Brassinin,a phytoalexin in cruciferous vegetables,suppresses obesity‐induced inflammatory responses through the Nrf2‐HO‐1 signaling pathway in an adipocyte‐macrophage co‐culture system

The aim of this study was to investigate the effect of brassinin (BR), a phytoalexin found in plants belonging to the Brassicaceae family, on the obesity‐induced inflammatory response and its molecular mechanism in co‐culture of 3T3‐L1 adipocytes and RAW264.7 macrophages. BR effectively suppressed lipid accumulation by down‐regulating the expression of adipogenic factors, which in turn, were regulated by early adipogenic factors such as CCAAT‐enhancer‐binding protein‐β and Kruppel‐like factor 2. Production of inflammatory cytokines and reactive oxygen species, induced by adipocyte‐conditioned medium, was significantly decreased in BR‐treated cells. This effect of BR was more prominent in contact co‐culture of adipocytes and macrophages with a 90% and 34% reduction in IL‐6 and MCP‐1 levels, respectively. BR also restored adiponectin expression, which was significantly reduced by culturing adipocytes in macrophage‐conditioned medium. In the transwell system, BR increased the protein levels of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and its target molecule, hemoxygenase‐1 (HO‐1), by 55%–93% and 45%–48%, respectively, and also increased Nrf2 translocation into the nucleus. However, knockdown of Nrf2 or HO‐1 in RAW264.7 cells restored this BR‐mediated inhibition of IL‐6 and MCP‐1 production. These results indicated that BR inhibited obesity‐induced inflammation via the Nrf2‐HO‐1 pathway.     

Eupatolide Inhibits PDGF‐induced Proliferation and Migration of Aortic Smooth Muscle Cells Through ROS‐dependent Heme Oxygenase‐1 Induction

The abnormal proliferation and migration of vascular smooth muscle cell (VSMC) contributes importantly to the pathogenesis of atherosclerosis and restenosis. Here, we investigated the effects of eupatolide (EuTL), a sesquiterpene lactone isolated from the medicinal plant Inula britannica, on platelet‐derived growth factor (PDGF)‐induced proliferation and migration of primary rat aortic smooth muscle cells (RASMCs), as well as its underlying mechanisms. EuTL remarkably inhibited PDGF‐induced proliferation and migration of RASMCs. Treatment of RASMCs with EuTL induced both protein and mRNA expression of heme oxygenase‐1 (HO‐1). SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor) and LY294002 (a PI3K inhibitor) did not suppress EuTL‐induced HO‐1 expression; however, N‐acetylcysteine (NAC, an antioxidant) blocked EuTL‐induced HO‐1 expression. Moreover, treatment of RASMCs with EuTL increased reactive oxygen species (ROS) accumulation and nuclear translocation of nuclear factor‐E2‐related factor 2 (Nrf2); however, this translocation was also inhibited by NAC. NAC or inhibition of HO‐1 significantly attenuated the inhibitory effects of EuTL on PDGF‐induced proliferation and migration of RASMCs. Taken together, these findings suggest that EuTL could suppress PDGF‐induced proliferation and migration of VSMCs through HO‐1 induction via ROS‐Nrf2 pathway and may be a potential HO‐1 inducer for preventing or treating vascular diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

Berberine ameliorates lipopolysaccharide‐induced acute lung injury via the PERK‐mediated Nrf2/HO‐1 signaling axis

