Antioxidant and Anti-Fatigue Effects of a Standardized Botanical Extract Fraction (HemoHIM) in Forced-Exercised Aged Mice.

HemoHIM is a standardized medicinal herbal preparation consisting of extracts of Angelica gigas Nakai, Cnidium officinale Makino, and Paeonia lactiflora Pallas that possesses immune regulatory activities. This study aimed to research the potential antioxidant effects of HemoHIM and its capacity for reducing fatigue in aged mice subjected to forced exercise. After administering HemoHIM 125 (500 mg/kg orally) for 4 weeks in 8-month-old female C57BL/6 mice (4 groups of 10 mice), various parameters were evaluated. The analyses revealed that HemoHIM enhanced swimming time and grip strength. In addition, it significantly reduced serum lactate levels and increased liver glutathione peroxidase (GPx) levels after exercise challenge. The expression levels of antioxidant enzymes and factors, including nuclear factor erythroid 2-related factor-2 (Nrf-2), heme oxygenase 1, superoxide dismutase, GPx, and glutathione reductase, were significantly higher in liver and muscle tissues of mice treated with HemoHIM. These results indicate that HemoHIM might function as an anti-fatigue and antioxidant agent by modulating the Nrf-2 signaling pathway.

Salvia miltiorrhiza suppresses cardiomyocyte ferroptosis after myocardial infarction by activating Nrf2 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferroptosis, a recently identified non-apoptotic form of cell death reliant on iron, is distinguished by an escalation in lipid reactive oxygen species (ROS) that are iron-dependent. This phenomenon has a strong correlation with irregularities in iron metabolism and lipid peroxidation. Salvia miltiorrhiza Bunge (DS), a medicinal herb frequently utilized in China, is highly esteemed for its therapeutic effectiveness in enhancing blood circulation and ameliorating blood stasis, particularly during the treatment of cardiovascular diseases (CVDs). Numerous pharmacological studies have identified that DS manifests antioxidative stress effects as well as inhibits lipid peroxidation. However, ambiguity persists regarding the potential of DS to impede ferroptosis in cardiomyocytes and subsequently improve myocardial damage post-myocardial infarction (MI). AIM OF THE STUDY: The present work focused on investigating whether DS could be used to prevent the ferroptosis of cardiomyocytes and improve post-MI myocardial damage. MATERIALS AND METHODS: In vivo experiments: Through ligation of the left anterior descending coronary artery, we constructed both a wild-type (WT) and NF-E2 p45-related factor 2 knockout (Nrf2-/-) mouse model of MI. Effects of DS and ferrostatin-1 (Fer-1) on post-MI cardiomyocyte ferroptosis were examined through detecting ferroptosis and myocardial damage-related indicators as well as Nrf2 signaling-associated protein levels. In vitro experiments: Erastin was used for stimulating H9C2 cardiomyocytes to construct an in vitro ferroptosis cardiomyocyte model. Effects of DS and Fer-1 on cardiomyocyte ferroptosis were determined based on ferroptosis-related indicators and Nrf2 signaling-associated protein levels. Additionally, inhibitor and activator of Nrf2 were used for confirming the impact of Nrf2 signaling on DS's effect on cardiomyocyte ferroptosis. RESULTS: In vivo: In comparison to the model group, DS suppressed ferroptosis in cardiomyocytes post-MI and ameliorated myocardial damage by inducing Nrf2 signaling-related proteins (Nrf2, xCT, GPX4), diminishing tissue ferrous iron and malondialdehyde (MDA) content. Additionally, it enhanced glutathione (GSH) levels and total superoxide dismutase (SOD) activity, effects that are aligned with those of Fer-1. Moreover, the effect of DS on alleviating cardiomyocyte ferroptosis after MI could be partly inhibited through Nrf2 knockdown. In vitro: Compared with the erastin group, DS inhibited cardiomyocyte ferroptosis by promoting the expression of Nrf2 signaling-related proteins, reducing ferrous iron, ROS, and MDA levels, but increasing GSH content and SOD activity, consistent with the effect of Fer-1. Additionally, Nrf2 inhibition increased erastin-mediated ferroptosis of cardiomyocytes through decreasing Nrf2 signaling-related protein expressions. Co-treatment with DS and Nrf2 activator failed to further enhance the anti-ferroptosis effect of DS. CONCLUSION: MI is accompanied by cardiomyocyte ferroptosis, whose underlying mechanism is probably associated with Nrf2 signaling inhibition. DS possibly suppresses ferroptosis of cardiomyocytes and improves myocardial damage after MI through activating Nrf2 signaling.

Cucurbitacin B attenuates osteoarthritis development by inhibiting NLRP3 inflammasome activation and pyroptosis through activating Nrf2/HO-1 pathway.

