Lipopolysaccharide-induced decreased protein S expression in liver cells is mediated by MEK/ERK signaling and NFκB activation: involvement of membrane-bound CD14 and toll-like receptor-4

BACKGROUND: The vitamin K-dependent protein S (PS), mainly synthesized in hepatocytes and endothelial cells, plays a critical role in the anticoagulant activity of plasma. The decreased plasma level of PS in sepsis is associated with thrombotic tendency, but the mechanism is unclear. OBJECTIVES: In the present study, we examined the effect of lipopolysaccharide (LPS) on PS expression in vivo in rat liver, and in vitro in isolated hepatocytes and sinusoidal endothelial cells (SECs) from normal rats. RESULTS: LPS induced a progressive decrease of plasma PS antigen level up to 12 h with a slight recovery at 24 h, and a transient decrease of liver PS mRNA level at 4-8 h with a complete recovery at 24 h. In the in vitro studies, LPS decreased PS antigen and mRNA levels in both hepatocytes and SECs. After LPS treatment, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) transiently increased in plasma. IL-6 increased the protein expression of PS from hepatocytes, while TNF-alpha decreased it from SECs. LPS increased CD14 in hepatocytes and decreased it in SECs, but did not affect toll-like receptor-4 (TLR-4) expression in both cells. Antirat CD14 and antirat TLR-4 antibodies inhibited LPS-induced NFkappaB activation, and a NFkappaB inhibitor suppressed LPS-induced decreased PS expression in both cells. Furthermore, MEK inhibitor blocked LPS-induced decreased PS expression in both cells. CONCLUSIONS: These findings suggest that LPS-induced decreased PS expression in hepatocytes and SECs is mediated by MEK/ERK signaling and NFkappaB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism. These findings may explain in part the decreased level of plasma PS and thrombotic tendency in sepsis.     

AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers

In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.     

肝硬化部分切除后肝再生及结构变化

背景：肝脏自身具有强大的再生能力,临床资料显示大部分肝癌患者合并有肝硬化,切除肿瘤后肝脏质量可逐渐恢复,提示硬化的肝脏仍具备修复潜能。目的：尝试用肝部分切除方法诱导小鼠硬化肝组织内具再生能力的细胞增殖,观察再生肝脏的结构变化。方法：使用CCl4皮下注射方法制备小鼠肝硬化模型。对肝硬化小鼠行肝部分切除,于切除后1,3,5,7,10,14及21 d取小鼠肝组织并称质量。观察比较肝部分切除后不同时间小鼠再生肝组织的结构变化;评估肝再生率;免疫组化方法分析肝星形细胞标志分子结蛋白及肝星形细胞活化标志分子α-平滑肌肌动蛋白的表达变化;5-溴脱氧尿核苷标记技术对增殖细胞进行定性、定位。结果与结论：肝硬化小鼠肝部分切除后第1,3,5,7,14,21天肝再生率分别为6.58%,22.03%,21.39%,29.05%,45.22%,50.98%。苏木精-伊红染色和Masson染色显示CCl4注射8周后呈早期肝硬化病理改变,肝部分切除后肝组织结构逐步改善。免疫组化结果显示肝部分切除后第1天再生肝组织内结蛋白表达减少,但第3天至第14天呈上升趋势,21 d则明显减少;肝再生过程中各时间点α-平滑肌肌动蛋白表达依时递减。5-溴脱氧尿核苷阳性细胞在肝部分切除后1-7 d主要为肝细胞,肝部分切除后14-21 d阳性细胞定位于肝血窦内皮及窦腔内。结果表明早期肝硬化小鼠大部肝切除后3 d出现明显的再生反应,并逐步恢复正常的肝组织结构。     

