Naoxintong capsule accelerates mitophagy in cerebral ischemia-reperfusion injury via TP53/PINK1/PRKN pathway based on network pharmacology analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. AIM OF THE STUDY: The aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. MATERIALS AND METHODS: Oxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. RESULTS: NXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. CONCLUSION: This study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.

Bone marrow mesenchymal stem cells-derived exosomal miR-381 alleviates lung ischemia-reperfusion injury by activating Treg differentiation through inhibiting YTHDF1 expression.

AIM: Our study aimed to investigate whether BMSCs-derived exosomal miR-381 promotes Treg cell differentiation in lung ischemia-reperfusion injury (LIRI), and the underlying mechanism. METHODS: The in vitro and in vivo models of LIRI were established by hypoxia/reoxygenation (H/R) treatment and lung ischemia/reperfusion (I/R) surgery, respectively. BMSCs-derived exosomes were isolated and identified by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Cell viability, proliferation, and apoptosis were assessed by CCK-8, EdU, and flow cytometry assay, respectively. IL-18 secretion level in lung microvascular endothelial cells (LMECs) and lung tissue homogenate was examined by ELISA. Treg cell differentiation was determined using flow cytometry. The relationships between miR-381, YTHDF1, and IL-18 were investigated using dual-luciferase reporter gene, RIP, and/or RNA pull-down assays. MeRIP assay was employed to determine m6A modification of IL-18 mRNA in LMECs. The ubiquitination level of Foxp3 protein in CD4+ T cells was analyzed by Co-IP assay. RESULTS: BMSCs-derived exosomes reduced LMECs injury and increased Treg cell differentiation in LIRI, whereas miR-381 inhibition in BMSCs weakened these impacts. Mechanistically, miR-381 inhibited IL-18 translation in LMECs by inhibiting YTHDF1 expression via binding to its 3'-UTR. As expected, YTHDF1 overexpression in LMECs abolished the effects of miR-381-overexpressed exosomes on LMECs injury and Treg cell differentiation. Moreover, LMECs-secreted IL-18 inhibited Treg cell differentiation by promoting the ubiquitination degradation of Foxp3 protein. CONCLUSION: BMSCs-derived exosomal miR-381 suppressed IL-18 translation in LMECs through binding to YTHDF1 3'-UTR, thus suppressing the ubiquitination degradation of Foxp3 in CD4+ T cells, which promoted Treg cell differentiation and mitigated LIRI development.

Correlation between prognosis and peripheral blood levels of NLRP3 and triglyceride-glucose index after myocardial ischemia-reperfusion injury.

OBJECTIVE: We aim to investigate the association between prognosis and outcomes following myocardial ischemia-reperfusion injury, as well as peripheral blood levels of NLRP3 and the triglyceride-glucose index (TyG). METHODS: A total of 100 patients who underwent emergency coronary intervention following myocardial infarction confirmed by coronary angiography at our hospital between October 2021 and May 2023 were included in this study. Patients were stratified into two groups based on their prognoses: the control group (n = 73), which did not experience new myocardial infarctions or require hospitalization for heart failure or suffer sudden cardiac death post-interventional treatment; and the observation group (n = 27), which experienced one or more cardiovascular events post-treatment. Patient demographics were obtained from clinical records while biochemical analyses assessed peripheral blood triglycerides, blood glucose levels, and TyG index. Additionally, ELISA measurements determined levels of NLRP3 as well as inflammatory factors IL-6, TNF-α, and CRP in peripheral blood samples. Cardiac function was evaluated according to NYHA standards. Univariable Cox regression analysis identified factors influencing patient prognosis while Pearson correlation analysis examined relationships among prognosis, outcomes following myocardial ischemia-reperfusion injury, TyG index, and peripheral blood NLRP3. RESULTS: No significant differences were observed in the general characteristics between the two patient groups (P > 0.05). However, the observation group exhibited higher levels of peripheral blood triglycerides, blood glucose, and TyG index compared to the control group (P < 0.05). Additionally, levels of NLRP3 and inflammatory factors IL-6, TNF-α, and CRP were elevated in the observation group compared to the control group (P < 0.05). Cardiac function impairment was more pronounced in the observation group (P < 0.05). Notably, TyG index and peripheral blood NLRP3 demonstrated higher risk ratios compared to other biomarkers (P < 0.05), indicating their significance in prognosis and outcomes. Elevated levels of NLRP3 and TyG index were associated with poorer recovery of cardiac function, increased rehospitalization rates, and higher mortality (P < 0.05). CONCLUSION: Elevated NLRP3 levels and an increased TyG index are strongly associated with impaired cardiac function and heightened risk of cardiovascular events. These findings suggest that these biomarkers may serve as crucial prognostic indicators following myocardial ischemia-reperfusion injury.

Curcumin pretreatment attenuates myocardial ischemia/reperfusion injury by inhibiting ferroptosis, autophagy and apoptosis via HES1.

