Punicalagin Protects against the Development of Methotrexate-Induced Hepatotoxicity in Mice via Activating Nrf2 Signaling and Decreasing Oxidative Stress,Inflammation, and Cell Death

Despite its effectiveness in treating inflammatory diseases and various malignancies, methotrexate (MTX) is well known to cause hepatotoxicity, which involves increased oxidative stress and inflammation, limiting its clinical use. Herein, we looked into the effect of punicalagin (PU), a polyphenolic molecule having a variety of health-promoting attributes, on MTX-induced hepatotoxicity in mice. PU (25 and 50 mg/kg/day) was given orally to the mice for 10 days, while a single dose of MTX (20 mg/kg) was injected intraperitoneally (i.p.) at day 7. The MTX-induced liver damage was demonstrated by remarkably higher transaminases (ALT and AST), ALP, and LDH, as well as significant histological alterations in hepatic tissues. MTX-injected mice also demonstrated increases in hepatic oxidative stress markers, including malondialdehyde (MDA) and nitric oxide (NO), with a concordant drop in glutathione (GSH) content and superoxide dismutase (SOD) and catalase (CAT) activities. PU significantly attenuated the MTX-induced serum transaminases, ALP and LDH elevations, and hepatic oxidative stress measures and boosted antioxidant defenses in the liver. Moreover, the liver of MTX-treated mice showed increases in NF-κB p65 expression, pro-inflammatory cytokine (IL-6 and TNF-α) levels, and pro-apoptotic protein (caspase-3 and Bax) expression, whereas Bcl-2 and Nrf2 expressions were reduced, which were all attenuated by PU treatment. Collectively, PU inhibits oxidative damage, inflammation, and apoptosis and upregulates Nrf2 in the liver of MTX-induced mice. Thus, these findings suggest that PU may have great therapeutic potential for the prevention of MTX-induced hepatotoxicity, pending further exploration in upcoming studies.     

Nrf2 Activation Attenuates Acrylamide-Induced Neuropathy in Mice

Acrylamide is a well characterized neurotoxicant known to cause neuropathy and encephalopathy in humans and experimental animals. To investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in acrylamide-induced neuropathy, male C57Bl/6JJcl adult mice were exposed to acrylamide at 0, 200 or 300 ppm in drinking water and co-administered with subcutaneous injections of sulforaphane, a known activator of the Nrf2 signaling pathway at 0 or 25 mg/kg body weight daily for 4 weeks. Assessments for neurotoxicity, hepatotoxicity, oxidative stress as well as messenger RNA-expression analysis for Nrf2-antioxidant and pro-inflammatory cytokine genes were conducted. Relative to mice exposed only to acrylamide, co-administration of sulforaphane protected against acrylamide-induced neurotoxic effects such as increase in landing foot spread or decrease in density of noradrenergic axons as well as hepatic necrosis and hemorrhage. Moreover, co-administration of sulforaphane enhanced acrylamide-induced mRNA upregulation of Nrf2 and its downstream antioxidant proteins and suppressed acrylamide-induced mRNA upregulation of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) in the cerebral cortex. The results demonstrate that activation of the Nrf2 signaling pathway by co-treatment of sulforaphane provides protection against acrylamide-induced neurotoxicity through suppression of oxidative stress and inflammation. Nrf2 remains an important target for the strategic prevention of acrylamide-induced neurotoxicity.     

Bisdemethoxycurcumin Attenuated Renal Injury via Activation of Keap1/Nrf2 Pathway in High-Fat Diet-Fed Mice

Bisdemethoxycurcumin (BDMC), a principal and active component of edible turmeric, was previously found to have beneficial effects on metabolic diseases. Chronic kidney disease (CKD) may benefit from its potential therapeutic use. Using a high-fat diet (HFD)-fed mouse model, we examined the effects of BDMC on renal injury and tried to determine how its associated mechanism works. A number of metabolic disorders are significantly improved by BDMC, including obesity, hyperglycemia, hyperinsulinemia, hyperlipidemia and inflammation. Further research on renal histopathology and function showed that BDMC could repair renal pathological changes and enhance renal function. Moreover, decreased serum malondialdehyde (MDA), elevated superoxide dismutase (SOD) activity, and the inhibition of renal reactive oxygen species (ROS) overproduction revealed the alleviation of oxidative stress after BDMC administration. In addition, renal Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) pathway was activated in BDMC-treated mice. In conclusion, these findings demonstrated BDMC as a potential therapy for HFD-induced CKD via the activation of the Keap1/Nrf2 pathway.     