原发性胆汁性肝硬化患者血清细胞因子表达及意义

目的探讨原发性胆汁性肝硬化(PBC)患者血清中16种相关炎性因子表达及其临床意义。方法以50例PBC患者和40名健康对照者作为研究对象,应用Luminex液态芯片系统检测16种炎性因子血清表达情况。分析表达存在明显差异细胞因子与PBC临床及实验室参数相关性。指标相关性采用Spearman相关性检验,数值资料比较采用t检验。结果与健康对照组相比,PBC组血清中IL-6、IL-8、IL-17A、IFN-γ和TNF-α表达显著升高,差异有统计学意义[(5.8±1.3)pg/ml,(69.7±22.1)pg/ml;(328.5±154.5)pg/ml,(874.5±678.5)pg/ml;(21.3±8.5)pg/ml,(68.5±32.5)pg/ml;(25.6±10.5)pg/ml,(104.5±46.8)pg/ml;(125.5±69.5)pg/ml,(408.5±185.6)pg/ml;P均<0.01]。IL-6、IL-17A、IFN-γ和TNF-α血清表达与PBC Mayo风险评分呈正相关,相关系数r分别为0.598、0.618、0.519和0.614,P均<0.05。IL-17A、IFN-γ和TNF-α血清表达与ALT、ALP及CRP水平呈正相关,相关系数r分别为:0.632、0.521、0.493;0.614、0.603、0.532;0.727、0.814、0.659;P均<0.05。结论 PBC患者血清炎性因子IL-6、IL-17A、IFN-γ和TNF-α表达显著升高,且与疾病活动指数呈相关。     

围手术期肠内免疫营养对肝硬化肝切除鼠免疫功能的影响

目的：观察围手术期使用肠内免疫营养剂对肝硬化肝切除大鼠免疫功能的调理作用。方法：48只肝硬化大鼠随机分为两组,A组为标准肠内营养组,B组为肠内免疫营养组,依标本采集时间不同,各组再分为4个亚组。大鼠用肠内营养剂喂养8d后行68%肝切除术,术后再用肠内营养剂喂养至取标本时间。分别于术前和术后1、4、8d取相应亚组大鼠全血,检测T细胞亚群分类、IgG和IL-6。结果：两组大鼠术后均有免疫功能降低和急性炎症反应的发生,但B组的程度明显低于A组。B组术后1dCD3、CD4、CD4/CD8和IgG,术后4dCD4、CD4/CD8和IgG,术后8dCD4/CD8和IgG均显著高于A组（P〈0.05）;B组术后1、4dIL-6显著低于A组（P〈0.05）。结论：围手术期肠内免疫营养支持与标准肠内营养相比,能减轻肝硬化肝切除大鼠术后的免疫抑制,增强术后的免疫功能,下调术后过度的急性炎症反应。     

Time-course changes in serum cytokine levels in two experimental acute pancreatitis models in rats

Activated leukocytes and cytokines have important roles in the multisystem involvement during acute pancreatitis. The changes in the serum level of tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) over time were investigated in two experimental acute pancreatitis models in rats. Mild edematous pancreatitis was induced with an overdose of cholecystokinin octapeptide (CCK-8), while a severe hemorrhagic form of pancreatitis was induced by ligation of the common bilio-pancreatic duct. The rats were examined 2, 4, 8, 16, 24 and 48 h after pancreatitis induction. The severity of the inflammation was assessed by measurement of the serum amylase activity, quantification of the edema, and histological examination. Serum TNF-α and IL-6 were determined by bioassay, using the TNF-sensitive WEHI 164 and the IL-6-dependent B9 cell lines, respectively. In CCK-8-induced acute pancreatitis, the pancreatic weight/body weight ratio (pw/bw) and amylase level were significantly elevated at 2 h, and the maximum levels were observed at 4 h (8.19±1.13 mg/g and 69.4±12.8×10³ U/ml, respectively). Both parameters subsequently decreased continuously during the observation period. The serum IL-6 level was significantly increased at 4 h relative to the controls (123.3±5.8 vs 37.5±15 pg/ml), and then decreased continuously. In this model, only a moderate level of serum TNF-α was observed at 2 h. In the biliary type of acute pancreatitis, the ratio pw/bw increased continuously during the study and reached the maximum level at 48 h relative to the sham-operated control (8.8±1.4 vs 5.3±0.8 mg/g). The serum amylase level was significantly elevated at 2 h (43.2±13×10³ U/ml), but then decreased continuously. The serum IL-6 reached its maximum level at 16 h (3800±447 pg/ml). In this model, increased TNF-α levels (75–300 U/ml) were measured 8, 16 and 24 h after pancreatitis induction. The results led to correlations between the serum IL-6 levels and the biochemical and morphological severity of acute pancreatitis in both experimental models. The data suggest that IL-6 and TNF-α may participate in the pathogenesis of these types of acute pancreatitis.     

