Gastrodin prevents homocysteine-induced human umbilical vein endothelial cells injury via PI3K/Akt/eNOS and Nrf2/ARE pathway

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine-induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3-kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200-800 μg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl-2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L-NAME but enhanced by Cav-1 knockdown, and MK-2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p-Akt, and eNOS and decreased Cav-1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine-induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.     

内皮细胞和平滑肌细胞氧化应激时Nrf2/ARE信号通路对抗氧化基因表达的调控:与动脉粥样硬化和先兆子痫的关系

动脉粥样硬化、糖尿病、慢性肾功能衰竭和先兆子痫等血管疾病时活性氧(reactive oxygen species,ROS)生成增加,容易导致内皮依赖性血管舒张功能的损害和血管损伤,而细胞可以诱导多种编码Ⅱ相解毒酶和抗氧化蛋白的基因表达,从而减轻ROS和亲电子物质介导的细胞损伤。一个被称为抗氧化反应元件(antioxidant response element,ARE)或亲电子反应元件(electrophile response element,EpRE)的顺式转录调控元件,可以介导诸如亚铁血红素加氧酶1、γ-谷氨酰半胱氨酸合成酶、硫氧还蛋白还原酶、谷胱甘肽-S转移酶和NAD(P)H:苯醌氧化还原酶等基因的转录。其他抗氧化酶,如超氧化物歧化酶、过氧化氢酶和非酶清除剂(如谷胱甘肽)等也参与ROS的清除。转录因子NF-E2相关因子2(nuclear factor-erythroid 2-related factor 2, Nrf2)是属于Cap‘n’Collar家族的转录因子,具有碱性亮氨酸拉链(basic region-leucine zipper,bZIP),它在ARE介导的抗氧化基因表达中起重要的作用。在正常情况下,Kelch样环氧氯丙烷相关蛋白-1(Kelch-like ECH-associated protein-1,Keapl)与Nrf2耦联,并与肌动蛋白细胞骨架结合被锚定于胞浆,但是在半胱氨酸残基发生氧化的情况下,Nrf2和Keapl解耦联,进入细胞核并与ARE结合,从而激活多种抗氧化基因和Ⅱ相解毒酶基因的转录。蛋白激酶C、丝裂原活化蛋白激酶和磷脂酰肌醇-3激酶参与Nrf2／ARE信号转导的调控。本文综述了有关Nrf2／ARE信号转导通路在血管稳态和动脉硬化、先兆子痫等疾病情况下内皮及平滑肌细胞对抗持续性氧化应激中起的作用。     

Pterostilbene protects against UVB-induced photo-damage through a phosphatidylinositol-3-kinase-dependent Nrf2/ARE pathway in human keratinocytes

Objective: Ultraviolet B (UVB) irradiation is the initial etiological factor for various skin disorders, including erythema, sunburn, photoaging, and photocarcinogenesis. Pterostilbene (Pter) displayed remarkable antioxidant, anti-inflammatory, and anticarcinogenic activities. This study aimed to investigate the effective mechanism of Pter against UVB-induced photodamage in immortalized human keratinocytes.

Methods: Human keratinocytes were pretreated with Pter (5 and 10?μM) for 24?h prior to UVB irradiation (300?mJ/cm²). Harvested cells were analyzed by MTT, DCFH-DA, comet, western blotting, luciferase promoter, small interference RNA transfection, and quantitative real-time polymerase chain reaction assay.

Results: Pter significantly attenuated UVB-induced cell death and reactive oxygen species (ROS) generation, and effectively increased nuclear translocation of NF-E2-related factor-2 (Nrf2), expression of Nrf2-dependent antioxidant enzymes, and DNA repair activity. Moreover, the protective effects of Pter were abolished by small interference RNA-mediated Nrf2 silencing. Furthermore, Pter was also found to induce the phosphorylation of Nrf2 and the known phosphatidylinositol-3-kinase (PI3K) phosphorylated kinase, Akt. The specific inhibitor of PI3K, LY294002, successfully abrogated Pter-induced Nrf2 phosphorylation, activation of Nrf2-antioxidant response element pathway, ROS scavenging ability, and DNA repair activity.

Conclusion: The present study indicated that Pter effectively protected against UVB-induced photodamage by increasing endogenous defense mechanisms, scavenging UVB-induced ROS, and aiding in damaged DNA repair through a PI3K-dependent activation of Nrf2/ARE pathway.     

