DIFFERENTIAL SIGNALING EFFECTS OF ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS IN HUMAN WHOLE BLOOD INDICATE DISTINCT REGULATION OF THE NRF2 PATHWAY.

ABSTRACT: Escherichia coli and Staphylococcus aureus are two of the most common bacterial species responsible for sepsis. While it is observed that they have disparate clinical phenotypes, the signaling differences elicited by each bacteria that drive this variance remain unclear. Therefore, we used human whole blood exposed to heat-killed E. coli or S. aureus and measured the transcriptomic signatures. Relative to unstimulated control blood, heat-killed bacteria exposure led to significant dysregulation (upregulated and downregulated) of >5,000 genes for each experimental condition, with a slight increase in gene alterations by S. aureus. While there was significant overlap regarding proinflammatory pathways, Gene Ontology overrepresentation analysis of the most altered genes suggested biological processes like macrophage differentiation and ubiquinone biosynthesis were more unique to heat-killed S. aureus, compared with heat-killed E. coli exposure. Using Ingenuity Pathway Analysis, it was demonstrated that nuclear factor erythroid 2-related factor 2 signaling, a main transcription factor in antioxidant responses, was predominately upregulated in S. aureus exposed blood relative to E. coli. Furthermore, the use of pharmacologics that preferentially targeted the nuclear factor erythroid 2-related factor 2 pathway led to differential cytokine profiles depending on the type of bacterial exposure. These findings reveal significant inflammatory dysregulation between E. coli and S. aureus and provide insight into the targeting of unique pathways to curb bacteria-specific responses.

Vincamine alleviates brain injury by attenuating neuroinflammation and oxidative damage in a mouse model of Parkinson's disease through the NF-κB and Nrf2/HO-1 signaling pathways.

Parkinson's disease (PD) is a neurodegenerative disease featured by progressive loss of nigrostriatal dopaminergic neurons, the etiology of which is associated with the existence of neuroinflammatory response and oxidative stress. Vincamine is an indole alkaloid that was reported to exhibit potent anti-inflammatory and antioxidant properties in many central and/or peripheral diseases. Nevertheless, the specific role of vincamine in PD development remains unknown. In our study, dopaminergic neuron loss was determined through immunohistochemistry staining and western blot analysis of tyrosine hydroxylase (TH) expression in the substantia nigra (SN) of PD mice. Reactive oxygen species (ROS) production and malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were detected through DHE staining and commercially available kits to assess oxidative stress. Pro-inflammatory cytokine (TNF-α, IL-1ß, and IL-6) levels in the SN were measured via RT-qPCR and western blot analysis. Microglial and astrocyte activation was examined through immunofluorescence staining of Iba-1 (microglia marker) and GFAP (astrocyte marker) in the SN. The regulation of vincamine on the NF-κB and Nrf2/HO-1 pathway was estimated through western blot analysis. Our results showed that vincamine treatment decreased TNF-α, IL-1ß, and IL-6 mRNA and protein levels, reduced GFAP and Iba-1 expression, decreased ROS production and MDA level, and increased SOD activity and GSH level in the SN of PD mice. Mechanically, vincamine repressed the phosphorylation levels of p65, IKKß, and IκBα but enhanced the protein levels of Nrf2 and HO-1 in PD mice. Collectively, vincamine plays a neuroprotective role in PD mouse models by alleviating neuroinflammation and oxidative damage via suppressing the NF-κB pathway and activating the Nrf2/HO-1 pathway.

Nrf2-mediated ferroptosis of spermatogenic cells involved in male reproductive toxicity induced by polystyrene nanoplastics in mice. / Nrf2ä»‹å¯¼çš„ç”Ÿç²¾ç»†èƒžé“æ­»äº¡å‚ä¸Žäº†èšè‹¯ä¹™çƒ¯çº³ç±³å¡‘æ–™å¯¼è‡´çš„å°é¼ é›„æ€§ç”Ÿæ®–æ¯’æ€§.

