Kelch-like ECH-associated Protein Research Articles

Biological insights in non-small cell lung cancer.

Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress. Several tumor types, including non-small cell lung cancer (NSCLC), upregulate the system xc- cystine/glutamate antiporter (xCT) through overexpression of the cystine transporter SLC7A11, thus sustaining intracellular cysteine levels to support glutathione synthesis. Nuclear factor erythroid 2-related factor 2 (NRF2) serves as a master regulator of oxidative stress resistance by regulating SLC7A11, whereas Kelch-like ECH-associated protein (KEAP1) acts as a cytoplasmic repressor of the oxidative responsive transcription factor NRF2. Mutations in KEAP1/NRF2 and p53 induce SLC7A11 activation in NSCLC. Extracellular cystine is crucial in supplying the intracellular cysteine levels necessary to combat oxidative stress. Disruptions in cystine availability lead to iron-dependent lipid peroxidation, thus resulting in a type of cell death called ferroptosis. Pharmacologic inhibitors of xCT (either SLC7A11 or GPX4) induce ferroptosis of NSCLC cells and other tumor types. When cystine uptake is impaired, the intracellular cysteine pool can be sustained by the transsulfuration pathway, which is catalyzed by cystathionine-B-synthase (CBS) and cystathionine g-lyase (CSE). The involvement of exogenous cysteine/cystine and the transsulfuration pathway in the cysteine pool and downstream metabolites results in compromised CD8+ T cell function and evasion of immunotherapy, diminishing immune response and potentially reducing the effectiveness of immunotherapeutic interventions. Pyroptosis is a previously unrecognized form of regulated cell death. In NSCLCs driven by EGFR, ALK, or KRAS, selective inhibitors induce pyroptotic cell death as well as apoptosis. After targeted therapy, the mitochondrial intrinsic apoptotic pathway is activated, thus leading to the cleavage and activation of caspase-3. Consequently, gasdermin E is activated, thus leading to permeabilization of the cytoplasmic membrane and cell-lytic pyroptosis (indicated by characteristic cell membrane ballooning). Breakthroughs in KRAS G12C allele-specific inhibitors and potential mechanisms of resistance are also discussed herein.

N-Acetyl-l-cysteine-Derived Carbonized Polymer Dots with ROS Scavenging via Keap1-Nrf2 Pathway Regulate Alveolar Bone Homeostasis in Periodontitis.

Periodontitis is a type of chronic inflammatory oral disease characterized by the destruction of periodontal connective tissue and progressive alveolar bone resorption. As oxidative stress is the key cause of periodontitis in the early periodontal microenvironment, antioxidative therapy has been considered a viable treatment for periodontitis. However, more stable and effective reactive oxygen species (ROS)-scavenging nanomedicines are still highly needed due to the instability of traditional antioxidants. Herein, a new type of N-acetyl-l-cysteine (NAC)-derived red fluorescent carbonized polymer dots (CPDs) has been synthesized with excellent biocompatibility, which can serve as an extracellular antioxidant to scavenge ROS effectively. Moreover, NAC-CPDs can promote osteogenic differentiation in human periodontal ligament cells (hPDLCs) under H2 O2 stimulation. In addition, NAC-CPDs are capable of targeted accumulation in alveolar bone in vivo, reducing the level of alveolar bone resorption in periodontitis mice, as well as performing fluorescence imaging in vitro and in vivo. In terms of mechanism, NAC-CPDs may regulate redox homeostasis and promote bone formation in the periodontitis microenvironment by modulating the kelch-like ECH-associated protein l (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. This study provides a new strategy for the application of CPDs theranostic nanoplatform for periodontitis.

Buspirone attenuated methotrexate-induced hippocampal toxicity in rats by regulating Nrf2/HO-1, PPAR-γ, NF-κB/nNOS, and ROS/NLRP3/caspase-1 signaling pathways.