Liver regeneration following partial hepatectomy and stimulation by hepatic stimulatory substance in cirrhotic and non-cirrhotic rats

The effects of hepatic stimulatory substance (HSS) on cirrhotic and noncirrhotic rats were studied after 70% partial hepatectomy. Liver cirrhosis was produced by weekly intragastric infusion of chloroform for 12–16 weeks. The HSS was prepared by extraction from the livers of weanling mice. Rats in the experimental group were injected with 5 ml HSS after 70% partial hepatectomy, and those in the control group received normal saline. The results showed that the³H-thymidine incorporation was higher in the HSS group 24 h after partial hepatectomy in both cirrhotic and non-cirrhotic rats, and persistently higher in the non-cirrhotic rats at 48h. Total DNA was significantly higher in the HSS group of non-cirrhotic rats 24 and 48 h after partial hepatectomy. The restituted liver volume and weight was significantly higher in non-cirrhotic rats 48 h after partial hepatectomy, while there was no significant difference between the HSS and the control groups in the cirrhotic rats. The HSS induced significant effects on³H-thymidine incorporation in the non-cirrhotic liver, resulting in increasing liver weight, volume and total DNA 48 h after partial hepatectomy. In cirrhotic rats, the³H-thymidine incorporation was higher in the HSS group at 24 h after partial hepatectomy, though not showing any increase at 48 h, but the regeneration of liver weight, volume and total DNA at 48 h showed no difference between the HSS group and the control group.     

Increase of tumour necrosis factor α synthesis and gene expression in peripheral blood mononuclear cells of children with idiopathic nephrotic syndrome

Abstract. The pathogenesis of idiopathic nephrotic syndrome (minimal change nephropathy and its variants) is not completely understood. In recent years it has been speculated that a cytokine released by circulating blood mononuclear cells could alter the permeability of the glomerular capillary wall. In this study we have explored the potential participation of tumour necrosis factor α (TNF α ), a cytokine mainly produced by monocytes, in 25 children with idiopathic nephrotic syndrome. TNF α was determined by cytotoxicity bioassay in the L-929 cell line and by double antibody/RIA. Patients in activity had higher serum TNF α levels and TNF α production by monocytes than patients in remission and controls. TNF α mRNA expression in blood mononuclear cells was analysed by Northern blot. The TNF α mRNA levels in patients in activity were increased compared to controls and to patients in remission. No significant differences in IL-1 β and IL-6 synthesis were found between patients and controls. Our results suggest that TNF α , but not other cytokines such as IL-1 β and IL-6, could play a role in the pathogenesis of the proteinuria in idiopathic nephrotic syndrome.     

Liver transduction with a simian virus 40 vector encoding insulin-like growth factor I reduces hepatic damage and the development of liver cirrhosis

Liver transplantation is the only treatment for advanced liver cirrhosis. Therapies halting the progression of the disease are urgently needed. Administration of recombinant insulin-like growth factor-I (rIGF-I) induces hepatoprotective effects in experimental cirrhosis. Therefore, we analyzed the efficacy of a recombinant simian virus 40 vector (rSV40) encoding IGF-I (rSVIGF-I) to prevent cirrhosis progression. First, transgene expression was evaluated in mice injected with rSV40 encoding luciferase, which showed long-term hepatic expression of the transgene. Interestingly, luciferase expression increased significantly in CCl(4)-damaged livers and upon IGF-I administration, thus liver injury and IGF-I expression from rSVIGF-I should favor transgene expression. rSVIGF-I therapeutic efficacy was studied in rats where liver cirrhosis was induced by CCl(4) inhalation during 36 weeks. At the end of the study, the hepatic levels of IGF-I and IGF-binding protein 3 were higher in rSVIGF-I-treated rats than in control cirrhotic animals. Cirrhotic rats treated with rSVIGF-I had reduced serum bilirubin, transaminases and liver fibrosis scores and increased hepatic expression of hepatocyte growth factor and STAT3alpha as compared to cirrhotic animals. Furthermore, cirrhotic animals showed testis atrophy and altered spermatogenesis, whereas testicular size and histology were normal in cirrhotic rats that received rSVIGF-I. Therefore, rSV40-mediated sustained expression of IGF-I in the liver slowed cirrhosis progression.     