A fundamental element of acute lung injury (ALI) is the inflammatory response, which can affect the entire respiratory system, including the respiratory tract and alveoli. Berberine has gained attention because of its anti‐inflammatory effects. Nuclear factor‐erythroid 2‐related factor 2 (Nrf2) and endoplasmic reticulum (ER) stress are involved in lung injury. Nrf2 also acts as a protein kinase‐like ER kinase (PERK) substrate in heart disease. Therefore, this study investigated the effect of berberine against lipopolysaccharide (LPS)‐induced ALI and the role of the PERK‐mediated Nrf2/HO‐1 signaling axis. Berberine promoted Nrf2 nuclear translocation and phosphorylation in vitro. After LPS stimulation, this effect was further enhanced, whereas inflammatory factor (IL‐6 and IL‐8) release and reactive oxygen species generation were significantly decreased. Berberine effectively alleviated lung injury by reducing lung edema and neutrophil infiltration. Berberine also significantly reduced histopathological inflammatory changes via inhibition of ER stress and activation of Nrf2 signaling. Thapsigargin‐induced ER stress and small interference RNA (siRNA)‐mediated Nrf2 inhibition abrogated the protective effects of berberine in vitro, whereas siRNA‐mediated suppression of ER stress and sulforaphane‐induced Nrf2 activation further improved those effects. Importantly, ER stress induction led to Nrf2 activation, whereas PERK depletion partly reduced the level of Nrf2 phosphorylation and translocation in LPS‐induced cells. Therefore, berberine inhibits LPS‐induced ALI through the PERK‐mediated Nrf2/HO‐1 signaling axis.     

Ampelopsin inhibits high glucose‐induced extracellular matrix accumulation and oxidative stress in mesangial cells through activating the Nrf2/HO‐1 pathway

Oxidative stress plays an important role in diabetic nephropathy (DN), which is a diabetic complication. Ampelopsin (AMP) is a natural flavonoid that has been found to possess antidiabetic and antioxidative activities. However, the effect of AMP on DN remains unclear. In this study, we aimed to evaluate the protective effect of AMP on glomerular mesangial cells (MCs) exposed to high glucose (HG). We found that AMP improved HG‐caused cell viability reduction in MCs. AMP significantly suppressed the intracellular ROS production and expression levels of ROS producing enzymes NADPH oxidase 2 (NOX2) and NOX4. Increased of NOX activity in HG‐stimulated MCs was suppressed by AMP. Pretreatment with AMP inhibited extracellular matrix (ECM) accumulation in HG‐stimulated MCs with decreased expression levels of fibronectin (FN) and collagen type IV (Col IV). Furthermore, AMP elevated the expression levels of nuclear Nrf2 and heme oxygenase‐1 (HO‐1), as well as increased the mRNA levels of Nrf2‐driven genes NAD(P)H dehydrogenase quinone‐1 (NQO‐1) and HO‐1 in HG‐treated MCs. Knockdown of Nrf2 reversed the protective effects of AMP against HG‐induced oxidative stress and EMC accumulation in MCs. In conclusion, these findings indicated that AMP protected MCs from HG‐induced oxidative damage and ECM accumulation, which might be mediated by Nrf2/HO‐1 pathway.     

Palbinone from Paeonia suffruticosa Protects Hepatic Cells via Up‐regulation of Heme Oxygenase‐1

Paeonia suffruticosa has been traditionally employed for vitalizing blood circulation and alleviating liver and inflammatory diseases. The pathways by which palbinone (PB) isolated from P. suffruticosa mediates heme oxygenase‐1 (HO‐1) induction were investigated using the specific inhibitors for PI3K and mitogen activated protein kinases pathways. The effect of PB‐treatment on Nrf2 translocalization and HO‐1‐antioxidant response element (ARE) regulation was examined employing Western blot and luciferase assays. PB induced HO‐1 expression via the activation of Nrf2 in the hepatic cells, and ARE‐dependent genes were stimulated via the PB‐mediated Nrf2 activation. PB‐mediated HO‐1 expression could be involved with PI3K/Akt and ERK1/2 pathways. Our study suggests the mechanism by which PB induces HO‐1 expression in the hepatic cells. This might substantiate the traditional applications of P. suffruticosa for the treatment of oxidative stress‐related diseases including oxidant and inflammatory‐mediated vascular and liver diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

Caffeoylserotonin Protects Human Keratinocyte HaCaT Cells against H2O2‐Induced Oxidative Stress and Apoptosis through Upregulation of HO‐1 Expression via Activation of the PI3K/Akt/Nrf2 Pathway