Methotrexate (MTX) is a chemotherapeutic agent widely used to treat a variety of tumors. Nonetheless, MTX-induced hippocampal neurotoxicity is a well-defined dose-limiting adverse effect that limits clinical utility. Proinflammatory cytokine production and oxidative stress are possible mechanisms for MTX-induced neurotoxicity. Buspirone (BSP), a partial agonist of the 5-HT1a receptor (5-HT1aR), has emerged as an anxiolytic drug. BSP has been shown to possess antioxidant and anti-inflammatory effects. The current study investigated BSP's potential anti-inflammatory and antioxidant effects in attenuating MTX-induced hippocampal toxicity. Rats received either BSP (1.5 mg/kg) orally for 10 days and MTX (20 mg/kg) i.p. on Day 5. BSP administration markedly protected hippocampal neurons from drastic degenerated neuronal changes induced by MTX. BSP significantly attenuated oxidative injury by downregulating Kelch-like ECH-associated protein 1 expression while potently elevating hippocampal Nrf2, heme oxygenase-1, and peroxisome proliferator-activated receptor expression. BSP dampened inflammation by reducing NO2 - , tumor necrosis factor-alpha, IL-6, and interleukin 1 beta levels mediated by downregulating NF-κB and neuronal nitric oxides synthase expression. Moreover, BSP potently counteracted hippocampal pyroptosis by downregulating NLRP3, ASC, and cleaved-caspase-1 proteins. Therefore, BSP may represent a promising approach to attenuate neurotoxicity in patients receiving MTX.

USP25 regulates KEAP1-NRF2 anti-oxidation axis and its inactivation protects acetaminophen-induced liver injury in male mice

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor responsible for mounting an anti-oxidation gene expression program to counter oxidative stress. Under unstressed conditions, Kelch-like ECH-associated protein 1 (KEAP1), an adaptor protein for CUL3 E3 ubiquitin ligase, mediates NRF2 ubiquitination and degradation. We show here that the deubiquitinase USP25 directly binds to KEAP1 and prevents KEAP1’s own ubiquitination and degradation. In the absence of Usp25 or if the DUB is inhibited, KEAP1 is downregulated and NRF2 is stabilized, allowing the cells to respond to oxidative stress more readily. In acetaminophen (APAP) overdose-induced oxidative liver damage in male mice, the inactivation of Usp25, either genetically or pharmacologically, greatly attenuates liver injury and reduces the mortality rates resulted from lethal doses of APAP.

Astaxanthin inhibits oxidative stress and apoptosis in diabetic retinopathy

BackgroundThe pathophysiology of diabetic retinopathy (DR) is thought to be influenced by oxidative stress. Astaxanthin (ASX) is a natural product with antioxidant effect, but it is not clear whether its mechanism of inhibiting the development of DR is related to anti-oxidation. MethodsRats were intraperitoneally injected with streptozotocin (60 mg/kg) to create DR rat models followed by ASX (20 mg/kg) for 45 days. Retinal tissue was examined by Hematoxylin and Eosin staining. By using Enzyme-linked immunosorbent assay (ELISA), 2,7-Dichlorodrhydrofluorescein diace (DCFH-DA) probes, immunohistochemistry and western blot, it was feasible to evaluate the contents of inflammation-related factors (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and macrophage inhibitory cytokine-1 (MIC-1)), oxidative stress-related indicators (glutathione (GSH), malonic dialdehyde (MDA), glutathione peroxidase (GPx), reactive oxygen species (ROS) and Total antioxidant capacity (T-AOC)), antioxidant enzymes (hemoxgenase-1(HO-1) and Quinone Oxidoreductase 1 (NQO1)), and apoptosis-related proteins (Bcl-2, Bcl2 Associated X Protein (BAX), and cleaved-caspase-3). Additionally, antioxidant proteins downstream of the nuclear factor E2 related factors (Nrf-2) pathway, expression levels of Nrf2/ Kelch-like ECH-associated protein 1(Keap 1) pathway-associated proteins, and nuclear and cytoplasmic levels of Nrf2 were assessed using immunohistochemistry, western blot, or quantitative real-time polymerase chain reaction (qRT-PCR). ResultsASX alleviated retinal tissue damage by increasing overall retina thickness and ganglion cell layer (GCL) cell numbers and exerted the anti-inflammatory, anti-oxidative stress, and anti-apoptosis effects in DR rats. Additionally, ASX could inhibit the expression of Keap1, promote the transport of Nrf2 from cytoplasm to nucleus and facilitate the expressions of HO-1, NQO1, γ-glutamylcysteine synthetase, (γ-GCS) and GPx. ConclusionASX exerted antioxidant effects through Nrf2/keap1 pathway, thereby alleviating apoptosis, inflammation, and oxidative stress in retinal tissues of DR rats.

