Neuroprotective effect of acetoxypachydiol against oxidative stress through activation of the Keap1-Nrf2/HO-1 pathway.

BACKGROUND: Excessive oxidative stress in the brain is an important pathological factor in neurological diseases. Acetoxypachydiol (APHD) is a lipophilic germacrane-type diterpene extracted as a major component from different species of brown algae within the genus Dictyota. There have been no previous reports on the pharmacological activity of APHD. The present research aims to explore the potential neuroprotective properties of APHD and its underlying mechanisms. METHODS: The possible mechanism of APHD was predicted using a combination of molecular docking and network pharmacological analysis. PC12 cells were induced by H2O2 and oxygen-glucose deprivation/reoxygenation (OGD/R), respectively. Western blot, flow cytometry, immunofluorescence staining, and qRT-PCR were used to investigate the antioxidant activity of APHD. The HO-1 inhibitor ZnPP and Nrf2 gene silencing were employed to confirm the influence of APHD on the signaling cascade involving HO-1, Nrf2, and Keap1 in vitro. RESULTS: APHD exhibited antioxidant activity in both PC12 cells subjected to H2O2 and OGD/R conditions by downregulating the release of LDH, the concentrations of MDA, and ROS, and upregulating SOD, GSH-Px, and GSH concentrations. APHD could potentially initiate the Keap1-Nrf2/HO-1 signaling cascade, according to the findings from network pharmacology evaluation and molecular docking. Furthermore, APHD was observed to increase Nrf2 and HO-1 expression at both mRNA and protein levels, while downregulating the protein concentrations of Keap1. Both Nrf2 silencing and treatment with ZnPP reversed the neuroprotective effects of APHD. CONCLUSIONS: APHD activated antioxidant enzymes and downregulated the levels of LDH, MDA, and ROS in two cell models. The neuroprotective effect is presumably reliant on upregulation of the Keap1-Nrf2/HO-1 pathway. Taken together, APHD from brown algae of the genus Dictyota shows potential as a candidate for novel neuroprotective agents.

Methyl 3,4-dihydroxybenzoate alleviates oxidative damage in granulosa cells by activating Nrf2 antioxidant pathway.

Oxidative damage induced granulosa cells (GCs) apoptosis was considered as a significant cause of compromised follicle quality, antioxidants therapy has emerged as a potential method for improving endometriosis pregnancy outcomes. Here, we found that GCs from endometriosis patients show increased oxidative stress level. Methyl 3,4-dihydroxybenzoate (MDHB), a small molecule compound that is extracted from natural plants, reversed tert-butyl hydroperoxide (TBHP) induced GCs oxidative damage. Therefore, the aim of this study was to assess the protective effect of MDHB for GCs and its potential mechanisms. TUNEL staining and immunoblotting of cleaved caspase-3/7/9 showed MDHB attenuated TBHP induced GCs apoptosis. Mechanistically, MDHB treatment decreased cellular and mitochondria ROS production, improved the mitochondrial function by rescuing the mitochondrial membrane potential (MMP) and ATP production. Meanwhile, MDHB protein upregulated the expression of vital antioxidant transcriptional factor Nrf2 and antioxidant enzymes SOD1, NQO1 and GCLC to inhibited oxidative stress state, further beneficial to oocytes and embryos quality. Therefore, MDHB may represent a potential drug candidate in protecting granulosa cells in endometriosis, which can improve pregnancy outcomes for endometriosis-associated infertility.

Human mesenchymal stem cells derived exosomes improve ovarian function in chemotherapy-induced premature ovarian insufficiency mice by inhibiting ferroptosis through Nrf2/GPX4 pathway.

BACKGROUND: Chemotherapy exposure has become a main cause of premature ovarian insufficiency (POI). This study aimed to evaluate the role and molecular mechanism of human umbilical cord mesenchymal stem cell-derived exosomes (hUMSC-Exos) in ovarian function protection after chemotherapy. METHODS: hUMSC-Exos were applied to cyclophosphamide-induced premature ovarian insufficiency mice and human ovarian granulosa tumor cells (KGN) to determine their effects on follicular development and granulosa cell apoptosis. Evaluation was done for iron ion and reactive oxygen species (ROS) production, lipid peroxidation levels, and changes in iron death-related molecules (nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Glutathione Peroxidase enzyme 4 (GPX4), and Solute carrier family 7 member 11 cystine glutamate transporter (SLC7A11; xCT)). Furthermore, rescue experiments using an Nrf2 inhibitor were performed to assess the therapeutic effects of hUMSC-Exos on granulosa cells. RESULTS: hUMSC-Exos promoted ovarian hormone levels and primary follicle development in POI mice and reduced granulosa cell apoptosis. After hUMSC-Exos treatment, the ROS production, free iron ions and lipid peroxidation levels of granulosa cells decreased, and the iron death marker proteins Nrf2, xCT and GPX4 also decreased. Furthermore, the Nrf2 inhibitor ML385 significantly attenuated the effects of hUMSC-Exos on granulosa cells. CONCLUSION: hUMSC-Exos inhibit ferroptosis and protect against CTX-induced ovarian damage and granulosa cell apoptosis through the Nrf2/GPX4 signaling pathway, revealing a novel mechanism of hUMSC-Exos in POI therapy.

