Protective Effect of Flavonoids from a Deep-Sea-Derived Arthrinium sp. against ox-LDL-Induced Oxidative Injury through Activating the AKT/Nrf2/HO-1 Pathway in Vascular Endothelial Cells

Oxidized low-density lipoprotein (ox-LDL)-induced oxidative injury in vascular endothelial cells is crucial for the progression of cardiovascular diseases, including atherosclerosis. Several flavonoids have been shown cardiovascular protective effects. Recently, our research group confirmed that the novel flavonoids isolated from the deep-sea-derived fungus Arthrinium sp., 2,3,4,6,8-pentahydroxy-1-methylxanthone (compound 1) and arthone C (compound 2) effectively scavenged ROS in vitro. In this study, we further investigated whether these compounds could protect against ox-LDL-induced oxidative injury in endothelial cells and the underlying mechanisms. Our results showed that compounds 1 and 2 inhibited ox-LDL-induced apoptosis and adhesion factors expression in human umbilical vein vascular endothelial cells (HUVECs). Mechanistic studies showed that these compounds significantly inhibited the ROS level increase and the NF-κB nuclear translocation induced by ox-LDL. Moreover, compounds 1 and 2 activated the Nrf2 to transfer into nuclei and increased the expression of its downstream antioxidant gene HO-1 by inducing the phosphorylation of AKT in HUVECs. Importantly, the AKT inhibitor MK-2206 2HCl or knockdown of Nrf2 by RNA interference attenuated the inhibition effects of these compounds on ox-LDL-induced apoptosis in HUVECs. Meanwhile, knockdown of Nrf2 abolished the effects of the compounds on ox-LDL-induced ROS level increase and the translocation of NF-κB to nuclei. Collectively, the data showed that compounds 1 and 2 protected endothelial cells against ox-LDL-induced oxidative stress through activating the AKT/Nrf2/HO-1 pathway. Our study provides new strategies for the design of lead compounds for related cardiovascular diseases treatment.     

茶黄素激活Nrf2/HO-1通路保护血管内皮细胞氧化应激损伤

为探讨茶黄素（Theaflavin,TF）对过氧化氢（H₂O₂）诱导的血管内皮细胞氧化应激损伤的保护效应及作用机制,将人脐静脉血管内皮细胞HUVEC分为对照组、损伤组（0.2 mmol·L^-1 H₂O₂处理）和TF预处理组（2.0、5.0、10.0 μmol·L^-1 TF和0.2 mmol·L^-1 H₂O₂处理）。损伤组和TF组均以H₂O₂处理24 h,其中TF组先以不同浓度TF预处理2 h,对照组均以定量溶剂处理。以MTT法测定细胞活力,DCFH-DA探针测定活性氧（ROS）水平,流式细胞术检测细胞凋亡,Western blot测定蛋白表达水平,相应试剂盒测定乳酸脱氢酶（LDH）、一氧化氮（NO）、丙二醛（MDA）含量及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化氢酶（GSH-Px）活性。结果显示,与对照组相比,损伤组细胞活力显著降低,LDH释放量增加,NO水平降低,胞内ROS水平升高,MDA增加,抗氧化酶活力降低,细胞凋亡升高;TF预处理能够显著提高细胞活力,降低LDH水平,维持NO水平,降低胞内ROS水平及MDA的产生,提高抗氧化酶活力,并抑制细胞凋亡;进一步研究显示,TF能够激活Nrf2/HO-1通路,且Nrf2抑制剂会显著降低TF的内皮细胞保护效应。可见,TF能够有效抑制H₂O₂诱导的血管内皮细胞氧化应激损伤,其机制至少部分是通过激活Nrf2/HO-1通路实现的。     

The Cytoprotective Effect of Petalonia binghamiae Methanol Extract against Oxidative Stress in C2C12 Myoblasts: Mediation by Upregulation of Heme Oxygenase-1 and Nuclear Factor-Erythroid 2 Related Factor 2

This study was designed to examine the protective effects of the marine brown algae Petalonia binghamiae against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms. P. binghamiae methanol extract (PBME) prevented hydrogen peroxide (H₂O₂)-induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species (ROS) induced by H₂O₂ in mouse-derived C2C12 myoblasts. PBME also significantly attenuated H₂O₂-induced comet tail formation in a comet assay, histone γH2A.X phosphorylation, and annexin V-positive cells, suggesting that PBME prevented H₂O₂-induced cellular DNA damage and apoptotic cell death. Furthermore, PBME increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2 related factor 2 (Nrf2). However, zinc protoporphyrin IX, a HO-1 competitive inhibitor, significantly abolished the protective effects of PBME on H₂O₂-induced ROS generation, growth inhibition, and apoptosis. Collectively, these results demonstrate that PBME augments the antioxidant defense capacity through activation of the Nrf2/HO-1 pathway.     

