Kelch-like ECH-associated Protein Research Articles

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.

Black tea bioactive phytoconstituents realign NRF2 for anticancer activity in lung adenocarcinoma.

Constitutive activation of nuclear factor erythroid 2-related factor 2 (NRF2) is pivotal in bestowing therapy resistance in cancer cells. Several phytochemicals have been reported with the potential of modulating NRF2. Therefore, it was hypothesized that NRF2-deregulated chemoresistance in lung adenocarcinoma (LUAD) may be counteracted by theaflavin-rich black tea (BT). A non-responsive LUAD cell line, A549, was the best sensitized towards cisplatin upon pre-treatment with BT. BT-mediated NRF2 reorientation was observed to be dependent on concentration and duration of treatment as well as on the mutational profile of NRF2 in A549 cells. Transient exposure of low-concentration BT hormetically downregulated NRF2, its downstream antioxidants, and drug transporter. BT also influenced the Kelch-like ECH-associated protein (KEAP1)-dependent cullin 3 (Cul3) and KEAP-1-independent signaling through epidermal growth factor receptor (EGFR) - rat sarcoma virus (RAS) - rapidly accelerated fibrosarcoma (RAF) - extracellular signal-regulated kinase 1/2 (ERK) - matrix metalloproteinase (MMP)-2 and MMP-9. The realignment of NRF2 in KEAP1-suppressed A549 cells enhanced the chemotherapeutic outcome. But a higher concentration of the same BT surprisingly upregulated NRF2 and its transcriptional targets with a subsequent decrease in the NRF2-regulatory machinery in NCI-H23 cells (a KEAP1-overexpressed LUAD cell line), ultimately resulting in a better anticancer response. The BT-mediated bidirectional NRF2 modulation was reconfirmed upon comparison with the action of a pharmacological NRF2 inhibitor, ML-385, in A549 and a known NRF2 activator, tertiary-butylhydroquinone, in NCI-H23 respectively. BT-mediated regulation of NRF2-KEAP1 and their upstream networks (EGFR/RAS/RAF/ERK) sufficed as a better anticancer agent than synthetic NRF2 modulators. Therefore, BT may be indicated as a potent multi-modal small molecule for increasing drug responsiveness in LUAD cells by maintaining NRF2/KEAP1 axis at an optimum level.

ATP2B3 Inhibition Alleviates Erastin-Induced Ferroptosis in HT-22 Cells through the P62-KEAP1-NRF2-HO-1 Pathway.

Ferroptosis participates in the occurrence and development of neurological disorders. Modulating ferroptosis may have therapeutic potential in nervous system diseases. Therefore, TMTbased proteomic analysis in HT-22 cells was performed to identify erastin-induced differentially expressed proteins. The calcium-transporting ATP2B3 (ATP2B3) was screened as a target protein. ATP2B3 knockdown markedly alleviated the erastin-induced decrease in cell viability and elevated ROS (p < 0.01) and reversed the up-regulation of oxidative stress-related proteins polyubiquitin-binding protein p62 (P62), nuclear factor erythroid 2-related factor2 (NRF2), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase-1 (NQO1) protein expression (p < 0.05 or p < 0.01) and the down-regulation of Kelch-like ECH-associated protein 1(KEAP1) protein expression (p < 0.01). Moreover, NRF2 knockdown, P62 inhibition, or KEAP1 overexpression rescued the erastin-induced decrease in cell viability (p < 0.05) and increase in ROS production (p < 0.01) in HT-22 cells, while simultaneous overexpression of NRF2 and P62 and knockdown of KEAP1 partially offset the relief effect of ATP2B3 inhibition. In addition, knockdown of ATP2B3, NRF2, and P62 and overexpression of KEAP1 significantly down-regulated erastin-induced high expression of the HO-1 protein, while HO-1 overexpression reversed the alleviating effects of ATP2B3 inhibition on the erastin-induced decrease in cell viability (p < 0.01) and increase in ROS production (p < 0.01) in HT-22 cells. Taken together, ATP2B3 inhibition mediates the alleviation of erastin-induced ferroptosis in HT-22 cells through the P62-KEAP1-NRF2-HO-1 pathway.