Development of KEAP1-targeting PROTAC and its antioxidant properties: In vitro and in vivo

Oxidative stress due to abnormal accumulation of reactive oxygen species (ROS) is an initiator of a large number of human diseases, and thus, the elimination and prevention of excessive ROS are important aspects of preventing the development of such diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is an essential transcription factor that defends against oxidative stress, and its function is negatively controlled by Kelch-like ECH-associated protein 1 (KEAP1). Therefore, activating NRF2 by inhibiting KEAP1 is viewed as a strategy for combating oxidative stress-related diseases. Here, we generated a cereblon (CRBN)-based proteolysis-targeting chimera (PROTAC), which we named SD2267, that induces the proteasomal degradation of KEAP1 and leads to NRF2 activation. As was intended, SD2267 bound to KEAP1, recruited CRBN, and induced the degradation of KEAP1. Furthermore, the KEAP1 degradation efficacy of SD2267 was diminished by MG132 (a proteasomal degradation inhibitor) but not by chloroquine (an autophagy inhibitor), which suggested that KEAP1 degradation by SD2267 was proteasomal degradation-dependent and autophagy-independent. Following KEAP1 degradation, SD2267 induced the nuclear translocation of NRF2, which led to the expression of NRF2 target genes and attenuated ROS accumulation induced by acetaminophen (APAP) in hepatocytes. Based on in vivo pharmacokinetic study, SD2267 was injected intraperitoneally at 1 or 3 mg/kg in APAP-induced liver injury mouse model. We observed that SD2267 degraded hepatic KEAP1 and attenuated APAP-induced liver damage. Summarizing, we described the synthesis of a KEAP1-targeting PROTAC (SD2267) and its efficacy and mode of action in vitro and in vivo. The results obtained suggest that SD2267 could be used to treat hepatic diseases related to oxidative stress.

Biological interactions and attenuation of MPTP-induced toxicity in Drosophila melanogaster by Trans-astaxanthin

Trans-astaxanthin (TA) is a carotenoid with amphipathic chemical structure found in yeast, and aquatic organisms. It is known to possess both antioxidative and anti-inflammatory properties. This study was carried out to investigate the ameliorative action of TA on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in Drosophila melanogaster (Fruit fly). The flies were orally treated with TA (2.5 mg/10 g diet) and/or MPTP (500 µM) for 5 days. Thereafter, we evaluated selected biomarkers of locomotor deficits (acetylcholinesterase (AChE) and negative geotaxis), oxidative stress (hydrogen peroxide (H2O2), protein carbonyls (PC)), antioxidants (total thiols (T-SH), non-protein thiols, glutathione-S-transferase (GST) and catalase), and inflammation (nitric oxide (nitrite/nitrate) in the flies. Furthermore, we investigated molecular docking analysis of TA against Kelch-like ECH-associated protein 1 (Keap1)) of Homo sapiens and D. melanogaster. The results indicated that TA increased MPTP-induced decreased activities of AChE, GST, and catalase, as well as levels of non-protein thiols and T-SH compared with MPTP-treated flies (p < 0.05). Furthermore, TA attenuated inflammation, and improved locomotor deficit in the flies. The molecular docking data showed that TA had docking scores for binding both the Human and Drosophila Keap1, nearly closer to or higher than the standard inhibitor. The attenuating effects of TA against MPTP-induced toxicity could arise from its antioxidative and anti-inflammatory properties as well as its chemical structure.

