Two neutral polysaccharides (BRNP-1, 6.9 kDa; BRNP-2, 4.8 kDa) were purified from the common edible plant Brassica rapa L. via the combined techniques of ion-exchange chromatography and high-performance gel permeation chromatography. Monosaccharide composition analysis showed that BRNP-1 and BRNP-2 were composed of glucosyl residues. Methylation and 1D- and 2D-NMR analyses revealed that both BRNP-1 and BRNP-2 contained a backbone chain that was composed of α-D-(1 → 4)-linked Glcp residues and side chains that were composed of terminally linked Glcp residues attached at the O-6 position of backbone-glycosyl residues. BRNP-1 and BRNP-2, however, differed in branch degree and molecular weight. Bioassay results showed that treatment with the higher dosage (400 μg/mL) of BRNP-1 and BRNP-2 stimulated the proliferation, NO release, and cytokine secretion (IL-6 and TNF-α) of RAW264.7 macrophages. These results suggested that BRNP-1 and BRNP-2 may enhance macrophage-mediated immune responses.

No abstract

Nrf2 (nuclear factor E2-related factor 2, encoded by Nfe2l2) acts as a master transcriptional regulator in mediating antioxidant, detoxification, and cytoprotective responses against oxidative, electrophilic, and metabolic stress, but also plays a crucial role in cancer metabolism and multiple oncogenic pathways, whereas the redox sensor Keap1 functions as a predominant inhibitor of Nrf2 and, hence, changes in its expression abundance directly affect the Nrf2 stability and transcriptional activity. However, nuanced functional isoforms of Keap1 α and β have rarely been identified to date. Herein, we have established four distinct cell models stably expressing Keap1−/−, Keap1β(Keap1Δ1–31), Keap1-Restored, and Keap1α-Restored aiming to gain a better understanding of similarities and differences of two Keap1 isoforms between their distinct regulatory profiles. Our experimental evidence revealed that although Keap1 and its isoforms are still localized in the cytoplasmic compartments, they elicited differential inhibitory effects on Nrf2 and its target HO-1. Furthermore, transcriptome sequencing unraveled that they possess similar but different functions. Such functions were further determined by multiple experiments in vivo (i.e., subcutaneous tumour formation in nude mice) and in vitro (e.g., cell cloning, infection, migration, wound healing, cell cycle, apoptosis, CAT enzymatic activity, and intracellular GSH levels). Of note, the results obtained from tumourigenesis experiments in xenograft model mice were verified based on the prominent changes in the PTEN signaling to the PI3K-AKT-mTOR pathways, in addition to substantially aberrant expression patterns of those typical genes involved in the EMT (epithelial–mesenchymal transition), cell cycle, and apoptosis.

In the past 25 years, Nrf2 (nuclear factor erythroid 2-related factor 2, also called NFE2L2) had been preferentially parsed as a master hub of regulating antioxidant, detoxification, and cytoprotective genes; albeit as a matter of fact that Nrf1 (nuclear factor erythroid 2-related factor 1, also called NFE2L1)—rather than Nrf2—is indispensable for cell homeostasis and organ integrity during normal growth and development. Herein, distinct genotypic cell lines (i.e., Nrf1α−/−, Nrf2−/−ΔTA, and caNrf2ΔN) are employed to determine differential yet integral roles of Nrf1 and Nrf2 in mediating antioxidant responsive genes to tert-butylhydroquinone (tBHQ) serving as a pro-oxidative stressor. In Nrf1α−/− cells, Nrf2 was highly accumulated but also could not fully compensate specific loss of Nrf1α’s function in its basal cytoprotective response against endogenous oxidative stress, though it exerted partially inducible antioxidant response, as the hormetic effect of tBHQ, against apoptotic damages. By contrast, Nrf2−/−ΔTA cells gave rise to a substantial reduction of Nrf1 in both basal and tBHQ-stimulated expression levels and hence resulted in obvious oxidative stress, but it can still be allowed to mediate a potent antioxidant response, as accompanied by a significantly decreased ratio of GSSG (oxidized glutathione) to GSH (reduced glutathione). Conversely, a remarkable increase of Nrf1 expression resulted from the constitutive active caNrf2ΔN cells, which were not manifested with oxidative stress, whether or not it was intervened with tBHQ. Such inter-regulatory effects of Nrf1 and Nrf2 on the antioxidant and detoxification genes (encoding HO-1, NQO1, GCLC, GCLM, GSR, GPX1, TALDO, MT1E, and MT2), as well on the ROS (reactive oxygen species)-scavenging activities of SOD (superoxide dismutase) and CAT (catalase), were further investigated. The collective results unraveled that Nrf1 and Nrf2 make distinctive yet cooperative contributions to finely tuning basal constitutive and/or tBHQ-inducible expression levels of antioxidant cytoprotective genes in the inter-regulatory networks. Overall, Nrf1 acts as a brake control for Nrf2’s functionality to be confined within a certain extent, whilst its transcription is regulated by Nrf2.