Therapeutic potential of targeting Nrf2 by panobinostat in pituitary neuroendocrine tumors.

We aimed to identify the druggable cell-intrinsic vulnerabilities and target-based drug therapies for PitNETs using the high-throughput drug screening (HTS) and genomic sequencing methods. We examined 9 patient-derived PitNET primary cells in HTS. Based on the screening results, the potential target genes were analyzed with genomic sequencing from a total of 180 PitNETs. We identified and verified one of the most potentially effective drugs, which targeted the Histone deacetylases (HDACs) both in in vitro and in vivo PitNET models. Further RNA sequencing revealed underlying molecular mechanisms following treatment with the representative HDACs inhibitor, Panobinostat. The HTS generated a total of 20,736 single-agent dose responses which were enriched among multiple inhibitors for various oncogenic targets, including HDACs, PI3K, mTOR, and proteasome. Among these drugs, HDAC inhibitors (HDACIs) were, on average, the most potent drug class. Further studies using in vitro, in vivo, and isolated PitNET primary cell models validated HDACIs, especially Panobinostat, as a promising therapeutic agent. Transcriptional surveys revealed substantial alterations to the Nrf2 signaling following Panobinostat treatment. Moreover, Nrf2 is highly expressed in PitNETs. The combination of Panobinostat and Nrf2 inhibitor ML385 had a synergistic effect on PitNET suppression. The current study revealed a class of effective anti-PitNET drugs, HDACIs, based on the HTS and genomic sequencing. One of the representative compounds, Panobinostat, may be a potential drug for PitNET treatment via Nrf2-mediated redox modulation. Combination of Panobinostat and ML385 further enhance the effectiveness for PitNET treatment.

Modifying Effects of Genetic Variations on the Association Between Dietary Isothiocyanate Exposure and Non-muscle Invasive Bladder Cancer Prognosis in the Be-Well Study.

SCOPE: Dietary isothiocyanate (ITC) exposure from cruciferous vegetable (CV) intake may improve non-muscle invasive bladder cancer (NMIBC) prognosis. This study aims to investigate whether genetic variations in key ITC-metabolizing/functioning genes modify the associations between dietary ITC exposure and NMIBC prognosis outcomes. METHODS AND RESULTS: In the Bladder Cancer Epidemiology, Wellness, and Lifestyle Study (Be-Well Study), a prospective cohort of 1472 incident NMIBC patients, dietary ITC exposure is assessed by self-reported CV intake and measured in plasma ITC-albumin adducts. Using Cox proportional hazards regression models, stratified by single nucleotide polymorphisms (SNPs) in nine key ITC-metabolizing/functioning genes, it is calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for recurrence and progression. The rs15561 in N-acetyltransferase 1 (NAT1) is alter the association between CV intake and progression risk. Multiple SNPs in nuclear factor E2-related factor 2 (NRF2) and nuclear factor kappa B (NFκB) are modify the associations between plasma ITC-albumin adduct level and progression risk (pint < 0.05). No significant association is observed with recurrence risk. Overall, >80% study participants are present with at least one protective genotype per gene, showing an average 65% reduction in progression risk with high dietary ITC exposure. CONCLUSION: Despite that genetic variations in ITC-metabolizing/functioning genes may modify the effect of dietary ITCs on NMIBC prognosis, dietary recommendation of CV consumption may help improve NMIBC survivorship.

Impact of circadian clock protein Bmal1 on experimentally-induced periodontitis-associated renal injury.