Osteoarthritis (OA) is a complicated joint disorder characterized by inflammation that causes joint destruction. Cucurbitacin B (CuB) is a naturally occurring triterpenoid compound derived from plants in the Cucurbitaceae family. The aim of this study is to investigate the potential role and mechanisms of CuB in a mouse model of OA. This study identified the key targets and potential pathways of CuB through network pharmacology analysis. In vivo and in vitro studies confirmed the potential mechanisms of CuB in OA. Through network pharmacology, 54 potential targets for CuB in treating OA were identified. The therapeutic potential of CuB is associated with the nod-like receptor pyrin domain 3 (NLRP3) inflammasome and pyroptosis. Molecular docking results indicate a strong binding affinity of CuB to nuclear factor erythroid 2-related factor 2 (Nrf2) and p65. In vitro experiments demonstrate that CuB effectively inhibits the expression of pro-inflammatory factors induced by interleukin-1ß (IL-1ß), including cyclooxygenase-2, inducible nitric oxide synthase, IL-1ß, and IL-18. CuB inhibits the degradation of type II collagen and aggrecan in the extracellular matrix (ECM), as well as the expression of matrix metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin motifs-5. CuB protects cells by activating the Nrf2/hemeoxygenase-1 (HO-1) pathway and inhibiting nuclear factor-κB (NF-κB)/NLRP3 inflammasome-mediated pyroptosis. Moreover, in vivo experiments show that CuB can slow down cartilage degradation in an OA mouse model. CuB effectively prevents the progression of OA by inhibiting inflammation in chondrocytes and ECM degradation. This action is further mediated through the activation of the Nrf2/HO-1 pathway to inhibit NF-κB/NLRP3 inflammasome activation. Thus, CuB is a potential therapeutic agent for OA.

TaoHe ChengQi decoction ameliorates sepsis-induced cardiac dysfunction through anti-ferroptosis via the Nrf2 pathway.

BACKGROUND: Sepsis-induced cardiac dysfunction (SICD) is a serious complication of sepsis that is associated with increased mortality. Ferroptosis has been reported in the SICD. TaoHe ChengQi decoction (THCQD), a classical traditional Chinese medicinal formula, has multiple beneficial pharmacological effects. The potential effects of THCQD on the SICD remain unknown. PURPOSE: To investigate the effect of THCQD on SICD and explore whether this effect is related to the regulation of myocardial ferroptosis through nuclear factor erythroid 2-related factor 2 (Nrf2) activation. METHODS: We induced sepsis in a mouse model using cecal ligation and puncture (CLP) and administered THCQD (2 and 4 g/kg) and dexamethasone (40 mg/kg). Mice mortality was recorded and survival curves were plotted. Echocardiography, hematoxylin and eosin staining, and analysis of serum myocardial injury markers and inflammatory factors were used to evaluate cardiac pathology. Myocardial ferroptosis was detected by quantifying specific biomarker content and protein levels. Through HPLC-Q-Exactive-MS analysis, we identified the components of the THCQD. Network pharmacology analysis and Cellular Thermal Shift Assay (CETSA) were utilized to predict the targets of THCQD for treating SICD. We detected the expression of Nrf2 using Western blotting or immunofluorescence. An RSL3-induced ferroptosis model was established using neonatal rat cardiomyocytes (NRCMs) to further explore the pharmacological mechanism of THCQD. In addition to measuring cell viability, we observed changes in NRCM mitochondria using electron microscopy and JC-1 staining. NRF2 inhibitor ML385 and Nrf2 knockout mice were used to validate whether THCQD exerted protective effects against SICD through Nrf2-mediated ferroptosis signaling. RESULTS: THCQD reduced mortality in septic mice, protected against CLP-induced myocardial injury, decreased systemic inflammatory response, and prevented myocardial ferroptosis. Network pharmacology analysis and CETSA experiments predicted that THCQD may protect against SICD by activating the Nrf2 signaling pathway. Western blotting and immunofluorescence showed that THCQD activated Nrf2 in cardiac tissue. THCQDs consistently mitigated RSL3-induced ferroptosis in NRCM, which is related to Nrf2. Furthermore, the pharmacological inhibition of Nrf2 and genetic Nrf2 knockout partially reversed the protective effects of THCQD on SICD and ferroptosis. CONCLUSION: The effect of THCQD on SICD was achieved by activating Nrf2 and its downstream pathways.

A clinical-stage Nrf2 activator suppresses osteoclast differentiation via the iron-ornithine axis.

Activating Nrf2 by small molecules is a promising strategy to treat postmenopausal osteoporosis. However, there is currently no Nrf2 activator approved for treating chronic diseases, and the downstream mechanism underlying the regulation of Nrf2 on osteoclast differentiation remains unclear. Here, we found that bitopertin, a clinical-stage glycine uptake inhibitor, suppresses osteoclast differentiation and ameliorates ovariectomy-induced bone loss by activating Nrf2. Mechanistically, bitopertin interacts with the Keap1 Kelch domain and decreases Keap1-Nrf2 binding, leading to reduced Nrf2 ubiquitination and degradation. Bitopertin is associated with less adverse events than clinically approved Nrf2 activators in both mice and human subjects. Furthermore, Nrf2 transcriptionally activates ferroportin-coding gene Slc40a1 to reduce intracellular iron levels in osteoclasts. Loss of Nrf2 or iron supplementation upregulates ornithine-metabolizing enzyme Odc1, which decreases ornithine levels and thereby promotes osteoclast differentiation. Collectively, our findings identify a novel clinical-stage Nrf2 activator and propose a novel Nrf2-iron-ornithine metabolic axis in osteoclasts.

