血管钙化时血管血红素加氧酶/一氧化碳系统的变化

目的:观察血管钙化时血管血红素加氧酶(HO)-一氧化碳(CO)-环磷酸鸟苷(cGMP)系统的改变,以探讨血管钙化发病的细胞分子机理。方法:利用维生素D3(VitD3)和尼古丁(nicotine)复制大鼠血管钙化模型,检测HO-1mRNA的相对含量,HO-1免疫组织化学染色,测定主动脉HO-1活性、碳氧血红蛋白(HbCO)的生成及血管cGMP含量。结果:用竞争性定量RT-PCR的方法发现,钙化动物血管组织的HO-1mRNA水平较对照组低34.9%(P＜0.05);免疫组织化学方法观察发现钙化血管的HO-1蛋白表达减少,仅在内膜略有表达,中膜无明显表达;HO-1活性低60.6%(P＜0.01);CO生成少53.9%(P＜0.01),cGMP的含量低77.1%(P＜0.01)。结论:钙化血管血红素-HO-CO-cGMP途径功能下调。     

内毒素攻击大鼠肺泡巨噬细胞时HO-1对高尔基体应激的影响

目的探讨内毒素诱导大鼠肺泡巨噬细胞损伤时血红素加氧酶1（HO-1）对高尔基体应激的影响。方法体外培养大鼠肺泡巨噬细胞,采用脂多糖（LPS）诱导大鼠肺泡巨噬细胞建立细胞损伤模型。使用CCK-8法检测细胞活力;使用DCFH-DA探针检测细胞内活性氧簇（ROS）的生成;使用生物化学方法检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平;使用TUNEL染色和凋亡相关蛋白caspase-3/7活性检测试剂盒检测细胞凋亡;使用RT-qPCR和Westernblot法检测HO-1和高尔基体磷蛋白3（GOLPH3）的表达;使用Westernblot法检测高尔基体结构相关蛋白GM130、golgin-97和mannosidaseII的表达。使用小干扰RNA（siRNA）沉默HO-1后,重复以上检测。结果LPS刺激肺泡巨噬细胞下调细胞活力、SOD活性及GM130、golgin-97和mannosidaseII表达水平,上调ROS和MDA含量及HO-1和GOLPH3表达水平,并导致TUNEL标记阳性细胞数增多,caspase-3/7活性增强（P<0.05）;HO-1基因沉默后,细胞活力、SOD活性及GM130、golgin-97和mannosidaseII表达显著下降,ROS和MDA含量及GOLPH3表达显著上升,TUNEL标记阳性细胞数增多,caspase-3/-7活性显著增强（P<0.05）。结论内毒素诱导大鼠肺泡巨噬细胞损伤时,HO-1可减轻氧化应激和高尔基体应激反应,减少细胞凋亡。     

苦参总黄酮调控Nrf2/HO-1通路对肝纤维化大鼠的机制研究

目的探究苦参总黄酮（TF-SF）是否通过调控核因子E2相关因子2/血红素加氧酶1（Nrf2/HO-1）通路影响大鼠肝纤维化（HF）。方法将SD大鼠随机分为对照组、HF组、低剂量苦参总黄酮组、中剂量苦参总黄酮组、高剂量苦参总黄酮组及高剂量苦参总黄酮+全反式维甲酸（Nrf2抑制剂）组,每组10只;四氯化碳-花生油溶液灌胃饲养8周构建HF大鼠模型,于第9周开始低、中、高剂量苦参总黄酮组及高剂量苦参总黄酮+全反式维甲酸组大鼠分别给予相应等剂量药物进行灌胃,而对照组和HF组大鼠予以等体积生理盐水灌胃（每天1次）,为期4周,实验结束后观察各组大鼠一般情况。ELISA法检测血清超氧化物歧化酶（SOD）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）、天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、透明质酸（HA）、Ⅳ型胶原（Ⅳ-C）、层黏连蛋白（LN）和Ⅲ型前胶原肽（PIIIP）水平;HE及Masson染色观察大鼠肝脏组织病理学情况;Westernblot检测大鼠肝脏组织中Nrf2/HO-1通路蛋白表达水平;real-timePCR检测肝脏组织中Nrf2和HO-1mRNA表达水平。结果与对照组相比,HF组大鼠活动迟钝、体态瘦弱、食欲不振,肝脏质地发硬、边缘钝化、颜色暗淡且被膜缺乏;与HF组相比,低、中、高剂量苦参总黄酮组大鼠活动、饮食及肝脏形态均得到改善;与高剂量苦参总黄酮组相比,高剂量苦参总黄酮+全反式维甲酸组大鼠活动、饮食及肝脏不良状况加重。与对照组相比,HF组大鼠饮食量,饮水量,体重,血清SOD和GSH-Px水平,以及肝脏组织Nrf2和HO-1蛋白及mRNA水平均显著降低,而肝脏重量,血清MDA、AST、ALT、HA、Ⅳ-C、LN和PIIIP水平,以及HF分期和半定量评分均显著增加（P<0.05）;与HF组相比,低、中、高剂量苦参总黄酮组大鼠饮食量,饮水量,体重,血清SOD和GSH-Px水平,以及肝脏组织Nrf2和HO-1蛋白及mRNA水平均显著增加,而肝脏重量,血清MDA、AST、ALT、HA、Ⅳ-C、LN和PIIIP水平,以及HF分期和半定量评分均显著降低,均呈苦参总黄酮剂量依赖性（P<0.05）;全反式维甲酸可逆转苦参总黄酮对肝脏纤维化的缓解。结论苦参总黄酮通过激活Nrf2/HO-1通路起到抗大鼠HF的作用。     

