Exosomes From Muscle-Derived Stem Cells Repair Peripheral Nerve Injury by Inhibiting Ferroptosis via the Keap1-Nrf2-Ho-1 Axis.

Currently, the clinical outcomes of peripheral nerve injuries are suboptimal, highlighting the urgent need to understand the mechanisms of nerve injury to enhance treatment strategies. Muscle-derived stem cells (MDSCs) are a diverse group of multipotent cells that hold promise for peripheral nerve regeneration due to their strong antioxidant and regenerative properties. Our research has revealed that severe ferroptosis occurs in the sciatic nerve and ipsilateral dorsal root ganglion following sciatic nerve injury. Interestingly, we have observed that MDSC-derived exosomes effectively suppress cell ferroptosis and enhance cell viability in Schwann cells and dorsal root ganglion cells. Treatment with exosomes led to increased expression of BDNF and P62 in Schwann cells, decreased expression of Keap1, Nrf2, and HO-1 in Schwann cells, and upregulated dorsal root ganglion cells. Rats treated with exosomes exhibited improvements in sciatic nerve function, sensitivity to stimuli, and reduced muscle atrophy, indicating a positive impact on post-injury recovery. In conclusion, our findings demonstrate the occurrence of ferroptosis in the sciatic nerve and dorsal root ganglion post-injury, with MDSC exosomes offering a potential therapeutic strategy by inhibiting ferroptosis, activating the Keap1-Nrf2-HO-1 pathway, and optimizing the post-injury repair environment.

Omaveloxolone Prevents Polystyrene Microplastic-Induced Ovarian Granulosa Cell Apoptosis via the Keap1/Nrf2/HO-1 Pathway in Rats.

Microplastics (MPs) are persistent environmental pollutants that enter the circulatory system and subsequently reduce sperm quantity and quality. However, the influence of polystyrene MPs (PS-MPs) on the ovary and relevant mechanisms remain elusive. Herein, we aimed to examine the impact of PS-MPs on oxidative disorders in ovarian tissues and elucidate the underlying mechanisms. Healthy female rats were treated with different concentrations of 0.5 µm PS-MPs (diluted in deionized H2O) for 90 days. Upon examination of hematoxylin-eosin-stained ovarian tissue sections, the number of growing follicles was reduced in PS-MP-treated rats when compared with that in control rats. Enzyme-linked immunosorbent assays revealed that PS-MP exposure markedly reduced anti-Müllerian hormone (AMH) levels. Treatment with PS-MPs downregulated superoxide dismutase, glutathione, and catalase activities in ovarian tissues while upregulating malondialdehyde levels. Furthermore, exposure to PS-MP blocked the Keap1/Nrf2/HO-1 signal transduction pathway. PS-MPs also triggered apoptosis in the ovarian tissue, as evidenced by increased TUNEL staining and expression levels of cleaved caspase-9, Bax, and Bcl-2. To reactivate the Keap1/Nrf2/HO-1 pathway, rats were co-administered PS-MPs and omaveloxolone (Oma), an Nrf2 activator, for 1 week. We found that Oma could counteract the PS-MP-mediated effects on oxidative disorder, apoptosis, AMH production, and follicle number in rat ovarian tissues. To develop an in vitro model, granulosa cells (GCs) were treated with 10 µM H2O2 for 12 h to induce oxidative stress. H2O2-stimulated GCs exhibited attenuated cell growth and upregulated apoptosis and oxidative stress. Oma administration could ameliorate the H2O2-induced effects in terms of regulating cell viability, apoptosis, and oxidative stress in GCs. In summary, PS-MPs could induce apoptosis and oxidative stress via the Keap1/Nrf2/HO-1 signaling pathway in both rats and GCs.

Neuroprotective effect of acetoxypachydiol against oxidative stress through activation of the Keap1-Nrf2/HO-1 pathway.