Protective effect of mussel polysaccharide on cyclophosphamide-induced intestinal oxidative stress injury via Nrf2-Keap1 signaling pathway.

The hard-shelled mussel (Mytilus coruscus) has been used as a traditional Chinese medicine and health food in China for centuries. Polysaccharides from mussel has been reported to have multiple biological functions, however, it remains unclear whether mussel polysaccharide (MP) exerts protective effects in intestinal functions, and the underlying mechanisms of action remain unclear. The aim of this study was to investigate the protective effects and mechanism of MP on intestinal oxidative injury in mice. In this study, 40 male BALB/C mice were used, with 30 utilized to produce an animal model of intestinal oxidative injury with intraperitoneal injection of cyclophosphamide (Cy) for four consecutive days. The protective effects of two different doses of MP (300 and 600 mg/kg) were assessed by investigating the change in body weight, visceral index, and observing colon histomorphology. Moreover, the underlying molecular mechanisms were investigated by measuring the antioxidant enzymes and related signaling molecules through ELISA, real-time PCR, and western blot methods. The results showed that MP pretreatment effectively protected the intestinal from Cy-induced injury: improved the colon tissue morphology and villus structure, increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, and reduced malondialdehyde (MDA) content in serum and colon tissues. Meanwhile, MP also significantly increased the expression levels of SOD, GSH-Px, heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) mRNA in colon tissues. Further, western blot results showed that the expression of Nrf2 protein was significantly upregulated while kelch-like ECH-associated protein 1 (Keap1) was significantly downregulated by MP in the colonic tissues. This study indicates that MP can ameliorate Cy-induced oxidative stress injury in mice, and Nrf2-Keap1 signaling pathway may mediate these protective effects.

The KEAP1–NRF2 pathway regulates TFEB/TFE3-dependent lysosomal biogenesis

The maintenance of redox and metabolic homeostasis is integral to embryonic development. Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress-induced transcription factor that plays a central role in the regulation of redox balance and cellular metabolism. Under homeostatic conditions, NRF2 is repressed by Kelch-like ECH-associated protein 1(KEAP1). Here, we demonstrate that Keap1 deficiency induces Nrf2 activation and postdevelopmental lethality. Loss of viability is preceded by severe liver abnormalities characterized by an accumulation of lysosomes. Mechanistically, we demonstrate that loss of Keap1 promotes aberrant activation of transcription factor EB(TFEB)/transcription factor binding to IGHM Enhancer 3(TFE3)-dependent lysosomal biogenesis. Importantly, we find that NRF2-dependent regulation of lysosomal biogenesis is cell autonomous and evolutionarily conserved. These studies identify a role for the KEAP1-NRF2 pathway in the regulation of lysosomal biogenesis and suggest that maintenance of lysosomal homeostasis is required during embryonic development.

Polysaccharides from sea buckthorn (Hippophae rhamnoides L.) berries ameliorate cognitive dysfunction in AD mice induced by a combination of d-gal and AlCl3 by suppressing oxidative stress and inflammation reaction.

The therapeutic properties of Hippophae rhamnoides L. were known in Ancient Greece and in Tibetan and Mongolian medicine, which commonly used it for the treatment of heart ailments, rheumatism, and brain disorders. Modern studies have indicated that Hippophae rhamnoides L. polysaccharide (HRP) can improve cognitive impairment in mice with Alzheimer's disease (AD) but the specific mechanisms of the protective effect of HRP have not been elucidated fully. Our results showed that Hippophae rhamnoides L. polysaccharide I (HRPI) improved pathological behaviors related to memory and cognition, and reduced 1 Beta-amyloid (Aβ) peptide deposition and neuronal cell necrosis. Pretreatment with Hippophae rhamnoides L. polysaccharide I (HRPI) also decreased the level of Toll-like receptor 4 (TLR4) and Myeloid differentiation factor 88 (MyD88), and reduced the release of inflammatory factors Tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6) in the brains of mice with AD. Treatment with HRPI also suppressed the expression level of Recombinant Kelch Like ECH Associated Protein 1 (KEAP1), and increased the levels of Nuclear factor erythroid 2-Related Factor 2 (Nrf2), antioxidant enzymes Superoxide dismutase (SOD) and Glutathione peroxidase (GSH-Px) in the brains of AD mice. On the whole, these findings revealed that HRPI could improve the learning and memory ability and attenuate pathologic impairment in AD mice, and the underlying mechanisms may involve mediating oxidative stress and inflammation, possibly through the regulation of the Keap1/Nrf2 and TLR4/MyD88 signaling pathways. © 2023 Society of Chemical Industry.

