体(LV-Tmub1),为研究Tmub1蛋白在肝细胞增殖过程中的作用提供实验材料.方法 化学合成Tmub1基因序列,用BamHI/AgeI酶切化学合成含有目的基因的质粒及GV287载体,PCR产物连接入线性化表达的载体.PCR鉴定引物,再对PCR鉴定阳性的克隆进行DNA测序和比对分析.使用构建的LV-Tmnb1,转染293T细胞,荧光法检测构建的慢病毒滴度.结果 成功构建了LV-Tmub1,并获得相应的病毒,病毒滴度为2×108 TU/mL.结论 LV-Tmub1为进一步研究Tmub1蛋白在肝细胞增殖中的作用提供了实验基础.

Resveratrol is a natural antitoxin which widely present in grapes,peanuts,nuts and other foods with anti-inflammatory,anti-oxidation,anti-aging,anti-tumor,immunomodulatory and other biological activities.In recent years,a large number of studies have showed that resveratrol have great effect on trauma and sepsis-induced damage.This paper mainly focuses on the protective effects of resveratrol and its potential mechanisms on trauma-hemorrhagic shock,traumatic brain injury,spinal cord injury,ischemiec reperfusion injury and sepsis injury.

目的 探讨胍丁胺对脓毒症小鼠血管内皮功能的影响.方法 将42只C57BL/6小鼠按随机数字表法分为假手术组、模型组、胍丁胺治疗组,每组14只.采用盲肠结扎穿孔术(CLP)复制脓毒症小鼠模型;假手术组只开腹不进行结扎穿孔术,术后腹腔注射磷酸盐缓冲液.胍丁胺组术后腹腔注射400mg/kg胍丁胺;其余两组术后腹腔注射等量磷酸盐缓冲液.于制模后24h处死小鼠取血,采用酶联免疫吸附试验(ELISA)检测血清细胞间黏附分子-1(ICAM-1)、血管间黏附分子-1(VCAM-1)、血管内皮生长因子(VEGF)、血管性血友病因子(vWF)、血管紧张素-Ⅱ(Ang-Ⅱ)、单核细胞趋化因子-1(MCP-1)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6、IL-10)的含量;通过异硫氰酸荧光素(FITC)-葡聚糖荧光示踪法检测肝、肺组织的血管通透性.结果 造模后24h,模型组小鼠腹部呈浅粉色、体温下降、精神萎靡、活动减少;胍丁胺组小鼠治疗后精神和活动状态均有明显好转.与假手术组比较,模型组血管内皮相关生物标志物的含量均显著升高[ICAM-1(ng/L):9887.12±897.90比4747.12±958.00,VCAM-1(ng/L):2884.09±230.00比1349.43±238.47,vWF(ng/L):48.61±0.69比31.82±2.38,VEGF(ng/L):45.14±1.76比20.65±0.57,Ang-Ⅱ(ng/L):13.26±0.84比8.87±0.80,MCP-1(ng/L):1881.31±106.71比66.21±6.68,均P＜0.01];胍丁胺组上述各指标含量均较模型组明显降低[ICAM-1 (ng/L):6380.45±1017.73比9887.12±897.90,VCAM-1(ng/L):1894.76±235.39比2884.09±230.00,vWF(ng/L):37.77±4.42比48.61±0.69,VEGF(ng/L):32.19±1.96比45.14±1.76,Ang-Ⅱ(ng/L):10.81±0.45比13.26±0.84,MCP-1 (ng/L):553.58±56.19比1881.31±106.71,均P＜0.01].同时,与假手术比较,模型组血清炎症因子水平显著升高[IL-6 (ng/L):5225.61±600.99比49.33±5.20,IL-10 (ng/L):1034.02±139.34比21.86±2.25,TNF-α(ng/L):135.84±18.86比68.64±17.85,均P＜0.01],而胍丁胺治疗后炎性介质的水平均较模型组明显降低[IL-6 (ng/L):1268.63±219.63比5225.61±600.99;IL-10(ng/L):240.84±75.20比1034.02±139.34;TNF-α(ng/L):68.67±20.30比135.84±18.86,均P＜0.01];模型组中肝、肺组织荧光强度较假手术组明显增强[肝:4998.00±616.33比2626.67±103.00;肺:495.33±67.11比255.33±28.67,均P＜0.01],而胍丁胺能显著降低肝、肺组织荧光强度[肝:3472.00±420.33比4998.00±616.33;肺:399.67±11.67比495.33±67.11,均P＜0.01].结论 胍丁胺通过抑制ICAM-1、VEGF及vWF等生物标志物和炎性因子的分泌,降低肝、肺组织血管通透性,从而缓解脓毒症小鼠的血管内皮损伤.