双环醇对肝硬化模型大鼠炎性因子、胃肠功能及肝功能的影响

[目的] 探讨双环醇对肝硬化模型大鼠炎性因子、胃肠功能及肝功能的影响.[方法] 选择雄性SD大鼠60只并分为对照组、模型组以及双环醇低剂量组、中剂量组、高剂量组,采用四氯化碳皮下注射的方式建立肝硬化模型.双环醇低剂量组、中剂量组、高剂量组分别给予0.1 g/kg、0.2 g/kg、0.3 g/kg双环醇灌胃.干预后8周时,检测并比较各组小鼠炎性因子[白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)] 、胃肠黏膜屏障功能指标[降钙素原(PCT)、D-乳酸、内毒素(LPS)] 、肝功能指标[丙氨酸氨基转氨酶(ALT)、天冬氨酸氨基转移酶(AST)] 的变化.[结果] 干预后8周时,模型组大鼠血清中IL-1β、IL-8、TNF-α、ALT、AST、PCT、LPS、D-乳酸水平均显著高于对照组(P<0.05);双环醇低剂量组、中剂量组、高剂量组大鼠血清中IL-1β、IL-8、TNF-α、ALT、AST、PCT、LPS、D-乳酸水平均低于模型组(P<0.05);双环醇剂量越大,血清中IL-1β、IL-8、TNF-α、ALT、AST、PCT、LPS、D-乳酸的水平越低(P<0.05).[结论] 双环醇对肝硬化模型大鼠的胃肠功能及肝功能具有保护作用,同时能够抑制炎性因子的合成,是治疗肝硬化的理想药物.     

血必净对心肺复苏后转录因子GATA-3和T-bet表达调节的实验研究

目的 探讨血必净对心肺复苏后转录因子GATA-3和T-bet表达的调节作用.方法 建立心肺复苏大鼠模型,30只SD大鼠动物随机(随机数字法)分为三组:假手术组(B组),仅进行麻醉和气管切开插管、血管穿刺,不进行窒息及心肺复苏;常规复苏组(C组),常规复苏+生理盐水4 mL/kg;血必净治疗组(D组),常规复苏+血必净4mL/kg.动态观察血清GATA-3和T-betmRNA表达及白介素-12(IL-12)、白介素-4(IL-4)、r-干扰素(IFN-γ)、肿瘤坏死因子-α(TNF-α)水平的变化.多组间均数比较采用单因素方差分析,方差齐则采用成组设计资料的t检验,两两比较用SNK法;如方差不齐,则采用随机区组设计的秩和检验(Wilcoxon检验),P<0.05为差异有统计学意义.结果 C,D组复苏后6 h与对照组、复苏后即刻比较,IL-12,IFN-γ,TNF-α水平均升高(P<0.01);C组复苏后6 h GATA表达下调(P>0.05),T-bel表达上调(P<0.01),GATA-3/T-bet下降(P<0.05).D组复苏后6 h T-bet表达上调(P>0.05),GATA表达上调(P<0.01),GATA-3/T-bet升高(P<0.05);D组复苏后6h与C组同时点比较,IL-12,IFN-γ,TNF-α水平低于C组(P<0.01),GATA-3表达上调、T-bet表达下调、GATA-3/T-bet升高均有统计学意义(P<0.01).结论 心肺复苏后存在转录因子T-bet和GATA-3失衡表达,血必净可调节GATA-3/T-bet趋向平衡及细胞因子IL-12,IFN-γ,TNF-α水平.     