[Essential oil of Cinnamomum camphora mitigates depression-like behavior in mice by regulating Nrf2/HO-1 pathway and inhibiting inflammatory cytokines].

This study aims to investigate the essential oil(EOL) of Cinnamomum camphora regarding its anti-depression effect and mechanism in regulating inflammatory cytokines and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) pathway. A mouse model of depression was established by intraperitoneal injection of lipopolysaccharide(LPS). Open field, elevated plus maze, and forced swimming tests were carried out to examine mouse behaviors. Western blot and qRT-PCR were employed to determine the expression of proteins and genes in the Nrf2/HO-1 pathway in the hippocampus. The levels of tumor necrosis factor(TNF)-α, interleukin(IL)-6, and IL-1ß in the serum were measured by enzyme-linked immunosorbent assay(ELISA). The changes of apoptosis in mouse brain were detected by Tunel staining. Compared with the blank control group, the model group showed shortened distance travelled and time spent in the central zone and reduced number of entries in the central zone in the open field test. In the elevated plus maze test, the model group showed reduced open arm time(OT%) and open arm entries(OE%). In the force swimming test, the model group showed extended duration of immobility compared with the blank control group. Compared with the model group, the treatment with EOL significantly increased the distance travelled and time spent in the central zone and increased the number of entries in the central zone in the open field test. In addition, EOL significantly increased the OT% and OE% in the elevated plus maze and shor-tened the immobility duration in the forced swimming test. The model group showed lower expression levels of Nrf2 and HO-1 and hig-her levels of TNF-α, IL-6, and IL-1ß than the blank control group. Compared with the model group, the treatment with EOL up-regulated the expression levels of Nrf2 and HO-1 and lowered the levels of TNF-α, IL-6, and IL-1ß. The Tunel staining results showed that the apoptosis rate in the brain tissue of mice decreased significantly after the treatment with EOL. To sum up, EOL can mitigate the depression-like behaviors of mice by up-regulating the expression of Nrf2 and HO-1 and preventing hippocampal inflammatory damage. The findings provide empirical support for the application of EOL and aromatherapy in the treatment of depression.

[Fuyu Decoction ameliorates myocardial fibrosis in rat model of heart failure by regulating Nrf2/GPX4-mediated ferroptosis].

This study aims to investigate the effect and mechanism of Fuyu Decoction(FYD) in the treatment of myocardial fibrosis in the rat model of heart failure(HF). Sixty Wistar rats were randomized into a modeling group(n=50) and a sham group(n=10). A post-myocardial infarction HF model was established by ligating the left anterior descending coronary artery in rats. The successfully modeled rats were assigned into model, low-dose(2.5 g·kg~(-1)) FYD(FYD-L), high-dose(5.0 g·kg~(-1)) FYD(FYD-H), and FYD+Nrf2 inhibitor(ML385, 30 mg·kg~(-1)) groups(n=10). FYD was administrated by gavage and ML385 by intraperitoneal injection. The rats in the sham and model groups were administrated with equal amounts of normal saline by gavage. After 8 weeks of intervention, the cardiac function indicators were measured, and the myocardial tissue morphology and collagen deposition were observed. The positive expression of collagens Ⅰ and Ⅲ, apoptosis, and oxidative stress were examined, and the levels of Fe~(2+) and reactive oxygen species(ROS) were determined. The protein levels of nuclear factor erythroid 2-related factor 2(Nrf2), solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4), and acyl-coenzyme A synthase long chain family member 4(ACSL4) in the myocardial tissue were determined. Compared with sham group, the model group showed decreased left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), increased left ventricular end internal dimension in systole(LVIDs), left ventricular internal diameter in diastole(LVIDd), and myocardial collagen deposition, positive expression of collagens Ⅰ and Ⅲ, elevated apoptosis rate and malondialdehyde(MDA), Fe~(2+), and ROS levels, lowered superoxide dismutase(SOD) and glutathione peroxidase(GSH) levels, down-regulated protein levels of Nrf2, SLC7A11, and GPX4, and up-regulated protein level of ACSL4. Compared with the model group, the above indicators were restored by FYD. Moreover, ML385 reversed the protective effect of FYD on myocardial fibrosis in HF rats. In conclusion, FYD can inhibit ferroptosis by activating the Nrf2/GPX4 pathway, thereby ameliorating myocardial fibrosis in HF rats.

[Ginsenoside Re regulates mitochondrial biogenesis through Nrf2/HO-1/PGC-1α pathway to reduce hypoxia/reoxygenation injury in H9c2 cells].