Nrf2 and NF-κB Signaling Pathways Contribute to Porphyra-334-Mediated Inhibition of UVA-Induced Inflammation in Skin Fibroblasts

In this study, we examined the protective effects of porphyra-334 against UVA-irradiated cellular damage and elucidated the underlying mechanisms. Porphyra-334 prevented UVA-induced cell death and exhibited scavenging activities against intracellular oxidative stress induced by UVA irradiation in skin fibroblasts. We found that porphyra-334 significantly reduced the secretion and expression of IL-6 and TNF-α, reduced nuclear expression of Nuclear factor-κB (NF-κB), and sustained NF-E2-related factor 2 (Nrf2) activation. Further mechanism research revealed that porphyra-334 promoted the Nrf2 signaling pathway in UVA-irradiated skin fibroblasts. Our results show that the antioxidant effect of porphyra-334 is due to the direct scavenging of oxidative stress and its inhibitory effects on NF-κB-dependent inflammatory genes, such as IL-6 and TNF-κ. Therefore, we hypothesize that boosting the Nrf2- NF-κB-dependent response to counteract environmental stress is a promising strategy for the prevention of UVA-related damage.     

Astaxanthin Provides Antioxidant Protection in LPS-Induced Dendritic Cells for Inflammatory Control

Astaxanthin, originating from marine organisms, is a natural bioactive compound with powerful antioxidant activity. Here, we evaluated the antioxidant ability of astaxanthin on dendritic cells (DCs), a key target of immune regulation, for inflammatory control in a sepsis model. Our results showed that astaxanthin suppressed nitric oxide (NO) production, reactive oxygen species (ROS) production, and lipid peroxidation activities in LPS-induced DCs and LPS-challenged mice. Moreover, the reduced glutathione (GSH) levels and the GSH/GSSG ratio were increased, suggesting that astaxanthin elevated the level of cellular reductive status. Meanwhile, the activities of antioxidant enzymes, including glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), were significantly upregulated. Astaxanthin also inhibited the LPS-induced secretions of IL-1β, IL-17, and TGF-β cytokines. Finally, we found that the expressions of heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) were significantly upregulated by astaxanthin in LPS-induced DCs, suggesting that the HO-1/Nrf2 pathway plays a significant role in the suppression of oxidative stress. These results suggested that astaxanthin possesses strong antioxidant characteristics in DC-related inflammatory responses, which is expected to have potential as a method of sepsis treatment.     

Protective Effect of Maize Silks (Maydis stigma) Ethanol Extract on Radiation-Induced Oxidative Stress in Mice

Maize silks, dried cut stigmata of maize female flowers, are a traditional medicinal plant. This study was conducted to investigate the antioxidant effect of maize silks ethanol extract (MSE) against oxidative damage in vivo. γ-radiation was employed to induce oxidative stress in mice and the variation of malondialdehyde (MDA), glutathione/glutathione disulfide ratio (GSH/GSSG), blood cells, NF-E2-related factor 2 (Nrf2) and related antioxidant enzymes were examined. The results showed that radiation elevate levels of MDA, induce hematological abnormalities and decrease levels of GSH/GSSG and Nrf2 expression in liver and kidney. MSE administration significantly abolished elevation of MDA levels in liver, maintained hepatic GSH/GSSG ratio and ameliorated hematological abnormalities dose dependently. Moreover, MSE up-regulated the hepatic protein expression of Nrf2 dose dependently and the activities as well as protein expression of Nrf2-related antioxidant enzymes were also increased. However, the antioxidant ability of MSE seemed not to be as effective in kidney as in liver. These findings firstly proved the protective role of MSE against oxidative stress, which was in part via up-regulation of Nrf2 and seemed to be tissue specific.     