BACKGROUND: Excessive oxidative stress in the brain is an important pathological factor in neurological diseases. Acetoxypachydiol (APHD) is a lipophilic germacrane-type diterpene extracted as a major component from different species of brown algae within the genus Dictyota. There have been no previous reports on the pharmacological activity of APHD. The present research aims to explore the potential neuroprotective properties of APHD and its underlying mechanisms. METHODS: The possible mechanism of APHD was predicted using a combination of molecular docking and network pharmacological analysis. PC12 cells were induced by H2O2 and oxygen-glucose deprivation/reoxygenation (OGD/R), respectively. Western blot, flow cytometry, immunofluorescence staining, and qRT-PCR were used to investigate the antioxidant activity of APHD. The HO-1 inhibitor ZnPP and Nrf2 gene silencing were employed to confirm the influence of APHD on the signaling cascade involving HO-1, Nrf2, and Keap1 in vitro. RESULTS: APHD exhibited antioxidant activity in both PC12 cells subjected to H2O2 and OGD/R conditions by downregulating the release of LDH, the concentrations of MDA, and ROS, and upregulating SOD, GSH-Px, and GSH concentrations. APHD could potentially initiate the Keap1-Nrf2/HO-1 signaling cascade, according to the findings from network pharmacology evaluation and molecular docking. Furthermore, APHD was observed to increase Nrf2 and HO-1 expression at both mRNA and protein levels, while downregulating the protein concentrations of Keap1. Both Nrf2 silencing and treatment with ZnPP reversed the neuroprotective effects of APHD. CONCLUSIONS: APHD activated antioxidant enzymes and downregulated the levels of LDH, MDA, and ROS in two cell models. The neuroprotective effect is presumably reliant on upregulation of the Keap1-Nrf2/HO-1 pathway. Taken together, APHD from brown algae of the genus Dictyota shows potential as a candidate for novel neuroprotective agents.

Salecan ameliorates LPS-induced acute lung injury through regulating Keap1-Nrf2/HO-1 pathway in mice.

Acute lung injury (ALI) is a severe clinical condition with high mortality, characterized by rapid onset and limited treatment options. The pathogenesis of ALI involves inflammation and oxidative stress. The polysaccharide salecan, a water-soluble ß-(1,3)-D-glucan, has been found to possess numerous pharmaceutical effects, including anti-inflammatory properties, inhibition of oxidative stress, and anti-fatigue effects. This study aims to investigate the protective effect and underlying mechanism of salecan against LPS-induced ALI in mice. Using an in vivo LPS-induced ALI mouse model and an in vitro RAW264.7 cell system, we investigated the role of salecan in ALI with various experimental approaches, including histological staining, quantitative real-time PCR, flow cytometry, western blot analysis, and other relevant assays. Pre-treatment with salecan effectively attenuated LPS-induced ALI in vivo, reducing the severity of pulmonary edema, inflammation, and oxidative stress. NMR-based metabolomic profiling analysis revealed that salecan attenuated LPS-induced metabolic imbalances associated with ALI. Furthermore, salecan downregulated Keap1 and upregulated Nrf2 and HO-1 protein levels, indicating its modulation of the Keap1-Nrf2/HO-1 signaling pathway as a potential mechanism underlying its protective effects against ALI. In vitro studies on RAW264.7 cells revealed that salecan exhibited binding affinity towards macrophages, thereby alleviating LPS-induced apoptosis and inflammation, which underpin its therapeutic potential against ALI. Our study suggests that salecan can alleviate LPS-induced ALI by modulating oxidative stress, inflammatory response, and apoptosis through the activation of the Keap1-Nrf2/HO-1 pathway. These findings provide novel insights into the potential therapeutic use of salecan for the treatment of ALI.

Tectorigenin inhibits oxidative stress by activating the Keap1/Nrf2/HO-1 signaling pathway in Th2-mediated allergic asthmatic mice.