The early restoration of hemodynamics/reperfusion in acute myocardial infarction (AMI) is an effective therapeutic strategy to reduce sudden death and improve patient prognosis. However, reperfusion induces additional cardiomyocyte damage and cardiac tissue dysfunction. In this context, turmeric­derived curcumin (Cur) has been shown to exhibit a protective effect against myocardial ischemia/reperfusion injury (I/RI). The molecular mechanism of its activity, however, remains unclear. The current study investigated the protective effect of Cur and its molecular mechanism via in vitro experiments. The Cell Counting Kit­8 and lactate dehydrogenase (LDH) assay kit were used to assess the cell viability and cytotoxicity. The contents of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase, glutathione (GSH)/glutathione disulfide (GSSG), total iron, ferrous iron, caspase­3 and reactive oxygen species (ROS) were measured using an appropriate kit. Western blotting was used to detect the expression of relevant proteins. The levels of apoptosis, mitochondrial permeability transition pore (MPTP) opening, and mitochondrial membrane potential (MMP) were detected by flow cytometry. The study findings indicated that anoxia/reoxygenation (A/R) injury significantly decreased cell viability, increased in LDH and caspase­3 activities, induced ferroptosis, increased apoptosis and overactivated autophagy. However, pretreatment with Cur or ferrostatin­1 (Fer­1, a ferroptosis inhibitor) significantly increased A/R­reduced cell viability, SOD, glutathione peroxidase activity, GSH/GSSH ratio and HES1 and glutathione peroxidase 4 protein expression; attenuated A/R­induced LDH, MDA, total iron, ferrous iron, prostaglandin­endoperoxide synthase 2 protein expression and prevented ROS overproduction and MMP loss. In addition, Cur inhibited caspase­3 activity, upregulated the Bcl­2/Bax ratio, reduced apoptotic cell number and inhibited MPTP over­opening. Furthermore, Cur increased P62, LC3II/I, NDUFB8 and UQCRC2 expression and upregulated the p­AMPK/AMPK ratio. However, erastin (a ferroptosis activator), pAD/HES1­short hairpin RNA, rapamycin (an autophagy activator) and Compound C (an AMPK inhibitor) blocked the protective effect of Cur. In conclusion, Cur pretreatment inhibited ferroptosis, autophagy overactivation and oxidative stress; improved mitochondrial dysfunction; maintained energy homeostasis; attenuated apoptosis; and ultimately protected the myocardium from A/R injury via increased HES1 expression.

Enforced HCELL Expression Enhances Bone Marrow Stromal Cells Homing and Amelioration of Cerebral Ischemia-Reperfusion Injury via induction of PGE2.

Ischemic stroke (IS) is a significant and potentially life-threatening disease with limited treatment options, often resulting in severe disability. Bone marrow stromal cells (BMSCs) transplantation has exhibited promising neuroprotection following cerebral ischemia-reperfusion injury (CIRI). However, the effectiveness is hindered by their low homing rate when administered through the vein. In this study, we aimed to enhance the homing ability of BMSCs through lentivirus transfection to express fucosyltransferase 7. This glycosylation engineered CD44 on BMSCs to express hematopoietic cell E-selectin/L-selectin ligand (HCELL), which is the most potent E-selectin ligand. Following enforced HCELL expression, the transplantation of BMSCs was then evaluated in a middle cerebral artery occlusion (MCAO) model. Results showed that HCELL+BMSCs significantly ameliorated neurological deficits and reduced the volume of cerebral infarction. Furthermore, the transplantation led to a decrease in apoptosis by up-regulating BCL-2 and down-regulating BAX, also reduced the mRNA levels of inflammatory factors, such as interleukin-1ß (IL-1ß), IL-2, IL-6 and tumor necrosis factor-alpha (TNF-α) in the ischemic brain tissue. Notably, enforced HCELL expression facilitated the migration of BMSCs towards cerebral ischemic lesions and their subsequent transendothelial migration through the up-regulation of PTGS-2, increased production of PGE2 and activation of VLA-4. In summary, our study demonstrates that transplantation of HCELL+BMSCs effectively alleviates CIRI, and that enforced HCELL expression enhances the homing of BMSCs to cerebral ischemic lesions and their transendothelial migration via PTGS-2/PGE2/VLA-4. These findings indicate that enforced expression of HCELL on BMSCs could serve as a promising therapeutic strategy for the treatment of ischemic stroke.

LncRNA ZFAS1/miR-186-5p axis is involved in oxidative stress inhibition of myocardial ischemia-reperfusion injury by targeting BTG2.

