Resveratrol protects human keratinocytes HaCaT cells from UVA-induced oxidative stress damage by downregulating Keap1 expression

Ultraviolet radiation A (UVA)-induced oxidative stress is recognized as an important factor in the development of skin carcinogenesis. Resveratrol is demonstrated to possess remarkable antioxidant activity in the organism. The aim of this study was to investigate the protective role of resveratrol in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism of the translocation of NF-E2-related factor-2 (Nrf2) into the nucleus. The HaCaT cells were UVA-irradiated and the effects of resveratrol on cell viability, reactive oxygen species generation and membrane-lipid peroxidation were measured. The proteins and mRNA of Nrf2 and Kelch-like-ECH-associated protein 1 (Keap1) were determined by immunofluorescence staining, Western blot and quantitative PCR, respectively. UVA exposure led to a decrease in viability and an increase in reactive oxygen species generation in HaCaT cells. Resveratrol could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, resveratrol increased the level of Nrf2 protein and facilitated Nrf2 accumulation in the nucleus; as a result, the activity of antioxidant enzymes was also upregulated. The main finding was that Keap1 protein, a repressor of Nrf2 in the cytoplasm, was clearly decreased by resveratrol treatment 12 h and beyond though the level of Keap1 mRNA still increased. Our results suggest that resveratrol can degrade Keap1 protein and facilitate Nrf2 accumulation in the nucleus, thereby protecting HaCaT cells from UVA-induced oxidative stress. Resveratrol could be a more useful natural medicine for the protection of epidermal cells from UVA-induced damage.     

Epigallocatechin‐3‐Gallate Attenuates Oxidative Stress and Inflammation in Obstructive Nephropathy via NF‐κB and Nrf2/HO‐1 Signalling Pathway Regulation

Oxidative stress and inflammation contribute importantly to the pathogenesis of chronic kidney disease (CKD). Epigallocatechin‐3‐gallate (EGCG), which is the most abundant and most active catechin polyphenol extracted from green tea, has been proved to have many bioactivities. In this study, the renoprotective effect of EGCG was evaluated in a widely used kidney disease model, the unilateral ureteral obstruction (UUO) mice model. After 14 days of EGCG administration, mean arterial blood pressure, body‐weight and obstructed kidney weight were measured. Levels of blood urea nitrogen (BUN) and creatinine (CR) and activities of glutamic–pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) in serum were estimated as indicators of renal function. Periodic acid–Schiff (PAS) staining was performed to observe the pathological changes of the obstructed kidney. Antioxidant enzymes and pro‐inflammatory cytokine production were estimated to reflect the oxidative stress and inflammatory state in the obstructed kidney. Finally, the main proteins in the NF‐κB and Nrf2 signalling pathway and DNA binding activity of NF‐κB and Nrf2 were measured to investigate the effect of EGCG on these two pathways. The results demonstrated that EGCG could restore UUO‐induced kidney weight loss and renal dysfunction. In addition, UUO‐induced oxidative stress and inflammatory responses in the obstructed kidney were also prevented by EGCG. Furthermore, EGCG could induce both NF‐κB and Nrf2 nuclear translocation in the UUO kidney and promote heme oxygenase‐1 (HO‐1) production. These results indicated that the renoprotective effect of EGCG might be through its NF‐κB and Nrf2 signalling pathway regulations.     

Regulation of Eclipta prostrata L. components on cigarette smoking‐induced autophagy of bronchial epithelial cells via keap1‐Nrf2 pathway