Asthma is a chronic obstructive airway condition and one of the most common non-communicable illnesses worldwide. Tectorigenin (Tec) is an isoflavonoid found in plants that possesses significant antioxidative and anti-inflammatory abilities. Nevertheless, the antioxidative properties of Tec have not yet been documented in allergic asthma. In this study, we created an asthmatic BALB/c mouse model induced by ovalbumin (OVA) and used it to assess the efficacy of Tec as a possible therapy agent. Tec decreased the serum OVA-specific immunoglobulin (Ig) E and IgG1 secretion levels. The total number of cells and the distribution of inflammatory cells decreased significantly in bronchoalveolar lavage fluid (BALF), with weakened inflammatory reaction in pulmonary tissues. Additionally, Tec regulated the T helper 1(Th1)/Th2 balance by increasing the expression of Th1- related factors (interleukin (IL)-12 and T-bet) and decreasing the expression of Th2-related factors (IL-4, IL-5, IL-13, and GATA binding protein 3. In addition, the pro-inflammatory cytokines such as IL-6, tumor necrosis factor-alpha, and IL-1ß were also inhibited by Tec. Tec also dramatically increased antioxidant (catalase and superoxide dismutase) concentrations while lowering the intensity of the indicators of oxidative stress such as reactive oxygen species and malondialdehyde in BALF. Finally, Tec effectively activated the Keap1/Nrf2/HO-1 signaling pathway and prevented the epithelial-mesenchymal transition. The results of the current study show that Tec may be useful in relieving the inflammatory and oxidative stress responses associated with asthma.

Intervention effect and mechanism of breviscapine on hepatic fibrosis in rats / 中国药房

OBJECTIVE To investigate the intervention effect and potential mechanism of breviscapine on hepatic fibrosis （HF） in rats based on the transforming growth factor-β（1 TGF-β1）/Smad2/extracellular signal-regulated protein kinase 1（ERK1） and Kelch-like epichlorohydrin-associated protein 1（Keap1）/nuclear factor-erythroid 2-related factor 2（Nrf2）/heme oxygenase-1（HO-1） pathways. METHODS Totally 60 rats were randomly divided into normal control group， model group， breviscapine low-dose， medium-dose and high-dose groups （5.4， 10.8， 21.6 mg/kg）， and colchicine group （positive control， 0.45 mg/kg）， with 10 rats in each group， half male and half female. Except for the normal control group， HF model of the other groups was induced by carbon tetrachloride. Subsequently， each drug group was given corresponding medicine by gavage once a day for 28 days. The liver appearance of rats in each group was observed and their liver coefficients were calculated. The levels of alanineaminotransferase （ALT） and aspartate aminotransferase （AST）in serum， those of ALT， AST， superoxide dismutase （SOD），malondialdehyde （MDA） and glutathione peroxidase （GSH- Px） in liver tissue were detected. The liver tissue inflammatory and fibrotic changes were observed. The protein and mRNA expressions of TGF-β1， Smad2， ERK1， Nrf2， Keap1 and HO-in liver tissue were detected. RESULTS Compared with the normal control group， the model group showed large areas of white nodular lesions in the liver， obvious inflammatory cell infiltration and collagen fiber deposition. The body weight， the levels of SOD and GSH-Px in liver tissue， the protein and mRNA expressions of Nrf2 and HO-1 were significantly lowered in the model group （P＜0.05）； the liver coefficient， the percentage of Masson staining positive area， ALT and AST levels of serum and liver tissue， MDA level of liver tissue， the protein and mRNA expressions of TGF-β1， Smad2， ERK1 and Keap1 were significantly increased （P＜0.05）. Compared with the model group， the liver lesions of rats in each drug group were improved， and the above quantitative indexes were generally reversed （P＜0.05）. CONCLUSIONS Breviscapine has a good intervention effect on HF rats， which may be related to inhibiting TGF-β1/Smad2/ERK1 pathway for anti-fibrosis and regulating Keap1/Nrf2/HO-1 pathway to inhibit oxidative stress.

Hatched Eggshell Membrane Can Be a Novel Source of Antioxidant Hydrolysates to Protect against H2O2-Induced Oxidative Stress in Human Chondrocytes.