Caffeoylserotonin (CaS) has strong radical scavenging activity as well as antioxidant activities, protecting cells from lipid peroxidation, intracellular reactive oxygen species generation, DNA damage, and cell death. The molecular mechanism by which CaS protects against oxidative stress is not well understood. Here, we analyzed the cytoprotective activity of CaS in hydrogen peroxide (H₂O₂)‐treated keratinocyte HaCaT cells. H₂O₂ induced apoptosis in the cells through activation of pro‐apoptotic p21, Bax, and caspase‐3. Pretreatment with CaS inhibited apoptotic gene expression and activated the anti‐apoptotic gene, Bcl‐xL. Although CaS did not directly affect heme oxygenase‐1 (HO‐1) expression, pretreatment with CaS augmented HO‐1 expression through an increase in NF‐E2‐related factor (Nrf2) stability and stimulation of Nrf2 translocation to the nucleus upon H₂O₂ exposure. H₂O₂ also induced the phosphorylation and subsequent activation of ERK, p38 MAPK, and Akt. Analysis using specific inhibitors of p38 MAPK and Akt demonstrated that only Akt activation was involved in HO‐1 and Nrf2 expressions. In addition, PI3K and PKC inhibitors suppressed HO‐1/Nrf2 expression and Akt phosphorylation. These results demonstrate that CaS protects against oxidative stress‐induced keratinocyte cell death in part through the activation of Nrf2‐mediated HO‐1 induction via the PI3K/Akt and/or PKC pathways, but not MAPK signaling. Copyright © 2013 John Wiley & Sons, Ltd.     

Xanthotoxin and umbelliferone attenuate cognitive dysfunction in a streptozotocin‐induced rat model of sporadic Alzheimer's disease: The role of JAK2/STAT3 and Nrf2/HO‐1 signalling pathway modulation

The aim of the present study was to assess the neuroprotective effects of xanthotoxin and umbelliferone in streptozotocin (STZ)‐induced cognitive dysfunction in rats. Animals were injected intracerebroventricularly (ICV) with STZ (3 mg/kg) once to induce a sporadic Alzheimer's disease (SAD)‐like condition. Xanthotoxin or umbelliferone (15 mg/kg, i.p.) were administered 5 hr after ICV‐STZ and daily for 20 consecutive days. Xanthotoxin or umbelliferone prevented cognitive deficits in the Morris water maze and object recognition tests. In parallel, xanthotoxin or umbelliferone reduced hippocampal acetylcholinestrase activity and malondialdehyde level. Moreover, xanthotoxin or umbelliferone increased glutathione content. These coumarins also modulated neuronal cell death by reducing the level of proinflammatory cytokines (tumour necrosis factor‐alpha and interleukin‐6), inhibiting the overexpression of inflammatory markers (nuclear factor κB [NF‐κB] and cyclooxygenase II), and upregulating the expression of NF‐κB inhibitor (IκB‐α). Interestingly, xanthotoxin diminished phosphorylated JAK2 and phosphorylated STAT3 protein expression, while umbelliferone markedly replenished nuclear factor erythroid‐derived 2‐like 2 (Nrf2) and haem oxygenase‐1 (HO‐1) levels. The current study provides evidence for the protective effect of xanthotoxin and umbelliferone in STZ‐induced cognitive dysfunction in rats. This effect may be attributed, at least in part, to inhibiting acetylcholinestrase and attenuating oxidative stress, neuroinflammation and neuronal loss.     