Differential tissue regulation of nrf2/keap1 crosstalk in response to Aeromonas infection in Nile tilapia: a comparative study

The antioxidant and detoxification systems involve intricate pathways in which nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) play pivotal roles. In the basal state, reactive oxygen species are generated and neutralized in a balanced manner. However, stressors can disrupt this equilibrium, resulting in oxidative stress and cellular damage. In this study, we analyzed the expression of nrf2 and keap1 in Nile tilapia (Oreochromis niloticus) under homeostasis and challenge with Aeromonas hydrophila. During homeostasis, the predominant expression of nrf2 was observed in the liver, blood, muscle, gut, and gills, while keap1 was highly expressed in the brain, liver, blood, spleen, eye, head kidney, and gills. After the challenge, the spleen demonstrated the highest keap1 expression, while the liver displayed the highest nrf2 levels among the tissues examined. Apparently, our findings suggest that the spleen may be susceptible to initial damage following infection, leading to the manifestation of the first lesion. This susceptibility could be attributed to the spleen’s high expression of keap1, acting as a negative regulator of nrf2. Notably, a positive correlation was observed between nrf2 and keap1 expression in several tissues, with the strongest association observed in the blood, gills, and head kidney under both normal and inflammatory conditions. Our findings indicate that blood may serve as a crucial mediator of Nrf2/Keap1 signaling in tissues like the liver and gut during normal and inflammatory states. By shedding light on the altered expression and correlation of nrf2 and keap1 in various tissues, this study elucidates their potential connection to antioxidant and immune responses, as well as the pathological features of A. hydrophila infection.

#4176 ROLE OF KIDNEY FUNCTION ON NRF2 MRNA LEVELS IN TYPE-2 DIABETES

Abstract Background and Aims Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces the expression of genes coding for antioxidant proteins and phase II detoxifying enzymes. Repressor molecule Kelch-like ECH-associated protein 1 (Keap1) and several microRNAs (miRNA) negatively regulate Nrf2 gene expression at post-transcriptional level. Oxidative stress is a major factor for kidney damage in diabetes but the role of Nrf2 in kidney dysfunction in diabetes is still unclear. Method To test whether the gene expression of Nrf-2 is downregulated in type-2 diabetes and whether kidney dysfunction per se further reduces Nrf2 in diabetic patients with diabetic nephropathy, we carried out a case-control study including 99 participants divided into three independent groups: 33 patients with diabetic nephropathy (DN), 33 patients with type 2 diabetes without nephropathy (D) and 33 control subjects (C), all accurately matched for age and sex. Standard ANOVA compared outcome measures in the three groups. Multiple linear regression analysis was used to test the expression levels of Keap1, miR-28-5p, miR-93-5p, miR-30e-5p, miR-125b-5p and miR-150-5p as potential mediators in the observed reduction of Nrf2 mRNA levels in patients with diabetic nephropathy. Results In patients with diabetic nephropathy, Nrf2 gene expression levels (0.82 arbitrary units, AU, IQR: 0.60-1.22 AU) were significantly lower than in diabetic patients without nephropathy (1.19 AU, 0.90-1.38 AU, P = 0.01) and control subjects (0.82 AU, 0.60-1.22 AU vs 1.04 AU, 0.87-1.66, P = 0.02) while there was no difference between diabetic patients without nephropathy and control subjects (P = 0.69). Keap1 gene expression levels were almost identical in patients with and without diabetic nephropathy (1.15 AU, 0.63-2.14 AU vs 1.13 AU, 0.79-1.73 AU, P = 0.91) and higher than in control subjects (1.15 AU, 0.63-2.14 AU vs 0.79 AU, 0.49-1.08 AU, P = 0.046). MiRNA expression levels were comparable in diabetic patients with and without nephropathy, but miRNA 30e-5p was lower in patients with diabetic nephropathy than in diabetic patients without nephropathy (0.91 AU, 0.76-1.10 AU vs 1.06 AU, 0.89-1.23 AU, P = 0.016). The expression levels of Nrf2 and miRNA 30e-5p were unrelated in patients with (r = 0.15, P = 0.44) and without (r = -0.02, P = 0.90) diabetic nephropathy. In linear regression analyses, eGFR emerged as the first factor in rank for explaining the difference in Nrf2 mRNA levels (explained variance 76%) among patients with and without diabetic nephropathy. Conclusion This case-control study comparing the gene expression level of Nrf2 in diabetic patients with and without nephropathy shows that kidney dysfunction is the key factor that explains the variability in Nrf2 mRNA levels in diabetes. These data suggest a primary role of Nrf2 in redox homeostasis and kidney damage in diabetic nephropathy.