Natural antioxidants derived from agricultural by-products have great promise and ecological advantages in the treatment of oxidative stress-related diseases. The eggshell membrane (ESM) from hatched eggs, i.e., the hatched ESM, is a globally abundant agricultural byproduct, and its high-value utilization has been rarely studied compared to the well-studied ESM from fresh eggs. In this research, we systematically characterized the hatched ESM as a novel source of antioxidant hydrolysates and explored their potential role in H2O2-induced human chondrocytes. The results showed that the hatched ESM is a protein-rich fibrous mesh material with a significantly different structure and composition from those of fresh ESM. Enzymatic hydrolysis of hatched ESM can produce antioxidant hydrolysates rich in low molecular weight (MW) peptides, which mainly derived from the Lysyl oxidase homolog by Nano-LC-MS/MS analysis. The peptide fraction with MW < 3 kDa (HEMH-I) exhibited the highest DPPH radical scavenging, Fe2+-chelating, and Fe3+-reducing abilities. In H2O2-induced human SW1353 chondrocytes, HEMH-I treatment significantly increased the cell viability and ameliorated oxidative stress, inflammatory response, and cartilage matrix degradation by reducing the level of ROS, matrix metalloprotease 3 (MMP3), MMP13, and IL-6, and by promoting the expression of SOD and type II collagen, potentially through activating the cellular Keap1/Nrf2/HO-1 pathway. This study provides a theoretical basis for the value-added application of hatched ESM waste to produce antioxidant hydrolysates and indicates their potential as functional food and pharmaceuticals.

Study on hypoglycemic effects of irradiated ginseng adventitious roots.

We aimed to explore the effects of the 60Co-γ irradiated ginseng adventitious root (GAR) with different radiation doses on the hypoglycemic effects of its extract (GARSE) through in vivo and in vitro experiments. The total saponin of GARSE was increased by 4.50% after 5 kGy irradiation, and the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability was enhanced by 5.10%. At 50 µg/mL, GARSE irradiated by 5 kGy displayed superior protective effects on human glomerular mesangial cells (HMCs) with high glucose damage. After feeding type 1 diabetes mellitus (T1DM) mice with GARSE irradiated by 5 kGy at 500 mg/kg·BW for 4 weeks, the glucose values was decreased by 16.0% compared with the unirradiated. The Keap1/Nrf2/HO-1 pathway was activated and the oxidative stress was attenuated, which further alleviated T1DM.

Ruscogenin Alleviates Myocardial Ischemia-Induced Ferroptosis through the Activation of BCAT1/BCAT2.

Ruscogenin (RUS), a natural steroidal sapogenin, exerts various biological activities. However, its effectiveness for preventing myocardial ischemia (MI) and its molecular mechanisms need further clarification. The model of MI mice and oxygen-glucose deprivation-induced cardiomyocytes injury was performed. RUS significantly alleviated MI, as evidenced by decreased infarct size, ameliorated biochemical indicators and cardiac pathological features, and markedly inhibited ferroptosis by means of the up-regulation of GPX4 and down-regulation of ACSL4 and FLC. Simultaneously, RUS notably mitigated cell injury and oxidative stress, and ameliorated ferroptosis in vitro. Subsequently, HPLC-Q-TOF/MS-based metabolomics identified BCAT1/BCAT2 as possible regulatory enzymes responsible for the cardioprotection of RUS. Importantly, RUS treatment significantly increased the expression of BCAT1 and BCAT2 in MI. Furthermore, we found that BCAT1 or BCAT2 siRNA significantly decreased cell viability, promoted ferroptosis, and increased Keap1 expression, and induced Nrf2 and HO-1 degradation in cardiomyocytes. Conversely, cardiac overexpression of BCAT1 or BCAT2 in MI mice activated the Keap1/Nrf2/HO-1 pathway. Moreover, RUS significantly activated the Keap1/Nrf2/HO-1 pathway in MI, whereas BCAT1 or BCAT2 siRNA partially weakened the protective effects of RUS, suggesting that RUS might suppress myocardial injury through BCAT1 and BCAT2. Overall, this study demonstrated that BCAT1/BCAT2 could alleviate MI-induced ferroptosis through the activation of the Keap1/Nrf2/HO-1 pathway and RUS exerted cardioprotective effects via BCAT1/BCAT2.

Sanghuangporus sanghuang Mycelium Prevents Paracetamol-Induced Hepatotoxicity through Regulating the MAPK/NF-κB, Keap1/Nrf2/HO-1, TLR4/PI3K/Akt, and CaMKKß/LKB1/AMPK Pathways and Suppressing Oxidative Stress and Inflammation.