This article explored the mechanism by which ginsenoside Re reduces hypoxia/reoxygenation(H/R) injury in H9c2 cells by regulating mitochondrial biogenesis through nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/peroxisome prolife-rator-activated receptor gamma coactivator-1α(PGC-1α) pathway. In this study, H9c2 cells were cultured in hypoxia for 4 hours and then reoxygenated for 2 hours to construct a cardiomyocyte H/R injury model. After ginsenoside Re pre-administration intervention, cell activity, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content, intracellular reactive oxygen species(Cyto-ROS), and intramitochondrial reactive oxygen species(Mito-ROS) levels were detected to evaluate the protective effect of ginsenoside Re on H/R injury of H9c2 cells by resisting oxidative stress. Secondly, fluorescent probes were used to detect changes in mitochondrial membrane potential(ΔΨ_m) and mitochondrial membrane permeability open pore(mPTP), and immunofluorescence was used to detect the expression level of TOM20 to study the protective effect of ginsenoside Re on mitochondria. Western blot was further used to detect the protein expression levels of caspase-3, cleaved caspase-3, Cyto C, Nrf2, HO-1, and PGC-1α to explore the specific mechanism by which ginsenoside Re protected mitochondria against oxidative stress and reduced H/R injury. Compared with the model group, ginse-noside Re effectively reduced the H/R injury oxidative stress response of H9c2 cells, increased SOD activity, reduced MDA content, and decreased Cyto-ROS and Mito-ROS levels in cells. Ginsenoside Re showed a good protective effect on mitochondria by increasing ΔΨ_m, reducing mPTP, and increasing TOM20 expression. Further studies showed that ginsenoside Re promoted the expression of Nrf2, HO-1, and PGC-1α proteins, and reduced the activation of the apoptosis-related regulatory factor caspase-3 to cleaved caspase-3 and the expression of Cyto C protein. In summary, ginsenoside Re can significantly reduce I/R injury in H9c2 cells. The specific mechanism is related to the promotion of mitochondrial biogenesis through the Nrf2/HO-1/PGC-1α pathway, thereby increasing the number of mitochondria, improving mitochondrial function, enhancing the ability of cells to resist oxidative stress, and alleviating cell apoptosis.

[Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice].

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1ß, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1ß in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1ß, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1ß in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.

Esculin induces endoplasmic reticulum stress and drives apoptosis and ferroptosis in colorectal cancer via PERK regulating eIF2α/CHOP and Nrf2/HO-1 cascades.

ETHNOPHARMACOLOGICAL RELEVANCE: Cortex fraxini (also known as Qinpi), the bark of Fraxinus rhynchophylla Hance and Fraxinus stylosa Lingelsh, constitutes a crucial component in several traditional Chinese formulas (e.g., Baitouweng Tang, Jinxiao Formula, etc.) and has demonstrated efficacy in alleviating intestinal carbuncle and managing diarrhea. Cortex fraxini has demonstrated commendable anticancer activity in the realm of Chinese ethnopharmacology; nevertheless, the underlying mechanisms against colorectal cancer (CRC) remain elusive. AIM OF THE STUDY: Esculin, an essential bioactive compound derived from cortex fraxini, has recently garnered attention for its ability to impede viability and induce apoptosis in cancer cells. This investigation aims to assess the therapeutic potential of esculin in treating CRC and elucidate the underlying mechanisms. MATERIALS AND METHODS: The impact of esculin on CRC cell viability was assessed using CCK-8 assay, Annexin V/PI staining, and Western blotting. Various cell death inhibitors, along with DCFH-DA, ELISA, biochemical analysis, and Western blotting, were employed to delineate the modes through which esculin induces HCT116 cells death. Inhibitors and siRNA knockdown were utilized to analyze the signaling pathways influenced by esculin. Additionally, an azomethane/dextran sulfate sodium (AOM/DSS)-induced in vivo CRC mouse model was employed to validate esculin's potential in inhibiting tumorigenesis and to elucidate its underlying mechanisms. RESULTS: Esculin significantly suppressed the viability of various CRC cell lines, particularly HCT116 cells. Investigation with diverse cell death inhibitors revealed that esculin-induced cell death was associated with both apoptosis and ferroptosis. Furthermore, esculin treatment triggered cellular lipid peroxidation, as evidenced by elevated levels of malondialdehyde (MDA) and decreased levels of glutathione (GSH), indicative of its propensity to induce ferroptosis in HCT116 cells. Enhanced protein levels of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and p-eIF2α suggested that esculin induced cellular endoplasmic reticulum (ER) stress, subsequently activating the Nrf2/ARE signaling pathway and initiating the transcriptional expression of heme oxygenase (HO)-1. Esculin-induced excessive expression of HO-1 could potentially lead to iron overload in HCT116 cells. Knockdown of Ho-1 significantly attenuated esculin-induced ferroptosis, underscoring HO-1 as a critical mediator of esculin-induced ferroptosis in HCT116 cells. Furthermore, utilizing an AOM/DSS-induced colorectal cancer mouse model, we validated that esculin potentially inhibits the onset and progression of colon cancer by inducing apoptosis and ferroptosis in vivo. CONCLUSIONS: These findings provide comprehensive insights into the dual induction of apoptosis and ferroptosis in HCT116 cells by esculin. The activation of the PERK signaling pathway, along with modulation of downstream eIF2α/CHOP and Nrf2/HO-1 cascades, underscores the mechanistic basis supporting the clinical application of esculin on CRC treatment.

