Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM: To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. METHODS: Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad-HO-1) or enhanced green fluorescent protein gene (Ad-EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS: After seven days culture, the viability of cultured rat islets decreased significantly (92% +/- 6% vs 52% +/- 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 +/- 0.55 mIU/L/30IEQ vs 4.57 +/- 0.40 mIU/L/30IEQ, 14.93 +/- 1.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% +/- 15% vs 52% +/- 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 +/- 2.17 mIU/L/30IEQ vs 8.87 +/- 0.65 mIU/L/30IEQ; 12.50 +/- 2.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 +/- 0.02 vs 2.08 +/- 0.05; 2.21 +/- 0.02 vs 2.11 +/- 0.03, P < 0.05). CONCLUSION: The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.     

Antioxidant role of heme oxygenase-1 in prehepatic portal hypertensive rats

AIM: To study the effect of bilirubin on the oxidative liver status and the activity and expression of heme oxy-genase-1 (HO-1) in rat liver injury induced by prehepatic portal hypertension. METHODS: Wistar male rats, weighing 200-250 g, were divided at random into two groups: one group with prehepatic portal hypertension (PH) induced by regulated prehepatic portal vein ligation (PPVL) and the other group corresponded to sham operated rats. Portal pressure, oxidative stress parameters, antioxidant enzymes, HO-1 activity and expression and hepatic sinusoidal va-sodilatation were measured. RESULTS: In PPVL rats oxidative stress was evidenced by a marked increase in thiobarbituric acid reactive substances (TBARS) content and a decrease in reduced glutathione (GSH) levels. The activities of liver antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were also diminished while activity and expression of HO-1 were enhanced. Administration of bilirubin (5μmol/kg body weight) 24 h before the end of the experiment entirely prevented all these effects. Pretreatment with Sn-pro-toporphyrin IX (Sn-PPIX) (100μg/kg body weight, i.p.), a potent inhibitor of HO, completely abolished the oxidative stress and provoked a slight decrease in liver GSH levels as well as an increase in lipid peroxidation. Besides, carbon monoxide, another heme catabolic product, induced a significant increase in sinusoidal hepatic areas in PPVL group. Pretreatment of PPVL rats with Sn-PPIX totally prevented this effect. CONCLUSION: These results suggest a beneficial role of HO-1 overexpression in prehepatic portal hypertensive rats.     

HO-1在CCK-8减轻脂多糖所致的急性肺损伤中的作用

目的： 探讨血红素氧合酶（HO）-1在八肽胆囊收缩素（CCK-8）减轻脂多糖（LPS）所致急性肺损伤（ALI）中的作用。方法: 将大鼠随机分为5组：正常对照组、LPS组、CCK-8+LPS组、LPS+Hm（氯血红素，CO供体）组、LPS+ZnPP（锌原卟啉，HO-1特异性阻断剂）组。各组给药后2 h、6 h、12 h行支气管肺泡灌洗、检测支气管肺泡灌洗液（BALF）中中性粒细胞（PMN）数目；进行肺组织的形态学观察；测定肺组织中丙二醛（MDA）含量和HO-1蛋白活性；应用RT-PCR和Western blotting技术检测给药后6h肺组织中HO-1 mRNA和蛋白的表达情况。结果: LPS组肺组织出现损伤性变化，同时BALF中PMN数目、肺组织中MDA含量、HO-1蛋白活性、HO-1 mRNA和蛋白的表达均高于相应对照组（均P<0.05）；CCK-8+LPS和LPS+Hm组肺组织损伤程度、BALF中PMN数目和肺组织中MDA含量低于相应LPS组，而肺组织中HO-1蛋白活性、HO-1 mRNA和蛋白的表达均高于相应LPS组（均P<0.05）；LPS+ZnPP组肺组织损伤程度、BALF中PMN数目和肺组织中MDA含量分别高于相应LPS组，而肺组织中HO-1蛋白活性、HO-1 mRNA和蛋白的表达分别低于相应LPS组（均P<0.05）。结论: CCK-8可部分通过HO-1介导的抗氧化、抑制PMN聚集等效应来发挥减轻LPS所致的肺损伤作用。     

