Sirtuin 1 in rat orthotopic liver transplantation:An I GL-1 preservation solution approach

AIM:To investigate the possible involvement of Sirtuin1(SIRT1)in rat orthotopic liver transplantation(OLT),when Institute Georges Lopez 1(IGL-1)preservation solution is enriched with trimetazidine(TMZ).METHODS:Male Sprague-Dawley rats were used as donors and recipients.Livers were stored in IGL-1 preservation solution for 8h at 4℃,and then underwent OLT according to Kamada’s cuff technique without arterialization.In another group,livers were stored in IGL-1 preservation solution supplemented with TMZ,at10-6 mol/L,for 8 h at 4℃and then underwent OLT.Rats were sacrificed 24 h after reperfusion,and liver and plasma samples were collected.Liver injury(transaminase levels),mitochondrial damage(glutamate dehydrogenase activity)oxidative stress(malondialdehyde levels),and nicotinamide adenine dinucleotide(NAD+),the cofactor necessary for SIRT1 activity,were determined by biochemical methods.SIRT1 and its substrates(acFox O1,ac-p53),the precursor of NAD+,nicotinamide phosphoribosyltransferase(NAMPT),as well as the phosphorylation of adenosine monophosphate activated protein kinase(AMPK),p-m TOR,p-p70S6K(direct substrate of m TOR),autophagy parameters(beclin-1,LC3B)and MAP kinases(p-p38 and p-ERK)were determined by Western blot.RESULTS:Liver grafts preserved in IGL-1 solution enriched with TMZ presented reduced liver injury and mitochondrial damage compared with those preservedin IGL-1 solution alone.In addition,livers preserved in IGL-1+TMZ presented reduced levels of oxidative stress.This was consistent with enhanced SIRT1 protein expression and elevated SIRT1 activity,as indicated by decreased acetylation of p53 and Fox O1.The elevated SIRT1 activity in presence of TMZ can be attributed to the enhanced NAMPT protein and NAD+/NADH levels.Up-regulation of SIRT1 was consistent with activation of AMPK and inhibition of phosphorylation of m TOR and its direct substrate(p-p70S6K).As a consequence,autophagy mediators(beclin-1 and LC3B)were overexpressed.Furthermore,MAP kinases were regulated in livers preserved with IGL-1+TMZ,as they were characterized by enhanced p-ERK and decreased p-p38protein expression.CONCLUSION:Our study shows that IGL-1 preservation solution enriched with TMZ protects liver grafts from the IRI associated with OLT,through SIRT1 up-regulation.     

缺血预处理抑制减体积肝移植大鼠早期肝细胞凋亡的研究

目的探讨缺血预处理（IPC）对减体积肝移植大鼠再灌注损伤早期细胞凋亡的影响。方法建立50%减体积大鼠肝移植模型,将72只成年雄性SD大鼠随机分为两组：对照组和IPC组,检测术后2、6、24 h血清丙氨酸转氨酶（ALT）水平变化及组织病理学变化;TUNEL法检测术后24 h肝细胞凋亡指数;免疫组织化学检测bcl-2和caspase-3蛋白表达。结果与对照组比较,IPC组术后6、24 h ALT水平下降,术后24 h肝损伤减轻,肝细胞凋亡指数、caspase-3表达均下降,而抗凋亡蛋白bcl-2表达增加（P均〈0.01）。结论 IPC明显减轻减体积肝移植术后再灌注损伤,其机制与通过上调bcl-2、下调caspase-3蛋白表达,从而抑制肝脏细胞凋亡相关。     

Long-term physical exercise induces changes in sirtuin 1 pathway and oxidative parameters in adult rat tissues