Veronica persica Ethanol Extract Ameliorates Dinitrochlorobenzene-Induced Atopic Dermatitis-like Skin Inflammation in Mice, Likely by Inducing Nrf2/HO-1 Signaling.

Atopic dermatitis (AD) is chronic allergic contact dermatitis with immune dysregulation. Veronica persica has pharmacological activity that prevents asthmatic inflammation by ameliorating inflammatory cell activation. However, the potential effects of the ethanol extract of V. persica (EEVP) on AD remain elusive. This study evaluated the activity and underlying molecular pathway of EEVP in two AD models: dinitrochlorobenzene (DNCB)-induced mice and interferon (IFN)-γ/tumor necrosis factor (TNF)-α-stimulated human HaCaT keratinocytes. EEVP attenuated the DNCB-induced increase in serum immunoglobulin E and histamine levels, mast cell counts in toluidine-blue-stained dorsal skin, inflammatory cytokine (IFN-γ, interleukin [IL]-4, IL-5, and IL-13) levels in cultured splenocytes, and the mRNA expression of IL6, IL13, IL31 receptor, CCR-3, and TNFα in dorsal tissue. Additionally, EEVP inhibited the IFN-γ/TNF-α-induced mRNA expression of IL6, IL13, and CXCL10 in HaCaT cells. Furthermore, EEVP restored the IFN-γ/TNF-α-induced downregulation of heme oxygenase (HO)-1 in HaCaT cells by inducing nuclear factor erythroid 2-related factor 2 (Nrf2) expression. A molecular docking analysis demonstrated that EEVP components have a strong affinity to the Kelch-like ECH-associated protein 1 Kelch domain. In summary, EEVP inhibits inflammatory AD by attenuating immune cell activation and inducing the Nrf2/HO-1 signaling pathway in skin keratinocytes.

AMG-510 and cisplatin combination increases antitumor effect in lung adenocarcinoma with mutation of KRAS G12C: a preclinical and translational research

BackgroundThe efficacy of monotherapy of AMG-510 is limited. This study explored whether the AMG-510 and cisplatin combination increases the anti-tumor effect in lung adenocarcinoma with the mutation of Kirsten rat sarcoma viral oncogene (KRAS) G12C.MethodsPatients’ data were used to analyze the proportion of KRAS G12C mutation. Besides, the next-generation sequencing data was used to uncover information about co-mutations. The cell viability assay, the concentration inhibiting 50% of cell viability (IC50) determination, colony formation, and cell-derived xenografts were conducted to explore the anti-tumor effect of AMG-510, Cisplatin, and their combination in vivo. The bioinformatic analysis was conducted to reveal the potential mechanism of drug combination with improved anticancer effect.ResultsThe proportion of KRAS mutation was 2.2% (11/495). In this cohort with KRAS mutation, the proportion of G12D was higher than others. Besides, KRAS G12A mutated tumors had the likelihood of concurrent serine/threonine kinase 11 (STK11) and kelch-like ECH-associated protein 1 (KEAP1) mutations. KRAS G12C and tumor protein p53 (TP53) mutations could appear at the same time. In addition, KRAS G12D mutations and C-Ros oncogene 1 (ROS1) rearrangement were likely to be present in one tumor simultaneously. When the two drugs were combined, the respective IC50 values were lower than when used alone. In addition, there was a minimum number of clones among all wells in the drug combination. In in vivo experiments, the tumor size reduction in the drug combination group was more than twice that of the single drug group (p < 0.05). The differential expression genes were enriched in the pathways of phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling and extracellular matrix (ECM) proteoglycans compared the combination group to the control group.ConclusionsThe anticancer effect of the drug combination was confirmed to be better than monotherapy in vitro and in vivo. The results of this study may provide some information for the plan of neoadjuvant therapy and the design of clinical trials for lung adenocarcinoma patients with KRAS G12C mutation.