慢性阻塞性肺疾病患者血红素加氧酶-1基因启动子多态性对其基因表达的影响

目的:探讨COPD患者血红素加氧酶-1(heme oxygenase-1,HO-1)基因启动子多态性对HO-1基因表达水平的影响.方法:利用PCR、免疫组织化学及原位杂交等技术,在50例肺癌合并COPD组(COPD组)与30例单纯肺癌组(对照组)的肺组织中,检测HO-1基因启动子5'端(GT)n重复序列不同基因型的频率分布特征,及分析其与HO-1基因信使核糖核酸(messenger ribonucleic acid,mRNA)表达及HO-1蛋白表达的关系.结果:①COPD组L/M基因型频率显著高于对照组,而S/S基因型频率显著低于对照组(均为P＜0.05),其它基因型频率比较差异无统计学意义(均为P＞0.05);②HO-1基因启动子(GT)n不同重复序列含有L等位基因样本(Ⅰ组)其HO-1 mRNA和蛋白表达水平显著低于不含有L等位基因的Ⅱ组(均为P＜0.01).结论:HO-1基因启动子5'端(GT)n大量重复序列可能降低了HO-1基因的表达水平,而与COPD的易感性有关.     

血脂异常者血清MCP-1、HO-1、APN和TNF-α水平的检测及临床价值

目的探讨血脂异常者血清单核细胞趋化蛋白-l(MCP-1)、血红素加氧酶-1(HO-1)、脂联素(APN)及肿瘤坏死因子α(TNF-α)水平的检测及临床价值。方法采用酶联免疫吸附法(ELISA)检测85例血脂异常者和35例健康对照者血清MCP-1、HO-1、APN及TNF-α的水平,同时检测血脂异常者血清其他脂类代谢指标[总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)及高密度脂蛋白胆固醇(HDL-C)]的水平,并分析MCP-1、HO-1、APN及TNF-α与脂类代谢指标的相关性。结果血脂异常者血清MCP-1、HO-1和APN水平较健康对照组均显著升高,差异具有统计学意义(P0.01),而TNF-α水平较健康对照组则明显降低,差异具有统计学意义(P0.01);同时血脂异常者血清HO-1水平与MCP-1及LDL-C水平均呈正相关关系;APN水平则与TNF-α呈负相关关系,与LDL-C水平呈正相关关系;而健康对照组的HO-1水平与MCP-1水平无明显关系。结论血脂异常者血清MCP-1、HO-1和APN水平显著升高,而TNF-α水平则明显降低,说明MCP-1、HO-1、APN及TNF-α可能参与了动脉粥样硬化的发生及发展,并可能成为动脉粥样硬化潜在的预防或治疗靶点。     

晚期糖基化终产物对健康成人外周血单核细胞肿瘤坏死因子α和血红素加氧酶-1表达的影响

目的:探讨不同浓度晚期糖基化终产物(advanced glycation end-product,AGE)对健康成人外周血单核细胞肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及血红素加氧酶-1(heme oxygenase-1,HO-1)表达的影响。方法:分离健康成人外周血单核细胞,随机分组,利用不同浓度体外制备的AGE修饰牛血清白蛋白(AGE-BSA)刺激,观察不同时间点每组细胞培养上清液中TNF-α表达量及细胞内HO-1mRNA的表达。结果:AGE-BSA可增加健康成人外周血单核细胞TNF-α的释放并可诱导健康人外周血单核细胞内HO-1mRNA的表达。结论:AGE可上调健康人外周血单核细胞TNF-α和细胞内HO-1mRNA的表达。     

阿司匹林预处理对原代培养大鼠Ⅱ型肺泡上皮细胞抗氧化损伤的影响研究

目的 观察阿司匹林对原代培养大鼠Ⅱ型肺泡上皮细胞(AEC Ⅱ)的保护效应,并探讨其抗氧化损伤的机制.方法 将原代分离、纯化、培养的离体大鼠AEC Ⅱ分为5组.过氧化氢损伤(H2O2)组在培养40 h后加入0.5 mmol/L H2O2,建立细胞氧化损伤模型;生理盐水(NS)组则加入NS ;阿司匹林预处理1、2、3(A1～3)组在加入H2O2前给予阿司匹林50、100、200μmol/L预处理.3 h后观察细胞形态学变化和贴壁细胞计数;采用四甲基偶氮唑盐(MTT)比色法检测细胞存活率;采用免疫组化法和聚合酶链反应法检测NS组、A1～3组在培养20、40、60 h AEC Ⅱ中血红素氧合酶-1(HO-1)的蛋白及mRNA表达.结果 采用胰蛋白酶消化、免疫黏附法每只鼠可收获(2.0～2.5)×107个AEC Ⅱ,纯度和活性均＞90%.与NS组比较,H2O2组细胞间隙增宽,贴壁细胞数减少,细胞皱缩,细胞存活率(A值)明显下降(0.054 6±0.004 0比0.103 8±0.009 9,P＜0.01);与H2O2组比较,A1～3组贴壁细胞数增多,细胞形态较完整,无明显皱缩,细胞存活率(A值)明显增加(0.066 9±0.003 9、0.071 0±0.006 5、0.078 7±0.009 2比0.054 6±0.004 0,均P＜0.01).与NS组比较,A1～3组培养20、40、60 h时HO-1的蛋白及mRNA表达均明显增加,60 h时达峰值[蛋白(积分A值):1.59±0.12、1.60±0.09、1.61±0.08比1.25±0.11;mRNA(Ct值比值):24.31±1.74、30.45±2.53、32.63±3.74比22.99±1.95,均P＜0.05];但A1～3组间HO-1蛋白表达无明显差异.结论 阿司匹林通过上调HO-1表达对离体培养氧化损伤的大鼠AEC Ⅱ起保护效应;HO-1可能是其中重要的保护调节因子.

