Extract of Ginkgo biloba induces phase 2 genes through Keap1-Nrf2-ARE signaling pathway

The standard extract of Ginkgo biloba (EGb) has been demonstrated to possess remarkable antioxidant activity in both cell lines and animals. However, the molecular mechanism underlying this effect is not fully understood. Phase 2 enzymes play important roles in the antioxidant system by reducing electrophiles and reactive oxygen species (ROS). We demonstrated that EGb induced typical phase 2 genes: glutamate cysteine ligase catalytic subunit (GCLC) and glutathione-S-transferase subunit-P1 (GST-P1), by real-time PCR. To investigate the molecular mechanism of this induction, we used quinone oxidoreductase 1 (NQO1) -- Antioxidant response element (ARE) reporter assay and found that EGb activated the activity of the wild type but not the one with ARE mutated. It indicated that EGb induced these genes through ARE, a cis-acting motif located in the promoter region of nearly all phase 2 genes. Since nuclear factor erythroid 2-related factor 2 (Nrf2) binds ARE to enhance the expression of phase 2 genes, we detected the Nrf2 content in nucleus and found an accumulation of Nrf2 stimulated by EGb. In a further test of Kelch-like ECH-associated protein 1 (Keap1), the repression protein of Nrf2 in the cytosol under resting condition, we found that Keap1 content was inhibited by EGb and then more Nrf2 would be released to translocate into nucleus. Thus, EGb was testified for the first time to induce the phase 2 genes through the Keap1-Nrf2-ARE signaling pathway, which is (or part of) the antioxidant mechanism of EGb.     

Dietary supplementation with sulforaphane ameliorates skin aging through activation of the Keap1-Nrf2 pathway

Visible impairments in skin appearance, as well as a subtle decline in its functionality at the molecular level, are hallmarks of skin aging. Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-pathway, which is important in controlling inflammation and oxidative stress that occur during aging, can be triggered by sulforaphane (SFN), an isothiocyanate found in plants from the Brassicaceae family. This study aimed to assess the effects of SFN intake on age-related skin alterations. Male C57BL6 young (2 months) and old (21 months) mice were treated for 3 months with SFN diet (442.5 mg per kg) or control diet. The antioxidant capacities of the skin were increased in old SFN-treated animals as measured by mRNA levels of Nrf2 (P<.001) and its target genes NQO1 (P<.001) and HO1 (P<.01). Protein expression for Nrf2 was also increased in old SFN fed animals (P<.01), but not the protein expression of NQO1 or HO1. Additionally, ROS and MMP9 protein levels were significantly decreased (P<.05) in old SFN fed animals. Histopathological analysis confirmed that there was no difference in epidermal thickness in old, when compared to young, SFN treated animals, while the dermal layer thickness was lower in old vs. young, treated animals (P<.05). Moreover, collagen deposition was improved with SFN treatment in young (P<.05) and structurally significantly improved in the old mice (P<.001). SFN dietary supplementation therefore ameliorates skin aging through activation of the Nrf2-pathway.     

Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2/Keap1 signaling pathway and decreasing ROS production

Oxidative stress (OS) plays a major role in the gastrointestinal disorders. Although probiotics were reported to repress OS, few researches compared the antioxidant ability of different Bacillus strains and deciphered the mechanisms. To select a Bacillus strain with higher antioxidant capacity, we used H₂O₂ to induce intestinal porcine epithelial cell 1 (IPEC-1) OS model. The most suitable H₂O₂ concentration and incubation time were determined by the half lethal dose and methyl thiazolyl tetrazolium. Correlation analysis was performed to choose a sensitive indicator for OS. As for the comparison of Bacillus, cells were divided into control, Bacillus treatment, H₂O₂ treatment, and Bacillus pre-protection + H₂O₂ treatment. Bacillus were co-cultured with IPEC-1 for 3 h in Bacillus and Bacillus pre-protection + H₂O₂ treatments. Then, based on OS model, 300 μmol/L H₂O₂ was added into medium of H₂O₂ and Bacillus pre-protection + H₂O₂ treatments for another 12 h. Antioxidant and apoptosis gene expressions were detected to screen the target strain. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) pathway, reactive oxygen species (ROS) production, mitochondrial membrane potential (Δψm), apoptosis, and necrosis were analyzed. Results revealed that heme oxygenase-1 (HO-1) gene expression had a positive correlation with H₂O₂ induction. Moreover, Bacillus amyloliquefaciens SC06 (SC06)-meditated IPEC-1 showed the best antioxidant capacity though modulating Nrf2 phosphorylation. Δψm was elevated, while ROS generation was reduced with SC06 pre-protection, resulting in decreased apoptosis and necrosis. Altogether, HO-1 expression could be regarded as an OS indicator. The regulation of Nrf2/Keap1 pathway and ROS production by SC06 are involved in alleviating OS of IPEC-1.