Microplastics (MPs) and nanoplastics (NPs) have become hazardous materials due to the massive amount of plastic waste and disposable masks, but their specific health effects remain uncertain. In this study, fluorescence-labeled polystyrene NPs (PS-NPs) were injected into the circulatory systems of mice to determine the distribution and potential toxic effects of NPs in vivo. Interestingly, whole-body imaging found that PS-NPs accumulated in the testes of mice. Therefore, the toxic effects of PS-NPs on the reproduction systems and the spermatocytes cell line of male mice, and their mechanisms, were investigated. After oral exposure to PS-NPs, their spermatogenesis was affected and the spermatogenic cells were damaged. The spermatocyte cell line GC-2 was exposed to PS-NPs and analyzed using RNA sequencing (RNA-seq) to determine the toxic mechanisms; a ferroptosis pathway was found after PS-NP exposure. The phenomena and indicators of ferroptosis were then determined and verified by ferroptosis inhibitor ferrostatin-1 (Fer-1), and it was also found that nuclear factor erythroid 2-related factor 2 (Nrf2) played an important role in spermatogenic cell ferroptosis induced by PS-NPs. Finally, it was confirmed in vivo that this mechanism of Nrf2 played a protective role in PS-NPs-induced male reproductive toxicity. This study demonstrated that PS-NPs induce male reproductive dysfunction in mice by causing spermatogenic cell ferroptosis dependent on Nrf2.

Propranolol Promotes Monocyte-to-Macrophage Differentiation and Enhances Macrophage Anti-Inflammatory and Antioxidant Activities by NRF2 Activation.

Adrenergic pathways represent the main channel of communication between the nervous system and the immune system. During inflammation, blood monocytes migrate within tissue and differentiate into macrophages, which polarize to M1 or M2 macrophages with tissue-damaging or -reparative properties, respectively. This study investigates whether the ß-adrenergic receptor (ß-AR)-blocking drug propranolol modulates the monocyte-to-macrophage differentiation process and further influences macrophages in their polarization toward M1- and M2-like phenotypes. Six-day-human monocytes were cultured with M-CSF in the presence or absence of propranolol and then activated toward an M1 pro-inflammatory state or an M2 anti-inflammatory state. The chronic exposure of monocytes to propranolol during their differentiation into macrophages promoted the increase in the M1 marker CD16 and in the M2 markers CD206 and CD163 and peroxisome proliferator-activated receptor É£ expression. It also increased endocytosis and the release of IL-10, whereas it reduced physiological reactive oxygen species. Exposure to the pro-inflammatory conditions of propranolol-differentiated macrophages resulted in an anti-inflammatory promoting effect. At the molecular level, propranolol upregulated the expression of the oxidative stress regulators NRF2, heme oxygenase-1 and NQO1. By contributing to regulating macrophage activities, propranolol may represent a novel anti-inflammatory and immunomodulating compound with relevant therapeutic potential in several inflammatory diseases.

Fisetin Alleviates Inflammation and Oxidative Stress in Deep Vein Thrombosis via MAPK and NRF2 Signaling Pathway.

Oxidative stress and inflammation play pivotal roles in the progression of deep vein thrombosis (DVT). Fisetin has demonstrated promising pharmacological features; however, its underlying mechanisms in DVT remain elusive. In our study, we investigated the effects and underlying mechanisms of Fisetin on a DVT mouse model. The protective effects of Fisetin on DVT were evaluated by comparing the size of thrombosis and detecting the mRNA expression levels of pro-inflammatory cytokines. After that, the biological processes were studied via transcriptomics after Fisetin administration. The antioxidant effect was evaluated and explained via NRF2 signaling pathway. Finally, the anti-inflammatory effect was explained according to KEGG analysis and the final mechanism was verified via Western blot. Our results found that the mRNA expression levels of pro-inflammatory cytokines were inhibited by Fisetin. Moreover, transcriptomic studies suggested that MAPK signaling pathway may be associated with the anti-inflammatory activity of Fisetin. Then, we confirmed that Fisetin administration significantly inhibited the activation of typical pro-inflammatory signaling pathways via Western blot. Finally, the results of Western blot showed that Fisetin significantly activated NRF2 signaling pathway and induced the expression of downstream antioxidant enzymes. Our findings suggested that Fisetin exhibits potential therapeutic effects on DVT through its ability to attenuate inflammation and oxidative stress. The underlying mechanism may involve the suppression of MAPK-mediated inflammatory signaling pathway and activation of NRF2-mediated antioxidant signaling pathway.

Mechanisms of PM10 Disruption of the Nrf2 Pathway in Cornea.