Nrf2参与MSCs调节大鼠梗死心肌氧化应激作用的实验研究

目的 探讨间充质干细胞（MSCs）移植对梗死心肌组织氧化应激反应的调节作用及其机制.方法 建立心肌梗死大鼠模型,利用随机数字表进行随机化分组,分为对照组（n=20）、MSC移植组（n=20）和MSC移植＋HO-1抑制剂组（n=20）.以密度梯度离心并贴壁培养获得骨髓源MSCs,于心肌梗死模型建立后经心肌局部多点注射MSCs悬液;细胞移植后不同时间点以测定血浆以及心肌梗死和梗死交界区组织超氧化物歧化酶（SOD）、还原型谷酰甘肽（GSH）和丙二醛（MDA）水平;Western Blot检测Nrf2和HO-1蛋白表达的变化.结果 细胞移植后8 h,3组大鼠梗死组织中SOD、GSH表达量开始下降,72 h时达到最低,以后逐渐开始回升,至两周时恢复接近心肌梗死前水平;同时,细胞移植后梗死组织中脂质氧化终产物MDA水平开始增高,3组均在72 h达到高峰（P〈0.05）,以后开始下降,至2周时降至梗死前水平.细胞移植后各时间点,与对照组比较,MSC移植组SOD和GSH含量下降幅度减小、恢复较快（P〈0.05）,且MDA含量减少（P〈0.05）.对照组梗死心肌组织中Nrf2及HO-1蛋白表达轻微升高,72 h时最显著;而MSC移植组中Nrf2和HO-1蛋白表达显著增高,且升高时间提前至细胞移植后24～72 h,且与对照组比较,细胞移植后24 h和72 h,Nrf2和HO-1表达水平均高于对照组（P〈0.05）;MSC移植＋HO-1抑制剂组与对照组比较,差异无统计学意义（P〉0.05）.结论 心肌梗死后移植MSCs可能通过促进局部Nrf2及其下游抗氧化酶靶基因HO-1蛋白表达而调节梗死心肌的氧化应激反应,进而减轻氧化应激诱导的心肌损伤.     

Neuroprotective effect of salidroside against central nervous system inflammation‐induced cognitive deficits: A pivotal role of sirtuin 1‐dependent Nrf‐2/HO‐1/NF‐κB pathway

Central nervous system (CNS) inflammation occurs in cognitive dysfunctions, but the underlying mechanisms remain unclear. Here, we investigated the role of sirtuin 1 (SIRT1) and salidroside in CNS inflammation‐induced cognitive deficits model. In vivo, CNS inflammation was initiated by a single intracerebroventricular injection of lipopolysaccharide (LPS). The levels of inflammatory cytokines and the capability of free radial scavenging were determined after the LPS challenge. In vivo, salidroside and nicotinamide, a SIRT1 inhibitor, were used in PC12 cell. Of note, with the treatment of salidroside, LPS‐induced learning and memory impairments were effectively improved. Salidroside also remarkably inhibited the inflammatory cytokines, up‐regulated the concentration of superoxide dismutase and inhibited the vitalities of malondialdehyde in serum, hippocampus, and cell supernatant. Besides, the expression of Sirt1, Nrf‐2, HO‐1, Bax, Bcl‐2, caspase‐9, and caspase‐3 and the phosphorylation of AMPK, NF‐κBp65, and IκBα were increased accompanying with the LPS‐induced cognitive impairments, which were significantly suppressed by salidroside treatment. In PC12 cell model, nicotinamide significantly abrogated the beneficial effects of salidroside, as indicated by the antioxidant, anti‐inflammatory, and antiapoptosis signaling. Together, our results showed that salidroside may be a novel therapy drug in neurodegenerative diseases, and the protective effect was involved in SIRT1‐dependent Nrf‐2/HO‐1/NF‐κB pathway.     

Thai herbal antipyretic 22 formula (APF22) inhibits UVA‐mediated melanogenesis through activation of Nrf2‐regulated antioxidant defense

Thai herbal antipyretic 22 formula (APF22), a polyherbal formula, has been traditionally used to treat dermatologic problems including hyperpigmentation. Exposure of the skin to ultraviolet A (UVA) causes abnormal melanin production induced by photooxidative stress. This study thus aimed to investigate the protective effects of APF22 extracts and phenolic compounds, ferulic acid (FA), and gallic acid (GA; used as positive control and reference compounds), on melanogenesis through modulation of nuclear factor E2‐related factor 2 (Nrf2) signaling and antioxidant defenses in mouse melanoma (B16F10) cells exposed to UVA. Our results revealed that the APF22 extracts, FA, and GA reduced melanin synthesis as well as activity and protein levels of tyrosinase in UVA‐irradiated B16F10 cells. Moreover, APF22 extracts and both FA and GA were able to activate Nrf2‐antioxidant response element signaling and promote antioxidant defenses including glutathione, catalase, glutathione peroxidase, and the glutathione‐S‐transferase at both mRNA and enzyme activity levels in irradiated cells. In conclusion, APF22 extracts suppressed UVA‐mediated melanogenesis in B16F10 cells possibly via redox mechanisms involving activation of Nrf2 signaling and upregulation of antioxidant defenses. Moreover, pharmacological action of the APF22 extracts may be attributed to the phenolic compounds, FA, and GA, probably serving as the APF22's active compounds.     

