Heme Oxygenase-1 Protein Expression Research Articles

Nootkatone mitigates periodontal inflammation and reduces alveolar bone loss via Nrf2/HO-1 and NF-κB pathways in rat model of periodontitis.

Periodontitis (PD) is a chronic inflammatory disease leading to alveolar bone loss. This study investigated the effect of nootkatone and regulatory mechanism in reducing periodontal inflammation and alveolar bone loss in a rat model. Twenty male Sprague-Dawley rats were divided into control, periodontitis, and nootkatone-treated groups (45 or 90 mg/kg). Ligature induction method was adopted to establish the PD model. After 21 days, rats received daily gavage of either saline or nootkatone for 10 days. Alveolar bone loss was assessed using micro-CT. Histological analyses included hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), and Masson's trichrome stainings. Immunohistochemistry for heme oxygenase 1 (HO-1) and nuclear factor erythroid-2 related factor 2 (Nrf2) were performed in periodontal tissues. Content of inflammatory cytokines IL-1β, IL-6, and TNF-α in gingival tissues around ligature were assessed using ELISA kits. Malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were analyzed and Western blot for NF-κB expression in gingival tissues were performed. Nootkatone significantly reduced the distance from cementoenamel junction to alveolar bone crest (CEJ-ABC), enhanced bone mineral density (BMD), bone volume (BV), and BV/total volume (TV) ratio in ligature-induced rats. Higher dose of nootkatone (90 mg/kg) did not show more significant therapeutic effect than lower dose (45 mg/kg). Histological staining showed decreased osteoclasts' number and improved bone architecture in the nootkatone group. Content of IL-1β, IL-6, and TNF-α and inflammatory cell infiltration level in gingival tissues around the ligature were decreased in the nootkatone-treatment rats. Nootkatone increased Nrf2 and HO-1 protein expression and decreased NF-κB protein level, suppressing MDA levels and enhancing SOD activity. In a rat model, nootkatone effectively mitigates periodontal inflammation and alveolar bone loss through the Nrf2/HO-1 and NF-κB pathways. These findings suggest nootkatone as a promising therapeutic agent for the treatment of periodontitis.

Evaluating the Role of Nrf-2/HO-1 Pathway in Glioblastoma Treatment Efficacy: A Co-Culture Study

Objective: Glioblastoma (GB) is a highly lethal form of brain tumor. Although standard therapy appears to be effective, the survival time is quite short, and the recurrence rate is high. Bortezomib (BTZ), is a proteasome inhibitor, used in GB therapies and resulted in serious off-target effects. Carfilzomib (CFZ), is an alternative for BTZ, has known with nonserious off-target effects. This study aimed to examine the potential off-target effects caused by BTZ and CFZ in terms of the therapy related activation of antioxidant mechanisms regarding to Nuclear factor (erythroid-derived 2)-like 2 (Nrf-2)/Heme Oxygenase-1 (HO-1)-dependent response. Methods: GB cells were co-cultured with heathy astrocyte (HA) cells to mimic the tumor microenvironment in some extent. Cell viability was determined following ionizing radiation (IR), temozolomide (TMZ), BTZ and CFZ alone and in combination. Nrf-2 and HO-1 protein expressions were analyzed by western blotting assay. Results: Co-culture results showed that the GB cells in the BTZ-treated groups expressed higher levels of Nrf-2 and HO-1 than in the CFZtreated groups. In the HAs, the group treated with CFZ showed higher Nrf-2 expression than the group treated with BTZ alone, while the same groups in combination with TMZ&IR showed exactly opposite results. HO-1 expression was also not seen in any of the HA groups. Conclusion: The significant increase in Nrf-2 levels in the CFZ-treated group in the HAs could also be interpreted as CFZ promoting the defence of healthy cells against therapy-induced stress conditions. Although further studies are needed, these preliminary results show that the evaluation of CFZ as a second-line therapy could be a milestone for the treatment of GB.

Berberine attenuates neuronal ferroptosis via the AMPK–NRF2–HO-1-signaling pathway in spinal cord-injured rats

Ferroptosis, characterized by iron-dependent accumulation of lipid peroxides, plays an important role in spinal cord injury (SCI). Berberine (BBR), as a lipid peroxide scavenger, has been widely used in treating other diseases; however, its role in ferroptosis has not been fully elucidated. Therefore, here, to test our hypothesis that BBR can reduce the severity of SCI and promote motor function recovery by inhibiting neuronal ferroptosis, we evaluated the changes in ferroptosis-related indicators after BBR administration by establishing a cellular ferroptosis model and an SCI contusion model. We found that BBR administration significantly reduces lipid peroxidation damage, maintains normal mitochondrial function, reduces excessive accumulation of iron ions, enhances antioxidant capacity, and activates the ferroptosis defense system in vivo and in vitro. Mechanistically, BBR alleviates neuronal ferroptosis by inducing adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and up-regulating nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) protein expression to promote glutathione production. BBR administration also significantly improves motor function recovery in SCI rats. Meanwhile, applying the AMPK inhibitor Compound C blocks the neuroprotective and all other effects of BBR. Collectively, our findings demonstrate that BBR can attenuate neuronal ferroptosis after SCI by activating the AMPK–NRF2–HO-1 pathway.