Quanzhen Yiqi decoction attenuates inflammation in mice with smoking-induced COPD by activating the Nrf2/HO-1 pathway and inhibiting the NLRP3 inflammasome.

OBJECTIVE: Quanzhen Yiqi decoction (QZYQ) is a traditional Chinese medicine for treating chronic obstructive pulmonary disease. METHODS: Mice were exposed to cigarette smoke (CS) 6 days/week (40 cigarettes/day) for 24 weeks and then intragastrically administered QZYQ (4.72, 9.45, or 18.89 g/kg) or dexamethasone (DEX, 0.6 mg/kg) for 6 weeks. We examined the lung function and collected bronchoalveolar lavage fluid for inflammatory cell and cytokine quantification. The pathological lung changes, ROS and oxidative biomarkers were measured. We used immunohistochemistry and western blotting to evaluate the levels of Nrf2/HO-1, NLRP3/ASC/Caspase1/IL-1ß/IL-18. RESULTS: The CS group showed significant increases in the forced vital capacity, lung resistance, and chord compliance and a lower FEV50/FVC compared with the control, and QZYQ improved these changes. In addition, QZYQ effectively reduced emphysema, immune cell infiltration, and airway remodeling. QZYQ stimulated HO-1 expression and reduced oxidative stress through the Nrf2 pathway. QZYQ inhibited the production of NLRP3/ASC/Caspase-1 to inhibit IL-1ß and IL-18. CONCLUSION: Our study suggested that QZYQ can improve the function and histology of the lungs and reduce inflammatory cell recruitment. QZYQ inhibits ROS production and NLRP3 inflammasome activation by upregulating Nrf2 to reduce lung injury. The anti-inflammatory effects of QZYQ are similar to those of DEX.

Ginsenoside Rg1 alleviates chronic inflammation-induced neuronal ferroptosis and cognitive impairments via regulation of AIM2 - Nrf2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng is a valuable herb in traditional Chinese medicine. Modern research has shown that it has various benefits, including tonifying vital energy, nourishing and strengthening the body, calming the mind, improving cognitive function, regulating fluids, and returning blood pressure, etc. Rg1 is a primary active component of ginseng. It protects hippocampal neurons, improves synaptic plasticity, enhances cognitive function, and boosts immunity. Furthermore, it exhibits anti-aging and anti-fatigue properties and holds great potential for preventing and managing neurodegenerative diseases (NDDs). AIM OF THE STUDY: The objective of this study was to examine the role of Rg1 in treating chronic inflammatory NDDs and its molecular mechanisms. MATERIALS AND METHODS: In vivo, we investigated the protective effects of Rg1 against chronic neuroinflammation and cognitive deficits in mice induced by 200 µg/kg lipopolysaccharide (LPS) for 21 days using behavioral tests, pathological sections, Western blot, qPCR and immunostaining. In vitro experiments involved the stimulation of HT22 cells with 10 µg/ml of LPS, verification of the therapeutic effect of Rg1, and elucidation of its potential mechanism of action using H2DCFDA staining, BODIPY™ 581/591 C11, JC-1 staining, Western blot, and immunostaining. RESULTS: Firstly, it was found that Rg1 significantly improved chronic LPS-induced behavioral and cognitive dysfunction in mice. Further studies showed that Rg1 significantly attenuated LPS-induced neuronal damage by reducing levels of IL-6, IL-1ß and ROS, and inhibiting AIM2 inflammasome. Furthermore, chronic LPS exposure induced the onset of neuronal ferroptosis by increasing the lipid peroxidation product MDA and regulating the ferroptosis-associated proteins Gpx4, xCT, FSP1, DMT1 and TfR, which were reversed by Rg1 treatment. Additionally, Rg1 was found to activate Nrf2 and its downstream antioxidant enzymes, such as HO1 and NQO1, both in vivo and in vitro. In vitro studies also showed that the Nrf2 inhibitor ML385 could inhibit the anti-inflammatory, antioxidant, and anti-ferroptosis effects of Rg1. CONCLUSIONS: This study demonstrated that Rg1 administration ameliorated chronic LPS-induced cognitive deficits and neuronal ferroptosis in mice by inhibiting neuroinflammation and oxidative stress. The underlying mechanisms may be related to the inhibition of AIM2 inflammasome and activation of Nrf2 signaling. These findings provide valuable insights into the treatment of chronic neuroinflammation and associated NDDs.

Ethanol extract of Eclipta prostrata induces multiple myeloma ferroptosis via Keap1/Nrf2/HO-1 axis.