     

Exploring the correlation between deltamethrin stress and Keap1-Nrf2-ARE pathway from Drosophila melanogaster RNASeq data

Deltamethrin (DM) is widely used in a variety of pest control, resulting in serious drug resistance. Keap1-Nrf2-ARE is the antioxidant stress pathway. We identified 268 genes differentially expressed (DEGs) in Drosophila Kc cells treated with DM, including up-regulated 180 genes and down-regulated 88 genes compared with the control group (fold-change≥2, qValue≤0.001) by RNA-seq, they are mainly linked to metabolic process, stimulation response, immune system process. When the cells are treated with DM in the case of overexpression of the Keap1 gene, the cytochrome P450 family genes were significantly down-regulated, and some diseases-related genes and non-coding genes also changed. Our data shown that Keap1-Nrf2-ARE pathway may play an important role in DM stress, which will provide a new direction for studying the mechanism of insect resistance.     

p62/SQSTM1 protects against cisplatin-induced oxidative stress in kidneys by mediating the cross talk between autophagy and the Keap1-Nrf2 signalling pathway

Acute kidney injury (AKI) is a major kidney disease associated with poor clinical outcomes. Oxidative stress is predominantly involved in the pathogenesis of AKI. Autophagy and the Keap1-Nrf2 signalling pathway are both involved in the oxidative-stress response. However, the cross talk between these two pathways in AKI remains unknown. Here, we found that autophagy is upregulated during cisplatin-induced AKI. In contrast with previous studies, we observed a marked increase in p62. We also found that p62 knockdown reduces autophagosome formation and the expression of LC3II. To explore the cross talk between p62 and the Keap1-Nrf2 signalling pathway, HK-2 cells were transfected with siRNA targeting Nrf2, and we found that Nrf2 knockdown significantly reduced cisplatin-induced p62 expression. Moreover, p62 knockdown significantly decreased the protein expression of Nrf2, as well as Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1), whereas the expression of kelch-like ECH-associated protein 1 (Keap1) was upregulated. These results indicate that p62 creates a positive feedback loop in the Keap1-Nrf2 signalling pathway. Finally, we examined the role of p62 in cell protection during cisplatin-induced oxidative stress, and we found that p62 silencing in HK-2 cells increases apoptosis and reactive oxygen species (ROS) levels, which further indicates the protective role of p62 under oxidative stress and suggests that the cytoprotection 62 mediated is in part by regulating autophagic activity or the Keap1-Nrf2 signalling pathway. Taken together, our results have demonstrated a reciprocal regulation of p62, autophagy and the Keap1-Nrf2 signalling pathway under oxidative stress, which may be a potential therapeutic target against AKI.     

LncRNA-cCSC1 modulates cancer stem cell properties in colorectal cancer via activation of the Hedgehog signaling pathway

Several long noncoding RNAs (lncRNAs) have been identified in various malignant tumors and determined to contribute to the process of tumorigenesis, including that of colorectal cancer (CRC). Cancer stem cells (CSCs) have been demonstrated to promote the expansion and maintain the invasion and metastasis of cancer cells, owing to their self-renewal capacity. However, the underlying modulation mechanism of CSC-associated lncRNAs in CRC remains largely unclear. Using integrated bioinformatic analysis, we identified a novel lncRNA (lncRNA-cCSC1) that is highly expressed in CRC and colorectal cancer stem cells (CRCSCs). The biological functions of lncRNA-cCSC1 were assessed in vitro and vivo through the silencing or upregulation of its expression. The depletion of lncRNA-cCSC1 markedly inhibited the self-renewal capacity of the CRCSCs and reduced their drug resistance to 5-fluorouracil. In contrast, lncRNA-cCSC1 overexpression increased the self-renewal effect. Furthermore, aberrant lncRNA-cCSC1 expression resulted in a concomitant alteration of smoothened (SMO) and GLI family zinc finger 1 (Gli1) expression in the Hedgehog (Hh) signaling pathway. Our study is the first to identify a novel lncRNA-cCSC1 in CRC and to indicate that it may regulate CSC-like properties via the Hh signaling pathway. Thus, lncRNA-cCSC1 could be a potential biomarker and promising therapeutic target for CRC.     

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages

Genipin, an aglycon of geniposide, has been reported to exhibit diverse pharmacological functions such as antitumor and anti-inflammatory effects. This study aimed to elucidate the anti-inflammatory mechanism of genipin, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. In RAW264.7 cells, genipin increased HO-1 expression and its enzyme activity via a NF-E2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. Additional experiments showed that the activation of c-Jun NH₂-terminal kinase 1/2 (JNK1/2) is required for genipin-induced phosphorylation and nuclear translocation of Nrf2 and antioxidant response element (ARE)-driven induction of HO-1, and acts as a downstream effector of PI 3-kinase. Furthermore, functional significance of HO-1 induction was revealed by genipin-mediated inhibition of lipopolysaccharide-stimulated inducible nitric oxide synthase expression or cyclooxygenase-2 promoter activity, the response was reversed by the blocking of HO-1 protein synthesis or HO-1 enzyme activity. Therefore, identification of PI 3-kinase-JNK1/2-Nrf2-linked signaling cascade in genipin-mediated HO-1 expression defines the signaling event that could participate in genipin-mediated anti-inflammatory response.     