Schisandrol A Alleviates Allergic Asthma in Mice via Regulating the NF-κB/IκBα and Nrf2/HO-1 Signaling Pathways.

Schisandra chinensis (Turcz) Baill (S. chinensis) is the key traditional Chinese medicine for the treatment of asthma used by ancient and modern medical practitioners. However, the material basis and the main mechanism of its antiasthmatic effect remain unclear. Our preliminary results showed that schisandrol A (SCA), a representative monomer of Schisandra lignans, had the best relaxation effect on tracheal rings in isolated rats. In this research, a mouse asthma model was prepared by combining ovalbumin (OVA) with Al (OH)3 for exploring the antiasthmatic action and the underlying mechanism of SCA. The study results demonstrated that SCA improved the behavior of mice with asthma and pathological changes in their lung tissues and airways, decreased serum immunoglobulin E (IgE) and OVA-IgE levels, interleukin-4 (IL-4), IL-5, IL-13, and eotaxin contents, and leukocytes number in bronchoalveolar lavage fluid. SCA downregulated the gene expressions of keratinocyte-derived protein chemokines and ILs and reduced the expressions of phosphorylated IκB kinase α (p-IKKα) and p-nuclear factor kappa-B (NF-κB) proteins in lung tissues. In addition, it was found that SCA could significantly increase T-superoxide dismutase and catalase activities, decrease malondialdehyde content, and elevate p-IκBα, NF-E2-related-factor 2 (Nrf2), and heme oxygenase-1 (HO-1) protein expressions. In summary, SCA treatment resulted in a significant improvement in the allergic bronchial asthma in mice, and its mechanisms may involve the regulation of the NF-κB/IκBα pathway to reduce inflammatory response and the Nrf2/HO-1 pathway to improve the body's antioxidant capacity. These results suggest that SCA is a key component of S. chinensis in exerting antiasthmatic effects.

Inhibitory Effect of Cayratia albifolia C. L. Li Extract on LPS-induced Inflammation of RAW264.7 Cells by Regulating the Nrf2/HO-1 Axis

Background: Cayratia albifolia C. L. Li (Jiao Mei Gu (JMG)) has potential therapeutic effects in inflammatory diseases. Purpose: The primary goal of the research was to focus on the antioxidant and anti-inflammatory qualities of C. albifolia and the underlying mechanisms that contribute to these benefits. Methods: To explore the anti-inflammatory abilities of JMG, we employed enzyme-linked immunosorbent assay and nitric oxide (NO) assay in a lipopolysaccharide (LPS)-induced macrophage inflammation model. Furthermore, flow cytometry was employed to assess the antioxidant effects of C. albifolia, and Western blotting was used to reveal the underlying mechanisms. Results: Ethyl acetate extract of C. albifolia effectively reduced the release of NO, interleukin-6, tumor necrosis factor-α, and prostaglandin E2 in macrophages. Additionally, it prevented the expression of reactive oxygen species. Mechanistically, the extract may downregulate the expression of inducible nitric oxide synthase, cyclooxygenase-2, the nuclear factor-erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) protein expression. Conclusion: The ethyl acetate extract of C. albifolia demonstrated remarkable anti-inflammatory and antioxidant effects by modulating the Nrf2/HO-1 axis.

Isoliensinine activated the Nrf2/GPX4 pathway to inhibit glutamate-induced ferroptosis in HT-22 cells.

Isoliensinine (ISO), a natural compound, is a bibenzyl isoquinoline alkaloid monomer in lotus seed, which has strong antioxidant and free radical scavenging activities. The oxidative toxicity caused by glutamic acid overdose is one of the important mechanisms of nerve cell injury, and the oxidative toxicity caused by glutamic acid is related to ferroptosis. This study aims to establish a glutamate-induced injury model of mouse hippocampal neurons HT-22 cells, and investigate the protective effect of ISO on the neurotoxicity of glutamate-induced HT-22 cells. The results showed that ISO inhibited glutamate-induced ferroptosis of neuronal cells through nuclear factor E2-related factor 2/glutathione peroxidase 4 (Nrf2/GPX4) signaling pathway. Pretreatment of HT-22 cells with ISO significantly reduced glutamate-induced cell death. Ferroptosis inhibitors have the same effect. ISO inhibited the decrease of mitochondrial membrane potential detection and the increase of iron content induced by glutamate, the increase of malondialdehyde and reactive oxygen species in cytoplasm and lipid, and protected the activities of GPx and superoxide dismutase enzymes. In addition, WB showed that glutamic acid could induce the upregulated expression of long-chain esteryl coA synthase 4 (ACSL4) protein and the downregulated expression of SLC7A11 and GPX4 protein in HT-22 cells, while ISO could prevent the abnormal expression of these proteins induced by glutamic acid. The nuclear translocation of Nrf2 in HT-22 cells was increased, and the expression of downstream heme oxygenase-1 protein was upregulated. In summary, ISO protects HT-22 cells from glutamate-induced ferroptosis through a novel mechanism of the Nrf2/GPX4 signaling pathway.