Cigarette smoking extract (CSE)‐induced autophagic injury has been regarded as an important contributor to the pathogenesis of lung cancer. We previously found that Eclipta prostrata L. component (CCE) reduced CSE‐induced bronchial epithelial cells damage. However, the mechanism remains unknown. Human normal bronchial epithelial cells (NHBE) were exposed to CSE to establish stress model. Nrf2‐siRNA and Keap1‐siRNA transfection were performed. mRFP‐GFP‐LC3 dual fluorescence and transmission electron microscopy were used to observe the autophagic characteristics. CCE prevented CSE‐induced Nrf2 transfer into cytoplasm and up‐regulated Keap1 level of NHBE cells. Furthermore, CCE significantly increased p‐p16, p‐p21 and p‐p53 phosphorylation levels in Nrf2‐siRNA‐ or Keap1‐siRNA‐transfected cells. As demonstrated by transmission electron microscopy and mRFP‐GFP‐LC3 dual fluorescence assays, CCE mitigated autophagic injury, and also down‐regulated autophagy‐related Beclin‐1, LC3II/LC3I ratio, Atg5 and ATF4 levels. Our findings showed the attenuation of CCE on CSE‐induced NHBE cells injury was associated with Nrf‐2‐mediated oxidative signaling pathway.     

Reduced Nrf2 activation in PI3K phosphorylation‐impaired vitiliginous keratinocytes increases susceptibility to ROS‐generating chemical‐induced apoptosis

Keratinocytes in affected epidermis of vitiligo patients are known to have impaired activation of the PI3K/AKT pathway. Based on critical roles of keratinocytes and oxidative stress in vitiligo development, this study examined whether keratinocytes with impaired PI3K activation were more vulnerable to apoptosis caused by oxidative stress from phenolic compounds, p‐tert‐butylphenol (4‐TBP) and hydroquinone (HQ). Cell viability assay, FACS analysis, ELISA for TNF‐α or IL‐1a, ROS assay, Western blot analysis for Nrf2 expression, and confocal microscopy with anti‐Nrf2 and phospho‐PI3K antibodies were done on primary cultured normal human keratinocytes with or without PI3K knockdown in the presence or absence of chemical treatment or antioxidant. Immunofluorescence staining using anti‐Nrf2, phospho‐PI3K, TNF‐ɑ, and IL‐1ɑ antibodies, ROS assay using dihydroethidium, and TUNEL assay were performed on sets of depigmented and normally pigmented skin from vitiligo patients. Results showed that 4‐TBP or HQ treatment increased apoptosis and the expression levels of TNF‐ɑ, IL‐1ɑ, and ROS in PI3K‐knockdown keratinocytes which reduced Nrf2 nuclear translocation compared to control keratinocytes. These changes were significantly recovered by an antioxidant treatment. Depigmented epidermis of vitiligo patients also showed lower levels of Nrf2 and phospho‐PI3K but higher levels of ROS, TNF‐ɑ, IL‐1ɑ, and ROS with more TUNEL‐positive cells. Therefore, impaired PI3K activation in keratinocytes in depigmented epidermis of vitiligo patients are vulnerable to apoptosis caused by ROS‐generating chemicals due to reduced Nrf2 activation.     

Di (2‐ethylhexyl) phthalate induces cytotoxicity in HEK‐293 cell line,implication of the Nrf‐2/HO‐1 antioxidant pathway

The di (2‐ethylhexyl) phthalate (DEHP) is a plasticizer used in the polyvinyl chloride industry. Human exposure to this plasticizer is inevitable and contributes to several side effects. In this study, we examined whether DEHP induces apoptosis and oxidative stress in embryonic kidney cells (HEK‐293) and whether the nuclear factor E2‐related factor 2 (Nrf‐2)/heme oxygenase‐1 (HO‐1) antioxidant pathway is involved in the pathogenesis of this process. We demonstrated that DEHP is cytotoxic to HEK‐293 cells. It causes oxidative damage through the generation of free radicals, induces lipid peroxidation, and alters superoxide dismutase and catalase activities. Simultaneously, DEHP treatment decreases the expression and the protein level of Nrf‐2 and HO‐1. Inhibition of the Nrf‐2/HO‐1 pathway is related to the mitochondrial pathway of apoptosis. This apoptotic process is characterized by a loss of mitochondrial transmembrane potential (ΔΨm) and upregulation of the expression of caspase‐3 mRNA as well as its protein level.     