Ferulic acid: Pharmacological and toxicological aspects

Ferulic acid (FA) belongs to the family of phenolic acids and is very abundant in fruits and vegetables. Over the past years, several studies have shown that FA acts as a potent antioxidant by scavenging free radicals and enhancing the cell stress response through the up-regulation of cytoprotective systems, e.g. heme oxygenase-1, heat shock protein 70, extracellular signal-regulated kinase 1/2 and the proto-oncogene Akt. Furthermore, FA was shown to inhibit the expression and/or activity of cytotoxic enzymes, including inducible nitric oxide synthase, caspases and cyclooxygenase-2. Based on this evidence, FA has been proposed as a potential treatment for many disorders including Alzheimer’s disease, cancer, cardiovascular diseases, diabetes mellitus and skin disease. However, despite the great abundance of preclinical research, only a few studies were carried out in humans, the majority of which used foods containing FA, and therefore the clinical efficacy of this mode of administration needs to be further documented. New efforts and resources are needed in clinical research for the complete evaluation of FA therapeutic potential in chronic diseases.     

Delayed remote preconditioning induces cardioprotection: role of heme oxygenase-1

Background

The role of heme oxygenase-1 (HO-1) in the cardioprotection induced by delayed remote ischemic preconditioning (DRIPC) has not been investigated. Therefore, this study was designed to investigate whether HO-1 is involved in DRIPC-mediated cardioprotection in an isolated perfused rat heart model.

Materials and methods

Isolated rat hearts were subjected to 30 min ischemia followed by 60 min reperfusion. DRIPC (four cycles 5-min occlusion and 5-min reflow at the unilateral hind limb once per day for 1, 2, or 3 d before heart isolation, abbreviated as D1RIPC, D2RIPC, or D3RIPC respectively). Infarct size, myocardial troponin levels, and heart function were measured. The protein and messenger RNA levels of HO-1 were determined.

Results

DRIPC facilitated postischemic cardiac functional recovery and decreased cardiac enzyme release. The infarct size-limiting effect of DRIPC was more pronounced in the D3RIPC group (10.22 ± 2.57%) than the D1RIPC group (22.34 ± 4.02%, P < 0.001) or the D2RIPC group (14.60 ± 3.13%, P = 0.034). These effects in the D1RIPC group could be blocked by Zinc Protoporphyrin IX (ZnPP) (an HO-1 specific inhibitor). DRIPC-mediated cardioprotection was associated with enhanced HO-1 protein expression (D1RIPC, 0.11 ± 0.03; versus 0.15 ± 0.06 in the D2RIPC group, P = 0.06; versus 0.20 ± 0.04 in the D3RIPC group, P = 0.04) and messenger RNA levels of HO-1 expression.

Conclusions

Our findings suggest that HO-1 is involved in the cardioprotection induced by DRIPC, and that increase in the number of preconditioning stimuli may enhance cardioprotective effects accompanied with increased HO-1 level.     