BACKGROUND: Multiple myeloma (MM) is an incurable hematological malignancy with limited therapeutic efficacy. Eclipta prostrata is a traditional Chinese medicinal plant reported to possess antitumor properties. However, the effects of E. prostrata in MM have not been explored. PURPOSE: The aim of this study was to define the mechanism of the ethanol extract of E. prostrata (EEEP) in treating MM and identify its major components. METHODS: The pro-ferroptotic effects of EEEP on cell death, cell proliferation, iron accumulation, lipid peroxidation, and mitochondrial morphology were determined in RPMI-8226 and U266 cells. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4), and 4-hydroxynonenal (4HNE) were detected using western blotting during EEEP-mediated ferroptosis regulation. The RPMI-8226 and U266 xenograft mouse models were used to explore the in vivo anticancer effects of EEEP. Finally, high performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry system (UPLC-Q/TOF-MS) were used to identify the major constituents of EEEP. RESULTS: EEEP inhibited MM cell growth and induced cell death in vitro and in vivo. By promoting malondialdehyde and Fe2+ accumulation, lipid peroxidation, and GSH suppression, EEEP triggers ferroptosis in MM. Mechanistically, EEEP regulates the Keap1/Nrf2/HO-1 axis and stimulates ferroptosis. EEEP-induced lipid peroxidation and malondialdehyde accumulation were blocked by the Nrf2 activator NK-252. In addition, HPLC and UPLC-Q/TOF-MS analysis elucidated the main components of EEEP, including demethylwedelolactone, wedelolactone, chlorogenic acid and apigenin, which may play important roles in the anti-tumor function of EEEP. CONCLUSION: In summary, EEEP exerts its anti-MM function by inducing MM cell death and inhibiting tumor growth in mice. We also showed that EEEP can induce lipid peroxidation and accumulation of ferrous irons in MM cells both in vivo and in vitro, leading to ferroptosis. In addition, this anti-tumor function may be achieved by the EEEP activation of Keap1/Nrf2/HO-1 axis. This is the first study to reveal that EEEP exerts anti-MM activity through the Keap1/Nrf2/HO-1-dependent ferroptosis regulatory axis, making it a promising candidate for MM treatment.

Kellerin alleviates cerebral ischemic injury by inhibiting ferroptosis via targeting Akt-mediated transcriptional activation of Nrf2.

BACKGROUND: Ischemic stroke (IS) is characterized as a detrimental cerebrovascular disease with high mortality and disability. Ferroptosis is a novel mechanism involved in neuronal death. There is a close connection between IS and ferroptosis, and inhibiting ferroptosis may provide an effective strategy for treating IS. Our previous investigations have discovered that kellerin, the active compound of Ferula sinkiangensis K. M. Shen, possesses the capability to shield against cerebral ischemia injury. PURPOSE: Our objective is to clarify the relationship between the neuroprotective properties of kellerin against IS and its ability to modulate ferroptosis, and investigate the underlying regulatory pathway. STUDY DESIGN: We investigated the impact and mechanism of kellerin in C57BL/6 mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) as well as SH-SY5Y cells exposed to oxygen-glucose deprivation/ re-oxygenation (OGD/R). METHODS: The roles of kellerin on neurological severity, cerebral infarction and edema were investigated in vivo. The regulatory impacts of kellerin on ferroptosis, mitochondrial damage and Akt/Nrf2 pathway were explored. Molecular docking combined with drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) were performed to analyze the potential target proteins for kellerin. RESULTS: Kellerin protected against IS and inhibited ferroptosis in vivo. Meanwhile, kellerin improved the neuronal damage caused by OGD/R and suppressed ferroptosis by inhibiting the production of mitochondrial ROS in vitro. Further we found that kellerin directly interacted with Akt and enhanced its phosphorylation, leading to the increase of Nrf2 nuclear translocation and its downstream antioxidant genes expression. Moreover, kellerin's inhibitory effect on ferroptosis and mitochondrial ROS release was eliminated by inhibiting Akt/Nrf2 pathway. CONCLUSIONS: Our study firstly demonstrates that the neuroprotective properties of kellerin against IS are related to suppressing ferroptosis through inhibiting the production of mitochondrial ROS, in which its modulation on Akt-mediated transcriptional activation of Nrf2 plays an important role. This finding shed light on the potential mechanism that kellerin exerts therapeutic effects in IS.

Echinacoside ameliorates post-stroke depression by activating BDNF signaling through modulation of Nrf2 acetylation.

BACKGROUND: Post-stroke depression (PSD) affects approximately one-third of stroke survivors, leading to adverse outcomes in rehabilitation, reduced quality of life, and increased mortality rates. Despite these implications, the underlying causes of PSD remain unclear, posing challenges for prevention and treatment. Echinacoside (ECH), a natural compound with known neuroprotective and antidepressant properties, holds significant therapeutic potential for PSD. However, the precise mechanism of its action remains unknown. PURPOSE: To unravel the specific mechanism through which ECH alleviates PSD by exploring the intricate interplay between ECH and Nrf2, as well as its impact on the BDNF/TrkB signaling axis. STUDY DESIGN AND METHODS: A rat PSD model was established though middle cerebral artery occlusion coupled with chronic unpredictable mild stress, followed by ECH treatment. The rats' depressive state was evaluated using the sucrose preference test and force swimming test. Brain damage was assessed through TTC staining, Nissl staining, and TUNEL assay. The multifaceted mechanism of ECH in PSD was investigated using immunofluorescence, immunohistochemistry, RT-qPCR, dual-luciferase assay, and western blotting. Additionally, the interaction between ECH and Nrf2 was explored through molecular docking and microscale thermophoresis. RESULTS: Our findings unveiled a novel facet of ECH action, demonstrating its unique ability to upregulate Nrf2 through acetylation within the hippocampus of PSD-affected rats (p < 0.05). Moreover, ECH showcased its distinctive potential by enhancing BDNF transcriptional activity, activating the BDNF/TrkB signaling axis, and orchestrating a comprehensive response against oxidative stress and apoptosis, thereby alleviating PSD symptoms in rats (p < 0.05). CONCLUSIONS: This study not only provides insights into the pivotal role of Nrf2 in mediating the BDNF/TrkB axis activation by ECH but also highlights the novelty of ECH's mechanism in addressing PSD. The elucidation of these unique aspects positions ECH as a groundbreaking candidate for further exploration and development in the realm of PSD intervention.