The protein deacetylase, sirtuin 1, is suggested as a master regulator of exercise-induced beneficial effects. Sirtuin 1 modulates mitochondrial biogenesis, primarily via its ability to deacetylate and activate proliferator-activated receptor-γ coactivator-1α (PGC-1α), interacting with AMPK kinase. Redox cell status can also influence this regulatory axis and together they form an important convergence point in hormesis during the aging process. Here, we tested whether treadmill training (36 weeks), as a paradigm of long-term moderate exercise, modifies the AMPK–sirtuin 1–PGC-1α axis and redox balance in rat gastrocnemius muscle, liver and heart. Physical activity induced increases in sirtuin 1 protein levels in all the aged rat tissues studied, as well as total PGC-1α levels. However, no changes in AMPK activation or significant differences in mitochondrial biogenesis (by measuring electron transport chain protein content) were found after exercise training. Parallel to these changes, we observed an improvement of oxidative stress defenses, mainly in muscle, with modification of the antioxidant enzyme machinery resulting in a reduction in lipid peroxidation and protein carbonylation. Thus, we demonstrate that moderate long-term exercise promotes tissue adaptations, increasing muscle, liver and heart sirtuin 1 protein content and activity and increasing PGC-1α protein expression. However, AMPK activation or mitochondrial biogenesis is not modified, but it cannot be discarded that its participation in the adaptive mechanism which prevents the development of the deleterious effects of age.     

内质网应激在肝脏疾病中的作用研究进展

内质网是细胞内蛋白质、脂类和糖类的重要合成基地,是细胞内钙离子的储存场所,与物质运输、物质交换、解毒作用密切相关。内质网应激是细胞的一种重要自我防御机制,能提高细胞对环境变化的适应能力,但过强过久的内质网应激则会引起不可逆的细胞损伤甚至凋亡。研究发现,内质网应激与多器官多种疾病相关。肝细胞中含有大量的内质网,对内质网应激更为敏感,许多肝脏疾病如肝细胞癌、药物性肝损伤、非酒精性脂肪性肝病、肝胰岛素抵抗等的发病机制均与内质网应激有关。本文就内质网应激对各种肝脏疾病的影响作一概述。     

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。     

Sirtuin 1 alleviates endoplasmic reticulum stress-mediated apoptosis of intestinal epithelial cells in ulcerative colitis

BACKGROUND Sirtuin 1(SIRT1) is a nicotinamide adenine dinucleotide(NAD~+)-dependent protein deacetylase that is involved in various diseases,including cancers,metabolic diseases,and inflammation-associated diseases.However,the role of SIRT1 in ulcerative colitis(UC) is still confusing.AIM To investigate the role of SIRT1 in intestinal epithelial cells(IECs) in UC and further explore the underlying mechanisms.METHODS We developed a coculture model using macrophages and Caco-2 cells.After treatment with the SIRT1 activator SRT1720 or inhibitor nicotinamide(NAM),the expression of occludin and zona occludens 1(ZO-1) was assessed by Western blot analysis.Annexin V-APC/7-AAD assays were performed to evaluate Caco-2 apoptosis.Dextran sodium sulfate(DSS)-induced colitis mice were exposed to SRT1720 or NAM for 7 d.Transferase-mediated dUTP nick-end labeling(TUNEL) assays were conducted to assess apoptosis in colon tissues.The expression levels of glucose-regulated protein 78(GRP78),CCAAT/enhancerbinding protein homologous protein(CHOP),caspase-12,caspase-9,and caspase-3 in Caco-2 cells and the colon tissues of treated mice were examined by quantitative real-time PCR and Western blot RESULTS SRT1720 treatment increased the protein levels of occludin and ZO-1 and inhibited Caco-2 apoptosis,whereas NAM administration caused the opposite effects.DSS-induced colitis mice treated with SRT1720 had a lower disease activity index(P 0.01),histological score(P 0.001),inflammatory cytokine levels(P 0.01),and apoptotic cell rate(P 0.01),while exposure to NAM caused the opposite effects.Moreover,SIRT1 activation reduced the expression levels of GRP78,CHOP,cleaved caspase-12,cleaved caspase-9,and cleaved caspase-3 in Caco-2 cells and the colon tissues of treated mice.CONCLUSION SIRT1 activation reduces apoptosis of IECs via the suppression of endoplasmic reticulum stress-mediated apoptosis-associated molecules CHOP and caspase-12 SIRT1 activation may be a potential therapeutic strategy for UC.     