The Metabolite of γ-Tocopherol, 2,7,8-Trimethyl-2-(2′-Carboxyethyl)-6-Hydroxychroman, Exerts Intracellular Antioxidant Activity via Up-Regulation of Heme Oxygenase-1 in Hepatocytes

γ-Tocopherol (γT) is the major form of vitamin E contained in plants and seed oils. Although it is readily metabolized in the liver, the function of the metabolites is not fully understood. This study investigated the antioxidant activities of the γT metabolite 2,7,8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman (γCEHC) in comparison to its parent compound. The pretreatment of mouse hepatoma Hepa1c1c7 cells with γCEHC showed a cytoprotective effect on the hydrogen peroxide-induced cytotoxicity to a lesser extent than that of γT. A mechanistic investigation revealed that both γ-CEHC and γT significantly up-regulated the gene and protein expressions of heme oxygenase-1 (HO-1) via the promotion of the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, the combination of γCEHC and γT significantly increased the gene and protein levels of HO-1 and the nuclear translocation of Nrf2, suggesting that it was an additive effect. Tin protoporphyrin IX (SnPP), a representative HO-1 inhibitor, significantly impaired the cytoprotection of γCEHC and γT against the hydrogen peroxide-induced cytotoxicity. These results suggested that not only γT but also its metabolite, γCEHC, are a promising cytoprotective factor against oxidative stress-induced cytotoxicity and that the cytoprotective effect is attributable to the cooperation of both compounds.

Molybdenum Nanoparticles Alleviate MC903-Induced Atopic Dermatitis-Like Symptoms in Mice by Modulating the ROS-Mediated NF-κB and Nrf2 /HO-1 Signaling Pathways.

Atopic dermatitis (AD) is a chronic inflammatory skin condition that can affect individuals of all ages. Recent research has shown that oxidative stress plays a crucial role in the development of AD. Therefore, inhibiting oxidative stress may be an effective therapeutic approach for AD. Nano-molybdenum is a promising material for use as an antioxidant. We aimed to evaluate the therapeutic effects and preliminary mechanisms of molybdenum nanoparticles (Mo NPs) by using a murine model of chemically induced AD-like disease. HaCaT cells, a spontaneously immortalized human keratinocyte cell line, were stimulated by tumor necrosis factor-alpha /interferon-gamma after pre-treatment with Mo NPs. Reactive oxygen species levels, production of inflammatory factors, and activation of the nuclear factor kappa-B and the nuclear factor erythroid 2-related factor pathways were then evaluated. Mo NPs was topically applied to treat a murine model of AD-like disease induced by MC903, a vitamin D3 analog. Dermatitis scores, pruritus scores, transepidermal water loss and body weight were evaluated. AD-related inflammatory factors and chemokines were evaluated. Activation of the nuclear factor kappa-B and nuclear factor erythroid 2-related factor / heme oxygenase-1 pathways was assessed. Our data showed that the topical application of Mo NPs dispersion could significantly alleviate AD skin lesions and itching and promote skin barrier repair. Further mechanistic experiments revealed that Mo NPs could inhibit the excessive activation of the nuclear factor kappa-B pathway, promote the expression of nuclear factor erythroid 2-related factor and heme oxygenase-1 proteins, and suppress oxidative stress reactions. Additionally, they inhibited the expression of thymic stromal lymphopoietin, inflammatory factors, and chemokines, thereby alleviating skin inflammation. Mo NPs present a promising alternative treatment option for patients with AD as they could address three pivotal mechanisms in the pathogenesis of AD concurrently.

Novel insights into the molecular mechanisms underlying anti-nociceptive effect of myricitrin against reserpine-induced fibromyalgia model in rats: Implication of SIRT1 and miRNAs