Mangiferin Inhibits Apoptosis in Doxorubicin-Induced Vascular Endothelial Cells via the Nrf2 Signaling Pathway

Doxorubicin increases endothelial permeability, hence increasing cardiomyocytes’ exposure to doxorubicin (DOX) and exposing myocytes to more immediate damage. Reactive oxygen species are major effector molecules of doxorubicin’s activity. Mangiferin (MGN) is a xanthone derivative that consists of C-glucosylxanthone with additional antioxidant properties. This particular study assessed the effects of MGN on DOX-induced cytotoxicity in human umbilical vein endothelial cells’ (HUVECs’) signaling networks. Mechanistically, MGN dramatically elevated Nrf2 expression at both the messenger RNA and protein levels through the upregulation of the PI3K/AKT pathway, leading to an increase in Nrf2-downstream genes. Cell apoptosis was assessed with a caspase-3 activity assay, transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) staining was performed to assess DNA fragmentation, and protein expression was determined by Western blot analysis. DOX markedly increased the generation of reactive oxygen species, PARP, caspase-3, and TUNEL-positive cell numbers, but reduced the expression of Bcl-2 and antioxidants’ intracellular concentrations. These were effectively antagonized with MGN (20 μM), which led to HUVECs being protected against DOX-induced apoptosis, partly through the PI3K/AKT-mediated NRF2/HO-1 signaling pathway, which could theoretically protect the vessels from severe DOX toxicity.     

Natural and Synthetic Xanthone Derivatives Counteract Oxidative Stress via Nrf2 Modulation in Inflamed Human Macrophages

Natural products have attracted attention due to their safety and potential effectiveness as anti-inflammatory drugs. Particularly, xanthones owning a unique 9H-xanthen-9-one scaffold, are endowed with a large diversity of medical applications, including antioxidant and anti-inflammatory activities, because their core accommodates a vast variety of substituents at different positions. Among others, α- and γ-mangostin are the major known xanthones purified from Garcinia mangostana with demonstrated anti-inflammatory and antioxidant effects by in vitro and in vivo modulation of the Nrf2 (nuclear factor erythroid-derived 2-like 2) pathway. However, the main mechanism of action of xanthones and their derivatives is still only partially disclosed, and further investigations are needed to improve their potential clinical outcomes. In this light, a library of xanthone derivatives was synthesized and biologically evaluated in vitro on human macrophages under pro-inflammatory conditions. Furthermore, structure–activity relationship (SAR) studies were performed by means of matched molecular pairs (MMPs). The data obtained revealed that the most promising compounds in terms of biocompatibility and counteraction of cytotoxicity are the ones that enhance the Nrf2 translocation, confirming a tight relationship between the xanthone scaffold and the Nrf2 activation as a sign of intracellular cell response towards oxidative stress and inflammation.     

MCFA alleviate H2O2-induced oxidative stress in AML12 cells via the ERK1/2/Nrf2 pathway

Oxidative stress is an important factor in the occurrence and development of liver disease. Medium-chain fatty acids (MCFAs) have potential antioxidant function, whereas the exact underlying mechanism of MCFA in oxidative injury of hepatocytes remains unclear. In our present study, three different MCFAs, 8-carbon octanoic acid (OA), 10-carbon capric acid (CA), and 12-carbon lauric acid (LA), have been performed to observe their protective action for hepatocyte under the H₂O₂ challenge. The result showed that MCFA treatment significantly increased the cell viability, T-AOC, and expression of antioxidant-related genes in AML12 cells under oxidative stress condition, and reduced reactive oxygen species (ROS) production. Moreover, MCFA treatment significantly increased the protein expression of Nrf2 and the phosphorylation level of ERK1/2; LA treatment significantly promoted the Nrf2 nuclear translocation. With a further test, the rescue ability of MCFA was blocked by treating with the ERK inhibitor U0126. Overall, our data suggested that MCFA treatment has positive impact on protecting AML12 cells against oxidative stress through ERK1/2/Nrf2 pathway.     