OBJECTIVES: To investigate the mechanism of circadian clock protein Bmal1 (Bmal1) on renal injury with chronic periodontitis, we established an experimental rat periodontitis model. METHODS: Twelve male Wistar rats were randomly divided into control and periodontitis groups (n=6, each group). The first maxillary molars on both sides of the upper jaw of rats with periodontitis were ligated by using orthodontic ligature wires, whereas the control group received no intervention measures. After 8 weeks, clinical periodontal parameters, including probing depth, bleeding index, and tooth mobility, were evaluated in both groups. Micro-CT scanning and three-dimensional image reconstruction were performed on the maxillary bones of the rats for the assessment of alveolar bone resorption. Histopatholo-gical observations of periodontal and renal tissues were conducted using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. Renal function indicators, such as creatinine, albumin, and blood urea nitrogen levels, and oxidative stress markers, including superoxide dismutase, glutathione, and malondialdehyde levels, were measured using biochemical assay kits. MitoSOX red staining was used to detect reactive oxygen species (ROS) content in the kidneys. The gene and protein expression levels of Bmal1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in rat renal tissues were assessed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. RESULTS: Micro-CT and HE staining results showed significant bone resorption and attachment loss in the maxillary first molar region of the periodontitis group. Histological examination through HE and PAS staining revealed substantial histopathological damage to the renal tissues of the rats in the periodontitis group. The findings of the assessment of renal function and oxidative stress markers indicated that the periodontitis group exhibited abnormal levels of oxidative stress, whereas the renal function levels showed abnormalities without statistical significance. MitoSOX Red staining results showed that the content of ROS in the renal tissue of the periodontitis group was significantly higher than that of the control group, and RT-qPCR and immunohistochemistry results showed that the expression levels of Bmal1, Nrf2, and HO-1 in the renal tissues of the rats in the periodontitis group showed a decreasing trend. CONCLUSIONS: Circadian clock protein Bmal1 plays an important role in the oxidative damage process involved in the renal of rats with periodontitis.

Differential squamous cell fates elicited by NRF2 gain of function versus KEAP1 loss of function.

Clinical evidence has revealed that high-level activation of NRF2 caused by somatic mutations in NRF2 (NFE2L2) is frequently detected in esophageal squamous cell carcinoma (ESCC), whereas that caused by somatic mutations in KEAP1, a negative regulator of NRF2, is not. Here, we aspire to generate a mouse model of NRF2-activated ESCC using the cancer-derived NRF2L30F mutation and cancer driver mutant TRP53R172H. Concomitant expression of NRF2L30F and TRP53R172H results in formation of NRF2-activated ESCC-like lesions. In contrast, while squamous-cell-specific deletion of KEAP1 induces similar NRF2 hyperactivation, the loss of KEAP1 combined with expression of TRP53R172H does not elicit the formation of ESCC-like lesions. Instead, KEAP1-deleted cells disappear from the esophageal epithelium over time. These findings demonstrate that, while cellular NRF2 levels are similarly induced, NRF2 gain of function and KEAP1 loss of function elicits distinct fates of squamous cells. The NRF2L30F mutant mouse model developed here will be instrumental in elucidating the mechanistic basis leading to NRF2-activated ESCC.

Psammaplin A and Its Analogs Attenuate Oxidative Stress in Neuronal Cells through Peroxisome Proliferator-Activated Receptor γ Activation.

Psammaplins are sulfur containing bromotyrosine alkaloids that have shown antitumor activity through the inhibition of class I histone deacetylases (HDACs). The cytotoxic properties of psammaplin A (1), the parent compound, are related to peroxisome proliferator-activated receptor γ (PPARγ) activation, but the mechanism of action of its analogs psammaplin K (2) and bisaprasin (3) has not been elucidated. In this study, the protective effects against oxidative stress of compounds 1-3, isolated from the sponge Aplysinella rhax, were evaluated in SH-SY5Y cells. The compounds improved cell survival, recovered glutathione (GSH) content, and reduced reactive oxygen species (ROS) release at nanomolar concentrations. Psammaplins restored mitochondrial membrane potential by blocking mitochondrial permeability transition pore opening and reducing cyclophilin D expression. This effect was mediated by the capacity of 1-3 to activate PPARγ, enhancing gene expression of the antioxidant enzymes catalase, nuclear factor E2-related factor 2 (Nrf2), and glutathione peroxidase. Finally, HDAC3 activity was reduced by 1-3 under oxidative stress conditions. This work is the first description of the neuroprotective activity of 1 at low concentrations and the mechanism of action of 2 and 3. Moreover, it links for the first time the previously described effects of 1 in HDAC3 and PPARγ signaling, opening a new research field for the therapeutic potential of this compound family.

[Microtubule-associated tumor suppressor 1 inhibits hemin-induced apoptosis of vascular endothelial cells via hemeoxygenase 1].