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages

A large majority of the 1000–1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K_D of 360 ± 30 nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated.     

Enhanced Keap1-Nrf2/Trx-1 axis by daphnetin protects against oxidative stress-driven hepatotoxicity via inhibiting ASK1/JNK and Txnip/NLRP3 inflammasome activation

BackgroundOxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF).AimsTo investigate the protective effect of daphnetin (Daph) on tert-butyl hydroperoxide (t-BHP) and acetaminophen (APAP)-induced hepatotoxicity through altering Nrf2/Trx-1 pathway activation.Materials and methodsIn vivo, male C57BL/6 mice with Wild-type (WT) and Nrf2^−/− were divided into five groups and acute liver injury model were established by APAP or LPS/GalN after injection with Daph (20, 40, or 80 mg/kg), seperately. Then, liver tissue and serum were collected for biochemical determination, TUNEL and H & E staining, and western blot analysis. In vitro, HepG2 cells were used to investigate the protective effect and mechanism of daphnetin against ROS and apoptosis induced by t-BHP via apoptosis detection, western blot, immunofluorescence analysis, and sgRNA transfection.ResultsOur results indicated that Daph efficiently inhibited t-BHP-stimulated hepatotoxicity, and modulated Trx-1 expression and Nrf2 activation which decreased Keap1-overexpression in HepG2 cells. Moreover, Daph inhibited t-BHP-excited hepatotoxicity and enhanced Trx-1 expression, which was reversed in Nrf2^−/− HepG2 cells. In vivo, a survival rate analysis first suggested that Daph significantly reduced the lethality induced by APAP or GalN/LPS in a Nrf2-dependent or -independent manner by using Nrf2^−/− mice, respectively. Next, further results implicated that Daph not only effectively alleviated APAP-induced an increase of ALT and AST levels, histopathological changes, ROS overproduction, malondialdehyde (MDA) formation and GSH/GSSG reduction, but it also relieved hepatic apoptosis by strengthening the suppression of cleaved-caspase-3 and expression of P53 protein. Additionally, Daph attenuated mitochondrial dysfunction by suppressing ASK1/JNK activation and decreasing apoptosis-inducing factor (AIF) and Cytochrome c release and Bax mitochondrial translocation. Daph inhibited inflammatory responses by inactivating the thioredoxin-interacting protein (Txnip)/NLRP3 inflammasome. Furthermore, Daph efficiently enhanced Nrf2 nuclear translocation and Trx-1 expression. However, these effects in WT mice were eliminated in Nrf2^−/− mice.ConclusionsThese investigations demonstrated that Daph treatment has protective potential against oxidative stress-driven hepatotoxicity by inhibition of ASK1/JNK and Txnip/NLRP3 activation, which may be strongly related to the Nrf2/Trx-1 upregulation.     

Hydrogen‐rich water alleviates cyclosporine A‐induced nephrotoxicity via the Keap1/Nrf2 signaling pathway

Oxidative stress induced by long‐term cyclosporine A (CsA) administration is a major cause of chronic nephrotoxicity, which is characterized by tubular atrophy, tubular cell apoptosis, and interstitial fibrosis in the progression of organ transplantation. Although hydrogen‐rich water (HRW) has been used to prevent various oxidative stress‐related diseases, its underlying mechanisms remain unclear. This study investigated the effects of HRW on CsA‐induced nephrotoxicity and its potential mechanisms. After administration of CsA (25 mg/kg/day), rats were treated with or without HRW (12 mL/kg) for 4 weeks. Renal function and vascular activity were investigated. Histological changes in kidney tissues were analyzed using Masson's trichrome and terminal deoxynucleotidyl transferase dUTP nick‐end labeling stains. Oxidative stress markers and the activation of the Kelch‐like ECH‐associated protein 1 (Keap1)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signaling pathway were also measured. We found that CsA increased the levels of reactive oxygen species (ROS) and malonaldehyde (MDA), but it reduced glutathione (GSH) and superoxide dismutase (SOD) levels. Such alterations induced vascular dysfunction, tubular atrophy, interstitial fibrosis, and tubular apoptosis. This was evident secondary to an increase in urinary protein, serum creatinine, and blood urea nitrogen, ultimately leading to renal dysfunction. Conversely, HRW decreased levels of ROS and MDA while increasing the activity of GSH and SOD. This was accompanied by an improvement in vascular and renal function. Moreover, HRW significantly decreased the level of Keap1 and increased the expression of Nrf2, NADPH dehydrogenase quinone 1, and heme oxygenase 1. In conclusion, HRW restored the balance of redox status, suppressed oxidative stress damage, and improved kidney function induced by CsA via activation of the Keap1/Nrf2 signaling pathway.