Ethnopharmacological relevanceManilkara zapota (L.) P. Royen, also termed sapodilla or chikoo, is a significant plant in ethnomedicine because of its long history of traditional medical applications. In diverse cultures, sapodilla is believed to protect against oxidative stress, inflammation, and some chronic diseases because of its high antioxidant content. The naturally occurring antioxidant myricitrin (MYR) flavonoid is primarily found in the leaves and other plant parts of sapodilla and it is well-known for having therapeutic qualities and possible health advantages. Aim of the studyTo appraise the possible impact of MYR on a rat model of reserpine-induced fibromyalgia (FM) and explore its mechanism of action. Materials and methodsIsolation and identification of MYR with more than 99% purity from Manilkara zapota leaves were primarily done and confirmed through chromatographic and spectrophotometric techniques. To develop FM model, reserpine (RSP) was injected daily (1 mg/kg, s.c.) for three successive days. Then, MYR (10 mg/kg, i.p.) and pregabalin (PGB, 30 mg/kg, p.o.) were given daily for another five days. Behavioral changes were assessed through open field test (OFT), hot plate test, and forced swimming test (FST). Further analyses of different brain parameters and signaling pathways were performed to assess monoamines levels, oxidative stress, inflammatory response, apoptotic changes as well as silent information regulator 1 (SIRT1) and micro RNAs (miRNAs) expressions. ResultsFrom High-Performance Liquid Chromatography (HPLC) analysis, the methanol extract of sapodilla leaves contains 166.17 μg/ml of MYR. Results of behavioral tests showed a significant improvement in RSP-induced nociceptive stimulation, reduced locomotion and exploration and depressive-like behavior by MYR. Biochemical analyses showed that MYR significantly ameliorated the RSP-induced imbalance in brain monoamine neurotransmitters. In addition, MYR significantly attenuated oxidative stress elicited by RSP via up-regulating nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expressions, enhancing superoxide dismutase (SOD) and catalase (CAT) activities, and reducing malondialdehyde (MDA) content in brain. The RSP-provoked inflammatory response was also diminished by MYR treatment as shown by a significant decreased NOD-like receptor protein 3 (NLRP3) inflammasome expression along with reduced levels of interleukin 1 beta (IL-1β) and nuclear factor-κB (NF-κB). Furthermore, the anti-apoptotic activity of MYR was demonstrated by a marked rise in Bcl-2-associated X protein (BAX)/B cell lymphoma-2 (Bcl-2) ratio by lowering Bcl-2 while increasing BAX levels. In addition, MYR treatment significantly boosted the expression of SIRT1 deacetylase in RSP-treated animals. Interestingly, molecular docking showed the ability of MYR to form a stable complex in the binding site of SIRT1. Regarding miRNAs, MYR effectively ameliorated RSP-induced changes in miR-320 and miR-107 gene expressions. ConclusionOur findings afford new insights into the anti-nociceptive profile of MYR in the RSP-induced FM model in rats. The underlying mechanisms involved direct binding and activation of SIRT1 to influence different signaling cascades, including Nrf2 and NF-κB/NLRP3 together with modulation of miRNAs. However, more in-depth studies are needed before proposing MYR as a new clinically relevant drug in the management of FM.

Probiotic Characteristics and Anti-Inflammatory Effects of Limosilactobacillus fermentum 664 Isolated from Chinese Fermented Pickles.

Limosilactobacillus fermentum (L. fermentum) is widely used in industrial food fermentations, and its probiotic and health-promoting roles attracted much attention in the past decades. In this work, the probiotic potential of L. fermentum 664 isolated from Chinese fermented pickles was assessed. In addition, the anti-inflammatory properties and mechanisms were investigated using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Results indicated that L. fermentum 664 demonstrated excellent acid and bile salt tolerance, adhesion capability, antimicrobial activity, and safety profile. L. fermentum 664 downregulated the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) stimulated with LPS. Moreover, L fermentum 664 inhibited the nuclear translocation of the nuclear factor κB (NF-κB) and the activation of mitogen-activated protein kinases (MAPKs) induced by LPS. This action was associated with a reduction in reactive oxygen species (ROS) levels and an enhanced expression of heme oxygenase-1 (HO-1) protein. Additionally, whole genome sequencing indicated that L. fermentum 664 contained genes that encode proteins with antioxidant and anti-inflammatory functions, including Cytochrome bd ubiquinol oxidase subunit I (CydA), Cytochrome bd ubiquinol oxidase subunit II (CydB), and NAD(P)H dehydrogenase quinone 1 (NQO1). In conclusion, our study suggested that L. fermentum 664 has the potential to become a probiotic and might be a promising strategy for the prevention of inflammation.

Protective effect and mechanism of quercetin on acute liver injury induced by diquat poisoning in mice