This study aimed to investigate the effects of microtubule associated tumor suppressor 1 (MTUS1) on hemeoxygenase 1 (HMOX1) expression and hemin-induced apoptosis of vascular endothelial cells and its regulatory mechanism. RNA sequencing, RT-qPCR and Western blot were used to assess altered genes of hemin binding proteins, the expression of cAMP response element-binding protein (CREB) and nuclear respiratory factor 2 (NRF2), hemin-induced HMOX1 expression in MTUS1 knockdown human umbilical vein endothelial cells (HUVEC), and the effect of overexpression of CREB and NRF2 on HMOX1 expression in MTUS1 knockdown 293T cells. The effect of MTUS1 or HMOX1 knockdown on hemin-induced apoptosis in HUVEC, and the overexpression of NRF2 on hemin-induced apoptosis in MTUS1 knockdown 293T cells were assayed with CCK8 and Western blot. The results showed that MTUS1 was knocked down significantly in HUVEC by siRNA (P < 0.01), accompanied by decreased HMOX1 expression (P < 0.01). The increased HMOX1 expression induced by hemin was also inhibited by MTUS1 knockdown (P < 0.01). And the apoptosis of HUVEC induced by hemin was amplified by MTUS1 or HMOX1 knockdown (P < 0.01). Moreover the expression of CREB and NRF2 were both inhibited by MTUS1 knockdown in HUVEC (P < 0.01). The decreased HMOX1 regulated by MTUS1 knockdown could be rescued partly by overexpression of NRF2 (P < 0.01), however, not by overexpression of CREB. And the MTUS1 knockdown mediated decreased 293T cells viability induced by hemin could be partly rescued by NRF2 overexpression (P < 0.01). These results suggest that MTUS1 can inhibit hemin-induced apoptosis of HUVEC, and the mechanism maybe related to MTUS1/NRF2/HMOX1 pathway.

ACOD1, rather than itaconate, facilitates p62-mediated activation of Nrf2 in microglia post spinal cord contusion.

BACKGROUND: Spinal cord injury (SCI)-induced neuroinflammation and oxidative stress (OS) are crucial events causing neurological dysfunction. Aconitate decarboxylase 1 (ACOD1) and its metabolite itaconate (Ita) inhibit inflammation and OS by promoting alkylation of Keap1 to induce Nrf2 expression; however, it is unclear whether there is another pathway regulating their effects in inflammation-activated microglia after SCI. METHODS: Adult male C57BL/6 ACOD1-/- mice and their wild-type (WT) littermates were subjected to a moderate thoracic spinal cord contusion. The degree of neuroinflammation and OS in the injured spinal cord were assessed using qPCR, western blot, flow cytometry, immunofluorescence, and trans-well assay. We then employed immunoprecipitation-western blot, chromatin immunoprecipitation (ChIP)-PCR, dual-luciferase assay, and immunofluorescence-confocal imaging to examine the molecular mechanisms of ACOD1. Finally, the locomotor function was evaluated with the Basso Mouse Scale and footprint assay. RESULTS: Both in vitro and in vivo, microglia with transcriptional blockage of ACOD1 exhibited more severe levels of neuroinflammation and OS, in which the expression of p62/Keap1/Nrf2 was down-regulated. Furthermore, silencing ACOD1 exacerbated neurological dysfunction in SCI mice. Administration of exogenous Ita or 4-octyl itaconate reduced p62 phosphorylation. Besides, ACOD1 was capable of interacting with phosphorylated p62 to enhance Nrf2 activation, which in turn further promoted transcription of ACOD1. CONCLUSIONS: Here, we identified an unreported ACOD1-p62-Nrf2-ACOD1 feedback loop exerting anti-inflammatory and anti-OS in inflammatory microglia, and demonstrated the neuroprotective role of ACOD1 after SCI, which was different from that of endogenous and exogenous Ita. The present study extends the functions of ACOD1 and uncovers marked property differences between endogenous and exogenous Ita. KEY POINTS: ACOD1 attenuated neuroinflammation and oxidative stress after spinal cord injury. ACOD1, not itaconate, interacted with p-p62 to facilitate Nrf2 expression and nuclear translocation. Nrf2 was capable of promoting ACOD1 transcription in microglia.