Nrf2 and Redox Status in Prediabetic and Diabetic Patients

The redox status associated with nuclear factor erythroid 2-related factor-2 (Nrf2) was evaluated in prediabetic and diabetic subjects. Total antioxidant status (TAS) in plasma and erythrocytes, glutathione (GSH) and malondialdehyde (MDA) content and activity of antioxidant enzymes were measured as redox status markers in 259 controls, 111 prediabetics and 186 diabetic type 2 subjects. Nrf2 was measured in nuclear extract fractions from peripheral blood mononuclear cells (PBMC). Nrf2 levels were lower in prediabetic and diabetic patients. TAS, GSH and activity of glutamate cysteine ligase were lower in diabetic subjects. An increase of MDA and superoxide dismutase activity was found in diabetic subjects. These results suggest that low levels of Nrf2 are involved in the development of oxidative stress and redox status disbalance in diabetic patients.     

Baicalin Ameliorates H2O2 Induced Cytotoxicity in HK-2 Cells through the Inhibition of ER Stress and the Activation of Nrf2 Signaling

Renal ischemia-reperfusion injury plays a key role in renal transplantation and greatly affects the outcome of allograft. Our previous study proved that Baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, protects kidney from ischemia-reperfusion injury. This study aimed to study the underlying mechanism in vitro. Human renal proximal tubular epithelial cell line HK-2 cells were stimulated by H₂O₂ with and without Baicalin pretreatment. The cell viability, apoptosis and oxidative stress level were measured. The expression of endoplasmic reticulum (ER) stress hallmarks, such as binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), were analyzed by western blot and real-time PCR. NF-E2-related factor 2 (Nrf2) expression was also measured. In the H₂O₂ group, cell viability decreased and cell apoptosis increased. Reactive Oxygen Species (ROS) and Glutathione/Oxidized Glutathione (GSH/GSSG) analysis revealed increased oxidative stress. ER stress and Nrf2 signaling also increased. Baicalin pretreatment ameliorated H₂O₂-induced cytotoxicity, reduced oxidative stress and ER stress and further activated the anti-oxidative Nrf2 signaling pathway. The inducer of ER stress and the inhibitor of Nrf2 abrogated the protective effects, while the inhibitor of ER stress and the inducer of Nrf2 did not improve the outcome. This study revealed that Baicalin pretreatment serves a protective role against H₂O₂-induced cytotoxicity in HK-2 cells, where the inhibition of ER stress and the activation of downstream Nrf2 signaling are involved.     

Downregulation of P300/CBP-Associated Factor Protects from Vascular Aging via Nrf2 Signal Pathway Activation

Increasing evidence has shown that vascular aging has a key role in the pathogenesis of vascular diseases. P300/CBP-associated factor (PCAF) is involved in many vascular pathological processes, but the role of PCAF in vascular aging is unknown. This study aims to explore the role and underlying mechanism of PCAF in vascular aging. The results demonstrated that the expression of PCAF was associated with age and aging, and remarkably increased expression of PCAF was present in human atherosclerotic coronary artery. Downregulation of PCAF could reduce angiotensin II (AngII)-induced senescence of rat aortic endothelial cells (ECs) in vitro. In addition, inhibition of PCAF with garcinol alleviated AngII-induced vascular senescence phenotype in mice. Downregulation of PCAF could alleviate AngII-induced oxidative stress injury in ECs and vascular tissue. Moreover, PCAF and nuclear factor erythroid-2-related factor 2 (Nrf2) could interact directly, and downregulation of PCAF alleviated vascular aging by promoting the activation of Nrf2 and enhancing the expression of its downstream anti-aging factors. The silencing of Nrf2 with small interfering RNA attenuated the protective effect of PCAF downregulation from vascular aging. These findings indicate that downregulation of PCAF alleviates oxidative stress by activating the Nrf2 signaling pathway and ultimately inhibits vascular aging. Thus, PCAF may be a promising target for aging-related cardiovascular disease.     