Liver damage induced by paracetamol overdose is the main cause of acute liver failure worldwide. In order to study the hepatoprotective effect of Sanghuangporus sanghuang mycelium (SS) on paracetamol-induced liver injury, SS was administered orally every day for 6 days in mice before paracetamol treatment. SS decreased serum aminotransferase activities and the lipid profiles, protecting against paracetamol hepatotoxicity in mice. Furthermore, SS inhibited the lipid peroxidation marker malondialdehyde (MDA), hepatic cytochrome P450 2E1 (CYP2E1), and the histopathological changes in the liver and decreased inflammatory activity by inhibiting the production of proinflammatory cytokines in paracetamol-induced acute liver failure. Moreover, SS improved the levels of glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase in the liver. Significantly, SS diminished mitogen-activated protein kinase (MAPK), Toll-like receptor 4 (TLR4), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and the nuclear factor-kappa B (NF-κB) axis, as well as upregulated the Kelch-like ECH-associated protein 1 (Keap1)/erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway, in paracetamol-induced mice. SS mainly inhibited the phosphorylation of the liver kinase B1 (LKB1), Ca2+/calmodulin-dependent kinase kinase ß (CaMKKß), and AMP-activated protein kinase (AMPK) protein expression. Furthermore, the protective effects of SS on paracetamol-induced hepatotoxicity were abolished by compound C, an AMPK inhibitor. In summary, we provide novel molecular evidence that SS protects liver cells from paracetamol-induced hepatotoxicity by inhibiting oxidative stress and inflammation.

Butyrate alleviates PTZ-induced mitochondrial dysfunction, oxidative stress and neuron apoptosis in mice via Keap1/Nrf2/HO-1 pathway.

This study aims to evaluate the neuroprotective effect of sodium butyrate against the pentylenetetrazol (PTZ)-induced kindling epilepsy. Sodium butyrate (SB) (5, 10 and 20 mg/kg) and sodium valproate for 40 days and PTZ (37 mg/kg) injection every day were conducted for Kunming mice, to investigate seizure intensity and latency, oxidative stress parameters, mitochondrial structure and function, histopathology, and Keap1/Nrf2/HO-1 expressions. It is shown that seizure latency was effectively increased and the intensity of seizures decreased by treatment with sodium butyrate. It was also found to reverse the structural disruption of the mitochondria, reduce the ROS level and improve the levels of NAD + and ATP in the brains of epileptic mice. Furthermore, pretreatment with SB led to an increase in antioxidant enzyme activity (CAT, SOD and GSH-PX) in the brain as well as conferred a neuroprotective effect against neuron loss and apoptosis. The activation of Keap1/Nrf2/HO-1 signals was also identified, in which the antiepileptic effect of SB may be partially due to its anti-mitochondrial injury and neuroprotective activities. Accordingly, the results of a series of functional tests indicate a significant improvement of neurological function following SB treatment. In a mouse model of seizures, brain injury and neurological deficits can be attenuated by treatment with butyrate through the activation of Nrf2 pathway and the improvement of mitochondrial function.

Preconditioning with rHMGB1 ameliorates lung ischemia-reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway.

BACKGROUND: Lung ischemia-reperfusion injury (LIRI) is a complex pathophysiological process that can lead to poor patient outcomes. Inflammasome-dependent macrophage pyroptosis contributes to organ damage caused by ischemia/reperfusion injury. Oxidative stress and antioxidant enzymes also play an important role in LIRI. In this study, we conducted experiments to investigate whether and how preconditioning with rHMGB1 could ameliorate LIRI in a mouse model. METHODS: Adult male BALB/c mice were anesthetized, the left hilus pulmonis was clamped, and reperfusion was performed. rHMGB1 was administered via intraperitoneal injection before anesthesia, and brusatol was given intraperitoneally every other day before surgery. We measured pathohistological lung tissue damage, wet/dry mass ratios of pulmonary tissue, and levels of inflammatory mediators to assess the extent of lung injury. Alveolar macrophage pyroptosis was evaluated by measuring release of lactate dehydrogenase, caspase-1 expression was assessed using flow cytometry, and gasdermin-D expression was analyzed using immunofluorescent staining. Levels of oxidative stress markers and antioxidant enzymes were also analyzed. RESULTS: Preconditioning with rHMGB1 significantly ameliorated lung injury induced by ischemia-reperfusion, based on measurements of morphology, wet/dry mass ratios, as well as expression of IL-1ß, IL-6, NF-κB, and HMGB1 in lung tissues. It also alleviated alveolar macrophage pyroptosis, reduced oxidative stress and restored the activity of antioxidant enzymes. These beneficial effects were mediated at least in part by the Keap1/Nrf2/HO-1 pathway, since they were reversed by the pathway inhibitor brusatol. CONCLUSIONS: Preconditioning with rHMGB1 may protect against LIRI by suppressing alveolar macrophage pyroptosis. This appears to involve reduction of oxidative stress and promotion of antioxidant enzyme activity via the Keap1/Nrf2/HO-1 pathway.