大剂量地塞米松对免疫性血小板减少性紫癜患者浆细胞样树突状细胞功能及Toll样受体9 mRNA表达的影响

本研究探讨大剂量地塞米松对免疫性血小板减少性紫癜(ITP)患者外周血浆细胞样树突状细胞(pDC)功能及Toll样受体9(TLR9)表达的影响。15例初诊的ITP患者给予地塞米松40 mg/d,连用4 d,采用免疫磁珠分离法体外分离15例正常对照及13例治疗有效患者治疗前后外周血中pDC;用CpG-ODN 2216刺激外周血pDC并与之共培养24 h,采用ELISA方法检测上清中IFN-α、IL-6、TNF-α的含量;用实时定量聚合酶链反应(RT-PCR)检测pDC的TLR9 mRNA表达量。结果表明,①治疗前pDC产生IFN-α、IL-6、TNF-α水平〔(960.83±164.65)pg/ml,(156.15±39.89)pg/ml,(137.31±35.44)pg/ml)〕明显高于正常对照组〔(616.67±105.98)pg/ml,(89.13±21.48)pg/ml,(88.53±25.81)pg/ml,P<0.05〕;治疗后pDC产生IFN-α、IL-6、TNF-α水平分别降至(678.46±128.88)pg/ml,(97.77±26.31)pg/ml,(103.08±26.42)pg/ml,与治疗前比较差异有统计学意(P<0.05),与正常对照组相比差异无统计学意义(P>0.05);②治疗前pDC的TLR9 mRNA的表达水平高于正常对照组(P<0.05);治疗后pDC的TLR9 mRNA的表达水平低于治疗前(P<0.05),与正常对照组比较差异无统计学显著性(P>0.05)。结论:pDC分泌的细胞因子及其表达的TLR9在ITP发病中起重要作用;地塞米松可能通过下调TLR9的表达,抑制pDC分泌细胞因子的功能,而对ITP起到治疗作用。     

Cytokines and Chemokines in Idiopathic Intracranial Hypertension

Background.— The pathogenesis of idiopathic intracranial hypertension (IIH) remains unclear and as such it remains a diagnosis of exclusion.
Objectives.— To identify cerebrospinal fluid (CSF) and serum cytokine and chemokine profiles associated with IIH.
Method.— Semiquantitative assessment with cytokine antibody arrays was used to detect the relative expression of 42 different cytokines and chemokines in the CSF and serum of 8 IIH patients and 8 controls. Subsequently, quantitative assay with enzyme linked immunosorbent assay was performed for chemokine CCL2, interleukin-1 alpha (IL-1α), and leptin.
Results.— Cytokine antibody array showed elevated levels of CCL2 in the CSF and CCL7, CCL8, IL-1α, and leptin levels in serum in IIH patients compared with controls. Subsequent quantitative assessment with enzyme linked immunosorbent assay showed significantly elevated CSF CCL2 in IIH patients compared with controls ( P  < .01) but there was no significant difference in leptin and IL-1α levels between the groups.
Conclusion.— This is the first report demonstrating differences in cytokine expression in the serum and CSF in IIH patients compared with controls. Since the pathogenesis of IIH is unclear, the heterogeneity of the cytokine expression reported here may help understand the pathogenesis of this condition.     

Relationship between vascular reactivity in vitro and blood flows in rats with cirrhosis.