沉香叶黄酮稳定性及对H2O2诱导HepG2细胞氧化损伤的保护作用

研究沉香叶黄酮稳定性及对H₂O₂诱导HepG2细胞氧化损伤的保护作用。通过测定溶液吸光值,探讨温度、光照、金属离子、pH值对沉香叶黄酮稳定性的影响。建立H₂O₂诱导HepG2细胞氧化损伤模型,将细胞分为正常组、模型组以及沉香叶黄酮低、中、高剂量组,采用水溶性四唑盐法检测细胞增殖率,2′,7′-二氢二氯荧光黄双乙酸钠法（DCFH-DA）测定细胞内活性氧（ROS）水平,生化试剂盒检测细胞乳酸脱氢酶（LDH）释放量、丙二醛（MDA）含量、细胞内总超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）活性。结果表明,沉香叶黄酮在温度70 ℃以下,pH 3~7范围内,Na⁺、K⁺、Mg²⁺、Ca²⁺金属离子中,黑暗环境与节能灯光中较稳定;能显著抑制H₂O₂造成的细胞死亡,降低胞内ROS与MDA的生成,减轻LDH向细胞外扩散程度,提高SOD、CAT、GSH-Px活性。沉香叶黄酮在低温、弱酸、部分金属离子、弱光条件下具有一定稳定性;对H₂O₂诱导HepG2氧化应激损伤具有保护作用,其作用可能与调节细胞氧化还原系统、清除胞内活性氧水平、提高细胞内抗氧化酶系活性有关。     

The Inhibition Effect of the Seaweed Polyphenol, 7-Phloro-Eckol from Ecklonia Cava on Alcohol-Induced Oxidative Stress in HepG2/CYP2E1 Cells

The liver is vulnerable to oxidative stress-induced damage, which leads to many diseases, including alcoholic liver disease (ALD). Liver disease endanger people’s health, and the incidence of ALD is increasing; therefore, prevention is very important. 7-phloro-eckol (7PE) is a seaweed polyphenol, which was isolated from Ecklonia cava in a previous study. In this study, the antioxidative stress effect of 7PE on HepG2/CYP2E1 cells was evaluated by alcohol-induced cytotoxicity, DNA damage, and expression of related inflammation and apoptosis proteins. The results showed that 7PE caused alcohol-induced cytotoxicity to abate, reduced the amount of reactive oxygen species (ROS) and nitric oxide (NO), and effectively inhibited DNA damage in HepG2/CYP2E1 cells. Additionally, the expression levels of glutathione (GSH), superoxide dismutase (SOD), B cell lymphoma 2 (Bcl-2), and Akt increased, while γ-glutamyltransferase (GGT), Bcl-2 related x (Bax), cleaved caspase-3, cleaved caspase-9, nuclear factor-κB (NF-κB), and JNK decreased. Finally, molecular docking proved that 7PE could bind to BCL-2 and GSH protein. These results indicate that 7PE can alleviate the alcohol-induced oxidative stress injury of HepG2 cells and that 7PE may have a potential application prospect in the future development of antioxidants.     

The Potential Protective Effect and Possible Mechanism of Peptides from Oyster (Crassostrea hongkongensis) Hydrolysate on Triptolide-Induced Testis Injury in Male Mice

Peptides from oyster hydrolysate (OPs) have a variety of biological activities. However, its protective effect and exact mechanism on testicular injury remain poorly understood. This study aimed to evaluate the protective effect of OPs on triptolide (TP)-induced testis damage and spermatogenesis dysfunction and investigate its underlying mechanism. In this work, the TP-induced testis injury model was created while OPs were gavaged in mice for 4 weeks. The results showed that OPs significantly improved the sperm count and motility of mice, and alleviated the seminiferous tubule injury. Further study showed that OPs decreased malonaldehyde (MDA) level and increased antioxidant enzyme (SOD and GPH-Px) activities, attenuating oxidative stress and thereby reducing the number of apoptotic cells in the testis. In addition, OPs improved the activities of enzymes (LDH, ALP and ACP) related to energy metabolism in the testis and restored the serum hormone level of mice to normal. Furthermore, OPs promoted the expression of Nrf2 protein, and then increased the expression of antioxidant enzyme regulatory protein (HO-1 and NQO1) in the testis. OPs inhibited JNK phosphorylation and Bcl-2/Bax-mediated apoptosis. In conclusion, OPs have a protective effect on testicular injury and spermatogenesis disorders caused by TP, suggesting the potential protection of OPs on male reproduction.     