We have previously shown that PM10 exposure causes oxidative stress and reduces Nrf2 protein levels, and SKQ1 pre-treatment protects against this damage in human corneal epithelial cells (HCE-2). The current study focuses on uncovering the mechanisms underlying acute PM10 toxicity and SKQ1-mediated protection. HCE-2 were pre-treated with SKQ1 and then exposed to 100 µg/mL PM10. Cell viability, oxidative stress markers, programmed cell death, DNA damage, senescence markers, and pro-inflammatory cytokines were analyzed. Nrf2 cellular location and its transcriptional activity were determined. Effects of the Nrf2 inhibitor ML385 were similarly evaluated. Data showed that PM10 decreased cell viability, Nrf2 transcriptional activity, and mRNA levels of antioxidant enzymes, but increased p-PI3K, p-NFκB, COX-2, and iNOS proteins levels. Additionally, PM10 exposure significantly increased DNA damage, phosphor-p53, p16 and p21 protein levels, and ß-galactosidase (ß-gal) staining, which confirmed the senescence. SKQ1 pre-treatment reversed these effects. ML385 lowered the Nrf2 protein levels and mRNA levels of its downstream targets. ML385 also abrogated the protective effects of SKQ1 against PM10 toxicity by preventing the restoration of cell viability and reduced oxidative stress. In conclusion, PM10 induces inflammation, reduces Nrf2 transcriptional activity, and causes DNA damage, leading to a senescence-like phenotype, which is prevented by SKQ1.

The Role of NRF2 in Cerebrovascular Protection: Implications for Vascular Cognitive Impairment and Dementia (VCID).

Vascular cognitive impairment and dementia (VCID) represents a broad spectrum of cognitive decline secondary to cerebral vascular aging and injury. It is the second most common type of dementia, and the prevalence continues to increase. Nuclear factor erythroid 2-related factor 2 (NRF2) is enriched in the cerebral vasculature and has diverse roles in metabolic balance, mitochondrial stabilization, redox balance, and anti-inflammation. In this review, we first briefly introduce cerebrovascular aging in VCID and the NRF2 pathway. We then extensively discuss the effects of NRF2 activation in cerebrovascular components such as endothelial cells, vascular smooth muscle cells, pericytes, and perivascular macrophages. Finally, we summarize the clinical potential of NRF2 activators in VCID.

Methylmercury Induces Apoptosis in Mouse C17.2 Neural Stem Cells through the Induction of OSGIN1 Expression by NRF2.

Methylmercury is a known environmental pollutant that exhibits severe neurotoxic effects. However, the mechanism by which methylmercury causes neurotoxicity remains unclear. To date, we have found that oxidative stress-induced growth inhibitor 1 (OSGIN1), which is induced by oxidative stress and DNA damage, is also induced by methylmercury. Therefore, in this study, we investigated the relationship between methylmercury toxicity and the induction of OSGIN1 expression using C17.2 cells, which are mouse brain neural stem cells. Methylmercury increased both OSGIN1 mRNA and protein levels in a time- and concentration-dependent manner. Moreover, these increases were almost entirely canceled out by pretreatment with actinomycin D, a transcription inhibitor. Furthermore, similar results were obtained from cells in which expression of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) was suppressed, indicating that methylmercury induces OSGIN1 expression via NRF2. Methylmercury causes neuronal cell death by inducing apoptosis. Therefore, we next investigated the role of OSGIN1 in methylmercury-induced neuronal cell death using the activation of caspase-3, which is involved in apoptosis induction, as an indicator. As a result, the increase in cleaved caspase-3 (activated form) induced by methylmercury exposure was decreased by suppressing OSGIN1, and the overexpression of OSGIN1 further promoted the increase in cleaved caspase-3 caused by methylmercury. These results suggest, for the first time, that OSGIN1 is a novel factor involved in methylmercury toxicity, and methylmercury induces apoptosis in C17.2 cells through the induction of OSGIN1 expression by NRF2.

Accelerated Aging Induced by an Unhealthy High-Fat Diet: Initial Evidence for the Role of Nrf2 Deficiency and Impaired Stress Resilience in Cellular Senescence.