内质网应激在肝脏疾病发病机制中的作用

内质网是细胞内蛋白质、脂类和糖类的重要合成基地，是细胞内钙离子的储存场所，与物质运输、物质交换、解毒作用密切相关。内质网应激是一种细胞的重要自我防御机制，持久强烈的内质网应激可引起细胞不可逆的损伤甚至凋亡。肝细胞中含有大量的内质网，许多肝脏疾病如病毒性肝炎、酒精性肝病、药物性肝炎、非酒精性脂肪肝等的发病机制均与内质网应激有关。本文从内质网应激的诱发因素、内质网应激反应的各个阶段及其对各种肝脏疾病的影响等方面作一综述。     

内质网应激与肝损伤研究进展

肝脏疾病组织学损伤,在细胞水平上可分为坏死和凋亡.近年来,大量研究表明,应激可能是肝损伤发生过程中的重要环节,如内质网应激、线粒体应激和氧化应激等,其中内质网应激是医学研究的一个新热点.本文将以内质网应激为切入点,探讨内质网应激的作用功能,内质网应激对细胞凋亡的影响,以及内质网应激与各种肝损伤之间的关系并对之进行综述.     

Hemodynamic and antifibrotic effects of losartan in rats with liver fibrosis and/or portal hypertension

BACKGROUND/AIMS: To assess the effects of the early and chronic administration of losartan--a specific angiotensin II receptor antagonist--in the prevention of hepatic fibrosis and portal hypertension. METHODS/RESULTS: (1) In CCl(4) rats, losartan at 5 and 10 mg/kg per day significantly decreased portal pressure (-11, -18%, respectively), splenorenal shunt blood flow (-60, -80%) and liver fibrosis (liver hydroxyproline and area of fibrosis) without significant changes in mortality and mean arterial pressure (MAP). (2) In bile duct ligated (BDL) rats, losartan at 5 mg/kg per day significantly decreased portal pressure (-14%), splenorenal shunt blood flow (-70%) and liver fibrosis. Losartan at 10 mg/kg per day significantly worsened liver and renal functions, mortality and liver fibrosis, without significant changes in portal pressure and splenorenal shunt blood flow. Losartan at 5 and 10 mg/kg per day significantly decreased MAP (-24, -30%). (3) In portal vein ligated (PVL) rats, losartan significantly decreased MAP (-12%) but did not change portal pressure or splenorenal shunt blood flow. CONCLUSIONS: In BDL and CCl(4) rats, losartan has beneficial effects on splanchnic hemodynamics and liver fibrosis. Losartan might decrease hepatic resistances in fibrotic liver. Losartan decreased MAP except in CCl(4) rats. Higher dosage of losartan had deleterious effects in BDL rats.     

Effect of cyclosporine on liver regeneration after orthotopic reduced-size hepatic transplantation in the rat

These experiments were undertaken to study the effects of cyclosporine A (CsA) on liver regeneration after an isogeneic orthotopic reduced-size hepatic transplantation (RSHT) in rats. Male Wistar rats were treated with or without a daily injection of CsA beginning 24 hr before surgery and were subjected to a 68% partial hepatectomy. A isogeneic orthotopic reduced-size hepatic transplantation was performed in recipient rats pretreated with or without CsA. A daily injection of CsA was continued until the recipient rats were sacrificed. Animals were sacrificed at various time points (12, 24, 36, 48, and 72 hr) postoperatively. The incorporation of bromodeoxyuridine (BrdU) into the DNA of the remnant hepatocytes was evaluated by immunohistochemical staining with a monoclonal antibody against BrdU. CsA (10 mg/kg/day) significantly augmented BrdU incorporation into hepatocytes after hepatectomy. The maximum labeling index (LI) was observed at 24 hr after hepatectomy. In contrast, the maximum LI in the recipient rats not receiving CsA was seen at 36 hr after RSHT, and 10 mg/kg/day of CsA decreased the LI at 36 hr after RSHT. A lower dose of CsA (3 mg/kg/day), however, significantly increased the LI in the recipient rats (P<0.01), and it reached a peak at 24 hr after RSHT when compared to the transplant recipients not receiving CsA. The time course of the increase in the LI in the transplant recipient rats receiving 3 mg/kg/day of CsA was similar to that observed in the rats after hepatectomy. This dosage improved the delay in the reduced-size hepatic transplant LI reaching its peak. These findings suggest that after RSHT the liver graft is more sensitive to both hepatotrophic and hepatotoxic effects of CsA.This work was supported in part by Mika Fund.     