Transcription factor Nrf2 (nuclear factor, erythroid 2-like 2, encoded by Nfe2l2) has been accepted as a key player in redox regulatory responses to oxidative or reductive stresses. However, relatively little is known about the potential role of Nrf1 (nuclear factor, erythroid 2-like 1, encoded by Nfe2l1) in the redox responses, particularly to reductive stress, although this ‘fossil-like’ factor is indispensable for cell homeostasis and organ integrity during the life process. Herein, we examine distinct roles of Nrf1 and Nrf2 in monitoring the defense response to 1,4–dithiothreitol (DTT, serving as a reductive stressor), concomitantly with unfolded protein response being induced by this chemical (also defined as an endoplasmic reticulum stressor). The results revealed that intracellular reactive oxygen species (ROS) were modestly increased in DTT-treated wild-type (WT) and Nrf1α−/− cell lines, but almost unaltered in Nrf2−/−ΔTA or caNrf2ΔN cell lines (with a genetic loss of transactivation or N-terminal Keap1-binding domains, respectively). This chemical treatment also enabled the rate of oxidized to reduced glutathione (i.e., GSSG to GSH) to be amplified in WT and Nrf2−/−ΔTA cells, but diminished in Nrf1α−/− cells, along with to or less extents changes in caNrf2ΔN cells. Consequently, Nrf1α−/−, but not Nrf2−/−ΔTA or caNrf2ΔN, cell viability was reinforced by DTT against its cytotoxicity, as accompanied by decreased apoptosis. Further experiments unraveled that Nrf1 and Nrf2 differentially, and also synergistically, regulated DTT-inducible expression of critical genes for defending against redox stress and endoplasmic reticulum stress. In addition, we also identified that Cys342 and Cys640 of Nrf1 (as redox-sensing sites within its N-glycodomain and DNA-binding domain, respectively) are required for its protein stability and transcription activity.

No abstract

Nrf2 plays a crucial role in the management of oxidative and electrophilic stress. Nrf2 acts as a master regulator of oxidative or metabolic stress responses and is involved in cancer cell metabolism and multiple oncogenic pathways. Keap1 is the main inhibitor of Nrf2, and its high or low expression also causes changes in Nrf2. The function of Keap1 isoforms has rarely been reported. Here, to gain a better understanding of their similarities and differences in distinct regulatory profiles, four distinct cell models stably expressing Keap1-/-, Keap1[beta]; (Keap1[Delta];1-31), Keap1-Restored or Keap1[alpha];-Restored have been established. Immunofluorescence localization and nucleocytoplasmic separation experiments found that Keap1 and its isoforms were localized in the cytoplasm and had inhibitory effects on Nrf2 and HO-1. Further transcriptome analysis revealed that they had similar and different functions. Their functions were further explored in vivo (subcutaneous tumour formation in nude mice) and in vitro (cell cloning, infection, migration, wound healing, CAT, GSH, cell cycle and apoptosis). Next, the results of tumorigenesis experiments in nude mice were verified based on changes in the PTEN and PI3K-mTOR pathways. Finally, some typical genes of EMT, cell cycle and apoptosis were detected, and the experimental results were verified by in vitro experiments.

Transcription factor Nrf2 (nuclear factor, erythroid 2-like 2, encoded by Nfe2l2) has been accepted as a key player in redox regulatory responses to oxidative or reductive stresses. However, it is less or not known about the potential role for Nrf1 (nuclear factor, erythroid 2-like 1, encoded by Nfe2l1) in the redox responses, particularly to reductive stress, albeit this fossil-like factor is indispensable for cell homeostasis and organ integrity during life process. Here, we examine distinct roles of Nrf1 and Nrf2 in monitoring the defense response to 1,4-dithiothreitol (DTT, serving as a reductive stressor), concomitantly with unfolded protein response being induced by this chemical (also as an endoplasmic reticulum stressor). The results revealed that intracellular reactive oxygen species (ROS) were modestly increased in DTT-treated wild-type (WT) and Nrf1α-∕- cell lines, but almost unaltered in Nrf2-∕-ΔTA or caNrf2ΔN cell lines (with a genetic loss of its transactivation or N-terminal Keap1-binding domains, respectively). This chemical treatment also enabled the rate of oxidized to reduced glutathione (i.e., GSSG to GSH) to be amplified in WT and Nrf2-∕-ΔTA cells, but diminished in Nrf1α-∕- cells, along with no changes in caNrf2ΔN cells. Consequently, Nrf1α-∕-, but not Nrf2-∕-ΔTA or caNrf2ΔN, cell viability was reinforced by DTT against its cytotoxicity, as accompanied by decreased apoptosis. Further experiments unraveled that Nrf1 and Nrf2 differentially, and also synergistically, regulated DTT-inducible expression of critical genes for defending redox stress and endoplasmic reticulum stress. In addition, we have also identified that Cys342 and Cys640 of Nrf1 (as redox-sensing sites within its N-glycodomain and DNA-binding domain, respectively) are required for its protein stability and transcription activity.