To investigate the protective effect of quercetin (QR) on acute liver injury induced by diquat (DQ) poisoning in mice and its mechanism. Eighty healthy male C57BL/6 mice with SPF grade were randomly divided into control group, DQ model group, QR treatment group, and QR control group, with 20 mice in each group. The DQ poisoning model was established by a one-time intraperitoneal injection of DQ solution (40 mg/kg); the control and QR control groups received equivalent amounts of distilled water through intraperitoneal injection. Four hours after modeling, the QR treatment group and the QR control group received 0.5 mL QR solution (50 mg/kg) through gavage. Meanwhile, an equivalent amount of distilled water was given orally to the control group and the DQ model group. The treatments above were administered once daily for seven consecutive days. Afterwards, the mice were anesthetized, blood and liver tissues were collected for following tests: changes in the structure of mice liver tissue were observed using transmission electron microscopy; the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using enzyme linked immunosorbent assay (ELISA); the levels of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in liver tissues were measured using the water-soluble tetrazolium-1 (WST-1) method, the thiobarbituric acid (TBA) method, and enzymatic methods, respectively; the protein expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), Kelch-like ECH-associated protein 1 (Keap1), and activated caspase-9 in liver tissues were detected using Western blotting. Severe mitochondrial damage was observed in the liver tissues of mice in the DQ model group using transmission electron microscopy, yet mitochondrial damage in the QR treatment group showed significant alleviation. Compared to the control group, the DQ model group had significantly increased levels of MDA in liver tissue, serum AST, and ALT, yet had significantly decreased levels of GSH and SOD in liver tissue. In comparison to the DQ model group, the QR treatment group exhibited significant reductions in serum levels of ALT and AST, as well as MDA levels in liver tissue [ALT (U/L): 52.60±6.44 vs. 95.70±8.00, AST (U/L): 170.45±19.33 vs. 251.10±13.09, MDA (nmol/mg): 12.63±3.41 vs. 18.04±3.72], and notable increases in GSH and SOD levels in liver tissue [GSH (μmol/mg): 39.49±6.33 vs. 20.26±3.96, SOD (U/mg): 121.40±11.75 vs. 81.67±10.01], all the differences were statistically significant (all P < 0.01). Western blotting results indicated that the protein expressions of Nrf2 and HO-1 in liver tissues of the DQ model group were significantly decreased compared to the control group. On the other hand, the protein expressions of Keap1 and activated caspase-9 were conspicuously higher when compared to the control group. In comparison to the DQ model group, the QR treatment group showed a significant increase in the protein expressions of Nrf2 and HO-1 in liver tissues (Nrf2/β-actin: 1.17±0.08 vs. 0.92±0.45, HO-1/β-actin: 1.53±0.17 vs. 0.84±0.09). By contrast, there was a notable decrease in the protein expressions of Keap1 and activated caspase-9 (Keap1/β-actin: 0.48±0.06 vs. 1.22±0.09, activated caspase-9/β-actin: 1.17±0.12 vs. 1.59±0.30), the differences were statistically significant (all P < 0.01). QR may reduce acute liver injury induced by DQ poisoning in mice via activating Keap1/Nrf2 signaling pathway.

Unveiling the protective role of sevoflurane in video-assisted thoracoscopic surgery associated-acute lung injury: Inhibition of ferroptosis

Acute lung injury (ALI) frequently occurs after video-assisted thoracoscopic surgery (VATS). Ferroptosis is implicated in several lung diseases. Therefore, the disparate effects and underlying mechanisms of the two commonly used anesthetics (sevoflurane (Sev) and propofol) on VATS-induced ALI need to be clarified. In the present study, enrolled patients were randomly allocated to receive Sev (group S) or propofol anesthesia (group P). Intraoperative oxygenation, morphology of the lung tissue, expression of ZO-1, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), superoxide dismutase (SOD), glutathione (GSH), Fe2+, glutathione peroxidase 4 (GPX4), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in the lung tissue as well as the expression of TNF-α and IL-6 in plasma were measured. Postoperative complications were recorded. Of the 85 initially screened patients scheduled for VATS, 62 were enrolled in either group S (n = 32) or P (n = 30). Compared with propofol, Sev substantially (1) improved intraoperative oxygenation; (2) relieved histopathological lung injury; (3) increased ZO-1 protein expression; (4) decreased the levels of TNF-α and IL-6 in both the lung tissue and plasma; (5) increased the contents of GSH and SOD but decreased Fe2+ concentration; (6) upregulated the protein expression of p-AKT, Nrf2, HO-1, and GPX4. No significant differences in the occurrence of postoperative outcomes were observed between both groups. In summary, Sev treatment, in comparison to propofol anesthesia, may suppress local lung and systemic inflammatory responses by activating the PI3K/Akt/Nrf2/HO-1 pathway and inhibiting ferroptosis. This cascade of effects contributes to the maintenance of pulmonary epithelial barrier permeability, alleviation of pulmonary injury, and enhancement of intraoperative oxygenation in patients undergoing VATS.