Changes in circulating sirtuin 1 after bariatric surgery

Background and aimsObesity is associated with chronic inflammation and oxidative stress. Weight loss after bariatric surgery improves the inflammatory state and risk of cardiovascular disease. Improvement in metabolic dysfunction might be associated with changes in the activity of sirtuin 1 (SIRT1) and we aimed to investigate the effect of bariatric surgery on its circulating levels.Methods and resultsThis is a sub-study of a prospective cohort study, including 110 subjects with morbid obesity. The surgical procedure was either laparoscopic Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG). Blood was sampled at inclusion and six and 12 months after surgery. SIRT1 was measured in EDTA plasma with an enzyme-linked immunosorbent assay. The mean age in the population was 43 years, 80% were women and mean body mass index (BMI) was 38.8 kg/m². RYGB and SG were performed in 89 and 21 subjects, respectively. SIRT1 concentration was significantly reduced from baseline to six and 12 months after surgery, with mean values (SD) 156.8 (82.6), 119.5 (65.6) and 94.9 (45.6) ng/mL, respectively, (p ≤ 0.002, all), accompanied by significant reductions in C-reactive protein (CRP), BMI and triglycerides from inclusion (p < 0.001, all). Type of surgery did not differently modify SIRT1 levels (p = 0.09). CRP and triglycerides were both positively predictive of SIRT1 levels (p ≤ 0.001, both).ConclusionSIRT1 concentration was significantly lower six and 12 months after bariatric surgery. CRP and triglycerides independently predicted SIRT1 levels, suggesting that reduction in SIRT1 levels might not intrinsically be related to weight reduction, but to improvement in metaflammation.     

Optimized postconditioning algorithm protects liver graft after liver transplantation in rats

Background: Ischemia reperfusion injury(IRI) causes postoperative complications and influences the outcome of the patients undergoing liver surgery and transplantation. Postconditioning(Post C) is a known manual conditioning to decrease the hepatic IRI. Here we aimed to optimize the applicable Post C protocols and investigate the potential protective mechanism.Methods: Thirty Sprague–Dawley rats were randomly divided into 3 groups: the sham group(n = 5),standard orthotopic liver transplantation group(OLT, n = 5), Post C group(OLT followed by clamping and re-opening the portal vein for different time intervals, n = 20). Post C group was then subdivided into 4 groups according to the different time intervals:(10 s × 3, 10 s × 6, 30 s × 3, 60 s × 3, n = 5 in each subgroup). Liver function, histopathology, malondialdehyde(MDA), myeloperoxidase(MPO), expressions of p-Akt and endoplasmic reticulum stress(ERS) related genes were evaluated.Results: Compared to the OLT group, the grafts subjected to Post C algorithm(without significant prolonging the total ischemic time) especially with short stimulus and more cycles(10 s × 6) showed significant alleviation of morphological damage and graft function. Besides, the production of reactive oxidative agents(MDA) and neutrophil infiltration(MPO) were significantly depressed by Post C algorithm. Most of ERS related genes were down-regulated by Post C(10 s × 6), especially ATF4, Casp12, hspa4, ATF6 and ELF2, while p-Akt was up-regulated.Conclusions: Post C algorithm, especially 10 s × 6 algorithm, showed to be effective against rat liver graft IRI. These protective effects may be associated with its antioxidant, inhibition of ERS and activation of p-Akt expression of reperfusion injury salvage kinase pathway.     