Dipeptidyl peptidase 3 is essential for maintaining osteoblastic differentiation under a high-glucose environment by inhibiting apoptosis, oxidative stress and inflammation through the modulation of the Keap1-Nrf2 pathway

Dipeptidyl peptidase 3 (Dpp3) has emerged as a pivotal mediator of bone homeostasis and bone loss pathology. However, whether Dpp3 plays a role in diabetic osteoporosis has not been addressed. Therefore, this work explored the possible role of Dpp3 in osteoblast dysfunction evoked by high glucose (HG), a cellular model for studying diabetic osteoporosis in vitro. Dpp3 expression was decreased in the pre-osteoblast MC3T3-E1 during osteoblastic differentiation under the HG environment. The osteoblastic differentiation impaired by HG was reversed in Dpp3-overexpressing MC3T3-E1 cells. The migration and invasion of MC3T3-E1 cells impeded by HG were reversed by Dpp3 overexpression. Moreover, HG-evoked apoptosis, oxidative stress and inflammation were ameliorated in Dpp3-overexpressing MC3T3-E1 cells. A mechanistic study showed that Dpp3 up-regulated the activation of nuclear factor E2-related factor 2 (Nrf2) depending on Kelch-like ECH-associated protein 1 (Keap1). The blockade of Nrf2 reversed Dpp3-mediated effects on osteoblastic differentiation, apoptosis, oxidative stress and inflammation of HG-stimulated MC3T3-E1 cells. Therefore, Dpp3 plays an essential role in maintaining osteoblastic differentiation under a HG environment associated with the regulation of the Keap1-Nrf2 pathway. This work indicates a possible relationship between Dpp3 and diabetic osteoporosis.

Role of Natural and Synthetic Compounds in Modulating NRF2/KEAP1 Signaling Pathway in Prostate Cancer.

Prostate cancer is the second most common cancer in men worldwide. Prostate cancer can be treated by surgery or active surveillance when early diagnosed but, when diagnosed at an advanced or metastatic stage, radiation therapy or androgen-deprivation therapy is needed to reduce cancer progression. However, both of these therapies can cause prostate cancer resistance to treatment. Several studies demonstrated that oxidative stress is involved in cancer occurrence, development, progression and treatment resistance. The nuclear factor erythroid 2-related factor 2 (NRF2)/KEAP1 (Kelch-Like ECH-Associated Protein 1) pathway plays an important role in protecting cells against oxidative damage. Reactive oxygen species (ROS) levels and NRF2 activation can determine cell fate. In particular, toxic levels of ROS lead physiological cell death and cell tumor suppression, while lower ROS levels are associated with carcinogenesis and cancer progression. On the contrary, a high level of NRF2 promotes cell survival related to cancer progression activating an adaptive antioxidant response. In this review, we analyzed the current literature regarding the role of natural and synthetic compounds in modulating NRF2/KEAP1 signaling pathway in prostate cancer.

Safflor Yellow A Protects Beas-2B Cells Against LPS-Induced Injury via Activating Nrf2.