In cirrhosis there is a hyperdynamic circulation, which occurs mainly in the systemic and splanchnic regions. Using isolated-vessel models, previous studies have shown reduced aortic reactivity to vasoconstrictors in rats with cirrhosis. The aim of the present study was to evaluate and compare the vascular responsiveness to phenylephrine in arterial rings and the blood flows from different regions in rats with cirrhosis and controls. Reactivity was studied in isolated thoracic aortic, superior mesenteric arterial and carotid arterial rings from sham-operated and bile-duct-ligated rats by measuring the cumulative concentration-dependent tension induced by phenylephrine (10(-9)-10(-4) M). Blood flows were measured by the radioactive microsphere method. In rats with cirrhosis, a significant hyporeactivity to phenylephrine was observed in both the aorta and the superior mesenteric artery compared with the corresponding arteries of normal rats. This hyporesponsiveness was corrected by N(omega)-nitro-L-arginine (0.1 mM). In contrast, carotid artery reactivity and the responses to N(omega)-nitro-L-arginine were similar in the cirrhotic and control groups. In each case, cardiac output and mesenteric arterial blood flow were significantly higher in cirrhotic than in normal rats. Cerebral blood flows were not significantly different between the two groups. In cirrhotic rats, arterial hyporeactivity may be a consequence of increased regional blood flow and increased production of nitric oxide.     

Total and acylated ghrelin in liver cirrhosis: Correlation with clinical and nutritional status

Abstract

Objectives. The pathogenesis of anorexia in cirrhotic patients is complex and the appetite-modulating hormone ghrelin could be involved. Acylated ghrelin is the biologically active form that modifies insulin sensitivity and body composition. The aim of the present study was to compare acylated and total ghrelin concentration in patients with liver cirrhosis and to investigate the possible relationship between ghrelin and clinical and nutritional parameters. Design and methods. Sixty patients with viral liver cirrhosis who did not have hepatocellular carcinoma or acute infections were studied. Twenty healthy volunteers were recruited after matching for age, gender, and body mass index with the patients and served as controls. Fasting levels of total, acylated ghrelin, leptin, TNF-α and insulin were measured in all subjects, in addition, clinical and nutrition parameters were assessed. Results. In cirrhotic patients, plasma levels of both acylated and total ghrelin were significantly higher than those in the controls. The mean plasma acylated ghrelin levels were significantly higher in Child C cirrhosis compared to Child A and B. Ghrelin (total and acylated) were negatively correlated with leptin in cirrhotic patients confirming the fact that leptin acts as a physiological counterpart of ghrelin. Conclusions. Nutritional and metabolic abnormalities in cirrhotic patients may be dependent on the changes in the ghrelin/leptin systems, mainly the acylated form of ghrelin.     

炎症因子的表达水平与心肌肥厚性改变的相关性研究

【目的】探讨炎症因子的表达与心肌肥厚性改变的相关性。【方法】将40只体重在180～200g的雄性SD大鼠随机分为两组,实验组（25只）制作压力负荷性心肌肥厚大鼠模型,对照组肾动脉不作处理。手术56d后用ELISA方法测量比较两组心肌组织肿瘤坏死因子（TNF-α）与白细胞介素-6（IL-6）的含量,并比较两组左室心肌重量。【结果】两组大鼠TNF-α和IL-6水平比较差异有显著性（P〈0．05）;且实验组大鼠的左室壁明显肥厚,重量明显增加（P〈0．05）。【结论】实验组大鼠心肌组织TNF-α、IL-6表达水平与左室心肌重量成正相关,并参与了左室肥厚的形成过程。     

Inhibition of the Ras oncoprotein reduces proliferation of hepatocytes in vitro and in vivo in rats