Ginsenoside Rg1 protects mice against streptozotocin-induced type 1 diabetic by modulating the NLRP3 and Keap1/Nrf2/HO-1 pathways.

Ginseng has been traditionally used to treat diabetes mellitus (DM) in China. Ginsenoside Rg1 is a major active ingredient in processed ginseng, which elicits proven biological and pharmacological effects. Although a correlation between nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and predisposition to type 1 diabetes mellitus (T1DM) has been identified, the mechanism underlying the potential function and activation of NLRP3 inflammasome in DM have not been elucidated to date. The present study aimed to elucidate the effects and underlying mechanism of Rg1 on streptozotocin (STZ)-induced T1DM in mice through short or long-term observation. Concurrently, we intended to explore the relationships between inflammasome, pyroptosis and oxidative stress and the role of NLRP3 and Keap1/Nrf2/HO-1 pathways in the development and progression of DM. Using ELISA and Western blot analysis, we found that Rg1 attenuated abnormally elevated blood glucose, reduced inflammatory factors IL-1ß and IL-18 in the blood, decreased ALT and AST levels, promoted insulin secretion, and weakened the function of NLRP3 in mouse liver and pancreas. In addition, Rg1 protected against STZ-induced reactive oxygen species-mediated inflammation by upregulating Nrf2/ARE pathway, which further activated antioxidant enzymes. Interestingly, Rg1 also regulated H3K9 methylation in liver and pancreas, as detected by immunohistochemistry. In summary, these data provide new understanding about the mechanism of Rg1 action, suggesting that it is a potential drug applied for preventing the occurrence and development of T1DM.

DC32, a Dihydroartemisinin Derivative, Ameliorates Collagen-Induced Arthritis Through an Nrf2-p62-Keap1 Feedback Loop.

Artemisinins have been reported to have diverse functions, such as antimalaria, anticancer, anti-inflammation, and immunoregulation activities. DC32 [(9α,12α-dihydroartemisinyl) bis(2'-chlorocinnmate)], a dihydroartemisinin derivative possessing potent immunosuppressive properties, was synthesized in our previous study. Collagen-induced arthritis (CIA) in DBA/1 mice and inflammatory model in NIH-3T3 cells were established to evaluate the effect of DC32 on RA and discover the underlying mechanisms. The results showed that DC32 could markedly alleviate footpad inflammation, reduce cartilage degradation, activate the Nrf2/HO-1 signaling pathway, and increase the transcription of p62 in DBA/1 mice with CIA. Further mechanistic exploration with NIH-3T3 cells indicated that DC32 could increase the transcription, expression, and nuclear translocation of Nrf2. In addition, DC32 promoted degradation of Keap1 protein and upregulated HO-1 and p62 expression. Furthermore, the effect of DC32 on Keap1 degradation could be prevented by p62 knockdown using siRNA. Administration of DC32 could inhibit the activation of Akt/mTOR and ERK, and pretreatment of NIH-3T3 cells with the autophagy inhibitor 3-methyladenine (3-MA) attenuated the degradation of Keap1 induced by DC32. These results suggest that DC32 inhibits the degradation of Nrf2 by promoting p62-mediated selective autophagy and that p62 upregulation contributed to a positive feedback loop for persistent activation of Nrf2. In summary, our present study demonstrated that DC32 significantly suppressed rheumatoid arthritis (RA) via the Nrf2-p62-Keap1 feedback loop by increasing the mRNA and protein levels of Nrf2 and inducing p62 expression. These findings provide new mechanisms for artemisinins in RA treatment and a potential strategy for discovering antirheumatic drugs.