(‐)‐Epigallocatechin Gallate Inhibits Endothelin‐1‐Induced C‐Reactive Protein Production in Vascular Smooth Muscle Cells

Abstract: Numerous studies have shown that C‐reactive protein (CRP), a pro‐inflammation cytokine, makes a direct contribution to atherosclerosis, and that (–)‐epigallocatechin gallate (EGCG) is able to exert an anti‐atherosclerotic effect by anti‐oxidative and anti‐inflammatory activities. Based on our previous study, the effect of EGCG on endothelin‐1 (ET‐1)‐induced CRP production in rat vascular smooth muscle cells (VSMCs) and the possible mechanism were observed. The in vitro experiments showed that EGCG concentration‐dependently inhibited ET‐1‐stimulated expression of CRP both in protein and mRNA levels in VSMCs as determined by the immunocytochemical staining, the enzyme‐linked immunosorbent assay and the real‐time quantitative polymerase chain reaction (RT‐qPCR). The in vivo investigation with the double‐labelled immunofluorescence staining and RT‐qPCR displayed that EGCG also prevented ET‐1‐induced CRP expression in protein and mRNA levels in the aortic VSMCs of rats receiving the subchronic infusion of ET‐1. In addition, EGCG suppressed reactive oxygen species (ROS) generation evoked by ET‐1 in VSMCs as observed by the fluorescence probe. These demonstrate that EGCG may inhibit ET‐1‐stimulated generation of CRP in VSMCs so to relieve the inflammatory response and oxidative stress via blocking ROS signal, which provides new evidence for an anti‐atherosclerotic effect of EGCG.     

The natural compound 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐d glucoside protects against adriamycin‐induced nephropathy through activating the Nrf2‐Keap1 antioxidant pathway

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐d ‐glucoside (THSG) is an active compound extracted from Polygonum multiflorum Thunb. This herb and radix Polygoni Multiflori preparata have been used to treat arteriosclerosis, hyperlipidemia, hypercholesterolemia, and diabetes for thousands of years. This study aimed to investigate the protective effects of THSG in an Adriamycin (AD)‐induced focal segmental glomerulosclerosis (FSGS) mouse model and the underlying mechanisms in an in vitro system. Mice were treated with THSG (2.5 and 10 mg/kg, oral gavage) for 24 consecutive days. On the third day, mice were intravenously given a single dose of AD (10 mg/kg). At the end of the experiment, plasma and kidney samples were harvested to evaluate the therapeutic effects of THSG. The potential mechanisms of THSG in protecting against AD‐induced cytotoxicity were examined using a real‐time polymerase chain reaction, immunoblots, lactate dehydrogenase assay, and a cellular oxidized‐thiol detection system in a mouse mesangial cell line. In this study, THSG showed concentration‐dependent protective effects in ameliorating the progression of AD‐induced FSGS. THSG suppressed albuminuria and hypercholesterolemia and reduced the status of lipid peroxidation in urine, plasma, and kidney tissue samples. Furthermore, THSG protected against podocyte damage, reduced renal fibrotic gene expressions, and alleviated the severity of glomerulosclerosis. Treatment of mouse mesangial cells with THSG induced nuclear factor erythroid‐derived 2‐like 2 (Nrf2) nuclear translocation, increased heme oxygenase‐1 and NAD(P)H:quinone oxidoreductase (NQO)‐1 gene expressions, and reduced cellular thiol oxidation and resistance to AD‐induced cytotoxicity. Silencing Nrf2 and its repressor protein, Kelch‐like ECH‐associated protein 1 (Keap1), abolished these protective effects of THSG. In conclusion, THSG can play a protective role in ameliorating the progression of FSGS in a mouse model through activation of the Nrf2‐Keap1 antioxidant pathway. Although a well‐designed therapeutic study is needed, THSG may be applied to manage chronic kidney disease.     