OBJECTIVE: To probe the involvement of long noncoding RNA zinc finger antisense 1 (ZFAS1)/microRNA (miR)-186-5p axis in inhibiting oxidative stress in myocardial ischemia-reperfusion injury (MIRI) by targeting B-cell translocation gene 2 (BTG2). METHODS: The MIRI mice model was established by ligating the left anterior descending branch of the left coronary artery in C57BL/6 mice. The in vitro MIRI model was constructed by hypoxia and reoxygenation of HL-1 cardiomyocytes. Cardiomyocyte apoptosis and the extent of myocardial injury in mice were detected. The apoptosis rates, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in HL-1 cells were assessed. The relationship among ZFAS1, miR-186-5p, and BTG2 was verified. RESULTS: High ZFAS1 and BTG2 levels and low miR-186-5p levels were demonstrated in I/R-injured myocardial tissues and in H/R-treated cardiomyocytes. Interference with ZFAS1 or elevation of miR-186-5p inhibited apoptosis and oxidative stress in H/R model cardiomyocytes and I/R-injured myocardial tissues. Overexpressing BTG2 impaired the ameliorative effects of miR-186-5p on MIRI. ZFAS1 negatively regulated miR-186-5p expression by acting as a molecular sponge. miR-186-5p targeted to regulate BTG2 negatively. CONCLUSION: Interfering with ZFAS1 can upregulate miR-186-5p and thus inhibit BTG2 expression, thereby ameliorating MIRI.

Exercise preconditioning mitigates brain injury after cerebral ischemia-reperfusion injury in rats by restraining TIMP1.

BACKGROUND: Cerebral ischemic disease is a common cerebrovascular disease, especially ischemic stroke. Exercise has protective functions on brain tissues following cerebral ischemia-reperfusion injury (CIRI), but its preventive effects and mechanisms in CIRI remain unclear. We aimed to investigate the effects and mechanisms of exercise preconditioning on CIRI. METHODS: The middle cerebral artery occlusion (MCAO) operation was prepared to establish CIRI rats. All rats were randomized into the MCAO, exercise (exercise preconditioning plus MCAO operation), vector (exercise preconditioning, MCAO operation plus intraventricular injection of empty vector), and tissue inhibitor of metalloprotease 1 overexpression (OE-TIMP1, exercise preconditioning, MCAO operation plus intraventricular injection of OE-TIMP1) groups. RESULTS: The results indicated that exercise preconditioning suppressed approximately 66.67% of neurological deficit scores and 73.79% of TIMP1 mRNA expression in MCAO rats, which were partially offset by OE-TIMP1. The protective effects of exercise against neuron death status and cerebral infarction size in MCAO rats were reversed by OE-TIMP1. It also confirmed that exercise weakened apoptosis and oxidative stress damage, with notable increases of B-cell lymphoma-2, superoxide dismutase, and glutathione peroxidase production, and evident decreases of BCL2-associated X, caspase 3, and malondialdehyde in MCAO rats, while these effects were partially reversed by OE-TIMP1. Additionally, the inhibitory effects of exercise on the protein levels of TIMP1, hypoxia-inducible factor-alpha, vascular endothelial growth factor receptor 2, vascular endothelial growth factor, and neurogenic locus notch homolog protein 1 in MCAO rats were partially reversed by OE-TIMP1. CONCLUSION: Altogether, exercise preconditioning had protective effects on CIRI by restraining TIMP1, which provided new therapeutic strategies for preventing CIRI.

Pharmacological upregulation of macrophage-derived itaconic acid by pubescenoside C attenuated myocardial ischemia-reperfusion injury.

INTRODUCTION: Myocardial ischemia-reperfusion injury (MIRI) remains a prevalent clinical challenge globally, lacking an ideal therapeutic strategy. Macrophages play a pivotal role in MIRI pathophysiology, exhibiting dynamic inflammatory and resolutive functions. Macrophage polarization and metabolism are intricately linked to MIRI, presenting potential therapeutic targets. Pubescenoside C (PBC) from Ilex pubescens showed significantly anti-inflammatory effects, however, the effect of PBC on MIRI is unknown. OBJECTIVES: This study aimed to assess the cardioprotective effects of PBC against MIRI and elucidate the underlying mechanisms. METHODS: Sprague-Dawley rats, H9c2 and RAW264.7 macrophages were used to establish the in vitro and in vivo models of MIRI. TTC/Evans blue staining, immunohistochemical staining, metabonomics analysis, chemical probe, surface plasmon resonance (SPR), co-immunoprecipitation (CO-IP) assays were used for pharmacodynamic and mechanism study. RESULTS: PBC administration effectively reduced myocardial infarct size, decreased ST-segment elevation, and lowered CK-MB levels, concurrently promoting macrophage M2 polarization in MIRI. Furthermore, PBC-treated macrophages and their conditioned culture medium attenuated the apoptosis of H9c2 cells induced by oxygen-glucose deprivation/reoxygenation (OGD/R). Metabonomics analysis revealed that PBC increased the production of itaconic acid (ITA) and malic acid (MA) in macrophages, which conferred protection against OGD/R injury in H9c2 cells. Mechanistic investigations indicated that ITA exerted its effects by covalently modifying pyruvate kinase M2 (PKM2) at Cys474, Cys424, and Lys151, thereby facilitating PKM2's mitochondrial translocation and enhancing the PKM2/Bcl2 interaction, subsequently leading to decreased degradation of Bcl2. SPR assays further revealed that PBC bound to HSP90, facilitating the interaction between HSP90 and GSK3ß and resulting in the inactivation of GSK3ß activity and upregulation of key metabolic enzymes for ITA and MA production (Acod1 and Mdh2). CONCLUSION: PBC alleviates MIRI-induced cardiomyocyte apoptosis by modulating the HSP90/ITA/PKM2 axis. Furthermore, pharmacological upregulation of ITA emerges as a promising therapeutic approach for MIRI, hinting at PBC's potential as a candidate drug for MIRI therapy.