Sargachromenol Purified from Sargassum horneri Inhibits Inflammatory Responses via Activation of Nrf2/HO-1 Signaling in LPS-Stimulated Macrophages

In this study, we isolated sargachromenol (SC) from Sargassum horneri and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. SC did not show cytotoxicity at all concentrations and effectively increased the cell viability by reducing the nitric oxide (NO) and intracellular reactive oxygen species (ROS) production in LPS-stimulated RAW 264.7 macrophages. In addition, SC decreased the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and inflammatory mediators (iNOS and COX-2). Moreover, SC suppressed the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and mitogen-activated protein kinase (MAPK) signaling, whereas activated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling in LPS-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effect of SC was abolished by the inhibition of HO-1 in LPS-stimulated RAW 264.7 macrophages. According to the results, this study suggests that the antioxidant capacity of SC leads to its anti-inflammatory effect and it potentially may be utilized in the nutraceutical and pharmaceutical sectors.     

Particulate Matter-Induced Inflammation/Oxidative Stress in Macrophages: Fucosterol from Padina boryana as a Potent Protector,Activated via NF-κB/MAPK Pathways and Nrf2/HO-1 Involvement

Fucosterol is a phytosterol that is abundant in marine brown algae and is a renowned secondary metabolite. However, its ability to protect macrophages against particulate matter (PM) has not been clarified with regard to inflammation; thus, this study aimed to illustrate the above. Padina boryana, a brown algae that is widespread in Indo–Pacific waters, was applied in the isolation of fucosterol. Isolation was conducted using silica open columns, while identification was assisted with gas chromatography-mass spectroscopy (GC-MS) and NMR. Elevated levels of PM led the research objectives toward the implementation of it as a stimulant. Both inflammation and oxidative stress were caused due the fact of its effect. RAW 264.7 macrophages were used as a model system to evaluate the process. It was apparent that the increased NO production levels, due to the PM, were mediated through the inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines (i.e., interleukin-6 (IL-6), interleukin-1 (IL-1β) and tumor necrosis factor-α (TNF-α), including prostaglandin E2 (PGE₂)). Further, investigations provided solid evidence regarding the involvement of NF-κB and mitogen-activated protein kinases (MAPKs) in the process. Oxidative stress/inflammation which are inseparable components of the cellular homeostasis were intersected through the Nrf2/HO-1 pathway. Conclusively, fucosterol is a potent protector against PM-induced inflammation in macrophages and hence be utilized as natural product secondary metabolite in a sustainable manner.     

Colored Potato Extracts Induce Superoxide Dismutase-2 mRNA Via ERK1/2 Pathway in HepG2 Cells

Rats fed a diet containing Shadow Queen (SQ), an anthocyanin-rich potato cultivar, previously showed an increase in the hepatic superoxide dismutase (SOD)-2 mRNA level. We investigated whether an extract of SQ would directly increase the hepatic SOD-2 mRNA level in HepG2 cells. Furthermore, we estimated the intracellular signaling pathway for the induction of SOD-2 mRNA expression. HepG2 cells were stimulated using extracts of four crops, including SQ, for 12 h; only extracts of colored potatoes induced SOD-2 mRNA expression significantly. This induction of SOD-2 mRNA expression was blocked by an inhibitor of the extracellular signal-related kinase (ERK) 1/2 pathway. Furthermore, an extract of SQ increased the phosphorylation of ERK1/2 after 15 or 30 min of stimulation. These data indicate that an extract of SQ directly induces hepatic SOD-2 mRNA expression via activation of ERK1/2 pathway in HepG2 cells.     