Ras oncoproteins are probably implicated in normal and malignant cell growth in various organs. Inhibition of Ras interferes with cell proliferation of non-hepatic cells in vitro and in vivo. A potential role for Ras in normal and malignant hepatocyte proliferation prompted us to evaluate the impact of Ras inhibition by FTS (S-farnesylthiosalicylic acid) on hepatocyte proliferation in vitro in the human hepatic tumour cell line HepG2 and in vivo after PH (partial hepatectomy) in rats. Rats were administered with FTS intraperitoneally (1, 8 and 16 h after PH) and killed 12, 24 and 48 h after PH. Cell proliferation, phosphorlyation of members of the MAPK (mitogen-activated protein kinase) pathway and levels and activity of cell cycle effectors (cyclin D, cyclin E, Cdk2 and Cdk4) were assessed in FTS-treated rats compared with controls. FTS significantly decreased overall cell count, PCNA (proliferating-cell nuclear antigen) expression and BrdU (bromodeoxyuridine) incorporation into HepG2 cells after 7 days of culture. FTS treatment significantly reduced BrdU incorporation and PCNA expression in hepatocytes after PH. Unlike control rats, cell-membrane expression of Ras was decreased in FTS-treated animals after PH, resulting in decreased Raf membrane recruitment and phosphorylation and in reduced phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2). The antiproliferative effect of FTS was linked to a decrease in expression and activity of the cyclin E/Cdk2 complex, without affecting cyclin D and Cdk4. Ras inhibition by FTS significantly decreased proliferation of HepG2 cells and normal hepatocytes after a strong and highly synchronized proliferation stimulus elicited by PH. The inhibitory effect was at least partially mediated by inhibition of Ras/Raf/MAPK signalling. It appears worthwhile to evaluate the impact of Ras inhibition on the development of hepatocarcinomas in vivo in adequate animal models.     

罗格列酮对原位肝移植胆道缺血-再灌注损伤的作用

目的 探讨过氧化物酶体增殖物活化受体-γ(PPAR-γ)配体罗格列酮在原位肝移植胆道缺血-再灌注损伤中作用的分子机制.方法 40只SD大鼠随机(随机数字法)分成假手术组(SO组)、缺血-再灌注组(I/R组)、罗格列酮组(ROS组)和GW9662组,每组10只.通过改良"双袖套法"建立大鼠自体原位肝移植胆道缺血-再灌注模型,通过肝脏及胆管组织病理学变化、血生化指标检测评价模型建立是否成功.SO组行自体原位肝移植术,I/R行建模缺血-再灌注,ROS在缺血-再灌注后以罗格列酮0.3mg/kg经门静脉注射,GW9662在ROS基础上10 min后经门静脉以0.3mg/kg注射GW9662,各组均于实验后4h取部分肝脏和胆管组织用于免疫组化检测;右心室穿刺抽血采用ELISA法测定细胞因子含量.采用方差分析进行统计学处理.结果 组织中细胞因子IL-1β,TNF-α,IL-6活性主要表达在肝细胞及胆管细胞胞浆中,转录因子NF-κB则在胞浆及胞核中均有表达;I/R组及GW9662组上述蛋白表达升高,和SO组以及ROS组比较明显增高(P＜0.05).血清中I/R组大鼠IL-1β,TNF-α及IL-6同步升高,较SO组明显增高(P＜0.05);罗格列酮干预后血清中IL-1β,TNF-α及IL-6和SO组比较无明显变化(P＞0.05);给予罗格列酮阻滞剂GW9662后,IL-1β,TNF-α及IL-6的含量和SO组比较则明显增高(P＜0.05).结论 PPAR-γ的配体罗格列酮对原位肝移植胆道缺血-再灌注损伤有保护作用,这种保护作用的机制与拮抗核因子-κB和抑制其表达,减少下游IL-6,IL-1β以及TNF-α等细胞因子的释放有关.