Objective:To investigate the protective effect of agmatine (AGM) against peritoneal inflammatory response and neutrophil (PMN) infiltration induced by zymosan (ZYM) in mice.Methods:Thirty-six adult male C57BL/6 mice were randomly divided into sham group, model group, and AGM treatment group. Peritonitis model was reproduced by intra-peritoneal injection of 1 mg/mL ZYM (0.5 mL), while equivalent phosphate buffer saline (PBS) was given to sham group. 200 mg/kg AGM was injected into peritoneal cavity after ZYM challenge in AGM treatment group. Six mice in each group were sacrificed at 2 hours and 6 hours, respectively, after reproduction of the model. Blood sample and peritoneal lavage fluid (PLF) were collected. The levels of keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), tumor necrosis factor-α (TNF-α), interleukins-6 (IL-6) in serum and PLF were determined by enzyme linked immunosorbent assay (ELISA). The number of leukocytes and PMN in PLF were determined by hemocytometer and flow cytometry, respectively.Results:Compared with sham group, all serum and PLF levels of KC, MIP-2, TNF-α and IL-6 were greatly elevated at 2 hours after ZYM injection in model group, while AGM treatment could dramatically reduce the levels of the above-mentioned cytokines in serum and PLF as compared with those of the model group [serum KC (ng/L): 990.7±137.9 vs. 2 053.2±262.7, MIP-2 (ng/L): 642.2±124.4 vs. 1 369.7±146.5, TNF-α (ng/L): 608.6±38.1 vs. 1 044.7±101.0, IL-6 (ng/L): 1 058.2±129.1 vs. 1 443.3±190.1; PLF KC (ng/L): 7 462.3±839.6 vs. 12 723.5±1 515.7, MIP-2 (ng/L): 1 570.8±193.4 vs. 3 471.4±384.7, TNF-α (ng/L): 1 115.8±156.7 vs. 1 499.2±231.2, IL-6 (ng/L): 2 646.5±223.2 vs. 3 126.7±291.4; all P < 0.05]. The expressions of KC, MIP-2 and TNF-α at 6 hours were significantly lower than those at 2 hours in model group and AGM treatment group, but IL-6 levels were further increased. The levels of KC and MIP-2 in serum and PLF at 6 hours were decreased to the levels of sham group. At 6 hours after the reproduction of the model, the number of total inflammatory cells and PMN of PLF in the model group was significantly higher than those of the sham group. In contrast, AGM notably lowered the number of inflammatory cells and PMN in peritoneal fluid after ZYM attack [total inflammatory cells (×109/L): 14.7±1.1 vs. 2.0±0.4, 10.1±1.2 vs. 14.7±1.1; PMN (×109/L): 11.37±1.22 vs. 0.18±0.05, 7.69±0.57 vs. 11.37±1.22, all P < 0.05].Conclusion:AGM can effectively alleviate acute peritoneal inflammatory injury induced by ZYM, mainly through reducing the secretion of inflammatory mediators and chemokines, and inhibiting the infiltration of leukocytes and neutrophils.

Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation.