Sanguisorbae Radix Protects Against 6‐Hydroxydopamine‐induced Neurotoxicity by Regulating NADPH Oxidase and NF‐E2‐related Factor‐2/Heme Oxygenase‐1 Expressions

6‐Hydroxydopamine (6‐OHDA) produces neuronal cell damage by generating reactive oxygen species (ROS). The major mechanisms of protection against ROS‐induced stress are inhibiting expression of ROS generating genes such as NADPH oxidase (NOX) and increasing expression of endogenous antioxidant genes such as heme oxygenase‐1 (HO‐1). This study investigated whether a standardized Sanguisorbae Radix extract (SRE), a medical herb commonly used in Asian traditional medicine, has a protective effect on 6‐OHDA‐induced cell toxicity by regulating ROS in SH‐SY5Y cells. SRE at 10 and 50 µg/mL significantly reduced 6‐OHDA‐induced cell damage dose dependently in the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and by Hoechst 33342 staining. SRE increased the B‐cell lymphoma 2 (Bcl‐2)/Bcl‐2‐associated X ratio and decreased cytochrome C release and caspase‐3 activity. SRE also abolished 6‐OHDA‐induced ROS by inhibiting NOX expression and by inducing HO‐1 expression via NF‐E2‐related factor‐2 activation. Taken together, these results demonstrate that SRE has protective effects against 6‐OHDA‐induced cell death by regulating ROS in SH‐SY5Y cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Paeonol extracted from Paeonia suffruticosa Andr. ameliorated UVB‐induced skin photoaging via DLD/Nrf2/ARE and MAPK/AP‐1 pathway

Paeonia suffruticosa Andr. (PS) has been used in traditional Chinese medicine for a long time. However, there are no studies that investigate the preventive effects of PS on ultraviolet B (UVB)‐induced photoaging. In this study, paeonol (PA) was detected the main compound in PS root. In vitro, PS and PA significantly inhibited UVB‐induced phosphorylation of mitogen‐activated protein kinase and activator protein 1 in keratinocytes, which consequently led to degradation of procollagen type I. On the other hand, PS and PA increased NAD(P)H:quinone oxidoreductase 1 and heme oxygenase‐1 expression, confirmed by greater nuclear accumulation of nuclear factor E2‐releated factor 2 (Nrf2). Furthermore, this study proved that the endogenous antioxidant system Nrf2/antioxidant response element was regulated by dihydrolipoamide dehydrogenase, a tricarboxylic acid (TCA) cycle‐associated protein whose level was decreased after UVB exposure. PS and PA promoted the production of dihydrolipoamide dehydrogenase, as well as the activation of Nrf2 and antioxidant response element, resulting in preventing procollagen type I ruined caused by UVB. In vivo, topical application of PS and PA attenuated UVB‐induced matrix metalloproteinase‐1 production and promoted procollagen type I in hairless mice. These results suggested PA a promising botanical in protecting skin from UVB‐induced photoaging.     