Nerolidol Alleviates Oxidative Stress and Inflammation in Diabetic Retinopathy Mediated by High Glucose through Ameliorating Nrf2/HO-1 Pathway

Background Diabetic retinopathy (DR) is the foremost microvascular problem that causes drastic visual impairment in diabetes patients. Hyperglycemia-triggered reaction cascade of inflammation and oxidative stress constitute the DR pathogenesis. The existing treatment options are not completely satisfactory. Materials and Methods We investigated the cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, inflammatory mediators, lactate dehydrogenase (LDH), superoxide dismutase, glutathione, and malonaldehyde (MDA) levels by ELISA and qRT-PCR assay, protein expression of Nrf2 and heme oxygenase-1 (HO-1) by western blotting assay were analyzed. Results According to our research, nerolidol (NRD) increases the proliferation and antioxidant activity of human retinal endothelial cells (HRECs) by inducing Nrf2/HO-1 signaling, while attenuating MDA, an oxidative stress marker, LDH, and inflammatory mediators. These outcomes suggest that a substantial reaction of inflammation and oxidative stress injury happened in DR, which might be correlated to the instigation of the signaling Nrf2/HO-1. Conclusion NRD effectively suppresses oxidative stress and inflammation in HG-induced HRECs. The primary mechanism of NRD on DR may be linked to the activation of the Nrf2/HO-1 pathway and may give a useful medicine for DR treatment.

Hyperoxia impacts heme oxygenase 1 and mitochondrial quality control in a rodent model of sarcopenic obesity

Critically ill sarcopenic obese (SO) patients may be at higher risk for other complications and require mechanical ventilation, extending length of stay and recovery time. It may be because in sarcopenia and obesity, oxidants, inflammation and mitochondrial fragmentation increase, impacting respiration. Heme oxygenase 1 (HO-1) is an enzyme critical for cellular antioxidant and anti-inflammatory defenses, and in the regulation of mitochondrial quality control (MQC). Whether HO-1 signaling is impaired in SO during heightened oxidant stress remains unknown, the hypothesis of this research. Young (7-week-old) and old (72-week-old) C57BL/6J mice consumed high-fat (58%) or control (11% fat) diets for 16-weeks. Mean body weight (g), glucose AUC and strength (N) (±SD) measured after dietary interventions were as follows: young lean (33±2, 540±220, 1.9±0.3), young obese (53±3, 710±116, 1.8±0.4), old lean (38±4, 597±148, 1.5±0.3), and old obese (57±10, 654±119, 1.4±0.2). To induce oxidant stress, mice were exposed to 100% oxygen for 48 h. Lower hindlimb skeletal muscle was harvested 16 h later. Oxidant stress and inflammation were determined by measuring superoxide (DHE), DNA oxidation (8-OHdG), mitochondrial DNA lesions and cytochrome b mRNA, and IL-10, IL-1β and TNF-α protein expression. MQC was assessed by measuring regulators of mitochondrial fission (DRP1) and fusion (MFN1 and 2), mitophagy (PINK1, Parkin and LC3-II), and mitochondrial biogenesis (NFE2L2, PGC-1α, NRF1 and 2, and TFAM). The mitochondrial membrane potential was measured using TMRM. In contrast to air controls where HO-1 protein expression was increased by aging and obesity, hyperoxia caused substantial reductions in both HO-1 protein and mRNA within old and obese mice. The greatest reductions were found in SO (25% of young lean). The changes in HO-1 were accompanied by the following responses to hyperoxia: One, superoxide and DNA oxidation increased in old and obese mice by as much as 200%, leading to elevated mitochondrial DNA lesions and reduced cytochrome b. Two, IL-1β and TNF-α increased in aged mice by 41-129% over young mice, whereas IL-10 was lower (-33%) only in SO. Three, regulators of mitochondrial fission and fusion and mitophagy were reduced by aging and obesity, being lower in sarcopenia which may explain why mitochondrial LC3-II was only reduced in these mice. Fourth, modulators of mitochondrial biogenesis were decreased by aging and obesity, suggesting a reduced mitochondrial population. Lastly, the increased mitochondrial membrane potential measured in young obese (78%) and old lean (58%) mice was not observed in SO, indicating reduced mitochondrial activity. In sarcopenic and obese mice exposed to hyperoxia, oxidant stress and mitochondrial DNA fragmentation increase without a concomitant change in MQC to maintain a healthy and functional mitochondrial network. Suppression of HO-1 may be a mechanism for these changes during periods of hyperoxic stress in sarcopenia and obesity that contribute to poor outcomes in critical illness. This work was supported by the Developing Research Excellence in Anesthesia Management Innovation Grant (2021). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Obesity aggravates acute kidney injury resulting from ischemia and reperfusion in mice

In critically ill patients, overweight and obesity are associated with acute respiratory distress syndrome and acute kidney injury (AKI). However, the effect of obesity on ischemia–reperfusion injury (IRI)-induced AKI is unknown. We hypothesized that obesity would aggravate renal IRI in mice. We fed mice a standard or high-fat diet for eight weeks. The mice were divided into four groups and submitted to sham surgery or IRI: obese, normal, normal + IRI, obese, and obese + IRI. All studies were performed 48 h after the procedures. Serum glucose, cholesterol, and creatinine clearance did not differ among the groups. Survival and urinary osmolality were lower in the obese + IRI group than in the normal + IRI group, whereas urinary neutrophil gelatinase-associated lipocalin levels, tubular injury scores, and caspase 3 expression were higher. Proliferating cell nuclear antigen expression was highest in the obese + IRI group, as were the levels of oxidative stress (urinary levels of thiobarbituric acid-reactive substances and renal heme oxygenase-1 protein expression), whereas renal Klotho protein expression was lowest in that group. Expression of glutathione peroxidase 4 and peroxiredoxin 6, proteins that induce lipid peroxidation, a hallmark of ferroptosis, was lower in the obese + IRI group. Notably, among the mice not induced to AKI, macrophage infiltration was greater in the obese group. In conclusion, greater oxidative stress and ferroptosis might aggravate IRI in obese individuals, and Klotho could be a therapeutic target in those with AKI.

Extraction, purification and in vitro assessment of the antioxidant and anti-inflammatory activity of policosanols from non-psychoactive Cannabis sativa L.

Policosanols (PCs) are bioactive compounds extracted from different natural waxes. In this work, the purification, characterization and assessment of the antioxidant and anti-inflammatory activity was carried out on PCs from an innovative source, i.e. a waxy material from supercritical-fluid extraction (SFE) of non-psychoactive Cannabis sativa L. (hemp) inflorescences. Starting from this material, PCs were obtained by microwave-assisted trans-esterification and hydrolysis, followed by preparative liquid chromatography under normal phase conditions. The purified product was characterized using high-performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). In vitro cell-free and cell-based antioxidant and anti-inflammatory assays were then performed to assess their bioactivity.HPLC-ELSED analysis of the purified mixture from hemp wax revealed C26OH and C28OH as the main compounds. In vitro assays indicated an inhibition of intracellular reactive oxygen species (ROS) production, a reduction of nuclear factor kappa B (NF-κB) activation and of the activity of the neutrophil elastase. Immunoblotting assays allowed us to hypothesize the mechanism of action of the compounds of interest, given the higher levels of MAPK-activated protein kinase 2 (MK2) and heme oxygenase-1 (HO-1) protein expression in the PC pretreated HaCaT cells. In conclusion, even if more research is needed to unveil other molecular mechanisms involved in hemp PC activity, the results of this work suggest that these compounds may have potential for use in oxinflammation processes.

Effects of M2-type macrophages and GKT137831 on oxidative stress in hepatic stellate cells

Objective: To investigate the effects of reduced nicotinamide adenine dinucleotide phosphooxidase 4 (NOX4) inhibitors GKT137831 and M2-type macrophages on oxidative stress markers NOX4, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the rat hepatic stellate cell line (HSC-T6). Methods: Rat bone marrow macrophages were extracted and induced using interleukin (IL)-4 to differentiate them into M2 phenotype macrophages. HSC-T6 activation was performed with 5 μg/L transforming growth factor β1 (TGF-β1). The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80 μmol/L after 48 hours was detected using the Cell Counting Kit-8 (CCK-8) assay. The optimal drug concentration was chosen and divided into an HSC co-culture group (the control group) and five experimental groups: the TGF-β1 stimulation group, the TGF-β1 +GKT137831 stimulation group, the M2-type macrophage + HSC co-culture group, the M2-type macrophage +TGF-β1 stimulation group, and the M2-type + TGF-β1 + GKT137831 stimulation group. Reactive oxygen species (ROS) production level was detected in each cell using the DCFH-DA probe method. NOX4, α-smooth muscle actin (α-SMA), Nrf2, and HO-1 levels in each group of HSC cells were detected using the qRT-PCR method and the Western blot method. The t-test was used to compare the two groups. The one-way ANOVA method was used to compare multiple groups. Results: Intracellular ROS increased significantly following TGF-β1 stimulation. ROS relative levels in each cell group were 1.03±0.11, 3.88±0.07, 2.90±0.08, 0.99±0.06, 3.30±0.05, 2.21±0.11, F = 686.1, P = 0.001, respectively. The mRNA and protein expressions of NOX4, α-SMA, Nrf2, and HO-1 were significantly increased (P < 0.05). After the addition of GKT137831, ROS, and NOX4, α-SMA mRNA and protein expression were comparatively decreased in the TGF-β1 stimulation group (P < 0.05), while mRNA and protein expressions of Nrf2 and HO-1 were increased (P < 0.05). The expression of ROS and NOX4, as well as α-SMA mRNA and protein, produced by HSC were significantly decreased in the co-culture group compared to the single culture group after TGF-β1 stimulation (P < 0.05). After the addition of GKT137831, ROS, NOX4, α-SMA mRNA, and protein expression were further reduced in the co-culture group compared with the single culture group (P < 0.05), while the mRNA and protein expression of Nrf2 and HO-1 were further increased (P < 0.05). Conclusion: NOX4 inhibitor GKT137831 can reduce RO, NOX4, and α-SMA levels while increasing Nrf2 and HO-1 levels in hepatic stellate cells. After M2-type macrophage co-culture, GKT137831 assists in lowering ROS, NOX4, and α-SMA levels while accelerating Nrf2 and HO-1 levels in hepatic stellate cells, which regulates the balance between oxidative stress and anti-oxidative stress systems, thereby antagonizing the fibrosis process.