Mild therapeutic hypothermic protection activates the PI3K/AKT signaling pathway to inhibit TRPM7 and suppress ferroptosis induced by myocardial ischemia­reperfusion injury.

The present study aimed to investigate the role of PI3K­mediated ferroptosis signaling induced by mild therapeutic hypothermia (MTH), which was defined as a temperature of 34˚C, in protecting against myocardial ischemia-reperfusion (I/R) injury (MIRI). To meet this aim, H9C2 cells underwent hypoxia­reperfusion (H/R) and/or MTH. The MTT assay was used to assess cell viability, cytotoxicity was measured using a lactate dehydrogenase cytotoxicity assay, and Annexin V­FITC/PI flow cytometric analysis was used to analyze early and late cell apoptosis. In addition, 84 healthy adult male Sprague­Dawley rats were randomly divided into seven groups (n=12), and underwent I/R and various treatments. Hemodynamics were monitored, and the levels of myocardial injury marker enzymes and oxidative stress markers in myocardial tissue were measured using ELISA. The expression levels of PI3K, AKT, transient receptor potential cation channel subfamily M member 7 (TRPM7), glutathione peroxidase 4 (GPX4) and acyl­CoA synthetase long chain family member 4 (ACSL4) in animals and cells were measured using western blot analysis. These experiments revealed that MTH could effectively reduce myocardial infarct size, improve hemodynamic performance following MIRI and suppress myocardial apoptosis, thereby contributing to the recovery from H/R injury. Mechanistically, MTH was revealed to be able to activate the PI3K/AKT signaling pathway in cells, upregulating GPX4, and downregulating the expression levels of TRPM7 and ACSL4. Treatment with 2­aminoethoxydiphenyl borate (an inhibitor of TRPM7) could further strengthen the myocardial protective effects of MTH, whereas treatment with erastin (promoter of ferroptosis) and wortmannin (inhibitor of PI3K) led to the effective elimination of the myocardial protective effects of MTH. Compared with in the I/R group, the PI3K/AKT activation level and the expression levels of GPX4 were both significantly increased, whereas the expression levels of TRPM7 and ACSL4 were significantly decreased in the I/R + MTH group. Taken together, the results of the present study indicated that MTH may activate the PI3K/AKT signaling pathway to inhibit TRPM7 and suppress ferroptosis induced by MIRI.

Exendin-4 promotes ischemia-reperfusion flap survival by upregulating Gpx4 to inhibit ferroptosis.

BACKGROUND: Effective drugs for preventing or treating skin flap necrosis remain elusive. In this study, we investigated the potential protective effect of exendin-4 against skin flap ischemia-reperfusion injury (IRI) through the inhibition of ferroptosis. METHOD: A rat abdomen was constructed with an island skin flap, and the superficial vascular pedicle of the abdominal wall was closed using a vascular clamp, which was removed after 8 h. Before surgery, RSL3 and ferrostatin-1 solutions were intraperitoneally injected. After the surgery, subcutaneous injections of exendin-4 were administered daily. The number of inflammatory cells, mean vascular density, collagen fiber content, and apoptosis and ferroptosis indicators were quantified 24 h after reperfusion. Survival, contraction rate, and blood perfusion of the skin flap were evaluated on days 1, 3, 5, and 7 after reperfusion. RESULTS: The flap survival rate was significantly higher in the exendin-4 group than that in the injury group, whereas the contraction rate was lower. Compared with the injury group, the exendin-4 group showed less inflammatory cell infiltration, higher vascular density, and less collagen fiber loss. At the molecular level, the exendin-4 group demonstrated opposite or elevated expression of apoptosis and ferroptosis indicators than those in the injury group, with significantly increased glutathione peroxidase 4 (Gpx4). Ferroptosis inhibitors and agonists enhanced and reversed the protective effects of exendin-4, respectively. CONCLUSION: Exendin-4 alleviates skin flap IRI by upregulating Gpx4 expression to inhibit ferroptosis. Therefore, exendin-4 may serve as a novel clinical treatment for skin flap IRI.