High-fat diets (HFDs) have pervaded modern dietary habits, characterized by their excessive saturated fat content and low nutritional value. Epidemiological studies have compellingly linked HFD consumption to obesity and the development of type 2 diabetes mellitus. Moreover, the synergistic interplay of HFD, obesity, and diabetes expedites the aging process and prematurely fosters age-related diseases. However, the underlying mechanisms driving these associations remain enigmatic. One of the most conspicuous hallmarks of aging is the accumulation of highly inflammatory senescent cells, with mounting evidence implicating increased cellular senescence in the pathogenesis of age-related diseases. Our hypothesis posits that HFD consumption amplifies senescence burden across multiple organs. To scrutinize this hypothesis, we subjected mice to a 6-month HFD regimen, assessing senescence biomarker expression in the liver, white adipose tissue, and the brain. Aging is intrinsically linked to impaired cellular stress resilience, driven by dysfunction in Nrf2-mediated cytoprotective pathways that safeguard cells against oxidative stress-induced senescence. To ascertain whether Nrf2-mediated pathways shield against senescence induction in response to HFD consumption, we explored senescence burden in a novel model of aging: Nrf2-deficient (Nrf2+/-) mice, emulating the aging phenotype. Our initial findings unveiled significant Nrf2 dysfunction in Nrf2+/- mice, mirroring aging-related alterations. HFD led to substantial obesity, hyperglycemia, and impaired insulin sensitivity in both Nrf2+/- and Nrf2+/+ mice. In control mice, HFD primarily heightened senescence burden in white adipose tissue, evidenced by increased Cdkn2a senescence biomarker expression. In Nrf2+/- mice, HFD elicited a significant surge in senescence burden across the liver, white adipose tissue, and the brain. We postulate that HFD-induced augmentation of senescence burden may be a pivotal contributor to accelerated organismal aging and the premature onset of age-related diseases.

Pazopanib stimulates senescence of renal carcinoma cells through targeting nuclear factor E2-related factor 2 (Nrf2).

Renal cell carcinoma (RCC) is the most common kidney cancer with high mortality rate. Pazopanib has been approved for the treatment of RCC. However, the underlying mechanism is not clear. Here, we report a novel finding by showing that treatment with Pazopanib could promote cellular senescence of the human RCC cell line ACHN. Cells were stimulated with 5, 10, and 20 µM Pazopanib, respectively. Cellular senescence was measured using senescence-associated ß-galactosidase (SA-ß-Gal) staining. Western blot analysis and real-time polymerase chain reaction were used to measure the mRNA and protein expression of nuclear factor E2-related factor 2 (Nrf2), γH2AX, human telomerase reverse transcriptase (hTERT), telomeric repeat binding factor 2 (TERF2), p53 and plasminogen activator inhibitor (PAI). First, we found that exposure to Pazopanib reduced the cell viability of ACHN cells. Additionally, Pazopanib induced oxidative stress  by increasing the production of reactive oxygen species, reducing the levels of glutathione peroxidase, and promoting nuclear translocation of Nrf2. Interestingly, Pazopanib exposure resulted in DNA damage by increasing the expression of γH2AX. Importantly, Pazopanib increased cellular senescence and reduced telomerase activity. Pazopanib also reduced the gene expression of hTERT but increased the gene expression of TERF2. Correspondingly, we found that Pazopanib increased the expression of p53 and PAI at both the mRNA and protein levels. To elucidate the underlying mechanism, the expression of Nrf2 was knocked down by transduction with Ad- Nrf2 shRNA. Results indicate that silencing of Nrf2 in ACHN cells abolished the effects of Pazopanib in stimulating cellular senescence and reducing telomerase activity. Consistently, knockdown of Nrf2 restored the expression of p53 and PAI in ACHN cells. Based on these results, we explored a novel mechanism whereby which Pazopanib displays a cytotoxicity effect in RCC cells through promoting cellular senescence mediated by Nrf2.

Endometrial stem cells alleviate cisplatin-induced ferroptosis of granulosa cells by regulating Nrf2 expression.

BACKGROUND: Premature ovarian failure (POF) caused by cisplatin is a severe and intractable sequela for young women with cancer who received chemotherapy. Cisplatin causes the dysfunction of granulosa cells and mainly leads to but is not limited to its apoptosis and autophagy. Ferroptosis has been also reported to participate, while little is known about it. Our previous experiment has demonstrated that endometrial stem cells (EnSCs) can repair cisplatin-injured granulosa cells. However, it is still unclear whether EnSCs can play a repair role by acting on ferroptosis. METHODS: Western blotting and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were applied to detect the expression levels of ferroptosis-related genes. CCK-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assays were used to evaluate cell viability. Transmission electron microscopy (TEM) was performed to detect ferroptosis in morphology. And the extent of ferroptosis was assessed by ROS, GPx, GSSG and MDA indicators. In vivo, ovarian morphology was presented by HE staining and the protein expression in ovarian tissue was detected by immunohistochemistry. RESULTS: Our results showed that ferroptosis could occur in cisplatin-injured granulosa cells. Ferroptosis inhibitor ferrostatin-1 (Fer-1) and EnSCs partly restored cell viability and mitigated the damage of cisplatin to granulosa cells by inhibiting ferroptosis. Moreover, the repair potential of EnSCs can be markedly blocked by ML385. CONCLUSION: Our study demonstrated that cisplatin could induce ferroptosis in granulosa cells, while EnSCs could inhibit ferroptosis and thus exert repair effects on the cisplatin-induced injury model both in vivo and in vitro. Meanwhile, Nrf2 was validated to participate in this regulatory process and played an essential role.

Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples (N = 90). With default filter settings, all but two potential MET exon 14 skipping variants were identified at similar allele frequencies. Both MET variants were found with an adapted calling filter. Three additional variants (KEAP1, STK11, TP53) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25 ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.

[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.

Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration­resistant prostate cancer cells.

Cancer cells are characterized by increased glycolysis, known as the Warburg effect, which leads to increased production of cytotoxic methylglyoxal (MGO) and apoptotic cell death. Cancer cells often activate the protective nuclear factor erythroid 2­related factor2 (Nrf2)/glyoxalase1 (Glo1) system to detoxify MGO. The effects of sodium butyrate (NaB), a product of gut microbiota, on Nrf2/Glos/MGO pathway and the underlying mechanisms in prostate cancer (PCa) cells were investigated in the present study. Treatment with NaB induced the cell death and reduced the proliferation of PCa cells (DU145 and LNCap). Moreover, the protein kinase RNA-like endoplasmic reticulum kinase/Nrf2/Glo1 pathway was greatly inhibited by NaB, thereby accumulating MGO-derived adduct hydroimidazolone (MG-H1). In response to a high amount of MGO, the expression of Nrf2 and Glo1 was attenuated, coinciding with an increased cellular death. NaB also markedly inhibited the Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (Stat3) pathway. Conversely, co­treatment with Colivelin, a Stat3 activator, significantly reversed the effects of NaB on Glo1 expression, MG-H1 production, and the cell migration and viability. As expected, overexpression of Stat3 or Glo1 reduced NaB­induced cell death. The activation of calcium/calmodulin dependent protein kinase II gamma and reactive oxygen species production also contributed to the anticancer effect of NaB. The present study, for the first time, demonstrated that NaB greatly increases MGO production through suppression of the JAK2/Stat3/Nrf2/Glo1 pathway in DU145 cells, a cell line mimicking castration­resistant PCa (CRPC), suggesting that NaB may be a potential agent for PCa therapy.

Icariin alleviates the apoptosis of chondrocytes in osteoarthritis through regulating SIRT-1-Nrf2-HO-1 signaling.

Icariin has shown the potential to treat osteoarthritis (OA), but the specific mechanism still needs further exploration. Therefore, this study attempted to reveal the effect and mechanism of icariin on OA based on in vitro and in vivo experiments. In vivo, a mouse model of OA was established by cutting the anterior cruciate ligament, and 10 mg/kg icariin was given to mice orally. Then, the OA injury and pathological changes of cartilage tissue in mice were identified by OA index and hematoxylin and eosin staining. In vitro, the viability of C28/I2 cells incubated with different concentrations of icariin was detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide assay. Subsequently, C28/I2 cells induced by IL-1ß were used as the cell model of OA, the expression of Sirtuin (SIRT)-1 in cells was knocked down, and icariin was added for intervention. Next, western blot was used to observe the expression level of sirtuin 1 (SIRT-1)-Nrf2-heme oxygenase 1 (HO-1) signaling pathway-related proteins in cells of each group. Besides, cell viability and apoptosis were detected by MTT and apoptosis assay, and DNA damage was observed by comet assay. In vivo experiments, intragastric administration of icariin could effectively reduce the OA index of mice, improve the pathological changes of cartilage tissue, and obviously activated the SIRT-1-Nrf2-HO-1 signaling pathway. In vitro experiments, icariin did not exhibit toxic effect on C28/I2 cells, but could activate the SIRT-1-Nrf2-HO-1 signaling pathway, improve the viability, reduce the level of apoptosis and relieve the DNA damage in OA cells; however, these effects were inhibited by si- SIRT-1. Icariin can improve the symptoms of OA by activating the SIRT-1-Nrf2-HO-1 signaling pathway.

Tetramethylpyrazine Nitrone Promotes the Clearance of Alpha-Synuclein via Nrf2-Mediated Ubiquitin-Proteasome System Activation.

Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson's disease (PD). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent enhancement of the expression of the 20S proteasome core particles (20S CPs) and regulatory particles (RPs) increases proteasome activity, which can promote α-syn clearance in PD. Activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) may reduce oxidative stress by strongly inducing Nrf2 gene expression. In the present study, tetramethylpyrazine nitrone (TBN), a potent-free radical scavenger, promoted α-syn clearance by the ubiquitin-proteasome system (UPS) in cell models overexpressing the human A53T mutant α-syn. In the α-syn transgenic mice model, TBN improved motor impairment, decreased the products of oxidative damage, and down-regulated the α-syn level in the serum. TBN consistently up-regulated PGC-1α and Nrf2 expression in tested models of PD. Additionally, TBN similarly enhanced the proteasome 20S subunit beta 8 (Psmb8) expression, which is linked to chymotrypsin-like proteasome activity. Furthermore, TBN increased the mRNA levels of both the 11S RPs subunits Pa28αß and a proteasome chaperone, known as the proteasome maturation protein (Pomp). Interestingly, specific siRNA targeting of Nrf2 blocked TBN's effects on Psmb8, Pa28αß, Pomp expression, and α-syn clearance. In conclusion, TBN promotes the clearance of α-syn via Nrf2-mediated UPS activation, and it may serve as a potentially disease-modifying therapeutic agent for PD.

LncRNA CARMN inhibits abdominal aortic aneurysm formation and vascular smooth muscle cell phenotypic transformation by interacting with SRF.

Phenotypic transformation of vascular smooth muscle cells (VSMCs) plays a crucial role in abdominal aortic aneurysm (AAA) formation. CARMN, a highly conserved, VSMC-enriched long noncoding RNA (lncRNA), is integral in orchestrating various vascular pathologies by modulating the phenotypic dynamics of VSMCs. The influence of CARMN on AAA formation, particularly its mechanisms, remains enigmatic. Our research, employing single-cell and bulk RNA sequencing, has uncovered a significant suppression of CARMN in AAA specimens, which correlates strongly with the contractile function of VSMCs. This reduced expression of CARMN was consistent in both 7- and 14-day porcine pancreatic elastase (PPE)-induced mouse models of AAA and in human clinical cases. Functional analyses disclosed that the diminution of CARMN exacerbated PPE-precipitated AAA formation, whereas its augmentation conferred protection against such formation. Mechanistically, we found CARMN's capacity to bind with SRF, thereby amplifying its role in driving the transcription of VSMC marker genes. In addition, our findings indicate an enhancement in CAMRN transcription, facilitated by the binding of NRF2 to its promoter region. Our study indicated that CARMN plays a protective role in preventing AAA formation and restrains the phenotypic transformation of VSMC through its interaction with SRF. Additionally, we observed that the expression of CARMN is augmented by NRF2 binding to its promoter region. These findings suggest the potential of CARMN as a viable therapeutic target in the treatment of AAA.

A novel insight into the neuroprotective effects of cannabidiol: maintained apelin/dopamine synthesis, NRF2 signaling, and AKT/CREB/BDNF gene expressions.

Neuroinflammation is a process associated with degeneration and loss of neurons in different parts of the brain. The most important damage mechanisms in its formation are oxidative stress and inflammation. This study aimed to investigate the protective effects of cannabidiol (CBD) against neuroinflammation through various mechanisms. Thirty­two female rats were randomly divided into 4 groups as control, lipopolysaccharide (LPS), LPS + CBD and CBD groups. After six hours following LPS administration, rats were sacrificed, brain and cerebellum tissues were obtained. Tissues were stained with hematoxylin­eosin for histopathological analysis. Apelin and tyrosine hydroxylase synthesis were determined immunohistochemically. Total oxidant status and total antioxidant status levels were measured, and an oxidative stress index was calculated. Protein kinase B (AKT), brain-derived neurotrophic factor (BDNF), cyclic­AMP response element­binding protein (CREB) and nuclear factor erythroid 2­related factor 2 (NRF2) mRNA expression levels were also determined. In the LPS group, hyperemia, degeneration, loss of neurons and gliosis were seen in all three tissues. Additionally, Purkinje cell loss in the cerebellum, as well as neuronal loss in the cerebral cortex and hippocampus, were found throughout the LPS group. The expressions of AKT, BDNF, CREB and NRF2, apelin and tyrosine hydroxylase synthesis all decreased significantly. CBD treatment reversed these changes and ameliorated oxidative stress parameters. CBD showed protective effects against neuroinflammation via regulating AKT, CREB, BDNF expressions, NRF2 signaling, apelin and tyrosine hydroxylase synthesis.