Itaconate alleviates anesthesia/surgery-induced cognitive impairment by activating a Nrf2-dependent anti-neuroinflammation and neurogenesis via gut-brain axis.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a common neurological complication of anesthesia and surgery in aging individuals. Neuroinflammation has been identified as a hallmark of POCD. However, safe and effective treatments of POCD are still lacking. Itaconate is an immunoregulatory metabolite derived from the tricarboxylic acid cycle that exerts anti-inflammatory effects by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. In this study, we investigated the effects and underlying mechanism of 4-octyl itaconate (OI), a cell-permeable itaconate derivative, on POCD in aged mice. METHODS: A POCD animal model was established by performing aseptic laparotomy in 18-month-old male C57BL/6 mice under isoflurane anesthesia while maintaining spontaneous ventilation. OI was intraperitoneally injected into the mice after surgery. Primary microglia and neurons were isolated and treated to lipopolysaccharide (LPS), isoflurane, and OI. Cognitive function, neuroinflammatory responses, as well as levels of gut microbiota and their metabolites were evaluated. To determine the mechanisms underlying the therapeutic effects of OI in POCD, ML385, an antagonist of Nrf2, was administered intraperitoneally. Cognitive function, neuroinflammatory responses, endogenous neurogenesis, neuronal apoptosis, and Nrf2/extracellular signal-related kinases (ERK) signaling pathway were evaluated. RESULTS: Our findings revealed that OI treatment significantly alleviated anesthesia/surgery-induced cognitive impairment, concomitant with reduced levels of the neuroinflammatory cytokines IL-1ß and IL-6, as well as suppressed activation of microglia and astrocytes in the hippocampus. Similarly, OI treatment inhibited the expression of IL-1ß and IL-6 in LPS and isoflurane-induced primary microglia in vitro. Intraperitoneal administration of OI led to alterations in the gut microbiota and promoted the production of microbiota-derived metabolites associated with neurogenesis. We further confirmed that OI promoted endogenous neurogenesis and inhibited neuronal apoptosis in the hippocampal dentate gyrus of aged mice. Mechanistically, we observed a decrease in Nrf2 expression in hippocampal neurons both in vitro and in vivo, which was reversed by OI treatment. We found that Nrf2 was required for OI treatment to inhibit neuroinflammation in POCD. The enhanced POCD recovery and promotion of neurogenesis triggered by OI exposure were, at least partially, mediated by the activation of the Nrf2/ERK signaling pathway. CONCLUSIONS: Our findings demonstrate that OI can attenuate anesthesia/surgery-induced cognitive impairment by stabilizing the gut microbiota and activating Nrf2 signaling to restrict neuroinflammation and promote neurogenesis. Boosting endogenous itaconate or supplementation with exogenous itaconate derivatives may represent novel strategies for the treatment of POCD.

Obacunone Alleviates Inflammatory Pain by Promoting M2 Microglial Polarization and by Activating Nrf2/HO-1 Signaling Pathway.

Background: Treating inflammatory pain (IP) continues to pose clinical challenge, because of the lack of effective pharmacological interventions. Microglial polarization serves as pivotal determinant in IP progress. Obacunone (OB), a low-molecular-weight compound with a diverse array of biological functions, having reported as an activator of nuclear factor E2-related factor 2 (Nrf2), exhibits anti-inflammatory property. However, it remains uncertain whether OB can alleviate IP by facilitating the transition of microglial polarization from the M1 to M2 state through modulating Nrf2/ heme oxygenase-1 (HO-1) pathway. Methods: We induced an mice IP model by subcutaneously administering Complete Freund's Adjuvant (CFA) into the hind paw. Paw withdrawal latency (PWL) in seconds (s) and paw withdrawal frequency (PWF) were employed to evaluate the establishment of the IP model, while a caliper was used to measure the maximal dorsoventral thickness of the mice paw. Nerve injury was assessed by Hematoxylin-Eosin (HE) Staining. Western blot and got conducted for detection of M1/M2 microglial polarization markers, Nrf2 and HO-1 in spinal cord tissues respectively. Results: In comparison to the control cohort, PWF, M1 phenotype marker iNOS, CD86, paw thickness increased significantly within CFA cohort, while PWL, M2 phenotype marker Arg-1, interleukin-10 (IL-10) decreased in the CFA group. In comparison to model cohort, OB treatment decreased PWF, paw thickness, M1 phenotype marker iNOS, CD86 significantly, while PWL, M2 phenotype marker Arg-1, IL-10, Nrf2, HO-1 increased significantly. The morphological injuries of sciatic nerve in CFA mice were obviously improved by OB treatment. OB inhibited the release of M1-related IL-1ß, CXCL1 but promoted M2-related TGF-ß, IL-10 in serum in CFA mice. The intervention of the Nrf2 inhibitor ML385 mitigated analgesic effect of OB. Conclusion: We demonstrate that OB is able to attenuate inflammatory pain via promoting microglia polarization from M1 to M2 and enhancing Nrf2/HO-1 signal. OB treatment may be a potential alternative agent in the treatment of IP.

Investigating the role of itaconate in macrophage activation and oxidative stress injury in sepsis-associated acute kidney injury.

BACKGROUND: Sepsis may be linked to oxidative stress and can be controlled by itaconate, an activator of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Nevertheless, the itaconate impact on sepsis-associated acute kidney injury (SA-AKI) has yet to be definitively established. METHODS: We employed SA-AKI mouse model through a cecal ligation and puncture (CLP) procedure for the in vivo investigation of the potential nephroprotective effect of itaconate in this study. A plasmid was transfected into RAW264.7 cells to examine the Nrf2 pathway function after itaconate administration. Finally, the immune-responsive gene 1-knockout (IRG1-/-) mice were used to study the itaconate impacts on oxidative stress-induced SA-AKI. RESULTS: We have shown that 4-octyl itaconate (OI) significantly reduced CD11b-positive macrophage aggregation and activated the Nrf2 pathway in the bone marrow-derived macrophages (BMDM). The impacts of Nrf2 inhibitor ML385 on the anti-inflammatory and antioxidant properties of itaconate were found to be partial. OI inhibited lipopolysaccharide-induced oxidative stress injury in RAW264.7 macrophages and activated Nrf2 in the nucleus to hinder the expression of nuclear factor kappa B p65, thereby suppressing oxidative stress injury in the macrophages. Additionally, the introduction of the transfected plasmid resulted in a partial inhibition of the anti-inflammatory impact of itaconate. The kidney injury caused by sepsis exhibited greater severity in the IRG1-/- mice than in the wild type mice. Exogenous OI partially attenuated the kidney injury induced by sepsis in the IRG1-/- mice and suppressed the oxidative stress injury in macrophages. CONCLUSIONS: This investigation offers new proof to support the itaconate function in the development and progression of SA-AKI and shows a new possible therapeutic agent for the SA-AKI treatment.

Interaction with B-type lamin reveals the function of Drosophila Keap1 xenobiotic response factor in nuclear architecture.

BACKGROUND: The Keap1-Nrf2 pathway serves as a central regulator that mediates transcriptional responses to xenobiotic and oxidative stimuli. Recent studies have shown that Keap1 and Nrf2 can regulate transcripts beyond antioxidant and detoxifying genes, yet the underlying mechanisms remain unclear. Our research has uncovered that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins can control high-order chromatin structure, including heterochromatin. METHODS AND RESULTS: In this study, we identified the molecular interaction between dKeap1 and lamin Dm0, the Drosophila B-type lamin responsible for the architecture of nuclear lamina and chromatin. Ectopic expression of dKeap1 led to an ectopic localization of lamin to the intra-nuclear area, corelated with the spreading of the heterochromatin marker H3K9me2 into euchromatin regions. Additionally, mis-regulated dKeap1 disrupted the morphology of the nuclear lamina. Knocking down of dKeap1 partially rescued the lethality induced by lamin overexpression, suggesting their genetic interaction during development. CONCLUSIONS: The discovered dKeap1-lamin interaction suggests a novel role for the Keap1 oxidative/xenobiotic response factor in regulating chromatin architecture.

MiR-497-5p ameliorates the oxyhemoglobin-induced subarachnoid hemorrhage injury in vitro by targeting orthodenticle homeobox protein 1 (Otx1) to activate the Nrf2/HO-1 pathway.

Subarachnoid hemorrhage (SAH) is a neurological disorder that severely damages the brain and causes cognitive impairment. MicroRNAs are critical regulators in a variety of neurological diseases. MiR-497-5p has been found to be downregulated in the aneurysm vessel walls obtained from patients with aneurysmal subarachnoid hemorrhage, but its functions and mechanisms in SAH have not been reported. Therefore, this study was designed to investigate the effect of miR-497-5p and its related mechanisms in SAH. We established an in vitro SAH model by exposing PC12 cells to oxyhemoglobin (oxyHb). We found that miR-497-5p was downregulated in SAH serum and oxyHb-treated PC12 cells, and its overexpression inhibited the oxyHb-induced apoptosis, inflammatory response and oxidative stress via activation of the Nrf2 pathway. Mechanistically, the targeting relationship between miR-497-5p and Otx1 was verified by luciferase reporter assays. Moreover, Otx1 upregulation abolished the protective effects of miR-497-5p upregulation against oxyHb-induced apoptosis, inflammation and oxidative stress in PC12 cells. Collectively, our findings indicate that miR-497-5p could inhibit the oxyHb-induced SAH damage by targeting Otx1 to activate the Nrf2/HO-1 pathway, which provides a potential therapeutic target for SAH treatment.

[Essential oil of Cinnamomum camphora mitigates depression-like behavior in mice by regulating Nrf2/HO-1 pathway and inhibiting inflammatory cytokines].

This study aims to investigate the essential oil(EOL) of Cinnamomum camphora regarding its anti-depression effect and mechanism in regulating inflammatory cytokines and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) pathway. A mouse model of depression was established by intraperitoneal injection of lipopolysaccharide(LPS). Open field, elevated plus maze, and forced swimming tests were carried out to examine mouse behaviors. Western blot and qRT-PCR were employed to determine the expression of proteins and genes in the Nrf2/HO-1 pathway in the hippocampus. The levels of tumor necrosis factor(TNF)-α, interleukin(IL)-6, and IL-1ß in the serum were measured by enzyme-linked immunosorbent assay(ELISA). The changes of apoptosis in mouse brain were detected by Tunel staining. Compared with the blank control group, the model group showed shortened distance travelled and time spent in the central zone and reduced number of entries in the central zone in the open field test. In the elevated plus maze test, the model group showed reduced open arm time(OT%) and open arm entries(OE%). In the force swimming test, the model group showed extended duration of immobility compared with the blank control group. Compared with the model group, the treatment with EOL significantly increased the distance travelled and time spent in the central zone and increased the number of entries in the central zone in the open field test. In addition, EOL significantly increased the OT% and OE% in the elevated plus maze and shor-tened the immobility duration in the forced swimming test. The model group showed lower expression levels of Nrf2 and HO-1 and hig-her levels of TNF-α, IL-6, and IL-1ß than the blank control group. Compared with the model group, the treatment with EOL up-regulated the expression levels of Nrf2 and HO-1 and lowered the levels of TNF-α, IL-6, and IL-1ß. The Tunel staining results showed that the apoptosis rate in the brain tissue of mice decreased significantly after the treatment with EOL. To sum up, EOL can mitigate the depression-like behaviors of mice by up-regulating the expression of Nrf2 and HO-1 and preventing hippocampal inflammatory damage. The findings provide empirical support for the application of EOL and aromatherapy in the treatment of depression.

[Fuyu Decoction ameliorates myocardial fibrosis in rat model of heart failure by regulating Nrf2/GPX4-mediated ferroptosis].

This study aims to investigate the effect and mechanism of Fuyu Decoction(FYD) in the treatment of myocardial fibrosis in the rat model of heart failure(HF). Sixty Wistar rats were randomized into a modeling group(n=50) and a sham group(n=10). A post-myocardial infarction HF model was established by ligating the left anterior descending coronary artery in rats. The successfully modeled rats were assigned into model, low-dose(2.5 g·kg~(-1)) FYD(FYD-L), high-dose(5.0 g·kg~(-1)) FYD(FYD-H), and FYD+Nrf2 inhibitor(ML385, 30 mg·kg~(-1)) groups(n=10). FYD was administrated by gavage and ML385 by intraperitoneal injection. The rats in the sham and model groups were administrated with equal amounts of normal saline by gavage. After 8 weeks of intervention, the cardiac function indicators were measured, and the myocardial tissue morphology and collagen deposition were observed. The positive expression of collagens Ⅰ and Ⅲ, apoptosis, and oxidative stress were examined, and the levels of Fe~(2+) and reactive oxygen species(ROS) were determined. The protein levels of nuclear factor erythroid 2-related factor 2(Nrf2), solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4), and acyl-coenzyme A synthase long chain family member 4(ACSL4) in the myocardial tissue were determined. Compared with sham group, the model group showed decreased left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), increased left ventricular end internal dimension in systole(LVIDs), left ventricular internal diameter in diastole(LVIDd), and myocardial collagen deposition, positive expression of collagens Ⅰ and Ⅲ, elevated apoptosis rate and malondialdehyde(MDA), Fe~(2+), and ROS levels, lowered superoxide dismutase(SOD) and glutathione peroxidase(GSH) levels, down-regulated protein levels of Nrf2, SLC7A11, and GPX4, and up-regulated protein level of ACSL4. Compared with the model group, the above indicators were restored by FYD. Moreover, ML385 reversed the protective effect of FYD on myocardial fibrosis in HF rats. In conclusion, FYD can inhibit ferroptosis by activating the Nrf2/GPX4 pathway, thereby ameliorating myocardial fibrosis in HF rats.