Acute lung injury and its severe form acute respiratory distress syndrome are lethal lung diseases. So far, effective therapy for the diseases is deficient and the prognosis is poor. Recently, it was found activating nuclear factor erythroid 2-related factor 2 could attenuate the injury including inflammation, oxidative stress, and apoptosis in those diseases. To discover novel therapy, we have evaluated safflor yellow A and explored the underlying mechanisms using Beas-2B cells injured by lipopolysaccharide. As a result, safflor yellow A could improve the viability of Beas-2B cells treated with lipopolysaccharide. Further investigations have revealed safflor yellow A suppressed oxidative stress induced by lipopolysaccharide via reducing reactive oxygen species and malondialdehyde, and elevating superoxide dismutase, catalase, and glutathione peroxidase. Meanwhile, the inflammation resulting from lipopolysaccharide was ameliorated through decreasing the pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6. It was also found nuclear factor κB was inactivated by safflor yellow A. In addition, safflor yellow A downregulated cysteinyl aspartate specific proteinase-3 and Bcl-2-associated X protein and upregulated B-cell lymphoma-2 to inhibited apoptosis of Beas-2B cells induced by lipopolysaccharide. The activation of nuclear factor erythroid 2-related factor 2 was observed in Beas-2B cells, which was associated with the protective effects of safflor yellow A. And molecular docking elucidated safflor yellow A interacted with Kelch-like ECH-associated protein 1 to activate nuclear factor erythroid 2-related factor 2. These results can provide evidences for the discovery of novel therapy for further evaluation of safflor yellow A in the treatment of acute lung injury and acute respiratory distress syndrome.

Insights into the Regulation of GFR by the Keap1-Nrf2 Pathway.

Literature data suggest that the activation of the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway increases glomerular filtration rate (GFR) in patients with type 2 diabetes and chronic kidney disease. However, the mechanisms whereby the Keap1-Nrf2 pathway regulates GFR are unknown. Methods: Various renal physiological parameters were assessed in C57BL/6 mice (WT), Nrf2-deficient mice, and Nrf2-activated Keap1-knockdown mice. Also, these parameters were assessed following the administration of RTA dh404 (CDDO-dhTFEA), a Nrf2 activator. Pharmacological and genetic Keap1-Nrf2 activation increased renal blood flow (p <0.05), glomerular volume (p <0.05) and GFR (p <0.05), but did not alter the afferent-to-efferent arteriolar diameter ratio or glomerular permeability. Calcium influx into the podocytes through TRPC channels in response to H2O2 was suppressed by Keap1-Nrf2 activation and TRPCs inhibition. Treatment with a TRPC6 and TRPC5 inhibitors increased single-nephron GFR in WT mice. In conclusion, the Keap1-Nrf2 pathway regulates GFR through changes in ultrafiltration by modulating redox-sensitive intracellular calcium signaling and cellular contractility, mediated through TRPC activity, in glomerular cells, in particular the podocytes.

Association between Kelch-like ECH-associated protein-1 and GRACE risk score in non-ST elevation myocardial infarction.

In this study, we measured the levels of Kelch-like ECH-associated protein 1 (KEAP1), which has the potential antioxidant capacity, among non-ST elevation myocardial infarction (NSTEMI) patients compared with healthy controls. We also investigated the possible association between KEAP1 levels and the GRACE score, which is a universal risk score commonly used for patients with acute myocardial infarction. As the patient group, 78 patients admitted to our center with a diagnosis of NSTEMI were included in the study. As the control group, 77 individuals found to have normal coronary arteries after coronary arteriography were included (155 patients in total). GRACE risk scores and left ventricular ejection fractions (LVEFs) were calculated, KEAP1 levels were measured, and the usual blood tests were performed. KEAP1 levels were significantly higher among the NSTEMI patients compared to the healthy control group (671.1 ± 120.7 vs. 262.7 ± 105.7, p < 0.001). We also found a moderate positive correlation between KEAP1 levels and GRACE risk scores among patients with NSTEMI (r = +0.521, p < 0.001). Additionally, a negative correlation between KEAP1 levels and LVEFs was detected (r = -0.264, p < 0.001). Elevated KEAP1 levels have the potential to be used as a risk factor for NSTEMI in terms of clinical adverse events and poor prognosis at admission.

Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet.

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)‍-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1‍‒‍14 and all infused with ETEC K88 1×1010 CFU on Days 15‍‒‍17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.