PYDDT,a novel phase 2 enzymes inducer,activates Keap1–Nrf2 pathway via depleting the cellular level of glutathione

Keap1–Nrf2 pathway has emerged as a regulator for the endogenous antioxidant response, which is critical in defending cells against carcinogenesis. Herein, we demonstrated that depleting the cellular level of glutathione (GSH) by a novel electrophilic agent 2-(pro-1-ynyl)-5-(5,6-dihydroxypenta-1,3-diynyl) thiophene (PYDDT) could activate Keap1–Nrf2 pathway. In above process, it was found that Keap1 was modified by S-glutathionylation, an important post-translational modification of protein cysteines with critical roles in oxidative stress and signal transduction. We concluded from our findings that conjugation with intracellular GSH by PYDDT might lead to Keap1 S-glutathionylation and was a key event involved in its Nrf2 inducing activity.     

Epigallocatechin gallate potentially abrogates fluoride induced lung oxidative stress,inflammation via Nrf2/Keap1 signaling pathway in rats: An in-vivo and in-silico study

BackgroundSince this Nrf2-dependent cellular defense response is able to protect multi-organs, including cancer, neurodegenerative diseases, cardiovascular diseases, inflammation and chronic lung injury. The antioxidant and anti-inflammatory potential of Epigallocatechin gallate (EGCG) and Nrf2/Keap1 signaling mechanisms in pulmonary toxicity have not been clarified. In the present study, we demonstrated that protective efficacy of EGCG against fluoride (Fl) induced oxidative stress mediated lung injury in rats.MethodsThe animals were divided in to four groups. Group 1: Control rats received normal saline; Group 2 rats received EGCG (40 mg/kg/bw) alone for four weeks; Group 3 rats received Fl (25 mg/kg/bw) alone for four weeks, Group 4 rats received EGCG (90 min before administration) along with Fl for four weeks.ResultsOral administration of Fl (25 mg/kg/bw) significantly (p < 0.05) increased the ROS, inflammatory cytokines, lung edema, melonaldehyde (MDA) and myeloperoxidase (MPO) in rats. In addition, upon administration of Fl significantly (p < 0.05) decreased the antioxidant status, Nrf2, and HO-1 with increased Keap1 protein. Histological and immunohistochemical (iNOS) study also revealed the Fl induced significant (p < 0.05) changes in the lung tissue of rats. Pre-administration of EGCG significantly (p < 0.05) improved the antioxidant status, and inhibited the oxidative stress, inflammatory cytokines, and Keap1 protein via the activation of Nrf2 translocation in to the nucleus. Moreover, the molecular docking studies also support the antioxidant potential of EGCG and Nrf2 activation.ConclusionTaken together, our data indicate that EGCG potentially abrogates Fl induced oxidative lung injury by activation of the Nrf2/Keap1 pathway in rats.     

Icaritin activates Nrf2/Keap1 signaling to protect neuronal cells from oxidative stress

Our earlier study indicated that icaritin (ICT) protected mice from cerebral ischemic injury by inhibiting oxidative stress, and this study aimed to investigate its mechanism using a H₂O₂‐treated SH‐SY5Y cells model. Cell viability was assessed by cell counting kit 8 (CCK‐8). Oxidative stress parameters were detected by flow cytometry, and signaling pathways were analyzed by immunoblotting. We found that ICT alleviated apoptosis and intracellular and mitochondrial reactive oxygen species (ROS) levels, decreased the expressions of Bax and cleaved caspase‐3, and increased the expressions of Bcl‐2 compared to H₂O₂ group. ICT increased mitochondrial membrane potential (ΔΨm) and blocked the opening of mitochondrial membrane permeability transporter (MPT), and increased the activity of glutathione peroxidase (GSH‐px), catalase (CAT), and superoxide dismutase (SOD), meanwhile, decreased the activity of malondialdehyde (MDA) compared to H₂O₂ group. Further investigation revealed that ICT significantly up‐regulated the expressions of nuclear factor erythroid 2‐related factor 2 (Nrf2), heme oxygenase 1 (HO‐1) and NAD(P)H‐quinone oxidoreductase 1 (NQO‐1). The anti‐apoptosis and antioxidative effects of ICT were blocked bay ML385, a Nrf2/Keap1 signaling pathway inhibitor. These results indicate that ICT can play a neuroprotective role against oxidative stress injury by activating Nrf2/Keap1 signaling pathway.     

Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation

Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6 h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H₂DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis.