Heme Oxygenase-1 Protein Levels Research Articles

Ethyl Acetate Extract of Cichorium glandulosum Activates the P21/Nrf2/HO-1 Pathway to Alleviate Oxidative Stress in a Mouse Model of Alcoholic Liver Disease

Background: Alcoholic liver disease (ALD) is a significant global health concern, primarily resulting from chronic alcohol consumption, with oxidative stress as a key driver. The ethyl acetate extract of Cichorium glandulosum (CGE) exhibits antioxidant and hepatoprotective properties, but its detailed mechanism of action against ALD remains unclear. This study investigates the effects and mechanisms of CGE in alleviating alcohol-induced oxidative stress and liver injury. Methods: Ultra-Performance Liquid Chromatography coupled with Quadrupole-Orbitrap Mass Spectrometry (UPLC-Q-Orbitrap-MS) was used to identify CGE components. A C57BL/6J mouse model of ALD was established via daily oral ethanol (56%) for six weeks, with CGE treatment at low (100 mg/kg) and high doses (200 mg/kg). Silibinin (100 mg/kg) served as a positive control. Liver function markers, oxidative stress indicators, and inflammatory markers were assessed. Transcriptomic and network pharmacology analyses identified key genes and pathways, validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Results: UPLC-Q-Orbitrap-MS identified 81 CGE compounds, mainly including terpenoids, flavonoids, and phenylpropanoids. CGE significantly ameliorated liver injury by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels and enhancing antioxidative markers such as total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) while lowering hepatic malondialdehyde (MDA) levels. Inflammation was mitigated through reduced levels of Tumor Necrosis Factor Alpha (TNF-α), Interleukin-1 Beta (IL-1β), and C-X-C Motif Chemokine Ligand 10 (CXCL-10). Transcriptomic and network pharmacology analysis revealed seven key antioxidant-related genes, including HMOX1, RSAD2, BCL6, CDKN1A, THBD, SLC2A4, and TGFβ3, validated by RT-qPCR. CGE activated the P21/Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) signaling axis, increasing P21, Nrf2, and HO-1 protein levels while suppressing Kelch-like ECH-associated Protein 1 (Keap1) expression. Conclusions: CGE mitigates oxidative stress and liver injury by activating the P21/Nrf2/HO-1 pathway and regulating antioxidant genes. Its hepatoprotective effects and multi-target mechanisms highlight CGE’s potential as a promising therapeutic candidate for ALD treatment.

Silencing PADI-2 induces antitumor effects by downregulating NF-κB, Nrf2/HO-1 and AKT1 in A549 lung cancer cells.

This study aimed to investigate the tumorigenic role and regulatory pathways of peptidyl arginine deiminase 2 (PAD-2) in A549 lung cancer cells following treatment with small interfering RNA (PADI-2 siRNA) or the pharmacological pan-PAD inhibitor BB-Cl amidine. A549 lung cancer cells were treated with PADI-2 siRNA to knock down PADI-2 expression or with BB-Cl amidine to inhibit PAD2 activity. The effects on cell proliferation, migration, invasion, and cell cycle phases were assessed. Additionally, the expression levels of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), AKT serine/threonine kinase 1 (AKT), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interleukin 6 (IL6), and p53 were analyzed to elucidate the underlying mechanisms involved. The manipulation of PAD-2 expression or activity significantly influenced tumor cell behavior. Knockdown of PADI-2 in A549 cells reduced cell proliferation by inhibiting the S and G2 phases and decreasing cell migration and invasion. Inhibition of PADI-2 expression also suppressed the protein levels of Nrf2 and HO-1 via suppression of the AKT/NF-κB pathway. Furthermore, this inhibition enhanced the senescence-associated secretory phenotype (SASP) through the regulation of IL6 and p53, resulted in significant upregulation of SASP factors mainly, p21, Lamin B1 and HMGB1. Downregulation of PADI-2 attenuated the proliferation, migration, and invasion of A549 lung cancer cells by modulating the Nrf2/HO-1/AKT signaling pathway. It also increased senescence in A549 lung cancer cells via IL6 and p53 key regulators. These findings highlight the potential of PADI-2 as a therapeutic target in lung cancer treatment.

Acupoint Autohemotherapy Alleviates Airway Inflammation in Asthmatic Rats via Upregulating Expression of Hemeoxygenase-1.

Acupoint autohemotherapy (AA), a therapeutic technique involving the subcutaneous injection of autologous blood into acupoints, has been empirically validated as safe and effective for treating asthma by alleviating symptoms and decreasing acute attacks, though its mechanism is not well understood. The role of heme oxygenase-1 (HO-1) in AA-induced suppression of asthmatic airway inflammation is examined. Twenty rats were assigned randomly to four groups, namely the Control, OVA, OVA + AA, and (OVA + Snpp) + AA. Rats in the OVA + AA and (OVA + Snpp) + AA received autologous blood injections into acupoints (BL13 and BL23) following OVA challenge. Rats in the (OVA + Snpp) + AA were concurrently subjected to intraperitoneal injections of Snpp, a inhibitor of HO-1. Airway inflammation was evaluated through HE staining, while the concentrations of cytokines in BALF were quantified using ELISA. The mRNA and protein levels of RORγt (Th17-specific transcription factor), Foxp3 (Treg-specific transcription factor), and HO-1 in lung tissue were assessed through qRT-PCR and WB. HE staining indicated that airway inflammation was alleviated in the OVA + AA. The OVA + AA displayed significantly lower counts of total cells and eosinophils in the BALF compared to both the OVA and (OVA + Snpp) + AA. The ELISA demonstrated a significant decrease in levels of pro-inflamatory cytokines (IL-4, IL-17A), and an increase in levels of anti-inflamatory cytokines (IFN-γ, IL-10), in the OVA + AA when compared to both OVA and (OVA + Snpp) + AA. The qRT-PCR and WB analyses revealed an upregulation of HO-1 and Foxp3 expression, and a downregulation of RORγt expression, in the OVA + AA when compared to OVA and (OVA + Snpp) + AA. The involvement of HO-1 in the underlying mechanism responsible for the anti-inflammatory effects of AA is evident.

Mevastatin-Induced HO-1 Expression in Cardiac Fibroblasts: A Strategy to Combat Cardiovascular Inflammation and Fibrosis.

Mevastatin (MVS) is known for its anti-inflammatory effects, potentially achieved by upregulating heme oxygenase-1 (HO-1), an enzyme involved in cytoprotection against oxidative injury. Nonetheless, the specific processes by which MVS stimulates HO-1 expression in human cardiac fibroblasts (HCFs) are not yet fully understood. In this study, we found that MVS treatment increased HO-1 mRNA and protein levels in HCFs. This induction was inhibited by pretreatment with specific inhibitors of p38 MAPK, JNK1/2, and FoxO1, and by siRNAs targeting NOX2, p47phox, p38, JNK1, FoxO1, Keap1, and Nrf2. MVS also triggered ROS generation and activated JNK1/2 and p38 MAPK, both attenuated by NADPH oxidase or ROS inhibitors. Additionally, MVS promoted the phosphorylation of FoxO1 and Nrf2, which was suppressed by p38 MAPK or JNK1/2 inhibitor. Furthermore, MVS inhibited TNF-α-induced NF-κB activation and vascular cell adhesion molecule-1 (VCAM-1) expression via the HO-1/CO pathway in HCFs. In summary, the induction of HO-1 expression in HCFs by MVS is mediated through two primary signaling pathways: NADPH oxidase/ROS/p38 MAPK, and JNK1/2/FoxO1 and Nrf2. This research illuminates the underlying processes through which MVS exerts its anti-inflammatory effects by modulating HO-1 in cardiac fibroblasts.

The BRD4 Inhibitor I-BET-762 Reduces HO-1 Expression in Macrophages and the Pancreas of Mice.

In pancreatic cancer, the tumor microenvironment (TME) accounts for up to 90% of the tumor mass. Pancreatitis, characterized by the increased infiltration of macrophages into the pancreas, is a known risk factor for pancreatic cancer. The NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor regulates responses to oxidative stress and can promote cancer and chemoresistance. NRF2 also attenuates inflammation through the regulation of macrophage-specific genes. Heme oxygenase 1 (HO-1) is expressed by anti-inflammatory macrophages to degrade heme, and its expression is dependent on NRF2 translocation to the nucleus. In macrophages stimulated with conditioned media from pancreatic cancer cells, HO-1 protein levels increased, which correlated with higher NRF2 expression in the nuclear fraction. Significant differences in macrophage infiltration and HO-1 expression were detected in LSL-KrasG12D/+; Pdx-1-Cre (KC) mice, Nrf2 whole-body knockout (KO) mice and wildtype mice with pancreatitis. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is therapeutically desirable. When the bromodomain inhibitor I-BET-762 was used to treat macrophages or mice with pancreatitis, high levels of HO-1 were reduced. This study shows that bromodomain inhibitors can be used to prevent physiological responses to inflammation that promote tumorigenesis.

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.

CircCHSY1 protects hearts against ischaemia/reperfusion injury by enhancing heme oxygenase 1 expression via miR-24-3p.

Circular RNAs (circRNAs) are important players involved in a variety of physiological and pathological processes. However, their functions and mechanisms during myocardial ischaemic injury and protection remain largely unknown. We recently found significant alterations of many circRNAs including circCHSY1 following myocardial ischaemia/reperfusion (I/R) injury, whereas their exact functions are unclear. Here, we investigated the roles of circCHSY1 in the acute myocardial I/R injury and the potential mechanisms involved. The expression of circCHSY1 was detected in cardiomyocytes from mouse, rat, and human embryonic stem cells (hESC-CMs). It was further up-regulated in mouse I/R (30 min/24 h) hearts, oxygen glucose deprivation/reperfusion (OGD/R, 6 h/2 h) primary neonatal rat ventricular cardiomyocytes (NRCMs) and OGD/R (48 h/2 h) hESC-CMs. Adenovirus-mediated circCHSY1 overexpression significantly decreased infarct size and lactate dehydrogenase (LDH) release in mouse I/R hearts. Consistently, circCHSY1 overexpression reduced the LDH release in the OGD/R NRCMs and hESC-CMs, improved cell viability, and preserved mitochondrial function in the OGD/R NRCMs, whereas there were no significant differences in cell viability and LDH release between the OGD/R NRCMs with and without small interfering RNA (siRNA)-mediated circCHSY1 knockdown. Mechanistically, circCHSY1 was detected to bind with miR-24-3p analysed by dual-luciferase assay and RNA pull-down assays. CircCHSY1 overexpression-mediated protective effects on cells and mitochondria in OGD/R NRCMs were reversed by the miR-24-3p mimic. Furthermore, dual-luciferase assay showed that miR-24-3p was directly bound to heme oxygenase 1 (HO1) via its 3'UTR. The protein level of HO1 was down-regulated by miR-24-3p mimic in OGD/R NRCMs but enhanced by the circCHSY1 overexpression in vitro and in vivo. Functionally, the HO1 knockdown by adenovirus in vivo and by siRNA in vitro eliminated cardioprotective effects of circCHSY1 overexpression. CircCHSY1 is up-regulated following myocardial I/R injury. The higher level of circCHSY1 protects I/R hearts and cardiomyocytes. The protection of circCHSY1 is mediated through enhancement of the HO1 level, resulting in preserving mitochondrial homoeostasis via targeting miR-24-3p in cardiomyocytes. These findings suggest circCHSY1 as a protective factor.

HMOX1 regulates ferroptosis via mic14 and its impact on chemotherapy resistance in small-cell lung cancer.

This study aimed to investigate the role and molecular mechanism of heme oxygenase-1 (HMOX1) in chemotherapy resistance in small-cell lung cancer (SCLC). Employed bioinformatics, qPCR, and Western Blot to assess HMOX1 levels in SCLC versus normal tissues and its prognostic relevance. CCK-8, flow cytometry, and thiobarbituric acid assays determined HMOX1's impact on SCLC chemosensitivity, ferroptosis markers, lipid peroxidation, and mic14's role in chemoresistance. In the GSE40275 and GSE60052 cohorts, HMOX1 expression was downregulated in SCLC tissues compared to normal tissues. Higher HMOX1 expression was associated with improved prognosis in the Sun Yat-sen University Cancer Hospital cohort and GSE60052 cohort. The RNA and protein levels of HMOX1 were reduced in drug-resistant SCLC cell lines compared to chemosensitive cell lines. Upregulation of HMOX1 increased chemosensitivity and reduced drug resistance in SCLC, while downregulation of HMOX1 decreased chemosensitivity and increased drug resistance. Upregulation of HMOX1 elevated the expression of ferroptosis-related proteins ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while decreasing the expression of GPX4 and xCT. Conversely, downregulation of HMOX1 decreased the expression of ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while increasing the expression of GPX4 and xCT. Upregulation of HMOX1 promoted cellular lipid peroxidation, whereas downregulation of HMOX1 inhibited cellular lipid peroxidation. Upregulation of HMOX1 reduced the RNA level of mic14, while downregulation of HMOX1 increased the RNA level of mic14. mic14 exhibited inhibitory effects on cellular lipid peroxidation in SCLC cells and contributed to reduced chemosensitivity and increased drug resistance in chemoresistant SCLC cell lines. HMOX1 plays a role in ferroptosis by regulating mic14 expression, thereby reversing chemoresistance in SCLC.

Combined exposure to lead and microplastics increased risk of glucose metabolism in mice via the Nrf2/NF-κB pathway.

The purpose of this study was to explore the effects of combined lead (Pb) and two types of microplastic (MP) (polyvinyl chloride [PVC] and polyethylene [PE]) exposure on glucose metabolism and investigate the role of the nuclear factor erythroid 2-related factor 2 (Nrf2)/nuclear factor-kappa B (NF-κB) signaling pathway in mediating these effects in mice. Adult C57BL/6J mice were randomly divided into four groups: control, Pb (100 mg/L), MPs (containing 10 mg/L PE and PVC), and Pb + MPs, each of which was treated with drinking water. Treatments were conducted for 6 weeks. Co-exposure to Pb + MPs exhibited increase glycosylated serum protein levels, insulin resistance, and damaged glucose tolerance compared with the control mice. Additionally, treatment with Pb + MPs caused more severe damage to hepatocytes than when exposed to them alone concomitantly, exposed to Pb + MPs exhibited improved the levels of interleukin-6, tumor necrosis factor-alpha, and malondialdehyde, but reduced superoxide dismutase, glutathione peroxidase, and catalase assay in livers. Furthermore, they increase the Kelch-like ECH-associated protein 1 (Keap1) and phosphorylated p-NF-κB protein levels but reduced the protein levels of heme oxygenase-1 and Nrf2, as well as increased Keap1 mRNA and Nrf2 mRNA. Co-exposure to Pb + MP impacts glucose metabolism via the Nrf2 /NF-κB pathway.

Crocin from saffron ameliorates allergic airway inflammation through NF-κB, IL-17, and Nrf2/HO-1 signaling pathways in mice.

Asthma is a complex inflammatory disorder with the infiltration of inflammatory cells in the lung airways. Saffron's active component, crocin, has been proven to possess anti-inflammatory and anti-oxidant effects. The objective of this current study was to explore the impact of crocin on NF-kB and nuclear erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) signaling pathways in ovalbumin (OVA)-sensitized mice, aiming to understand its mechanism. Four different groups were formed by dividing forty male BALB/C mice: control group, OVA-sensitized group (OVA), OVA combined with crocin 30 mg/kg (OVA-Cr30), and the OVA combined with crocin 60 mg/kg (OVA-Cr60). In order to determine the total number of WBC and inflammatory cells infiltrating the lung, we utilized the bronchoalveolar lavage fluid for counting purposes. The mRNA and protein levels of Nrf2, HO-1, IL-17, and NF-κB in lung tissue were assessed through real-time PCR and western blot techniques. Crocin significantly prevented the increase of total WBC and inflammatory cells in the lung tissue (P<0.001 for all) and histopathological changes in OVA-sensitized mice. Furthermore, crocin displayed suppressive effects on the enhancement of NF-kB (P<0.01) and IL-17 (P<0.05) mRNA and protein levels in OVA-sensitized mice while preserving Nrf2 (P<0.01) and HO-1 (P<0.05) expression levels. Crocin effects became increasingly apparent when utilized at high concentrations. Crocin decreased airway inflammation, partially by inhibiting NF-κB and IL-17 and up-regulating Nrf2/HO-1 mRNA and protein expression levels.

Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose.

To determine whether the microRNA-27b-3p (miR-27b-3p)/NF-E2-related factor 2 (Nrf2) pathway plays a role in human retinal pigment epithelial (hRPE) cell response to high glucose, how miR-27b-3p and Nrf2 expression are regulated, and whether this pathway could be specifically targeted. hRPE cells were cultured in normal glucose or high glucose for 1, 3, or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species (ROS) levels using a dihydroethidium kit. miR-27b-3p, Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunocytofluorescence (ICF), respectively. Western blot analyses were performed to determine nuclear and total Nrf2 protein levels. Nrf2, NQO1, and HO-1 expression levels by RT-qPCR, ICF, or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection. Finally, the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine. Persistent exposure to high glucose gradually suppressed hRPE Nrf2, NQO1, and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels. High glucose also promoted ROS release and inhibited cellular proliferation. Nrf2, NQO1, and HO-1 mRNA levels decreased after miR-27b-3p overexpression and, conversely, both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor. After treating hRPE cells exposed to high glucose with pyridoxamine, ROS levels tended to decreased, proliferation rate increased, Nrf2, NQO1, and HO-1 mRNA and protein levels were upregulated, and miR-27b-3p mRNA levels were suppressed. Nrf2 is a downstream target of miR-27b-3p. Furthermore, the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

Shenqi Dihuang Decoction inhibits high-glucose induced ferroptosis of renal tubular epithelial cells via Nrf2/HO-1/GPX4 pathway

This study aims to explore the effects of Shenqi Dihuang Decoction on high-glucose induced ferroptosis and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4) axis in human renal tubular epithelial cells(HK-2) and to clarify the underlying mechanism. The cell injury model was established by exposing HK-2 to high glucose, and the Shenqi Dihuang Decoction-medicated serum was prepared. The optimal concentration and intervention time of Shenqi Dihuang Decoction were determined. HK-2 were divided into normal, high glucose, and low-, medium-, and high-dose Shenqi Dihuang Decoction groups. After interventions, the cell proliferation rate in each group was determined and the cell morphology and mitochondrial ultrastructure were observed. Then, the levels of intracellular reactive oxygen species(ROS), ferrous ion(Fe~(2+)), glutathione(GSH), and malondialdehyde(MDA) and the protein levels of Nrf2, HO-1, GPX4, and xCT were measured. The optimal concentration and intervention time of Shenqi Dihuang Decoction-medicated serum were determined to be 10% and 24 h, respectively. Compared with the high glucose group, high-dose Shenqi Dihuang Decoction promoted the proliferation of HK-2. The cells in the low-, medium-, and high-dose Shenqi Dihuang Decoction groups presented tight arrangement, an increased cell count, improved morphology from a spindle-fiber shape to a cobblestone shape, and improved morphology and structure of mitochondrial membrane and cristae, compared with those in the high glucose group. Meanwhile, all the doses of Shenqi Dihuang Decoction inhibited ROS elevation to mitigate the peroxidation damage, lowered the Fe~(2+) and MDA levels and elevated the GSH level to inhibit lipid peroxidation, and activated the antioxidant pathway to upregulate the protein levels of Nrf2, HO-1, xCT, and GPX4. In conclusion, Shenqi Dihuang Decoction-medicated serum can inhibit high-glucose induced ferroptosis of HK-2 in vitro, which involves the antioxidant effect and the activation of the Nrf2/HO-1/GPX4 pathway.

Licochalcone a improves cardiac functions after ischemia-reperfusion via reduction of ferroptosis in rats

Myocardial ischemia-reperfusion (I/R) injury triggers several cell death types, including apoptosis, autophagy, and ferroptosis. Licochalcone A (LCA), a natural flavonoid compound isolated from the root of Glycyrrhiza glabra, has been demonstrated to exert potential pharmacological benefits, such as antioxidant, antitumor, and anti-inflammatory activities. The present study aimed to investigate the involvement of ferroptosis in the pathogenesis of I/R and determine whether LCA can inhibit ferroptosis to prevent the myocardial I/R injury in rats. The effects of LCA on myocardial I/R injury were detected by examining the left ventricular-developed pressure and triphenyltetrazolium chloride staining. We conducted Western blotting analyses, ELISA assay, and quantitative real-time PCR to determine the levels of ferroptosis-related molecules. To demonstrate the cardioprotective effect of LCA in vitro, H9c2 and primary neonatal rat cardiomyocytes were co-treated with ferroptosis inducers (erastin, RSL3, or Fe-SP) and LCA for 16 and 24 h. Our ex vivo study showed that LCA increased the cardiac contractility, and reduced the infarct volume and ferroptosis-related biomarkers in rat hearts after I/R. Moreover, LCA reduced the levels of ferroptosis inducers-induced reactive oxygen species generation, lipid peroxidation, and ferroptosis-related biomarkers in cultured H9c2 cells and cardiomyocytes. LCA also reduced the Fe-SP-increased nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 protein levels in cultured cardiomyocytes. In the present study, we showed that the LCA-induced cardioprotective effects in attenuating the myocardial I/R injury were correlated with ferroptosis regulation, and provided a possible new therapeutic strategy for prevention or therapy of the myocardial I/R injury.

Total triterpenes of Euphorbium alleviates rheumatoid arthritis via Nrf2/HO-1/GPX4 pathway

This study aims to investigate the effect and mechanism of total triterpenes of Euphorbium in treating rheumatoid arthritis(RA). The rat model of RA was established with Freund's complete adjuvant(FCA). Male rats were randomly assigned into control, model, Tripterygium glycosides(7.5 mg·kg~(-1)), and low-, medium-, and high-dose total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1), respectively) groups, with 10 rats in each group. In other groups except the control group, 0.2 mL FCA was injected into the right hind toe. Rats in the intervention groups were administrated with corresponding drugs by gavage, and the control group and the model group with the same volume of 0.5% CMC-Na solution once a day. During the treatment period, the swelling degree of the hind paw was measured and the arthritis was scored until day 30. At the end of drug administration, the pathological changes of the joint tissue were observed by hematoxylin-eosin staining. The content of malondialdehyde(MDA), glutathione(GSH), and Fe~(2+) and the activity of superoxide dismutase(SOD) in the joint tissue were measured by biochemical colorimetry. RT-PCR was performed to determine the mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2), glutathione peroxidase 4(GPX4), and acyl-CoA synthetase long chain family member 4(ACSL4) in the joint tissue. Western blot was employed to determine the protein levels of Nrf2, Kelch-like ECH-associated protein 1(Keap1), heme oxygenase-1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), SOD2, GPX4, and ACSL4 in the joint tissue. The results showed that the treatment with Tripterygium glycosides(7.5 mg·kg~(-1)) and total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1)) alleviated the swelling degree of bilateral hind limbs, decreased the arthritis score, reduced joint tissue lesions and the content of MDA and Fe~(2+) in the joint tissue, and increased GSH content and SOD activity. Furthermore, the interventions up-regulated the protein and mRNA levels of Nrf2 and GPX4, down-regulated the protein and mRNA levels of ACSL4, and up-regulated the protein levels of Keap1, NQO1, HO-1, and SOD2 in the Nrf2/HO-1/GPX4. In summary, the total triterpenes of Euphorbium can treat RA by inhibiting lipid peroxidation and abnormal ferroptosis, which may involve the Nrf2/HO-1/GPX4 signaling pathway.

Dimethyl Fumarate Protects Rats against Testicular Ischemia–Reperfusion Injury

Ischemia–reperfusion injury (IRI) after testicular torsion is linked to significant damage in testicular tissue. Dimethyl fumarate (DMF) activates the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, thereby inducing antioxidant and anti-inflammatory effects. We analyzed the usefulness of DMF in preventing IRI following testicular torsion/detorsion in Sprague-Dawley rats ( n = 32 ). The animals were classified into control (sham), DMF (200 mg/kg/day), IRI, and IRI+DMF (IRI with 200 mg/kg/day DMF) groups. Testicular IRI was induced by detorsion after 1.5 h of torsion. DMF was administered via oral gavage daily from 1 h before testicular detorsion until day 7, when orchiectomy was performed. The testis-to-body weight ratio was calculated. Histopathological evaluation was performed using the Johnsen and Cosentino scores for seminiferous tubules. Malondialdehyde, superoxide dismutase, and total glutathione levels were determined in testicular tissues. Moreover, Nrf2, heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), nuclear factor kappa B (NF-κB), and inflammatory cytokine (interleukin 1b (IL1b), IL6, and tumor necrosis factor alpha (TNF-α)) levels were determined through quantitative polymerase chain reaction. Nuclear Nrf2 and cytoplasmic HO-1 and NQO1 protein levels were also evaluated. DMF significantly improved the testis-to-body weight ratio and reduced histopathological damage in the testes. Moreover, it significantly improved the concentration of malondialdehyde, superoxide dismutase, and total glutathione. Furthermore, it inhibited NF-κB and inflammatory cytokine mRNA expression compared with the findings obtained in untreated rats with IRI (all p &lt; 0.05 ). Nrf2, HO-1, and NQO1 expressions (mRNA and protein) were markedly elevated following DMF treatment in rats with IRI (all p &lt; 0.05 ). DMF administration activated the Nrf2 signaling pathway and induced antioxidant and anti-inflammatory effects, thereby improving IRI-induced testicular damage. Thus, DMF may prevent IRI following testicular torsion.

Role and mechanism of SIRT1 in regulating Nrf2/HO-1 signaling pathway in septic liver injury

To investigate the role and mechanism of silent information regulator 1 (SIRT1) in regulating nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in oxidative stress and inflammatory response to sepsis-induced liver injury. A total of 24 male Sprague-Dawley (SD) rats were randomly divided into sham operation (Sham) group, cecal ligation and puncture (CLP) group, SIRT1 agonist SRT1720 pretreatment (CLP+SRT1720) group and SIRT1 inhibitor EX527 pretreatment (CLP+EX527) group, with 6 rats in each group. Two hours before operation, SRT1720 (10 mg/kg) or EX527 (10 mg/kg) were intraperitoneally injected into the CLP+SRT1720 group and CLP+EX527 group, respectively. Blood was collected from the abdominal aorta at 24 hours after modeling and the rats were sacrificed for liver tissue. The serum levels of interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by microplate method. Hematoxylin-eosin (HE) staining was used to observe the pathological injury of rats in each group. The levels of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), glutathione (GSH) and superoxide dismutase (SOD) in liver tissue were detected by corresponding kits. The mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Compared with the Sham group, the serum levels of IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were significantly increased; histopathological results showed that liver cords were disordered, hepatocytes were swollen and necrotic, and a large number of inflammatory cells infiltrated; the contents of MDA and 8-OHdG in liver tissue increased, while the contents of GSH and SOD decreased; and the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were significantly decreased. These results suggest that sepsis rats have liver dysfunction, and the levels of SIRT1, Nrf2, HO-1 and antioxidant protein in liver tissues were decreased, while the levels of oxidative stress and inflammation were increased. Compared with the CLP group, the levels of inflammatory factors and oxidative stress were significantly decreased in the CLP+SRT1720 group, the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 were significantly increased [IL-6 (ng/L): 34.59±4.21 vs. 61.84±3.78, IL-1β (ng/L): 41.37±2.70 vs. 72.06±3.14, TNF-α (ng/L): 76.43±5.23 vs. 130.85±5.30, ALT (U/L): 30.71±3.63 vs. 64.23±4.59, AST (U/L): 94.57±6.08 vs. 145.15±6.86, MDA (μmol/g): 6.11±0.28 vs. 9.23±0.29, 8-OHdG (ng/L): 117.43±10.38 vs. 242.37±11.71, GSH (μmol/g): 11.93±0.88 vs. 7.66±0.47, SOD (kU/g): 121.58±5.05 vs. 83.57±4.84, SIRT1 mRNA (2-ΔΔCt): 1.20±0.13 vs. 0.46±0.02, Nrf2 mRNA (2-ΔΔCt): 1.21±0.12 vs. 0.58±0.03, HO-1 mRNA (2-ΔΔCt): 1.71±0.06 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.89±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.87±0.08 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.93±0.14 vs. 0.54±0.12, all P < 0.05], these results indicated that SIRT1 agonist SRT1720 pretreatment could improve liver injury in sepsis rats. However, pretreatment with SIRT1 inhibitor EX527 showed the opposite effect [IL-6 (ng/L): 81.05±6.47 vs. 61.84±3.78, IL-1β (ng/L): 93.89±5.83 vs. 72.06±3.14, TNF-α (ng/L): 177.67±5.12 vs. 130.85±5.30, ALT (U/L): 89.33±9.52 vs. 64.23±4.59, AST (U/L): 179.59±6.44 vs. 145.15±6.86, MDA (μmol/g): 11.39±0.51 vs. 9.23±0.29, 8-OHdG (ng/L): 328.83±11.26 vs. 242.37±11.71, GSH (μmol/g): 5.07±0.34 vs. 7.66±0.47, SOD (kU/g): 59.37±4.28 vs. 83.57±4.84, SIRT1 mRNA (2-ΔΔCt): 0.34±0.03 vs. 0.46±0.02, Nrf2 mRNA (2-ΔΔCt): 0.46±0.04 vs. 0.58±0.03, HO-1 mRNA (2-ΔΔCt): 0.21±0.03 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.47±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.32±0.07 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.19±0.09 vs. 0.54±0.12, all P < 0.05]. SIRT1 can inhibit the release of proinflammatory factors and alleviate the oxidative damage of hepatocytes by activating Nrf2/HO-1 signaling pathway, thus playing a protective role against CLP-induced liver injury.

The Effect and Mechanism of Syringa pinnatifolia Hemsl. Ligans on Cerebral Ischemia-Reperfusion Injury and Oxidative Stress in Mice.

Lignans are the main components of Syringa pinnatifolia Hemsl. (SP). Previous studies have shown that SP lignans (SPL) can considerably improve CCl4-induced acute liver injury in mice by the anti-oxidative stress (OS) mechanism. In this study, we investigated the antioxidant effects of SPL on cerebral ischemia/reperfusion injury (CIRI) and its underlying molecular mechanism. We developed a middle cerebral artery occlusion/reperfusion (MCAO/R) model in mice to achieve CIRI and orally administered SPL daily for 1-3 days. We evaluated neurological function deficits and performed hematoxylin and eosin staining. We further calculated the infarct volume. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the brain were detected using corresponding kits. The transcription and protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H quinone dehydrogenase 1 (NQO1) in brain tissues were analyzed by real-time reverse transcription polymerase chain reaction and western blotting, respectively. The results showed that SPL could remarkably ameliorate neurological functions and pathological damage in brain tissues, reducing the cerebral infarct volume. It also increased the SOD and GPx activities decreased the MDA levels as well as inhibited the expression of (NOX)2 and NOX4. We also found that the mRNA and protein levels of Nrf2, HO-1, and NQO1 in the CIRI mice increased transiently and peaked at 24h of reperfusion, and then began to decline. SPL could reverse decreasing Nrf2 and HO-1 levels after 24h. In conclusion, SPL can alleviate CIRI and OS by activating the Nrf2/HO-1 pathway.

Targeting the Nrf2-dependent mechanism of b-Ecdysterone in attenuating the motor dysfunction in the MPTP/Pro-induced Parkinson's disease mice model.

Oxidative stress is a pivotal stimulating factor in neurocyte apoptosis and has been involved in the pathogenesis of Parkinson's disease (PD). In this study, we have demonstrated that the improvement in the motor disorder of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/Pro-induced mice caused by b-Ecdysterone (b-Ecd) treatment is due to its antioxidant properties. Using open field, rotarod, and pole climbing tests, we have found that b-Ecd alleviates motor disorder in MPTP/Pro-induced mice and ultimately reduces the impairment of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra (SN). Notably, these effects of b-Ecd were not observed in Nrf2-KO mice. In addition, b-Ecd significantly reduced the formation of ROS and the level of MDA, blocked the increase of LPO, and partially reversed the GSH/GSSG ratio in MPTP/Pro-induced WT mice; however, these results were also not observed in MPTP/Pro-induced Nrf2-KO mice. Mechanistically, b-Ecd enhanced the expression levels of heme oxygenase 1 (HO-1) and GCLc, but not NQO1 (NAD(P)H quinone dehydrogenase 1) and GCLm expression. Interestingly, b-Ecd failed to increase the protein and mRNA levels of HO-1 and GCLc in Nrf2-KO mice, suggesting that b-Ecd attenuates oxidative stress through an Nrf2-dependent mechanism. Furthermore, b-Ecd promoted the expressions of PI3K/Akt phosphorylation (activity) and GSK-3b phosphorylation (inactivity). Conversely, administration of b-Ecd markedly decreased Fyn phosphorylation levels. Collectively, our findings suggest that b-Ecd focuses on Nrf2 in reducing MPTP/Pro-induced oxidative stress and subsequent motor deficits by inhibiting its nuclear export through PI3K/Akt/GSK-3b/Fyn pathway regulation. These further indicate that b-Ecd may be an absorbing therapeutic agent for PD.

Evaluating the Anticancer Activity of Blocking TNF Type 1 Receptors in Thioacetamide-Induced Hepatocellular Carcinoma in a Rat Model.

Background Hepatocellular carcinoma (HCC) can explicate about 90% of the total primary liver cancer cases, with approximately 800,000 new cases identified each year worldwide. In addition, any changes in the expression of the tumor necrosis factor α (TNF-α) type 1 receptor (TNFR1) might impact many biological processes, which may lead to cancer. Aims We conducted the following study to investigate the ability of CAY10500, a TNF-α inhibitor that prevents binding to the TNF receptor 1, to produce anticancer effects against hepatocellular carcinoma experimentally induced in rats and to discover its effect on nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Materials and methods HCC was induced in rats via 200 mg/kg thioacetamide followed by treating some rats with IV 1 mg/kg CAY10500. Assessment of the liver impairment was by measuring the serum α-fetoprotein (AFP) and investigation of liver sections stained with hematoxylin/eosin. The hepatic expression of both the messenger RNA (mRNA) and protein levels ofTNF-α, TNFR1, Nrf2, and HO-1 was assessed. Results We found that CAY10500 increased the survival percent of rats associated with a reduction in serum AFP and the number of hepatic nodules. Besides, CAY10500 reduced the expression of TNFR1 without affecting the expression of TNF-α. Finally, CAY10500 increased the expression of both Nrf2 and HO-1. Conclusions Inhibition of TNFR1 expression in HCC by using CAY10500 produced therapeutic effects as indicated by increasing the survival rate, reducing the serum AFP level, decreasing liver nodules, and improving hepatocytes' structure. In addition, TNFR1 significantly enhanced the expression of Nrf2 and HO-1.

XANTHOHUMOL REDUCES THE EXPRESSION OF PRO-INFLAMMATORY AND OXIDATIVE STRESS MARKERS IN THE LUNG OF SENESCENCE-ACCELERATED PRONE MICE (SAMP8)

<h3>Objective</h3> Aging consists of a series of deleterious changes in cells and tissues along time, which lead to altered homeostasis. Nowadays we consider oxidative stress and inflammation the most relevant mechanisms of cell damage and dysfunction during aging. Both clinical and subclinical antigen exposure along life trigger a chronic inflammatory and oxidative response in the host. The lung, due to its position between the internal and the external milieu, is particularly exposed to pathogens and pollutants. An upregulation of oxidative stress and inflammation in lung tissue is expected. Xanthohumol, the major prenylated chalcone found in hops, has been shown to present anti-inflammatory and antioxidant properties in several organs, including liver, brain, and the reproductive system. Its effect on the aging lung has yet to be elucidated. For this reason, the aim of the present study was to evaluate the possible anti-inflammatory role of xanthohumol on a model of male senescence-accelerated prone mice (SAMP8). Design and method: Animals: young and old animals, aged 2 and 10 months respectively, were maintained under a 12:12 h light-dark cycle in an environmentally-controlled room and fed ad libitum. They were divided into 4 experimental groups: young non-treated, old non-treated, old treated with 1 mg/kg/day xanthohumol, and old treated with 5 mg/kg/day xanthohumol. Young and old non-treated male senescence-accelerated resistant mice (SAMR1) were used as controls. Treatment: xanthohumol was dissolved in ethanol and added to the drinking water. Only ethanol was administered to the water of non-treated animals. After 30 days of treatment, animals were sacrificed and lungs were collected and immediately frozen in liquid nitrogen. Inflammation and oxidative stress markers: both mRNA and protein levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and heme oxygenase 1 (HO-1) were analyzed. mRNA nuclear factor NF-kappa-B (NFκB) levels were also quantified. mRNA and protein expressions were measured by RT-PCR and Western blotting, respectively. The results were analyzed by using ANOVA followed by Fisher's post-hoc test. <h3>Results</h3> Compared to the young animals, old non-treated SAMP8 mice presented significantly elevated (p<0.05) mRNA and protein levels of TNF-α, IL-1β, and HO-1. mRNA NFκB2 levels were also significantly increased (p<0.05). SAMR1 mice showed significantly decreased values (p<0.05). <h3>Conclusions</h3> Treatment with xanthohumol was able to decrease significantly (p<0.05) the age-induced elevation at any of the doses administered. Supplementing the diet with foodstuffs containing xanthohumol may be used as a potential tool for counteracting inflammation and oxidative stress in the aging lung.