Esculin inhibits hepatic stellate cell activation and CCl4-induced liver fibrosis by activating the Nrf2/GPX4 signaling pathway.

BACKGROUND: Liver fibrosis (LF) is a pathological process of the liver that threatens human health. Currently, effective treatments are still lacking. Esculin, a prominent constituent found in the Fraxinus rhynchophylla. (bark), Aesculus hippocastanum. (bark), and Cichorium intybus. (herb), has been shown to possess significant anti-inflammatory, antioxidant, and antibacterial properties. However, to date, there have been no studies investigating its potential efficacy in the treatment of LF. OBJECTIVE: The study aims to investigate the therapeutic effect of esculin on LF and elucidate its potential molecular mechanism. METHODS: Carbon tetrachloride (CCl4) was injected intraperitoneally to induce LF in mice, and transforming growth factor ß1 (TGF-ß1) was injected to induce LX-2 cells to investigate the improvement effect of esculin on LF. Kit, histopathological staining, immunohistochemistry (IHC), immunofluorescence (IF), polymerase chain reaction (PCR), and western blot (WB) were used to detect the expression of fiber markers and nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway in liver tissue and LX-2 cells. Finally, molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) were used to verify the targeting between Nrf2 and esculin. RESULTS: Esculin significantly inhibited CCl4-induced hepatic fibrosis and inflammation in mice. This was evidenced by the improvement of liver function indexes, fibrosis indicators, and histopathology. Additionally, esculin treatment prominently reduced the levels of pro-inflammatory factors, oxidative stress, and liver Fe2+ in CCl4-induced mice. In vitro studies also showed that esculin treatment significantly inhibited TGF-ß1-induced LX-2 cell activation and decreased alpha-smooth muscle actin (α-SMA) and collagen I expression. Mechanism experiments proved that esculin can activate the Nrf2/GPX4 signaling pathway and inhibit liver ferroptosis. However, when LX-2 cells were treated with the Nrf2 inhibitor (ML385), the therapeutic effect of esculin significantly decreased. CONCLUSION: This study is the first to demonstrate that esculin is a potential natural active ingredient in the treatment of LF, which can inhibit the activation of hepatic stellate cells (HSC) and improve LF. Its therapeutic effect is related to the activation of the Nrf2/GPX4 signaling pathway.

Celastrol inhibits oligodendrocyte and neuron ferroptosis to promote spinal cord injury recovery.

BACKGROUND: Spinal cord injury (SCI) is a traumatic injury to the central nervous system and can cause lipid peroxidation in the spinal cord. Ferroptosis, an iron-dependent programmed cell death, plays a key role in the pathophysiology progression of SCI. Celastrol, a widely used antioxidant drug, has potential therapeutic value for nervous system. PURPOSE: To investigate whether celastrol can be a reliable candidate for ferroptosis inhibitor and the molecular mechanism of celastrol in repairing SCI by inhibiting ferroptosis. METHODS: First, a rat SCI model was constructed, and the recovery of motor function was observed after treatment with celastrol. The regulatory effect of celastrol on ferroptosis pathway Nrf2-xCT-GPX4 was detected by Western blot and immunofluorescence. Finally, the ferroptosis model of neurons and oligodendrocytes was constructed in vitro to further verify the mechanism of inhibiting ferroptosis by celastrol. RESULTS: Our results demonstrated that celastrol promoted the recovery of spinal cord tissue and motor function in SCI rats. Further in vitro and in vivo studies showed that celastrol significantly inhibited ferroptosis in neurons and oligodendrocytes and reduced the accumulation of ROS. Finally, we found that celastrol could inhibit ferroptosis by up-regulating the Nrf2-xCT-GPX4 axis to repair SCI. CONCLUSION: Celastrol effectively inhibits ferroptosis after SCI by upregulating the Nrf2-xCT-GPX4 axis, reducing the production of lipid ROS, protecting the survival of neurons and oligodendrocytes, and improving the functional recovery.