Eupatilin Suppresses OVA-Induced Asthma by Inhibiting NF-κB and MAPK and Activating Nrf2 Signaling Pathways in Mice

To investigate the effect of eupatilin in asthma treatment, we evaluated its therapeutic effect and related signal transduction in OVA-induced asthmatic mice and LPS-stimulated RAW264.7 cells. The BALF was tested for changes in lung inflammatory cells. Th2 cytokines in the BALF and OVA-IgE in the serum were measured by ELISA. H&E and PAS staining were used to evaluate histopathological changes in mouse lungs. The key proteins NF-κB, MAPK, and Nrf2 in lung tissues were quantitatively analyzed by Western blotting. Finally, we evaluated the effect of eupatilin on cytokines and related protein expression in LPS-stimulated RAW 264.7 cells in vitro. In OVA-induced asthmatic mice, eupatilin reduced the numbers of inflammatory cells, especially neutrophils and eosinophils. Eupatilin also decreased the levels of IL-5, IL-13 in the BALF and OVA-IgE in the serum. Furthermore, eupatilin inhibited the activation of NF-κB and MAPK pathways and increased the expression of Nrf2 in OVA-induced asthmatic mice. In vitro, eupatilin significantly reduced LPS-stimulated NO, IL-6, and ROS production. Additionally, the NF-κB, MAPK, and Nrf2 protein expression in LPS-stimulated RAW264.7 cells was consistent with that in OVA-induced asthmatic lung tissues. In summary, eupatilin attenuated OVA-induced asthma by regulating NF-κB, MAPK, and Nrf2 signaling pathways. These results suggest the utility of eupatilin as an anti-inflammatory drug for asthma treatment.     

Cardiac Protective Effect of Kirenol against Doxorubicin-Induced Cardiac Hypertrophy in H9c2 Cells through Nrf2 Signaling via PI3K/AKT Pathways

Kirenol (KRL) is a biologically active substance extracted from Herba Siegesbeckiae. This natural type of diterpenoid has been widely adopted for its important anti-inflammatory and anti-rheumatic properties. Despite several studies claiming the benefits of KRL, its cardiac effects have not yet been clarified. Cardiotoxicity remains a key concern associated with the long-term administration of doxorubicin (DOX). The generation of reactive oxygen species (ROS) causes oxidative stress, significantly contributing to DOX-induced cardiac damage. The purpose of the current study is to investigate the cardio-protective effects of KRL against apoptosis in H9c2 cells induced by DOX. The analysis of cellular apoptosis was performed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining assay and measuring the modulation in the expression levels of proteins involved in apoptosis and Nrf2 signaling, the oxidative stress markers. Furthermore, Western blotting was used to determine cell survival. KRL treatment, with Nrf2 upregulation and activation, accompanied by activation of PI3K/AKT, could prevent the administration of DOX to induce cardiac oxidative stress, remodeling, and other effects. Additionally, the diterpenoid enhanced the activation of Bcl2 and Bcl-xL, while suppressing apoptosis marker proteins. As a result, KRL is considered a potential agent against hypertrophy resulting from cardiac deterioration. The study results show that KRL not only activates the IGF-IR-dependent p-PI3K/p-AKT and Nrf2 signaling pathway, but also suppresses caspase-dependent apoptosis.     

Prunella vulgaris Suppresses HG-Induced Vascular Inflammation via Nrf2/HO-1/eNOS Activation

Vascular inflammation is an important factor which can promote diabetic complications. In this study, the inhibitory effects of aqueous extract from Prunella vulgaris (APV) on high glucose (HG)-induced expression of cell adhesion molecules in human umbilical vein endothelial cells (HUVEC) are reported. APV decreased HG-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. APV also dose-dependently inhibited HG-induced adhesion of HL-60 monocytic cells. APV suppressed p65 NF-κB activation in HG-treated cells. APV significantly inhibited the formation of intracellular reactive oxygen species (ROS). HG-stimulated HUVEC secreted gelatinases, however, APV inhibited it. APV induced Akt phosphorylation as well as activation of heme oxygenase-1 (HO-1), eNOS, and nuclear factor E2-related factor 2 (Nrf2), which may protect vascular inflammation caused by HG. In conclusion, APV exerts anti-inflammatory effect via inhibition of ROS/NF-κB pathway by inducing HO-1 and eNOS expression mediated by Nrf2, thereby suggesting that Prunella vulgaris may be a possible therapeutic approach to the inhibition of diabetic vascular diseases.     