FRI-405 - Kelch-like ECH-associated protein-1 deletion rescues cell death associated with glutathione-glutathione peroxidase 4 knockdown in hepatocytes in acute models of liver injury

FRI-405 - Kelch-like ECH-associated protein-1 deletion rescues cell death associated with glutathione-glutathione peroxidase 4 knockdown in hepatocytes in acute models of liver injury

RRx-001: a chimeric triple action NLRP3 inhibitor, Nrf2 inducer, and nitric oxide superagonist.

RRx-001 is a shape shifting small molecule with Fast Track designation for the prevention/amelioration of chemoradiation-induced severe oral mucositis (SOM) in newly diagnosed Head and Neck cancer. It has been intentionally developed or "engineered" as a chimeric single molecular entity that targets multiple redox-based mechanisms. Like an antibody drug conjugate (ADC), RRx-001 contains, at one end a "targeting" moiety, which binds to the NLRP3 inflammasome and inhibits it as well as Kelch-like ECH-associated protein 1 (KEAP1), the negative regulator of Nrf2, and, at the other end, a conformationally constrained, dinitro containing 4 membered ring, which fragments under conditions of hypoxia and reduction to release therapeutically active metabolites i.e., the payload. This "payload", which is delivered specifically to hypoperfused and inflamed areas, includes nitric oxide, nitric oxide related species and carbon-centered radicals. As observed with ADCs, RRx-001 contains a backbone amide "linker" attached to a binding site, which correlates with the Fab region of an antibody, and to the dinitroazetidine payload, which is microenvironmentally activated. However, unlike ADCs, whose large size impacts their pharmacokinetic properties, RRx-001 is a nonpolar small molecule that easily crosses cell membranes and the blood brain barrier (BBB) and distributes systemically. This short review is organized around the de novo design and in vivo pro-oxidant/pro-inflammatory and antioxidant/anti-inflammatory activity of RRx-001, which, in turn, depends on the reduced to oxidized glutathione ratio and the oxygenation status of tissues.

Exosomal miR-141-3p from PDLSCs Alleviates High Glucose-Induced Senescence of PDLSCs by Activating the KEAP1-NRF2 Signaling Pathway.

Human periodontal ligament stem cells (PDLSCs) are the most promising stem cells for periodontal tissue engineering. Senescent PDLSCs have diminished abilities to proliferate and differentiate, affecting the efficiency of periodontal tissue repair and regeneration. Stem cell-derived exosomes are important participants in intercellular information exchange and can help ameliorate senescence. In this study, we investigated PDLSC senescence in a high glucose microenvironment as well as the ability of human periodontal ligament stem cell-derived exosomes (PDLSC-Exos) to alleviate cellular senescence and the underlying mechanisms. Herein, PDLSCs and PDLSC-Exos were isolated and extracted. Then, cellular senescence indicators were evaluated after high glucose (25 mM) treatment of cultured PDLSCs. PDLSC-Exos were cocultured with senescent PDLSCs to further explore the role of PDLSC-Exos in cellular senescence and determine the differences in cellular oxidative stress levels after PDLSC-Exo treatment. Next, we investigated whether PDLSC-Exos alleviated cellular senescence by restoring the balance of oxidative stress signals and explored the underlying molecular pathways. We discovered that PDLSCs underwent premature senescence due to high glucose culture, but they were rejuvenated by PDLSC-Exos. The rejuvenating effects of PDLSC-Exos were notably reversed by cotreatment with ML385, an inhibitor of nuclear factor erythroid 2-related factor 2 (NRF2), indicating that this recovery depended on NRF2 activation. Further analyses revealed that microRNA-141-3p (miR-141-3p) was expressed at relatively high levels in PDLSC-Exos and was instrumental in PDLSC-Exo-mediated restoration by downregulating Kelch-like ECH-associated protein 1 (KEAP1), which is a negative regulator of NRF2 expression. Our findings suggest that PDLSC-Exos alleviate high glucose-induced senescence of PDLSCs by transferring miR-141-3p to activate the KEAP1-NRF2 signaling pathway. Based on this research, PDLSC-Exos may behave similarly to their parental PDLSCs and have significant effects on cellular senescence by delivering their encapsulated bioactive chemicals to target cells.