[Ginsenoside Re regulates mitochondrial biogenesis through Nrf2/HO-1/PGC-1α pathway to reduce hypoxia/reoxygenation injury in H9c2 cells].

This article explored the mechanism by which ginsenoside Re reduces hypoxia/reoxygenation(H/R) injury in H9c2 cells by regulating mitochondrial biogenesis through nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/peroxisome prolife-rator-activated receptor gamma coactivator-1α(PGC-1α) pathway. In this study, H9c2 cells were cultured in hypoxia for 4 hours and then reoxygenated for 2 hours to construct a cardiomyocyte H/R injury model. After ginsenoside Re pre-administration intervention, cell activity, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content, intracellular reactive oxygen species(Cyto-ROS), and intramitochondrial reactive oxygen species(Mito-ROS) levels were detected to evaluate the protective effect of ginsenoside Re on H/R injury of H9c2 cells by resisting oxidative stress. Secondly, fluorescent probes were used to detect changes in mitochondrial membrane potential(ΔΨ_m) and mitochondrial membrane permeability open pore(mPTP), and immunofluorescence was used to detect the expression level of TOM20 to study the protective effect of ginsenoside Re on mitochondria. Western blot was further used to detect the protein expression levels of caspase-3, cleaved caspase-3, Cyto C, Nrf2, HO-1, and PGC-1α to explore the specific mechanism by which ginsenoside Re protected mitochondria against oxidative stress and reduced H/R injury. Compared with the model group, ginse-noside Re effectively reduced the H/R injury oxidative stress response of H9c2 cells, increased SOD activity, reduced MDA content, and decreased Cyto-ROS and Mito-ROS levels in cells. Ginsenoside Re showed a good protective effect on mitochondria by increasing ΔΨ_m, reducing mPTP, and increasing TOM20 expression. Further studies showed that ginsenoside Re promoted the expression of Nrf2, HO-1, and PGC-1α proteins, and reduced the activation of the apoptosis-related regulatory factor caspase-3 to cleaved caspase-3 and the expression of Cyto C protein. In summary, ginsenoside Re can significantly reduce I/R injury in H9c2 cells. The specific mechanism is related to the promotion of mitochondrial biogenesis through the Nrf2/HO-1/PGC-1α pathway, thereby increasing the number of mitochondria, improving mitochondrial function, enhancing the ability of cells to resist oxidative stress, and alleviating cell apoptosis.

[Tanshinone â ¡_A inhibits ferroptosis via Nrf2 signaling pathway to protect liver in rats of non-alcoholic fatty liver disease].

This study investigated the protective effect of tanshinone Ⅱ_A(TSⅡ_A) on the liver in the rat model of non-alcoholic fatty liver disease(NAFLD) and the mechanism of TSⅡ_A in regulating ferroptosis via the nuclear factor E2-related factor 2(Nrf2) signaling pathway. The rat model of NAFLD was established with a high-fat diet for 12 weeks. The successfully modeled rats were assigned into model group, low-and high-dose TSⅡ_A groups, and inhibitor group, and normal control group was set. Enzyme-linked immunosorbent assay was employed to determine the content of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum of rats in each group. A biochemical analyzer was used to measure the content of aspartate aminotransferase(AST), alaninl aminotransferase(ALT), total cholesterol(TC), and triglycerides(TG). Hematoxylin-eosin(HE) staining was used to detect pathological damage in liver tissue. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling(TUNEL) was employed to examine the apoptosis of the liver tissue. Oil red O staining, MitoSOX staining, and Prussian blue staining were conducted to reveal lipid deposition, the content of reactive oxygen species(ROS), and iron deposition in liver tissue. Western blot was employed to determine the expression of Nrf2, heme oxygenase-1(HO-1), glutathione peroxidase 4(GPX4), ferroptosis suppressor protein 1(FSP1), B cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) in the liver tissue. The result showed that TSⅡ_A significantly reduced the content of MDA, AST, ALT, TC, and TG in the serum, increased the activity of SOD, decreased the apoptosis rate, lipid deposition, ROS, and iron deposition in the liver tissue, up-regulated the expression of Nrf2, HO-1, FSP1, GPX, and Bcl-2, and inhibited the expression of Bax in the liver tissue of NAFLD rats. However, ML385 partially reversed the protective effect of TSⅡ_A on the liver tissue. In conclusion, TSⅡ_A could inhibit ferroptosis in the hepatocytes and decrease the ROS and lipid accumulation in the liver tissue of NAFLD rats by activating the Nrf2 signaling pathway.

[Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice].

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1ß, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1ß in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1ß, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1ß in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.