目的 探讨芳香烃受体(aryl hydrocarbon receptor,AhR)内源性配体6-甲酰基吲哚并[3,2-b]咔唑(6-formylindolo[3,2-b] carbazole,FICZ)对脂多糖(lipopolysaccharide,LPS)诱导的大鼠急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)的保护作用及其机制.方法 按照随机数字表法将24只SD大鼠分为正常对照组、FICZ对照组、LPS模型组和FICZ治疗组,每组6只.通过气管滴注LPS(3 mg/kg)制备ARDS模型,并于建模后6h收集各组肺泡灌洗液(bronchoalveolar lavage fluid,BALF)及肺组织,通过肺湿/干质量比值、HE染色考察各组肺组织的损伤情况;采用酶联免疫吸附试验(ELISA)检测BALF和肺组织中炎症因子(IL-1β、TNF-α)水平及肺组织中髓过氧化物酶(my-eloperoxidase,MPO)含量;利用Western blot检测肺组织中AhR和NF-κB p65的蛋白表达.结果 与LPS模型组相比,FICZ可有效减轻LPS所致的肺水肿及组织病理学损伤,减少BALF和肺组织中炎症因子TNF-α、IL-1β的释放及肺组织中MPO的含量.另外,FICZ在激活AhR的同时对LPS作用下肺组织胞核中NF-κB p65的升高起到明显的抑制作用.结论 FICZ对LPS诱导的大鼠ARDS具有保护作用,其作用可能是通过激活AhR以减少NF-κB p65的入核来实现的.

Objective To observe the effect of agmatine (AGM) on inflammatory factor in Kupffer cells of liver,and to investigate the protective effects of AGM on severe trauma-induced liver injury in mice and its possible mechanism.Methods Forty-two adult male BALB/c mice were randomly divided into sham group,model group,and AGM treatment group,with 14 mice in each group.The mice model of trauma-hemorrhage was reproduced by hindlimbs fracture combined with 35％ of orbital bleeding.The mice in the sham group were only anesthetized without other treatments.The mice in AGM treatment group were given intraperitoneal injection of 200 mg/kg AGM when limited recovery was performed,and the mice in model group were given the equal amount of normal saline.Seven mice in each group were sacrificed at 12 hours and 24 hours,respectively,after modeling,and blood samples and liver tissue were harvested,and liver Kupffer cells were isolated.Serum alanine aminotransferase (ALT),aspartate transaminase (AST)and lactic dehydrogenase (LDH) were determined with automatic biochemistry analyzer.Hepatic pathological changes were observed with light microscope using hematoxylin and eosin (HE) staining.The levels of tumor necrosis factor-α(TNF-o) and interleukin-6 (IL-6) in serum,hepatic homogenate and Kupffer cell supernatant were determined with enzyme linked immunosorbent assay (ELISA).The mRNA expressions of pro-inflammatory cytokines TNF-α and IL-6 in the Kupffer cell were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR).Results ① The normal liver tissue structure was found in sham group.At 24 hours after modeling in the model group,the changes in pathobiology were found as following:neutrophil infiltration,hepatocytes swelling,hyperemia,and necrosis,as well as the abnormality of parameters reflecting liver function.AGM could significantly improve the pathological changes in liver tissue caused by severe trauma,and ameliorate the liver function.② There were no significant differences in the levels of TNF-α and IL-6 in serum and hepatic tissue at 12 hours after modeling,and the parameters at 24 hours in model group were higher than those at 12 hours,which were significantly higher than those of the sham group [serum TNF-α (ng/L):80.8±4.7 vs.34.7±4.7,IL-6 (ng/L):104.0±9.0 vs.55.4±3.3;liver TNF-α (ng/mg):405.2± 19.6 vs.57.2±10.0,IL-6 (ng/mg):58.4±7.7 vs.14.3±2.1,all P ＜ 0.01].AGM could effectively reduce the levels of TNF-o and IL-6 in serum and hepatic tissue [serum TNF-α (ng/L):58.2 ± 3.1 vs.80.8 ± 4.7,IL-6 (ng/L):74.1 ± 6.6 vs.104.0± 9.0;liver TNF-α (ng/mg):248.7 ± 22.5 vs.405.2 ± 19.6,IL-6 (ng/mg):22.5 ± 3.1 vs.58.4 ± 7.7,all P ＜ 0.01].③ The levels of TNF-o and IL-6 in Kupffer cells supernatant were significantly higher than those of the sham group,and they were further increased after lipopolysaccharide (LPS) stimulation for 24 hours.AGM could effectively reduce the levels of TNF-α and IL-6 in Kupffer cells [TNF-α (ng/L):256.6 ± 5.6 vs.465.5 ± 5.2,IL-6 (ng/L):1 185.5 ± 64.4 vs.2 018.8 ± 53.2,both P ＜ 0.01],and also decreased the mRNA expressions of TNF-α and IL-6 [TNF-α mRNA (2-△△Ct):7.2±0.4 vs.13.5±0.4,IL-6 mRNA (2-△△Ct):13.2±0.7 vs.21.3 ± 1.6,both P ＜ 0.01].Conclusion Agmatine can reduce trauma-induced acute hepatic injury via suppression of cytokines release in Kupffer cells,and can ameliorate the liver function.