Cytoprotective Potential of Fucoxanthin in Oxidative Stress-Induced Age-Related Macular Degeneration and Retinal Pigment Epithelial Cell Senescence In Vivo and In Vitro

Oxidative stress is identified as a major inducer of retinal pigment epithelium (RPE) cell dysregulation and is associated with age-related macular degeneration (AMD). The protection of RPE disorders plays an essential role in the pathological progress of retinal degeneration diseases. The pharmacological functions of fucoxanthin, a characteristic carotenoid, including anti-inflammatory and antioxidant properties, may ameliorate an outstanding bioactivity against premature senescence and cellular dysfunction. This study demonstrates that fucoxanthin protects RPE cells from oxidative stress-induced premature senescence and decreased photoreceptor cell loss in a sodium iodate-induced AMD animal model. Similarly, oxidative stress induced by hydrogen peroxide, nuclear phosphorylated histone (γH2AX) deposition and premature senescence-associated β-galactosidase staining were inhibited by fucoxanthin pretreatment in a human RPE cell line, ARPE-19 cells. Results reveal that fucoxanthin treatment significantly inhibited reactive oxygen species (ROS) generation, reduced malondialdehyde (MDA) concentrations and increased the mitochondrial metabolic rate in oxidative stress-induced RPE cell damage. Moreover, atrophy of apical microvilli was inhibited in cells treated with fucoxanthin after oxidative stress. During aging, the RPE undergoes well-characterized pathological changes, including amyloid beta (Aβ) deposition, beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) expression and tight junction disruption, which were also reduced in fucoxanthin-treated groups by immunofluorescence. Altogether, pretreatment with fucoxanthin may protect against premature senescence and cellular dysfunction in retinal cells by oxidative stress in experimental AMD animal and human RPE cell models.     

Valproic Acid Ameliorates Locomotor Function in the Rat Model of Contusion via Alteration of Mst1, Bcl-2, and Nrf2 Gene Expression

Background:In animal models of inflammatory diseases, Mst1 facilitates the programmed cell death as a novel pro-apoptotic kinase. This research aimed to determine the expression level of Mst1 gene in a rat model of SCI treated with VPA. Methods:Severe rat model contusion was used for evaluation of the neuroprotective effect of valproic acid. The BBB test, was performed to determine locomotor functions. H&E staining and TUNEL assay were performed to detect cavity formation and apoptosis, respectively. The mRNA levels of the genes Mst1, Nrf2, and Bcl-2 were evaluated, using quantitative RT-PCR. Results:The results revealed that Mst1 gene expression and TUNEL-positive cells in the VPA-treated group were significantly reduced as compared to the untreated group (p ≤ 0.05). Conclusion:Our findings indicate that VPA has therapeutic potential and can be a candidate for the treatment of neurodegenerative disorders and traumatic injury as a promising drug. Key Words: Bcl-2, Contusion, Mst1, Nrf2, Valproic acid     

油酸在黄曲霉毒素诱导肝细胞损伤中的保护作用

为了解油酸在黄曲霉毒素B1（aflatoxin B1,AFB1）诱发的肝损伤中所起的保护作用,以体外培养的肝细胞（L-02）作为靶细胞,研究油酸的保护机制。设置3组实验：对照组（无处理）、AFB1组（只有AFB1）、油酸组（AFB1和油酸共同处理）,药物处理24h后显微镜观察细胞形态,细胞增殖-毒性检测试剂盒（Cell Counting Kit-8, CCK-8）检测细胞活力,荧光法检测活性氧（reactive oxygen species,ROS）水平,Annexin V-FITC/PI检测细胞凋亡率,蛋白质免疫印迹检测血红素加氧酶-1（heme oxygenase 1,HO-1）、Caspase-3以及Survivin蛋白的表达。采用t检验或单因素方差分析进行统计分析。结果发现：与对照组相比,AFB1暴露能够明显抑制细胞活力（P<0.05）,促进细胞凋亡（P<0.001）,升高ROS水平（P<0.001）;与AFB1组相比,油酸作用后细胞活力显著增加（P<0.01）,细胞凋亡减少（P<0.01）,ROS水平降低（P<0.001）。与AFB1组相比,油酸作用后可显著提高 HO-1（P<0.05）和抑凋亡蛋白Survivin的表达（P<0.05）,降低凋亡蛋白Caspase-3（P<0.01）表达。因此认为油酸对AFB1引发的肝细胞损伤具有保护作用,其机制可能与其通过促进抗氧化酶蛋白HO-1的表达,从而降低氧化应激水平,降低细胞凋亡有关。