Effect of sulforaphane on testicular ischemia-reperfusion injury induced by testicular torsion-detorsion in rats.

Testicular ischemia-reperfusion induces enhanced concentration of reactive oxygen species. The increased reactive oxygen species harm cellular lipids, nucleic acids, proteins, and carbohydrates, and ultimately cause testicular injury. Sulforaphane, a kind of natural dietary isothiocyanate, exists predominantly in some cruciferous vegetables, like broccoli and cabbage. It can protect tissues from oxidative stress-induced damage. Herein, we analyzed the effectiveness of sulforaphane in treating ischemia-reperfusion injury occurring after testicular torsion-detorsion. Male rats (n = 60) were grouped as follows: sham-operated group, unilateral testicular ischemia-reperfusion group, and unilateral testicular ischemia-reperfusion group receiving sulforaphane treatment at 5 mg/kg. No testicular torsion-detorsion was performed in the sham group. Unilateral testicular ischemia-reperfusion model was created by detorsion after 2 h of left testicular torsion. In the sulforaphane-treated group, intraperitoneal sulforaphane (5 mg/kg) was administered at left testicular detorsion. Biochemical assay, Western blot, and hematoxylin and eosin staining were used to evaluate testicular malondialdehyde content (an important marker of reactive oxygen species), protein levels of superoxide dismutase and catalase (intracellular antioxidant defense mechanism), and testicular reproductive function, respectively. In testicular tissues, malondialdehyde content was significantly promoted, while protein levels of superoxide dismutase and catalase, and testicular reproductive function were significantly reduced in ipsilateral testes by testicular ischemia-reperfusion. Nevertheless, sulforaphane administration partially reversed the effect of testicular ischemia-reperfusion on these indexes. It can be concluded that sulforaphane elevates protein levels of superoxide dismutase and catalase, and suppresses reactive oxygen species content, thereby preventing ischemia-reperfusion injury in testis.

Naringenin alleviates intestinal ischemia/reperfusion injury by inhibiting ferroptosis via targeting YAP/STAT3 signaling axis.

BACKGROUND: Intestinal ischemia/reperfusion injury (IRI) is a significant clinical emergency, and investigating novel therapeutic approaches and understanding their underlying mechanisms is essential for improving patient outcomes. Naringenin (Nar), a flavanone present in tomatoes and citrus fruits, is frequently consumed in the human diet and recognized for having immunomodulatory, anti-inflammatory, and antioxidant properties. Despite Nar being able to alleviate intestinal IRI, the exact molecular mechanisms remain elusive. PURPOSE: To investigate Nar's protective properties on intestinal IRI and elucidate the mechanisms, a comprehensive approach that combines network pharmacology analysis with experimental verification in vitro and in vivo was adopted. METHODS: The oxygen-glucose deprivation/reoxygenation (OGD/R) model in IEC-6 cells and a murine model of intestinal IRI were used. Nar's effects on intestinal IRI were assessed through histological analysis using H&E staining and tight junction (TJ) protein expression. Ferroptosis-related parameters, including iron levels, superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial morphology, were analyzed. Network pharmacology was utilized to predict the pathways through which Nar exerts its anti-ferroptosis effects. Further mechanistic insights were obtained through si-RNA transfection, YAP inhibitor (verteporfin, VP) treatment, ferroptosis inhibitor (Ferrostatin-1) and ferroptosis inducer (Erastin) application, co-immunoprecipitation (Co-IP) and Western blotting. RESULTS: Our results revealed that pretreatment with Nar significantly mitigated intestinal tissue damage and improved gut barrier function, as evidenced by increased TJ proteins (ZO-1 and Occludin). Nar reduced iron, MDA, and ROS, while it increased GSH and SOD levels. Additionally, Nar alleviated mitochondrial damage in mice. Nar treatment increased GPX4 and SLC7A11, while decreasing ACSL4 levels both in vivo and in vitro. Network pharmacology analysis suggested that Nar may target the Hippo signaling pathway. Notably, YAP, a key transcriptional co-activator within the Hippo pathway, was downregulated in intestinal IRI mice and OGD/R-induced IEC-6 cells. Nar pretreatment activated YAP, thereby augmenting anti-ferroptosis effects. The inhibition of YAP activation by VP or YAP knockdown increased p-STAT3 expression, thereby diminishing Nar's efficacy. Co-IP and immunofluorescence studies confirmed the interaction between YAP and STAT3. CONCLUSION: This study shows that Nar can inhibit ferroptosis in intestinal IRI via activating YAP, which in turn suppresses STAT3 phosphorylation, thereby unveiling a novel mechanism and supporting Nar's potential to be a promising therapeutic agent for intestinal IRI.

Nrf2 inhibits M1 macrophage polarization to ameliorate renal ischemia-reperfusion injury through antagonizing NF-κB signaling.