Abstract：

Objective To explore the effective molecular mechanism of PPAR-γligands rosiglitazone to biliary ischemia-reperfusion injury in autologous liver transplantation. Method A total of 40 SD rats were randomly (random number) divided into sham operation group (SO), ischemia - reperfusion group (Ⅰ/R), rosiglitazone (ROS) and GW9662 group, with 10 ones in each. The models, rat biliary ischemiareperfusion injury of autologous liver transplantation, were made by modified two-cuff technique. Tissues of the liver and bile ducts and blood of those models were evaluated by pathological and biochemical methods to make sure the models were made successfully or not. SO group suffered autologous orthotopic liver transplantation, and L/R group suffered both that and ischemia-reperfusion. ROS group were injected rosiglitazone (0.3mg/kg) via portal vein after having been done all as I/R. GW9662 group suffered all as ROS, and 10min later ,they were injected GW9662(0.3mg/kg) via portal vein. 4h after the experiment, tissues of livers and bilary ducts were taken to be tested by immunohistochemistry method, and the blood punctured from the right ventricular were taken to be determined by ELISA. ANOVA was used for statistical analysis.Results IL-1β, TNF-α and IL-6 were mainly expressed in the cytoplasm of hepatocytes and bile duct cells,while NF-κB was expressed both in the cytoplasm and nuclei. Expression of those proteins in L/R and GW9662 group was increased, significantly higher when compared to the SO and ROS (P ＜ 0.05). IL-1β,TNF-α and IL-6 in rat serum were simultaneously increased, and significantly higher than SO(P ＜0.05).Compared with the SO, expressions of the IL-1 β,TNF-α and IL-6 were not significantly changed in ROS (P＞ 0.05 )but significantly increased in GW9662. Conclusions PPAR-γ ligand rosiglitazone took protective role in biliary ischemia-reperfusion injury in autologous liver transplantation. The mechanism correlates with the release of the IL-lα, IL-1β and TNF-α and other inflammatory mediators, which decreased as the expression of NF-κB inhibited by its antagonist.     

Foxp3磷酸化通过调控调节性T细胞功能对类风湿关节炎大鼠TNF-α抑制状态影响的分子机制

目的探究Foxp3磷酸化通过调节性T细胞功能改善类风湿关节炎大鼠肿瘤坏死因子-α(TNF-α)抑制状态的分子机制。方法建立类风湿关节炎大鼠模型,分组为正常组(无特殊处理)、模型组(类风湿关节炎模型)和实验组(类风湿关节炎模型+尾静脉注射TNF-α阻滞剂进行治疗)。酶联免疫吸附(ELISA)检测炎症因子白介素10(IL-10)、白介素17(IL-17)和转化生长因子-β(TGF-β)水平;流式细胞术检测T细胞阳性率;Western Blot检测TNF-α表达以及试剂盒检测Foxp3磷酸酶活性。结果与正常组相比,模型组IL-10、TGF-β水平显著降低,IL-17水平显著升高(P<0.05);与模型组相比,实验组IL-10、TGF-β的水平显著升高,IL-17的水平显著降低(P<0.05)。与正常组相比,模型组CD4^+CD25^+T细胞和CD4^+CD25^+Foxp3^+T细胞检测阳性率均显著降低(P<0.05);与模型组相比,实验组以上指标检测比率均显著增加(P<0.05);与正常组相比,模型组的TNF-α蛋白相对表达量显著增加(P<0.05);与模型组相比,实验组TNF-α蛋白的相对表达量显著降低(P<0.05)。与正常组相比,模型组的Foxp3磷酸酶活性显著降低(P<0.05);与模型组相比,实验组Foxp3磷酸酶活性显著升高(P<0.05)。结论通过尾静脉注射TNF-α阻滞剂治疗后,大鼠炎症反应水平、TNF-α蛋白表达及FOXP^3磷酸酶活性均降低,机制可能是Foxp3磷酸化通过调控调节性T细胞数量及炎症因子的变化参与对类风湿关节炎大鼠抑制状态,从而一定程度缓解类风湿关节炎不良症状。     

心肺复苏大鼠脑TNF—α、IL一1、IL一6和MMP9的表达及意义

目的探讨心肺复苏早期大鼠脑组织TNF-α、IL-1、IL-6和MMP9的表达及意义。方法将动物分为假手术组和复苏组,在即刻及3、9、24h和48h时间点分5个时间点处死大鼠后,取样,测定脑组织TNF-α、IL一1、IL-6和MMP9的表达,使用电镜观察脑组织的超微结构。结果假手术组心肺复苏后脑组织TNF—α、IL一1、IL-6、MMP9含量无明显变化,电镜观察脑组织超微结构也无明显变化。复苏组心肺复苏后脑脑组织TNF—α、IL一1、IL-6、MMP9含量明显升高,电镜超微结构观察均发生改变。结论心肺复苏后大鼠脑组织TNF-α、IL-1、IL-6和MMP9动态变化,血脑屏障的损伤与MMP9相关。