Activation of Transcription Factor Nrf2 in HeLa Cells under the Action of Nitrosyl Iron Complex with N-Ethylthiourea.

We studied the effect of an NO donor, nitrosyl iron complex with N-ethylthiourea, on Nrf2-dependent antioxidant system activation of tumor cells in vitro. The complex increased intracellular accumulation of Nrf2 transcription factor and induced its nuclear translocation. It was shown that both heme oxygenase-1 gene and protein expression increased significantly under the influence of the complex. Nrf2 activation was accompanied by a decrease in the intracellular accumulation of proinflammatory transcription factor NF-κB p65 subunit and expression of its target genes. The cytotoxic effect of N-ethylthiourea leads to induction of Nrf2/HO-1 antioxidant response and suppression of NF-κB-dependent processes in tumor cells.

Small-Molecule Peptides from Lactiplantibacillus plantarum SCS2 Attenuate H2O2-Induced Oxidative Damage in INS-1 Cells via Regulating the Keap1-Nrf2 Signaling Pathway

Objective. This study aimed to delve into the antioxidant potential of small-molecule peptides derived from Lactiplantibacillus plantarum SCS2 (L. plantarum SCS2), targeting hydrogen peroxide- (H2O2-) induced rat insulinoma (INS-1) cells. Methods. INS-1 cells were pretreated with small-molecule peptides of distinct molecular weights, specifically P1 (≤1 kDa), P2 (1–5 kDa), and P3 (5–10 kDa), which were isolated from L. plantarum SCS2, followed by H2O2 to induce oxidative damage in INS-1 cells. The oxidative status of the cells was assessed by measuring reactive oxygen species (ROS), malondialdehyde (MDA) concentrations, and antioxidant enzyme activities. In addition, the expression levels of proteins and genes associated with the Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway were examined to elucidate the underlying antioxidative mechanisms of the small-molecule peptides from L. plantarum SCS2. Results. The cellular activity and the activities of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, were higher after pretreatment with P1 than those after pretreatment with P2 and P3, which led to the suppression of ROS and MDA production. In addition, P1 upregulated mRNA and protein expression of Nrf2 and heme oxygenase-1, significantly decreased mRNA and protein expression of Keap1, and promoted the entry of Nrf2 into the nucleus compared with that in the model group. Conclusion. P1 from L. plantarum SCS2 could activate the Keap1-Nrf2 signaling pathway, thereby ameliorating oxidative damage in INS-1 cells.

Neferine inhibits the progression of diabetic nephropathy by modulating the miR-17-5p/nuclear factor E2-related factor 2 axis.

To investigate the effect of Neferine (Nef) on diabetic nephropathy (DN) and to explore the mechanism of Nef in DN based on miRNA regulation theory. A DN mouse model was constructed and treated with Nef. Serum creatinine (Crea), blood urea (UREA) and urinary albumin were measured in mice by kits, and renal histopathological changes and fibrosis were observed by hematoxylin-eosin staining and Masson staining. Renal tissue superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) activities were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to detect the expression of nuclear factor E2-related factor 2 (Nrf2)/ heme oxygenase 1 (HO-1) signaling pathway-related proteins in kidney tissues. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-17-5p in kidney tissues. Subsequently, a DN in vitro model was constructed by high glucose culture of human mesangial cells (HMCs), cells were transfected with miR-17-5p mimic and/or treated with Nef, and we used qRT-PCR to detect cellular miR-17 expression, flow cytometry to detect apoptosis, ELISAs to detect cellular SOD, MDA, and GSH-Px activities, Western blots to detect Nrf2/HO-1 signaling pathway-related protein expression, and dual luciferase reporter gene assays to verify the targeting relationship between Nrf2 and miR-17-5p. Administration of Nef significantly reduced the levels of blood glucose, Crea, and UREA and the expression of miR-17-5p, improved renal histopathology and fibrosis, significantly reduced MDA levels, elevated SOD and GSH-Px activities, and activated Nrf2 expression in kidney tissues from mice with DN. Nrf2 is a post-transcriptional target of miR-17-5p. In HMCs transfected with miR-17-5p mimics, the mRNA and protein levels of Nrf2 were significantly suppressed. Furthermore, miR-17-5p overexpression and Nef intervention resulted in a significant increase in high glucose-induced apoptosis and MDA levels in HMCs and a significant decrease in the protein expression of HO-1 and Nrf2. Collectively, these results indicate that Nef has an ameliorative effect on DN, and the mechanism may be through the miR-17-5p/Nrf2 pathway.