Adrenomedullin protects rat cerebral endothelial cells from oxidant damage in vitro

Increased permeability and reduced cerebral endothelial cell (CEC) viability induced by oxidative stress are the hallmarks of the blood-brain barrier disruption. In our experiments hydrogen peroxide (H2O2, 0.5 mM) induced a continuous decrease of the transendothelial electrical resistance (TEER) and resulted in intercellular gap formations in cultured rat CECs. Adrenomedullin (AM) increased TEER, enhanced peripheral localization of F-actin bands and attenuated the increased permeability induced by H2O2. Furthermore, AM treatment preserved mitochondrial membrane potential, attenuated cytochrome c release, and consequently improved CEC viability in H2O2 treated cultures. These results suggest that AM treatment protects CECs against oxidative injury.     

Nrf2/ARE通路介导右美托咪定减轻肢体缺血/再灌注损伤中的作用

目的：观察Nrf2/ARE通路在右美托咪定（DEX）预处理减轻大鼠肢体缺血/再灌注损伤中的作用。方法：28只成年雄性SD大鼠随机分为4组（n=7）：假手术组（Sham组）、缺血再灌注组（I/R组）、I/R+右美托咪定预处理组（DEX组）、I/R+DEX+阿替美唑组（Atip组）。Atip组在麻醉后腹腔一次性给予Atip （250 μg/kg）和DEX （25 μg/kg）,Sham组和I/R组在麻醉后腹腔给予相应体积生理盐水,DEX组给予相应体积DEX和生理盐水,30 min后单侧股部切口,无创动脉夹夹闭股动脉,侧支循环用橡皮筋以恒定张力结扎,缺血3 h后去除动脉夹及橡皮筋,开放2 h后,取大鼠血清测乳酸脱氢酶（LDH）、肌酸激酶（CK）;取部分腓肠肌,测量丙二醛（MDA）、超氧化物歧化酶（SOD）以及Western blot检测胞核核因子E2相关因子2（Nrf2）、胞浆HO-1蛋白;免疫组化检测胞核Nrf2、胞浆HO-1蛋白和光镜观察骨骼肌形态;同时切取少量腓肠肌进行湿干比检测。结果：与Sham组相比,I/R组湿干比、MDA、LDH、CK、Nrf2、HO-1蛋白表达明显升高（P<0.05）,SOD活性显著降低（P<0.05）;与I/R组相比,DEX组湿干比、MDA、LDH、CK明显降低（P<0.05）,SOD、Nrf2、HO-1蛋白表达显著增多（P<0.05）;与DEX组相比,Atip恰能扭转DEX的这种作用,Atip组各指标与DEX组有显著差异（P<0.05）。结论：Nrf2蛋白存在于大鼠的骨骼肌中并且DEX可以通过α2受体上调核内Nrf2水平,使Nrf2下游的HO-1保护蛋白增多,起到抗氧化的作用。     

Maslinic acid protects vascular smooth muscle cells from oxidative stress through Akt/Nrf2/HO-1 pathway

Maslinic acid (MA) is a natural triterpenoid widely distributed in edible and medicinal plants and has been demonstrated to possess bioactivity. However, its effect on vascular smooth muscle cells (VSMC) has not been explored yet. In this study, we found that heme oxygenase-1 (HO-1) expression was increased in VSMCs treated with MA. Furthermore, MA was found to induce Akt activation in a dose- and time-dependent manner. Wortmannin suppression of Akt was able to abolish HO-1 upregulation in VSMCs, suggesting the requirement of Akt activation for MA effect on HO-1. Further investigation indicated that Akt activation resulted in the elevated expression of Nrf2, a HO-1 promoter, in MA-treated VMSCs. Finally, we found that MA was able to protect VSMCs from oxidative stress induced by H₂O₂. Blocking the activation of Akt/Nrf2/HO-1 was able to compromise the protective effect of MA on VSMCs. Collectively, we provided evidence that MA protected VMSCs from oxidative stress through Akt/Nrf2/HO-1 pathway.     