Sufentanil induces delayed myocardial preconditioning mediated by HO-1

Purpose. The objective of this study was to examine whether sufentanil also confers delayed cardioprotection and whether this effect is mediated through HO-1.
Methods. Male Sprague-Dawley rats received either delayed ischemic preconditioning （DIPC） or sufentanilinduced preconditioning （SPC; with 3 μg/kg, 15 μg/kg, 30 μg/kg, 60 μg/kg, or 120 μg/kg sufentanil） or an ischemic reperfusion（CON）. After 24 h, all animals were subjected to a 30 min coronary occlusion followed by a 2 h reperfusion. In the group treated with 120 μg/kg sufentanil, the selective HO-1 inhibitor Zinc protoporphyrin IX （Znpp IX） was administered. The infarct size （IS） was determined with 2,3,5-triphenyltetrazolium chloride staining. Western blotting analysis was used to examine HO-1 expression.
Results. The IS/AAR ratios in the animals treated with DIPC （0.33±0.07） or with SPC （0.44±0.08, 0.32±0.10, 0.32±0.06, and 0.28±0.07 for the groups treated with 15 μg/kg, 30 μg/kg, 60 t~g/kg, or 120 μg/kg sufentanil, respectively） were significantly reduced compared with control （CON） group （0.54±0.06; P〈0.05）. The ED50 of sufentanil was found to be 13.83 μg/kg according to the sigmoid equation. Znpp IX abolished the effect of the 120 μg/kg sufentanil treatment （the IS/ AAR values were 0.54±0.04 for the SPC±Znpp IX group and 0.28±0.07 for the group treated with 120μg/kg SPC; P〈0.05）. The 120 μg/kg SPC treatment increased the expression of HO-1 compared with the CON group（P〈0.05）, and this effect was prevented by Znpp IX （P〈0.05）.
Conclusion. These results indicate that sufentanil produces delayed cardioprotection in anaesthetized rats and HO-1 may be involved in it.     

Assessment of ultra-structure,viability and function of lipopolysaccharides-stimulated human dermal fibroblasts treated with chrysin and exosomes (in vitro study)

BackgroundLipopolysaccharides (LPS) stimulate production of inflammatory cytokines. Chrysin is flavonoid beneficial for treatment of inflammatory conditions. Bone marrow mesenchymal stem cell (BM-MSC) exosomes have regenerative ability in different tissues.ObjectiveTo assess potential role of chrysin and BM-MSC exosomes on ultra-structure, viability and function of human dermal fibroblasts-adult (HDFa) stimulated by LPS.MethodsHDFa cells were divided into: Group I: Cells received no treatment. Group II: Cells were stimulated with LPS. Group III: LPS stimulated cells were treated with chrysin. Group IV: LPS stimulated cells were treated with exosomes.ResultsAfter 48 h, ultrastructural examination of HDFa cells in Group I revealed intact plasma membrane and numerous cytoplasmic organelles. Group II displayed destructed plasma membrane and apoptotic bodies. Group III showed intact plasma membrane with loss of its integrity at some areas. Group IV demonstrated intact plasma membrane that showed fusion with exosomes at some areas. Statistical analysis of MTT represented highest mean value of cell viability% in Group IV followed by Groups III, I and II respectively. Statistical analysis of enzyme-linked immunosorbent assay (ELISA) showed the highest mean value of interleukin-1β (IL-1β) was in Group II followed by Groups III, IV and I, while highest mean values of interleukin-10 (IL-10), nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins were in Group I, followed by Groups IV, III and II respectively.ConclusionsLPS have harmful consequences on ultra-structure, viability and function of HDFa cells. BM-MSC exosomes have better regenerative action on inflamed fibroblasts in comparison to chrysin.     

Heme oxygenase protects hippocampal neurons from ethanol-induced neurotoxicity

Ethanol has deleterious effects on neuronal cells both in vivo and in vitro, but the mechanisms are unknown. Here, treatment with increasing doses of ethanol (from 20 up to 600mM) decreased the viability of a mouse hippocampal neuroblastoma cell line, HT22. The glutathione concentration decreased and intracellular reactive oxygen species (ROS) increased in a dose-and time-dependent manner, suggesting that the neurotoxicity was due to oxidative stress. Expression of heme oxygenase (HO)-1, a redox regulator and heat shock protein, increased with time after ethanol treatment, but HO-2 was expressed constitutively. The addition of 5microM zinc protoporphyrin IX (ZnPP IX), a competitive HO inhibitor, with the ethanol further reduced cell viability and increased intracellular ROS, but these effects were reversed by co-treatment with 50nM bilirubin, a well-known antioxidant and a product of HO catalysis. These results suggest that HO has a protective role in hippocampal neurons as an intrinsic factor against ethanol-induced oxidative stress and the protection depends on the degree of oxidative stress.