Abstract：

Objective To investigate the protective effect of aspirin on primary cultured type Ⅱ alveolar epithelial cell (AEC Ⅱ ), and the mechanism of its effect on anti-oxidation damage. Methods The original generation of adult rat AEC Ⅲ were cultured and purified. They were divided into normal saline (NS) group,hydrogen peroxide injury group (H2O2 group), and 1, 2, 3 aspirin pretreatment groups (Al -3 groups). In H2O2 group, 0. 5 mmol/L H2O2 was added to AEC Ⅱ after 40 hours of culture to reproduce a cell oxidative injury model. In NS group, only NS was added to AEC Ⅱ culture. To the A1 - 3 groups aspirin 50, 100 and 200 μmol/L were added respectively. Cell form, cell count and cell survival rate were observed at 3 hours after H2O2 was given. Immunohistochemical and polymerase chain reaction (PCR) methods were used for the determination of heme oxygenase-1 (HO-1) protein and HO-1 mRNA (20, 40, 60 hours of culture). Results With trypsin digestion and immune adherence method AEC Ⅱ could be harvested (2.0- 2. 5)× 107, and the purity and activity were both over 90%. Compared with NS group, gaps between cells were widened in H2O2 group, cell account was reduced, and the survival rate (A value) was reduced significantly (0. 054 6±0. 004 0 vs. 0. 103 8±0. 009 9, P＜0. 01). Compared with H2O2 group, in A1 - 3 groups the number of adherent cells was increased, cell morphology was intact, and no obvious cell shrinkage was found. Higher survival rate (A value) was found in A1 - 3 groups than that of H2O2 group (0. 066 9±0. 003 9, 0. 071 0±0. 006 5,0. 078 7±0. 009 2 vs. 0. 054 6±0. 004 0, all P＜0. 01). Compared with NS group, HO-1 protein and HO-1 mRNA expression in AEC Ⅱ after 20, 40 and 60 hours of culture reached peak level at 60 hours, and they were increased significantly in A1 - 3 groups [protein (A value) : 1.59±0. 12, 1.60±0. 09, 1.61±0. 08 vs.1.25±0. 11; mRNA (the ratio of Ct value: 24.31±1.74, 30. 45±2. 53, 32. 63±3. 74 vs. 22.99±1.95, all P＜0. 05]. There was no significant difference in HO-1 protein expression among A1 - 3 groups. Conclusion There are significant protective effects of aspirin against anti-oxidative damage in cultured AEC Ⅱ cell.As expression of HO-1 is increased in aspirin groups, it may be considered as a protective factor agninst anti-oxidative damage in AEC Ⅱ cell culture.     

缬沙坦对高血压病患者血清过氧化物酶体增生物激活受体-γ及血红素氧合酶-1水平的影响及其肾脏保护作用

目的观察缬沙坦对高血压病患者血清过氧化物酶体增生物激活受体-γ（PPAR-γ）、血红素氧合酶-1（HO-1）水平的影响及其肾脏保护作用关系。方法将35～65岁高血压病患者60例随机分为缬沙坦组（80～160 mg/d）30例和常规治疗组30例,接受16周的治疗。治疗期间监测血压,分别于治疗前和治疗16周后采用Elisa方法检测血清PPAR-γ、HO-1水平;并检测尿β2-微球蛋白和尿微量蛋白含量。结果缬沙坦治疗可显著降低高血压病患者血压水平（P〈0.01）,升高血清HO-1和PPAR-γ水平（P〈0.01）,降低尿β2-微球蛋白和尿微量蛋白含量（P〈0.01）。结论缬沙坦在血压治疗达标的同时可显著降低尿蛋白,作用机制推测可能与其炎症调控作用有关。