Renal ischemia-reperfusion injury (IRI) is a condition that arises from a sudden interruption of the blood flow to the kidney for a period of time followed by restoration of the blood supply. This process contributes to acute kidney injury (AKI), increases morbidity and mortality, and is a major risk factor for chronic kidney disease (CKD). Nuclear factor erythroid-derived 2-like 2 (Nrf2) has been shown to exhibit strong anti-oxidative and anti-inflammatory effects, which are reciprocally regulated by the pro-inflammatory actions of nuclear factor-kappa B (NF-κB) signaling. In this study, we established a model of AKI caused by renal IRI in mice lacking the Nrf2 gene (KO-Nrf2) and mice pre-injected with ML385 (Nrf2 inhibitor). In addition, LPS- or IL-4-induced M1- or M2-type polarized macrophages (RAW264.7), respectively, were also treated with Nrf2 activation and inhibition. The results demonstrated a more pronounced activation of the NF-κB signaling pathway in the Nrf2 inhibition model, accompanied by a more severe inflammatory effect. In cultured macrophages and renal IRI mice, Nrf2 inhibition activated M1 macrophage polarization, thereby increasing the release of proinflammatory cell factors (iNOS and TNF-α) and aggravating renal IRI. Notably, the inhibitory effect of Nrf2 on M1 macrophage polarization was related to the downregulation of the NF-κB signaling pathway activity, resulting in partial relief of renal IRI. Consequently, our findings indicated that Nrf2 inhibits M1 macrophage polarization to ameliorate renal IRI through antagonizing NF-κB signaling. Targeted activation of Nrf2 may be one of the important strategies for renal IRI treatment.

Fibroblast growth factor 21 attenuates pulmonary ischemia/reperfusion injury via inhibiting endoplasmic reticulum stress-induced ferroptosis though FGFR1/PPARδ signaling pathway.

BACKGROUND: Acute lung injury is a critical life-threatening complication of pulmonary and cardiac surgery with a high rate of morbidity and mortality. Fibroblast growth factor 21 (FGF21) has been reported to play an important role in protecting vital organs from damage. This study aims to investigate the potential protective role and mechanism of FGF21 in pulmonary ischemia/reperfusion (I/R)-induced acute lung injury. METHODS: A pulmonary epithelial cell line was treated with hypoxia/regeneration (H/R) in vitro and a mouse model of acute lung injury was induced with pulmonary I/R in vivo. Lung injury after pulmonary I/R was compared between FGF21-konckout (KO) mice and wild-type (WT) mice. Recombinant FGF21 was administrated in vivo and in vitro to determine its therapeutic effect. RESULTS: Circulating levels of FGF21 in mice with pulmonary I/R injury were significantly higher than in those without pulmonary I/R injury. Lung injury was aggravated in FGF21-KO mice compared with WT mice and the administration of FGF21 alleviated lung injury in mouse treated with I/R and pulmonary epithelial cell injury treated with H/R. FGF21 treatment decreased endoplasmic reticulum (ER) stress, Fe2+ and lipid reactive oxygen species (ROS) contents and GPX4 expression and increased PTGS2 levels. Mechanistically, FGF21 upregulated the expression of FGFR1 and PPARδ, ameliorated ER stress and ER stress induced-ferroptosis. Furthermore, FGF21 increased the expression level of PPARδ in pulmonary epithelial cell exposed to H/R, which was inhibited by FGFR1 inhibitor (PD173074). The protective effects of FGF21 were abolished by co-treatment with PPARδ inhibitor (GSK0660), indicating FGF21 attenuated ER stress-induced ferroptosis by dependent on FGFR1/PPARδ signaling pathway. CONCLUSION: Our study reveals that FGF21 protects against pulmonary I/R injury via inhibiting ER stress-induced ferroptosis though FGFR1/PPARδ signaling pathway. Boosting endogenous FGF21 or the administration of recombinant FGF21 could be promising therapeutic strategies for pulmonary IRI.

The combination of post-mortem sevoflurane ventilation and in situ topical cooling provides improved 6h lung preservation in an uncontrolled DCD porcine model.