目的 比较腹腔注射脂多糖(lipopolysaccharide,LPS)、气管滴注LPS和盲肠结扎穿刺(cecal ligation and puncture,CLP)诱导的3种急性肺损伤模型的差异.方法 70只成年SD大鼠随机分为7组:正常组、腹腔注射LPS组(10 mg/kg,LPS-IP组)及其假手术组(SHAM-1);气管滴注LPS组(5 mg/kg,LPS-IT组)及其假手术组(SHAM-2);盲肠结扎穿刺组(CLP)及其假手术组(SHAM-3).于造模后24 h,收集各组大鼠的动脉血用于血气分析,并通过二喹啉甲酸(bicinchoninic acid,BCA)蛋白测定法、血细胞计数板以及瑞氏-姬姆萨染色分别对肺泡灌洗液中的蛋白含量、细胞总数和类别进行统计,同时利用湿干比和苏木精-伊红(HE)染色法来评价肺组织的损伤程度.结果 LPS-IT组和LPS-IP组的肺灌洗液总蛋白浓度、总细胞数均高于正常组和假手术组(P＜0.05),湿干比明显增高(P＜0.05),PaO2低于正常组及相应的假手术组(P＜0.05);LPS-IT组和LPS-IP组肺组织有明显病理变化,包括肺水肿和肺泡壁增厚,炎性细胞浸润.CLP组未见明显改变.结论 在急性肺损伤的3种大鼠模型中,LPS-IT组和LPS-IP组肺损伤较为严重,CLP组未见明显肺损伤.提示腹腔注射LPS及气管给予LPS更适用于建立急性肺损伤模型以及后续应用药物的动物实验.

目的 探讨胍丁胺(agmatine,AGM)对脂多糖(lipopolysaccharides,LPS)诱导小鼠巨噬细胞系RAW264.7氧化应激的抑制作用及其机制.方法 RAW264.7细胞经LPS(10 μg/mL)刺激发生氧化应激,同时AGM(1 mmol/L)对其进行干预.分为对照组、LPS组、LPS+AGM组、AGM组.药物作用24 h,以2,7-二氢二氯荧光素二乙酸酯(2 ',7'-dichlorofluoresce in diacetate,DCFH-DA)为荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;Griess法检测细胞内一氧化氮(NO)水平;Western blot检测细胞内的iNOS蛋白及细胞质细胞核内Nrf2蛋白表达;RT-PCR检测Nrf2下游抗氧化酶血红素氧合酶(heme oxygenase-1,HO-1)mRNA的表达.结果 与对照组相比,LPS可显著增加RAW264.7细胞中ROS、NO以及iNOS蛋白水平,而AGM干预后上述指标含量均明显降低(P＜0.05).AGM干预组中细胞核的Nrf2表达及其下游分子HO-1 mRNA表达水平较LPS单独刺激组显著升高,表明Nrf2信号通路在AGM的作用下被活化.结论 AGM可通过活化转录因子Nrf2促进抗氧化酶HO-1的表达,进而减轻细胞内的氧化应激损伤.