Nuclear factor-κB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-κB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft.METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-κB decoy ODNs or scrambled ODNs. NF-κB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation,respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-κB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-α, IFN-γand intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-α and IFN-γ were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed.RESULTS: NF-κB activation in liver graft was induced in a time-dependent manner, and NF-κB remained activated for 16 h after graft reperfusion. NF-κB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-κB decoy ODNs significantly suppressed NF-κB activation as well as mRNA expression of TNF-α, IFN-γ and ICAM-1 in the liver graft. The hepatic NF-κB DNA binding activity [presented as integral optical density (IOD) value] in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16±0.78 vs 36.78±6.35 and 3.06±0.84 vs 47.62± 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P＜0.001).The hepatic mRNA expression level of TNF-α, IFN-y and ICAM-1 [presented as percent of β-actin mRNA(%)] in the NF-κBdecoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31 ±3.48 vs 46.37±10.65 and 7.46± 3.72 vs 74.82±12.25for hepatic TNF-α mRNA, 5.58±2.16 vs 50.46±9.35 and6.47±2.53 vs 69.72±13.41 for hepatic IFN-γ mRNA, 6.79±2.83 vs 46.23±8.74 and 5.28±2.46 vs 67.44±10.12for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P＜0.001). Administration of NF-κB decoy ODNs almost completely abolished the increase of serum level of TNF-α and IFN-γ induced by hepatic ischemia/reperfusion, the serum level (pg/mL)of TNF-α and IFN-γ in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7±13.6 vs 176.7±15.8 and 48.4±15.1 vs216.8±17.6 for TNF-α level, 31.5±12.1 vs 102.1±14.5and 40.2±13.5 vs 118.6±16.7 for IFN-γ level after 4 and8 h of reperfusion, respectively, P＜0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-κB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17±0.07 vs 1.12±0.25 and 0.46±0.17 vs 1.46±0.32 for hepatic MPO content, 71.7±33.2 vs 286.1±49.6 and 84.3±39.7 vs467.8±62.3 for ALT level after 4 and 8 h of reperfusion,respectively, P＜ 0.001).CONCLUSION: The data suggest that NF-κB decoy ODNs protects against I/R injury in liver graft by suppressing NF-κB activation and subsequent expression of proinflammatory mediators.     

内质网及氧化应激在大鼠慢性肝损伤中的变化

目的研究内质网应激相关基因、氧化应激指标在四氯化碳诱导的大鼠慢性肝损伤中的变化。方法四氯化碳制备大鼠慢性肝损伤模型,通过测定大鼠血清ALT、AST水平,采用HE染色和TUNEL法观察肝组织病理形态和肝细胞凋亡改变,评价成模效果。检测大鼠肝脏葡萄糖调节蛋白78（GRP78）、GRP94、血红素加氧酶（HO）-1 mRNA表达及超氧化物歧化酶（SOD）、丙二醛（MDA）、还原型谷胱甘肽（GSH）变化。结果四氯化碳成功诱导了大鼠慢性肝损伤,与对照组相比,大鼠肝脏GRP78、GRP94、HO—1的表达量、MDA含量均明显增加,SOD活性、还原型GSH显著降低。结论四氯化碳诱导大鼠慢性肝损伤时,内质网应激相关基因的表达增加,氧化应激指标亦发生改变,表明内质网应激、氧化应激共同参与了大鼠慢性肝损伤过程。     

内质网应激与肝脏疾病研究进展

内质网应激是一种重要的细胞自我防御机制,内质网应激时,首先启动生存途径,但是持续的内质网应激将启动细胞凋亡途径.肝细胞内有大量的内质网,许多肝脏疾病均与内质网应激及其介导的细胞凋亡有关,如病毒性肝炎、酒精性肝病、非酒精性脂肪性肝病、药物性肝病、急性肝衰竭、肝癌等,针对内质网应激途径来进行凋亡保护或促进凋亡以寻找新靶点药物来治疗肝脏疾病在理论上成为可能.本文将从内质网应激反应的生存途径、凋亡途径,内质网应激及其介导的细胞凋亡在肝脏疾病发病机制中的作用以及在肝病干预治疗上的意义等方面进行综述.     