BACKGROUND: Recent clinical series on donation after uncontrolled cardiovascular death (uDCD) reported successful transplantation of lungs preserved by pulmonary inflation up to 3h post-mortem. This study aims to investigate the additive effects of in situ lowering of intrathoracic temperature and sevoflurane preconditioning on lung grafts in a porcine uDCD model. METHODS: After uDCD induction, donor pigs were allocated to one of the following groups: Control - static lung inflation only (SLI); TC - SLI + continuous intrapleural topical cooling (TC); or TC+Sevo - SLI + TC + sevoflurane. Lungs were retrieved 6h post-asystole and evaluated via ex vivo lung perfusion (EVLP) for 6h. A left single lung transplant was performed using lungs from the best performing group, followed by 4h of graft evaluation. RESULTS: Animals that received topical cooling achieved intrathoracic temperature < 15°C within 1 hour after chest filling of coolant. Only lungs from donors that received TC and TC+Sevo completed the planned post-preservation 6h EVLP assessment. Despite similar early performance of the two groups on EVLP, the TC+Sevo group was superior - associated with overall lower airway pressures, higher pulmonary compliances, less edema development, and less release of inflammatory cytokines. Transplantation was performed using lungs from the TC+Sevo group, and excellent graft function was observed post-reperfusion. CONCLUSION: Preservation of uDCD lungs with a combination of static lung inflation, topical cooling and sevoflurane treatment maintains good pulmonary function up to 6h post-mortem with excellent early post-lung transplant function. These interventions may significantly expand the clinical utilization of uDCD donor lungs.

Quercetin as a therapeutic agent activate the Nrf2/Keap1 pathway to alleviate lung ischemia-reperfusion injury.

Lung ischemia-reperfusion injury (LIRI) causes oxidative stress, inflammation, and immune system activation. The Nrf2/Keap1/HO-1 pathway is important in cellular defense against these effects. Quercetin, a flavonoid with antioxidant, anti-inflammatory, and anti-cancer properties, has been investigated. Our aim in this study was to investigate the effect of quercetin on preventing lung ischemia-reperfusion injury and the role of the Nrf2/Keap1/HO-1 pathway. Sixty-four male Wistar rats were divided into four distinct groups(n = 16). Sham, lung ischemia-reperfusion (LIR), Saline + LIR, Quercetin + LIR (30 mg/kg i.p for a week before LIR). LIR groups were subjected to 60 min of ischemia (left pulmonary artery, vein, and bronchus) and 120 min of reperfusion. Our assessment encompassed a comprehensive analysis of various factors, including the evaluation of expression Nrf2, Keap1, and Heme Oxygenase-1 (HO-1) levels and NF-κB protein. Furthermore, we examined markers related to inflammation (interleukin-1ß and tumor necrosis factor alpha), oxidative stress (malondialdehyde, total oxidant status, superoxide dismutase, glutathione peroxidase, total antioxidant capacity), lung edema (Wet/dry lung weight ratio and total protein concentration), apoptosis (Bax and Bcl2 protein), and histopathological alterations (intra-alveolar edema, alveolar hemorrhage, and neutrophil infiltration). Our results show that ischemia-reperfusion results in heightened inflammation, oxidative stress, apoptosis, lung edema, and histopathological damage. Quercetin showed preventive effects by reducing these markers, acting through modulation of the Nrf2/Keap1 pathway and inhibiting the NF-κB pathway. This anti-inflammatory effect, complementary to the antioxidant effects of quercetin, provides a multifaceted approach to cell protection that is important for developing therapeutic strategies against ischemia-reperfusion injury and could be helpful in preventive strategies against ischemia-reperfusion.

Cardiovascular events in crush syndrome: on-site therapeutic strategies and pharmacological investigations.

Crush syndrome often occurs after severe crush injury caused by disasters or accidents, and is associated with high mortality and poor prognosis. Cardiovascular complications, such as cardiac arrest, hypovolemic shock, and hyperkalemia-related cardiac dysfunction, are the primary causes of on-site death in crush syndrome. Prehospital evaluation, together with timely and correct treatment, is of great benefit to crush syndrome patients, which is difficult in most cases due to limited conditions. Based on current data and studies, early fluid resuscitation remains the most important on-site treatment for crush syndrome. Novel solutions and drugs used in fluid resuscitation have been investigated for their effectiveness and benefits. Several drugs have proven effective for the prevention or treatment of cardiovascular complications in crush syndrome, such as hypovolemic shock, hyperkalemia-induced cardiac complications, myocardial ischemia/reperfusion injury, ventricular dysfunction, and coagulation disorder experimentally. Moreover, these drugs are beneficial for other complications of crush syndrome, such as renal dysfunction. In this review, we will summarize the existing on-site treatments for crush syndrome and discuss the potential pharmacological interventions for cardiovascular complications to provide clues for clinical therapy of crush syndrome.

Evaluation of the effect of carvedilol orodispersible tablets on ischemia-reperfusion injury and flap viability in rats: An in vivo study.