Park7 protects retinal ganglion cells and promotes functional preservation after optic nerve crush via regulation of the Nrf2 signaling pathway.

We aim to investigate the effect of Park7 on mice RGC survival and function following optic nerve crush (ONC), and to explore its potential mechanism. Wild-type male C57BL/6J mice were subjected to optic nerve crush. Six weeks before ONC, mice received rAAV-shRNA (Park7)-EGFP or rAAV-EGFP intravitreally. Western blotting was used to detect Park7 levels. RGC survival was measured using immunofluorescence. Retinal cell apoptosis was detected using terminal deoxynucleotidyl transferase nick-end-labelling. An electroretinogram (ERG) and the optomotor response (OMR) were used to assess RGC function. Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor (Nrf2), and heme oxygenase 1 (HO-1) levels were assessed using western blotting. ONC injury increased the relative expression of Park7 significantly and decreased RGC survival, the amplitude of the photopic negative response (PhNR), and OMR. Intravitreal injection of rAAV-shRNA(Park7)-EGFP downregulated Park7 expression and was clearly demonstrated by the green fluorescence protein in many retinal layers. Moreover, Park7 downregulation aggravated the decrease in RGC survival and amplitude of PhNR as well as the visual acuity after ONC. However, inhibition of Park7 significantly increased Keap1 levels, decreased the total and nuclear Nrf2 levels, and reduced HO-1 levels. Park7 downregulation enhanced RGC injury and decreased retinal electrophysiological response and OMR after ONC in mice via the Keap1-Nrf2-HO-1 signaling pathway. Park7 may have neuroprotective effects and could represent a novel way to treat optic neuropathy.

Gentisic acid ameliorates type 2 diabetes induced by Nicotinamide-Streptozotocin in male mice by attenuating pancreatic oxidative stress and inflammation through modulation of Nrf2 and NF-кB pathways

AimsThere is a close link between oxidative stress, inflammation, and type 2 diabetes mellitus (T2DM). Gentisic acid (GA) is a di-phenolic compound and an active metabolite of aspirin that possesses antioxidant and anti-inflammatory properties, but its potential anti-diabetic effects have not been evaluated so far. Therefore, this study aimed to evaluate GA's potential antidiabetic effects through the Nuclear Factor Erythroid 2-Related Factor (Nrf2) and Nuclear Factor Kappa Beta (NF-кB) signaling pathways. Material and methodsIn this study, T2DM induced by a single intraperitoneal injection of STZ (65 mg/kg B.W) after 15 min nicotinamide (120 mg/kg B.W) injection. After seven days of injections, fasting blood glucose (FBS) was measured. Seven days after FBS monitoring treatments started. Grouping and treatments were as follows: 1) Normal control group; NC, 2) Diabetic control group; DC, 3) Metformin group; MT (150 mg/kg B.W, daily), 4) Test group; GA (100 mg/kg B.W, daily). Treatments continued for 14 consecutive days. Key findingsDiabetic mice treatment with GA significantly decreased FBS, improved plasma lipid profiles and pancreatic antioxidant status. GA modulated Nrf2 pathway by upregulation of Nrf2 protein, NAD(P)H: quinone oxidoreductase 1 (Nqo1), and p21, and downregulation of miR-200a, Kelch-like ECH-associated protein 1 (Keap1), and nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2). Also, GA attenuated inflammation by upregulation of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and interleukin-10 (IL-10) and downregulation of miR-125b, NF-кB, tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß). SignificanceGA attenuates T2DM, possibly by improving antioxidant status through the Nrf2 pathway and attenuation of inflammation.