目的 研究胰岛素样生长因子-1受体(insulin-like growth factor 1 receptor,IGF-1R)的选择性抑制剂PQ401在体内外的抗炎效应.方法 体外实验:小鼠RAW264.7细胞株经0.1、1、10 μmol/LPQ401预作用1h后,加入10 μg/mL脂多糖(LPS)作用12h,采用酶联免疫吸附实验(ELISA)检测细胞上清肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-1 β、IL-6)的含量.另将小鼠RAW264.7细胞株经10 μmol/L PQ401预作用1h后,加入10 μg/mL LPS作用6、12、24 h后再次检测以上指标的变化.体内实验:将50只成年雄性C57BL/6小鼠按随机数字表法分为对照组、模型组和不同剂量PQ401组,每组10只.腹腔注射LPS 10 mg/kg制备急性炎症反应小鼠模型,对照组腹腔注射等量二甲基亚砜(DMSO),PQ401组分别腹腔注射LPS 10 mg/kg和PQ401 25、50、100 mg/kg.于给药12 h后通过ELISA检测血清和肝组织中TNF-ot、IL-1β、IL-6的含量;用全自动生化分析仪测定血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)及总胆红素(TBil)水平;采用Western blot检测肝细胞核内核转录因子-κB p65(NF-κB p65)的含量.结果 与LPS单独作用组相比,PQ401呈浓度和时间依赖性降低RAW264.7细胞上清中TNF-α、IL-1β、IL-6的水平.在LPS诱导的小鼠急性腹膜炎模型中,小鼠经LPS注射12 h后表现为精神萎靡、活动减少,而PQ401组小鼠的精神状态、活动均好于模型组.PQ401治疗后能有效降低LPS所致急性腹膜炎小鼠血清中TNF-α、IL-1β、IL-6的含量,降低肝组织TNF-α、IL-1β的水平和NF-κB p65的含量.结论 PQ401在体内外对LPS诱导的炎症因子分泌具有明显的抑制效应.

The enzyme complex IκB kinase (IKK) is an essential activator of NF-κB signaling pathway involved in propagating the cellular response to inflammation. The complex contains two functional subunits IKKα and IKKβ, which are structurally conserved kinases and selective inhibition of them would result in distinct biological effects. However, most existing IKK inhibitors show moderate or high promiscuity for the two homologous kinases. Understanding of the molecular mechanism and biological implication underlying the specific interactions in IKK-ligand recognition is thus fundamentally important for the rational design of selective IKK inhibitors. In the current work, we integrated molecular docking, quantum mechanics/molecular mechanics calculation and Poisson-Boltzmann/surface area analysis to investigate the structural basis and energetic property of the selective binding of small-molecule ligands to IKKα and IKKβ. It was found that the selectivity is primarily determined by the size and topology difference in ATP-binding pocket of IKKα and IKKβ kinase domains; bulky inhibitor molecules commonly have, respectively, low and appropriate affinities towards IKKα and IKKβ, and thus exhibit relatively high selectivity for IKKβ over IKKα, whereas small ligands can only bind weakly to both the two kinases with low selectivity. In addition, the conformation, arrangement and distribution of residues in IKK pockets are also responsible for constituting the exquisite specificity of ligand binding to KKα and IKKβ. Next, a novel quantitative structure-selectivity relationship model was developed to characterize the relative contribution of each kinase residue to inhibitor selectivity and to predict the selectivity and specificity for a number of known IKK inhibitors. Results showed that the active-site residues contribute significantly to the selectivity by directly interacting with inhibitor ligands, while those protein portions far away from the kinase active sites may also play an important role in determining the selectivity through long-range non-bonded forces and indirect allosteric effect.