Bioactive compound schaftoside from Clinacanthus nutans attenuates acute liver injury by inhibiting ferroptosis through activation the Nrf2/GPX4 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Clinacanthus nutans (Burm. f.) Lindau, a traditional herb renowned for its anti-tumor, antioxidant, and anti-inflammatory properties, has garnered considerable attention. Although its hepatoprotective effects have been described, there is still limited knowledge of its treatment of acute liver injury (ALI), and its mechanisms remain unclear. AIM OF THE STUDY: To assess the efficacy of Clinacanthus nutans in ALI and to identify the most effective fractions and their underlying mechanism of action. METHODS: Bioinformatics was employed to explore the underlying anti-hepatic injury mechanisms and active compounds of Clinacanthus nutans. The binding ability of schaftoside, a potential active ingredient in Clinacanthus nutans, to the core target nuclear factor E2-related factor 2 (Nrf2) was further determined by molecular docking. The role of schaftoside in improving histological abnormalities in the liver was observed by H&E and Masson's staining in an ALI model induced by CCl4. Serum and liver biochemical parameters were measured using AST, ALT and hydroxyproline kits. An Fe2+ kit, transmission electron microscopy, western blotting, RT-qPCR, and DCFH-DA were used to measure whether schaftoside reduces ferroptosis-induced ALI. Subsequently, specific siRNA knockdown of Nrf2 in AML12 cells was performed to further elucidate the mechanism by which schaftoside attenuates ferroptosis-induced ALI. RESULTS: Bioinformatics analysis and molecular docking showed that schaftoside is the principal compound from Clinacanthus nutans. Schaftoside was shown to diminish oxidative stress levels, attenuate liver fibrosis, and forestall ferroptosis. Deeper investigations revealed that schaftoside amplified Nrf2 expression and triggered the Nrf2/GPX4 pathway, thereby reversing mitochondrial aberrations triggered by lipid peroxidation, GPX4 depletion, and ferroptosis. CONCLUSION: The lead compound schaftoside counters ferroptosis through the Nrf2/GPX4 axis, providing insights into a novel molecular mechanism for treating ALI, thereby presenting an innovative therapeutic strategy for ferroptosis-induced ALI.

Corynoline Suppresses Osteoclastogenesis and Attenuates ROS Activities by Regulating NF-κB/MAPKs and Nrf2 Signaling Pathways.

Declining estrogen production in postmenopausal females causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. Although clinical drugs are currently available for the treatment of osteoporosis, sustained medication use is accompanied by serious side effects. Corydalis bungeana Herba, a famous traditional Chinese herb listed in the Chinese Pharmacopoeia Commission, constitutes various traditional Chinese Medicine prescriptions, which date back to thousands of years. One of the primary active components of C. bungeana Turcz. is Corynoline (Cor), a plant isoquinoline alkaloid derived from the Corydalis species, which possesses bone metabolism disease therapeutic potential. The study aimed at exploring the effects as well as mechanisms of Cor on osteoclast formation and bone resorption. TRAcP staining, F-actin belt formation, and pit formation were employed for assessing the osteoclast function. Western blot, qPCR, network pharmacology, and docking analyses were used for analyzing the expression of osteoclast-associated genes and related signaling pathways. The study focused on investigating how Cor affected OVX-induced trabecular bone loss by using a mouse model. Cor could weaken osteoclast formation and function by affecting the biological receptor activators of NF-κB and its ligand at various concentrations. Mechanistically, Cor inhibited the NF-κB activation, and the MAPKs pathway stimulated by RANKL. Besides, Cor enhanced the protein stability of the Nrf2, which effectively abolished the RANKL-stimulated ROS generation. According to an OVX mouse model, Cor functions in restoring bone mass, improving microarchitecture, and reducing the ROS levels in the distal femurs, which corroborated with its in vitro antiosteoclastogenic effect. The present study indicates that Cor may restrain osteoclast formation and bone loss by modulating NF-κB/MAPKs and Nrf2 signaling pathways. Cor was shown to be a potential drug candidate that can be utilized for the treatment of osteoporosis.

Dihydropashanone Isolated from Lindera erythrocarpa, a Potential Natural Product for the Treatment of Neurodegenerative Diseases.

Lindera erythrocarpa, a flowering plant native to eastern Asia, has been reported to have neuroprotective activity. However, reports on the specific bioactive compounds in L. erythrocarpa are finite. The aim of this study was to investigate the anti-neuroinflammatory and neuroprotective effects of the compounds isolated from L. erythrocarpa. Dihydropashanone, a compound isolated from L. erythrocarpa extract, was found to have protected mouse hippocampus HT22 cells from glutamate-induced cell death. The antioxidant and anti-inflammatory properties of dihydropashanone in mouse microglial BV2 and HT22 cells were explored in this study. The results reveal that dihydropashanone inhibits lipopolysaccharide-induced inflammatory response and suppresses the activation of nuclear factor (NF)-κB in BV2 cells. In addition, dihydropashanone reduced the buildup of reactive oxygen species in HT22 cells and induced activation of the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 signaling pathway in BV2 and HT22 cells. Our results suggest that dihydropashanone reduces neuroinflammation by decreasing NF-κB activation in microglia cells and protects neurons from oxidative stress via the activation of the Nrf2/HO-1 pathway. Thus, our data suggest that dihydropashanone offers a broad range of applications in the treatment of neurodegenerative illnesses.