1,2,3,6‐tetra‐O‐galloyl‐β‐D‐allopyranose gallotannin isolated,from Euphorbia jolkini,attenuates LPS‐induced nitric oxide production in macrophages

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation and macrophage‐mediated immunity. Macrophages express inducible NO synthase (iNOS) and produce NO after lipopolysaccharide (LPS) stimulation. Gallotannins are water‐soluble polyphenols with wide‐ranging biological activities. Various chemical structures of gallotannins occurring in medicinal and food plants that are used worldwide showed several remarkable biological and pharmacological activities. In the present study, we examined the inhibitory effects of gallotannin 1,2,3,6‐tetra‐O‐galloyl‐β‐D‐allopyranose (GT24) isolated from Euphorbia jolkini on the LPS‐induced NO production and underlying mechanisms of action. GT24 dose‐dependently decreased LPS‐induced NO production and iNOS expression in J774A.1 macrophages. In addition, GT24 inhibited LPS‐induced activation of nuclear factor (NF)‐κB as indicated by inhibition of degradation of I‐κBα, nuclear translocation of NF‐κB, and NF‐κB dependent gene reporter assay. Our results suggest that GT24 possesses an inhibitory effect on the LPS‐induced inflammatory reaction. Copyright © 2010 John Wiley & Sons, Ltd.     

Gastrodin Ameliorates Oxidative Stress and Proinflammatory Response in Nonalcoholic Fatty Liver Disease through the AMPK/Nrf2 Pathway

This study was designed to investigate the antioxidative, antiinflammatory and metabolism‐regulating effects of gastrodin (GSTD) in the treatment of nonalcoholic fatty liver disease (NAFLD). Oleic acid (OA) was used to induce steatosis in HL‐7702 cells; a high‐fat or high‐fat and high‐cholesterol diet was used to induce NAFLD in mice and rats. Our results showed that GSTD significantly increased hepatic superoxide dismutase (SOD) but decreased reactive oxygen species (ROS)/malondialdehyde (MDA) and reduced the mRNA levels of proinflammatory cytokines both in vitro and in vivo. GSTD promoted the phosphorylation of nuclear factor erythroid‐2‐related factor‐2 (Nrf2) at serine (Ser) 40, stimulated its nuclear translocation and increased hepatic expression of heme oxygenase‐1 (HO‐1). GSTD activated AMP‐activated protein kinase (AMPK), suppressed hepatic steatosis, lowered serum triglyceride (TG)/glucose and decreased body weight gain in animals with NAFLD. The stimulating effects of GSTD on the Nrf2 pathway as well as its antioxidative/antiinflammatory activities were abolished by compound C in OA‐treated HL‐7702 cells. In summary, our results demonstrate that GSTD activates the AMPK/Nrf2 pathway, ameliorates oxidative stress/proinflammatory response and improves lipid metabolism in NAFLD. Our findings may support the future clinical application of GSTD for the treatment of NAFLD to reduce hepatic steatosis, oxidative stress and proinflammatory response. Copyright © 2015 John Wiley & Sons, Ltd.     

Antipyretic and Anti‐inflammatory Effects of Asiaticoside in Lipopolysaccharide‐treated Rat through Up‐regulation of Heme Oxygenase‐1

Asiaticoside (AS), a triterpenoid isolated from Centella asiatica, has been found to exhibit antioxidant and anti‐inflammatory activities in several experimental animal models. However, the underlying mechanisms remain elusive. In this study, we provide experimental evidences that AS dose‐dependently inhibited lipopolysaccharide (LPS)‐induced fever and inflammatory response, including serum tumor necrosis factor (TNF)‐α and interleukin (IL)‐6 production, liver myeloperoxidase (MPO) activity, brain cyclooxygenase‐2 (COX‐2) protein expression and prostaglandin E₂ (PGE₂) production. Interestingly, AS increased serum IL‐10 level, liver heme oxygenase‐1 (HO‐1) protein expression and activity. Furthermore, we found that the suppressive effects of AS on LPS‐induced fever and inflammation were reversed by pretreatment with ZnPPIX, a HO‐1 activity inhibitor. In summary, our results suggest that AS has the antipyretic and anti‐inflammatory effects in LPS‐treated rat. These effects could be associated with the inhibition of pro‐inflammatory mediators, including TNF‐α and IL‐6 levels, COX‐2 expression and PGE₂ production, as well as MPO activity, which might be mediated by the up‐regulation of HO‐1. Copyright © 2012 John Wiley & Sons, Ltd.     