Flap surgery is an integral part of plastic surgery, and ischemia-reperfusion (I/R) injury significantly affects the viability of the flap. Carvedilol (CRV), a nonselective beta-blocker with alpha-1 blocking and antioxidant properties, and known for its potential in reducing I/R damage, was chosen as the active substance for our study. The aim of this study was to investigate the vasodilator and antioxidant effects of CRV on rat inferior epigastric artery skin flap using orally disintegrating tablets (ODTs). The optimized ODT formulation was subjected to in vivo experiments using Sprague-Dawley female rats (n = 24) divided into three groups: Group I (control, I/R), Group II (treatment, I/R + CRV), and Group III (treatment, I/R), I/R + CRV ODT). Reperfusion was then observed following the release of the microclamp from the pedicle, and the flap was then re-adapted to its original position. Control rats were given oral isotonic solution via gavage and were subjected to 8 h of ischemia and 12 h of reperfusion. Group II was given 2 mg/kg CRV oral tablets for 7 days before and after surgery. Group III was given 2 mg/kg/day CRV ODT for the same period. Biopsies were taken from the flap and histopathological and biochemical analyses including superoxide dismutase, glutathionenitric oxide, malondialdehyde, paraoxonase 1, total oxidant, and total antioxidant capacities were performed. This study demonstrates that CRV ODTs significantly increased flap viability by approximately 25% compared to the control group, highlighting their promising therapeutic potential.

The role and possible mechanism of the ferroptosis-related SLC7A11/GSH/GPX4 pathway in myocardial ischemia-reperfusion injury.

BACKGROUND: Myocardial ischemia-reperfusion injury (MI/RI) is an unavoidable risk event for acute myocardial infarction, with ferroptosis showing close involvement. We investigated the mechanism of MI/RI inducing myocardial injury by inhibiting the ferroptosis-related SLC7A11/glutathione (GSH)/glutathione peroxidase 4 (GPX4) pathway and activating mitophagy. METHODS: A rat MI/RI model was established, with myocardial infarction area and injury assessed by TTC and H&E staining. Rat cardiomyocytes H9C2 were cultured in vitro, followed by hypoxia/reoxygenation (H/R) modeling and the ferroptosis inhibitor lipoxstatin-1 (Lip-1) treatment, or 3-Methyladenine or rapamycin treatment and overexpression plasmid (oe-SLC7A11) transfection during modeling. Cell viability and death were evaluated by CCK-8 and LDH assays. Mitochondrial morphology was observed by transmission electron microscopy. Mitochondrial membrane potential was detected by fluorescence dye JC-1. Levels of inflammatory factors, reactive oxygen species (ROS), Fe2+, malondialdehyde, lipid peroxidation, GPX4 enzyme activity, glutathione reductase, GSH and glutathione disulfide, and SLC7A11, GPX4, LC3II/I and p62 proteins were determined by ELISA kit, related indicator detection kits and Western blot. RESULTS: The ferroptosis-related SLC7A11/GSH/GPX4 pathway was repressed in MI/RI rat myocardial tissues, inducing myocardial injury. H/R affected GSH synthesis and inhibited GPX4 enzyme activity by down-regulating SLC7A11, thus promoting ferroptosis in cardiomyocytes, which was averted by Lip-1. SLC7A11 overexpression improved H/R-induced cardiomyocyte ferroptosis via the GSH/GPX4 pathway. H/R activated mitophagy in cardiomyocytes. Mitophagy inhibition reversed H/R-induced cellular ferroptosis. Mitophagy activation partially averted SLC7A11 overexpression-improved H/R-induced cardiomyocyte ferroptosis. H/R suppressed the ferroptosis-related SLC7A11/GSH/GPX4 pathway by inducing mitophagy, leading to cardiomyocyte injury. CONCLUSIONS: Increased ROS under H/R conditions triggered cardiomyocyte injury by inducing mitophagy to suppress the ferroptosis-related SLC7A11/GSH/GPX4 signaling pathway activation.

Ultra-Small Copper-Based Multienzyme-Like Nanoparticles Protect Against Hepatic Ischemia-Reperfusion Injury Through Scavenging Reactive Oxygen Species in Mice.

Hepatic ischemia-reperfusion injury (IRI) is a severe complication that occurs in the process of liver transplantation, hepatectomy, and other end-stage liver disease surgery, often resulting in the failure of surgery operation and even patient death. Currently, there is no effective way to prevent hepatic IRI clinically. Here, it is reported that the ultra-small copper-based multienzyme-like nanoparticles with catalase-like (CAT-like) and superoxide dismutase-like (SOD-like) catalytic activities significantly scavenge the surge-generated endogenous reactive oxygen species (ROS) and effectively protects hepatic IRI. Density functional theory calculations confirm that the nanoparticles efficiently scavenge ROS through their synergistic effects of the ultra-small copper SOD-like activity and manganese dioxides CAT-like activity. Furthermore, the results show that the biocompatible CMP NPs significantly protected hepatocytes from IRI in vitro and in vivo. Importantly, their therapeutic effect is much stronger than that of N-acetylcysteamine acid (NAC), an FDA-approved antioxidative drug. Finally, it is demonstrated that the protective effects of CMP NPs on hepatic IRI are related to suppressing inflammation and hepatocytic apoptosis and maintaining endothelial functions through scavenging ROS in liver tissues. The study can provide insight into the development of next-generation nanomedicines for scavenging ROS.