Corynoline protects against zearalenone-induced liver injury by activating the SIRT1/Nrf2 signaling pathway

Corynoline has been reported to have anti-inflammatory and antioxidative effects. In the present study, the potential protective effects of corynoline against zearalenone (ZEA)-induced liver injury were investigated. ZEA was administered daily for 5 days. Then, liver tissues were used for subsequent experiments. Corynoline attenuated liver histopathological changes induced by ZEA. The production of tumor necrosis factor-α and interleukin-1β in liver tissues, as well as aspartate aminotransferase and alanine aminotransferase in serum, was also inhibited by corynoline. Meanwhile, ZEA-induced MPO activity and MDA content were both attenuated by corynoline. ZEA-induced NF-κB p65 and IκBα phosphorylation were inhibited by corynoline. Furthermore, SIRT1, Nrf2, and HO-1 expression were increased by corynoline. In addition, the protective effects of corynoline against liver injury were reversed by the SIRT1 inhibitor EX-527. Taken together, corynoline protected against ZEA-induced liver injury by activating the SIRT1/Nrf2 signaling pathway.     

Enhancement of intracellular glutathione protects endothelial cells against oxidant damage

We studied the role of glutathione in the endothelial cell defense against H2O2 damage. Treatment of endothelial cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, depleted the cells of GSH, while L-2-oxothiazolidine-4-carboxylate, an effective intracellular cysteine delivery agent, markedly enhanced endothelial cell GSH concentration. Depletion of intracellular GSH sensitized the endothelial cells to injury by H2O2 either preformed or generated by the glucose-glucose oxidase system. In contrast, an increase of intracellular GSH protected the cells from H2O2 damage. There was an inverse, linear relationship between the intracellular GSH concentrations and killing of endothelial cells by H2O2. Our results suggest that enhancement of endothelial cell GSH may be an alternative approach toward the prevention of oxidant-induced endothelial damage such as adult respiratory distress syndrome.     

Cardioprotective effects of melatonin against myocardial ischaemia/reperfusion injury: Activation of AMPK/Nrf2 pathway

Although reperfusion is the most effective therapy for patients with acute myocardial infarction, reperfusion injury limits the therapeutic effects of early reperfusion. Oxidative stress plays a crucial role in myocardial ischaemia/reperfusion (I/R) injury. Melatonin, a circulating hormone, is well-known as an antioxidant in cardiovascular diseases. In this short communication, we show that melatonin significantly improves post-ischaemic cardiac function, reduces infarct size and decreases oxidative stress. Furthermore, melatonin markedly increases AMPK activation and Nrf2 nuclear translocation. Nevertheless, these melatonin-induced changes are abrogated by compound C. In addition, ML-385, an Nrf2 inhibitor, also withdraws the antioxidative effects of melatonin but has little effect on AMPK activation. In conclusion, our results demonstrate that melatonin alleviates myocardial I/R injury by inhibiting oxidative stress via the AMPK/Nrf2 signalling pathway.     

A novel compound C12 inhibits inflammatory cytokine production and protects from inflammatory injury in vivo

Inflammation is a hallmark of many diseases. Although steroids and cyclooxygenase inhibitors are main anti-inflammatory therapeutical agents, they may cause serious side effects. Therefore, developing non-steroid anti-inflammatory agents is urgently needed. A novel hydrosoluble compound, C12 (2,6-bis(4-(3-(dimethylamino)-propoxy)benzylidene)cyclohexanone), has been designed and synthesized as an anti-inflammatory agent in our previous study. In the present study, we investigated whether C12 can affect inflammatory processes in vitro and in vivo. In mouse primary peritoneal macrophages, C12 potently inhibited the production of the proinflammatory gene expression including TNF-α, IL-1β, IL-6, iNOS, COX-2 and PGE synthase. The activity of C12 was partly dependent on inhibition of ERK/JNK (but p38) phosphorylation and NF-κB activation. In vivo, C12 suppressed proinflammatory cytokine production in plasma and liver, attenuated lung histopathology, and significantly reduced mortality in endotoxemic mice. In addition, the pre-treatment with C12 reduced the inflammatory pain in the acetic acid and formalin models and reduced the carrageenan-induced paw oedema and acetic acid-increased vascular permeability. Taken together, C12 has multiple anti-inflammatory effects. These findings, coupled with the low toxicity and hydrosolubility of C12, suggests that this agent may be useful in the treatment of inflammatory diseases.     

A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis

Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.