Technique for orthotopic reduced-size hepatic transplantation combined withex vivo liver cut down in the rat

A technique is described for orthotopic reduced-size hepatic transplantation combined withex vivo liver cut down in the rat. Following perfusion of the donor liver with cold heparinized saline, the portal veins, bile ducts, and hepatic arteries to the median and left lobes together were dissectedin situ, encircled, and divided. After harvesting the donor liver, a hepatectomy was performed byex vivo liver cut down of the median and left lobes. The remnant amounted to 32% of the whole liver. As a result, the suprahepatic vena cava could be well visualized with adequate exposure for vascular anastomosis. Orthotopic reduced-size hepatic transplantation was performed using the right and caudate lobes of the liver. The suprahepatic vena cava was anastomosed with a 7-0 silk running suture. A simplified cuff without processes was made with an obliquely cut polyethylene tube and used for the portal and infrahepatic caval anastomoses. A Teflon tube stent was used for the biliary anastomosis. The newly devised angled clamp and flexible arm were used for the cuff attachment and operative procedure. Transplant survival followingex vivo liver cut down was as good as that with whole liver transplantation. Reestablishment of the hepatic artery restores liver function following transplantation. The maximum hepatocyte labeling index (LI) occurs 24 hr after a 68% hepatectomy, and at 36 hr following a reduced-size hepatic transplantation with or without hepatic arterialization. Possible explanations for the slight delay in achieving the maximal LI may include damage that is induced by the operation itself, pregraft preservation, and reperfusion injuries. In conclusion, the anatomical features of the hepatic lobes in rats are well suited to successful completion ofex vivo liver cut down.This work was supported in part by the Mika Memorial Fund for Medical Research at Kumamoto University Medical School.     

Nuclear factor-kappaB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). CONCLUSION: The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.     

血管紧张素1-7与糖尿病及糖尿病肾脏内质网应激关系的研究

目的 调查糖尿病患者血管紧张素1-7(Ang1-7)水平和评价Ang1-7对糖尿病大鼠肾脏内质网应激的影响.方法 选取2014年12月至2015年6月于天津市海滨人民医院未诊断为糖尿病的95名体检人员为研究对象,所有受试者均进行口服葡萄糖耐量试验,根据血糖水平分为健康组(n=31)、血糖异常组(n=32)和糖尿病组(n=32).检测其体重、腰围、血压、肝功能、肾功能、Ang1-7、血管紧张素转换酶(ACE)2等指标.分析血糖与Ang1-7、ACE2的相关性.另外,选取10只150～200 g健康雄性Sprague-Dawley (SD)大鼠,通过腹腔注射链脲佐菌素制备糖尿病模型,全部成功,根据随机数字法,分为糖尿病组和Ang1-7组,每组5只,分别皮下泵人生理盐水和Ang1-7,另选取5只健康SD大鼠作为正常组.干预2周后处死所有大鼠,留取肾组织标本,Western印迹和实时荧光定量PCR检测葡萄糖调节蛋白78(GRP78)、转录活性因子4(ATF4)、增强子结合蛋白同源蛋白(CHOP)水平.结果 与健康组相比,血糖异常组和糖尿病组甘油三酯、ACE2、Ang1-7水平均升高;糖尿病组较健康组升高更明显(F=5.57,6.12,5.44,P均＜0.01).Pearson相关分析显示,糖尿病组、血糖异常组的空腹血糖、餐后血糖均与Ang1-7呈正相关(r=0.69,0.45,P均＜0.01;r =0.54,0.52,P均＜0.01).与正常组相比,糖尿病组大鼠肾脏GRP78、ATF4和CHOP水平明显升高;与糖尿病组相比,Ang1-7组GRP78、ATF4和CHOP水平明显下降(t=20.3,25.7,19.6,P均＜0.05).与同期正常组比较,糖尿病组大鼠肾组织GRP78、ATF4和CHOP的mRNA均显著上调.与糖尿病组比较,Ang1-7组大鼠肾组织GRP78、ATF4和CHOP的mRNA的表达受到显著抑制(t=15.1,11.6,12.3,P均＜0.05).结论 Ang1-7水平随着受试者血糖浓度的升高而升高,另外,Ang1-7可降低糖尿病大鼠肾脏内质网应激蛋白水平.