Ethanol extract of Prunella vulgaris var. lilacina inhibits HMGB1 release by induction of heme oxygenase-1 in LPS-activated RAW 264.7 cells and CLP-induced septic mice

The ethanol extract of the flower of P. vulgaris var. lilacina (EEPV) has been used traditionally as an antiinflammatory agent in many countries. Inducers of heme oxygenase‐1 (HO‐1) reduce high mobility group box 1 (HMGB1), a late phase cytokine, in sepsis. Although EEPV has been used as an antiinflammatory agent, no report is available as to whether it modifies HMGB1 in sepsis due to HO‐1 induction. It was found that EEPV increased HO‐1 protein expression in RAW 264.7 cells, which was significantly inhibited by LY294002, but not PD98059, SB203580 or SP600125. In addition, EEPV activated NF‐E2‐related factor (Nrf2) to move from the cytosol to the nucleus, and EEPV‐induced HO‐1 and activation of ARE‐luciferase activity were significantly reduced by siNrf2 transfection and LY294002 but not SB203508. EEPV also significantly inhibited NF‐κB luciferase activity, and decreased both iNOS/NO and COX‐2/PGE₂ production in lipopolysaccharide (LPS)‐stimulated macrophages which was reversed by siHO‐1 RNA transfection. Importantly, EEPV inhibited HMGB1 release in LPS‐activated macrophages in a PI3K‐sensitive manner and reduced serum HMGB1 level and lung HMGB1 expression in cecal ligation and puncture (CLP)‐induced septic mice. It is concluded that EEPV induces HO‐1 expression through PI3K/Nrf2 signal pathways, which may be beneficial for the treatment of sepsis due to a reduction of HMGB1 release. Copyright © 2011 John Wiley & Sons, Ltd.     

Hawthorn (Crataegus oxyacantha L.) Bark Extract Regulates Antioxidant Response Element (ARE)‐Mediated Enzyme Expression Via Nrf2 Pathway Activation in Normal Hepatocyte Cell Line

Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti‐carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE‐2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE‐2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2‐dependent genes as glutathione S‐transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase‐1 (HO‐1) (0.2–1.1‐fold change, p < 0.05), however, only in normal THLE‐2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21–0.42‐fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin‐mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals. Copyright © 2013 John Wiley & Sons, Ltd.     

Paeoniflorin ameliorates ANIT‐induced cholestasis by activating Nrf2 through an PI3K/Akt‐dependent pathway in rats

Cholestasis causes hepatic accumulation of bile acids leading to liver injury, fibrosis and liver failure. Paeoniflorin, the major active compound isolated from the roots of Paeonia lactiflora pall and Paeonia veitchii Lynch, is extensively used for liver diseases treatment in China. However, the mechanism of paeoniflorin's hepatoprotective effect on cholestasis has not been investigated yet. In this study, we administered paeoniflorin to rats for 3 days prior to alpha‐naphthylisothiocyanate (ANIT) administration for once, then went on administering paeoniflorin to rats for 3 days. The data demonstrated that paeoniflorin significantly prevented ANIT‐induced change in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphates (ALP), serum total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA) and gamma‐glutamyl transpeptidase (γ‐GT). Histology examination revealed that paeoniflorin treatment rats relieved more liver injury and bile duct proliferation than ANIT‐administered rats. Moreover, our data indicated that paeoniflorin could restore glutathione (GSH) and its related synthase glutamate‐cysteine ligase catalytic subunit (GCLc) and glutamate‐cysteine ligase modifier subunit (GCLm) in ANIT‐treated group. In addition, the RNA and protein expression of Akt and nuclear factor‐E2‐related factor‐2 (Nrf2) were also activated by paeoniflorin in ANIT‐induced rats. These findings indicated that paeoniflorin protected ANIT‐induced cholestasis and increased GSH synthesis by activating Nrf2 through PI3K/Akt‐dependent pathway. Therefore, paeoniflorin might be a potential therapeutic agent for cholestasis. Copyright © 2015 John Wiley & Sons, Ltd.