Acetaminophen-Induced Rat Hepatotoxicity Based on M1/M2-Macrophage Polarization,in Possible Relation to Damage-Associated Molecular Patterns and Autophagy

Overdose of acetaminophen (APAP), an antipyretic drug, is an important cause of liver injury. However, the mechanism in the rat model remains undetermined. We analyzed APAP-induced hepatotoxicity using rats based on M1/M2-macrophage functions in relation to damage-associated molecular patterns (DAMPs) and autophagy. Liver samples from six-week-old rats injected with APAP (1000 mg/kg BW, ip, once) after 15 h fasting were collected at hour 10, and on days 1, 2, 3, and 5. Liver lesions consisting of coagulation necrosis and inflammation were seen in the affected centrilobular area on days 1 and 2, and then, recovered with reparative fibrosis by day 5. Liver exudative enzymes increased transiently on day 1. CD68⁺ M1-macrophages increased significantly on days 1 and 2 with increased mRNAs of M1-related cytokines such as IFN-γ and TNF-α, whereas CD163⁺ M2-macrophages appeared later on days 2 and 3. Macrophages reacting to MHC class II and Iba1 showed M1-type polarization, and CD204⁺ macrophages tended to be polarized toward M2-type. At hour 10, interestingly, HMGB1 (representative DAMPs) and its related signals, TLR-9 and MyD88, as well as LC3B⁺ autophagosomes began to increase. Collectively, the pathogenesis of rat APAP hepatotoxicity, which is the first, detailed report for a rat model, might be influenced by macrophage functions of M1 type for tissue injury/inflammation and M2-type for anti-inflammatory/fibrosis; particularly, M1-type may function in relation to DAMPs and autophagy. Understanding the interplayed mechanisms would provide new insight into hepato-pathogenesis and contribute to the possible development of therapeutic strategies.     

Microbe-Derived Antioxidants Reduce Lipopolysaccharide-Induced Inflammatory Responses by Activating the Nrf2 Pathway to Inhibit the ROS/NLRP3/IL-1β Signaling Pathway

Inflammation plays an important role in the innate immune response, yet overproduction of inflammation can lead to a variety of chronic diseases associated with the innate immune system; therefore, modulation of the excessive inflammatory response has been considered a major strategy in the treatment of inflammatory diseases. Activation of the ROS/NLRP3/IL-1β signaling axis has been suggested to be a key initiating phase of inflammation. Our previous study found that microbe-derived antioxidants (MA) are shown to have excellent antioxidant and anti-inflammatory properties; however, the mechanism of action of MA remains unclear. The current study aims to investigate whether MA could protect cells from LPS-induced oxidative stress and inflammatory responses by modulating the Nrf2-ROS-NLRP3-IL-1β signaling pathway. In this study, we find that MA treatment significantly alleviates LPS-induced oxidative stress and inflammatory responses in RAW264.7 cells. MA significantly reduce the accumulation of ROS in RAW264.7 cells, down-regulate the levels of pro-inflammatory factors (TNF-α and IL-6), inhibit NLRP3, ASC, caspase-1 mRNA, and protein levels, and reduce the mRNA, protein levels, and content of inflammatory factors (IL-1β and IL-18). The protective effect of MA is significantly reduced after the siRNA knockdown of the NLRP3 gene, presumably related to the ability of MA to inhibit the ROS-NLRP3-IL-1β signaling pathway. MA is able to reduce the accumulation of ROS and alleviate oxidative stress by increasing the content of antioxidant enzymes, such as SOD, GSH-Px, and CAT. The protective effect of MA may be due to its ability of MA to induce Nrf2 to enter the nucleus and initiate the expression of antioxidant enzymes. The antioxidant properties of MA are further enhanced in the presence of the Nrf2 activator SFN. After the siRNA knockdown of the Nrf2 gene, the antioxidant and anti-inflammatory properties of MA are significantly affected. These findings suggest that MA may inhibit the LPS-stimulated ROS/NLRP3/IL-1β signaling axis by activating Nrf2-antioxidant signaling in RAW264.7 cells. As a result of this study, MA has been found to alleviate inflammatory responses and holds promise as a therapeutic agent for inflammation-related diseases.