Mitochondrial Permeability Uncouples Elevated Autophagy and Lifespan Extension.

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.

Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity.

The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We determined a 3.2 Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational analysis of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a molecular basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.

Multi-Omics Profiling Identifies Microglial Annexin A2 as a Key Mediator of NF-κB Pro-inflammatory Signaling in Ischemic Reperfusion Injury.

Cerebral stroke is one of the leading causes of mortality and disability worldwide. Restoring the cerebral circulation following a period of occlusion and subsequent tissue oxygenation leads to reperfusion injury. Cerebral ischemic reperfusion (I/R) injury triggers immune and inflammatory responses, apoptosis, neuronal damage, and even death. However, the cellular function and molecular mechanisms underlying cerebral I/R-induced neuronal injury are incompletely understood. By integrating proteomic, phosphoproteomic, and transcriptomic profiling in mouse hippocampi after cerebral I/R, we revealed that the differentially expressed genes and proteins mainly fall into several immune inflammatory response-related pathways. We identified that Annexin 2 (Anxa2) was exclusively upregulated in microglial cells in response to cerebral I/R in vivo and oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. RNA-seq analysis revealed a critical role of Anxa2 in the expression of inflammation-related genes in microglia via the NF-κB signaling. Mechanistically, microglial Anxa2 is required for nuclear translocation of the p65 subunit of NF-κB and its transcriptional activity upon OGD/R in BV2 microglial cells. Anxa2 knockdown inhibited the OGD/R-induced microglia activation and markedly reduced the expression of pro-inflammatory factors, including TNF-α, IL-1ß, and IL-6. Interestingly, conditional medium derived from Anxa2-depleted BV2 cell cultures with OGD/R treatment alleviated neuronal death in vitro. Altogether, our findings revealed that microglia Anxa2 plays a critical role in I/R injury by regulating NF-κB inflammatory responses in a non-cell-autonomous manner, which might be a potential target for the neuroprotection against cerebral I/R injury.

Control of tissue oxygenation by S-nitrosohemoglobin in human subjects.

S-Nitrosohemoglobin (SNO-Hb) is unique among vasodilators in coupling blood flow to tissue oxygen requirements, thus fulfilling an essential function of the microcirculation. However, this essential physiology has not been tested clinically. Reactive hyperemia following limb ischemia/occlusion is a standard clinical test of microcirculatory function, which has been ascribed to endothelial nitric oxide (NO). However, endothelial NO does not control blood flow governing tissue oxygenation, presenting a major quandary. Here we show in mice and humans that reactive hyperemic responses (i.e., reoxygenation rates following brief ischemia/occlusion) are in fact dependent on SNO-Hb. First, mice deficient in SNO-Hb (i.e., carrying C93A mutant Hb refractory to S-nitrosylation) showed blunted muscle reoxygenation rates and persistent limb ischemia during reactive hyperemia testing. Second, in a diverse group of humans-including healthy subjects and patients with various microcirculatory disorders-strong correlations were found between limb reoxygenation rates following occlusion and both arterial SNO-Hb levels (n = 25; P = 0.042) and SNO-Hb/total HbNO ratios (n = 25; P = 0.009). Secondary analyses showed that patients with peripheral artery disease had significantly reduced SNO-Hb levels and blunted limb reoxygenation rates compared with healthy controls (n = 8 to 11/group; P < 0.05). Low SNO-Hb levels were also observed in sickle cell disease, where occlusive hyperemic testing was deemed contraindicated. Altogether, our findings provide both genetic and clinical support for the role of red blood cells in a standard test of microvascular function. Our results also suggest that SNO-Hb is a biomarker and mediator of blood flow governing tissue oxygenation. Thus, increases in SNO-Hb may improve tissue oxygenation in patients with microcirculatory disorders.

CK-666 protects against ferroptosis and renal ischemia-reperfusion injury through a microfilament-independent mechanism.

Ferroptosis is a type of regulated cell death driven by iron-dependent accumulation of lipid peroxidation, exhibiting unique morphological changes. While actin microfilaments are crucial for various cellular processes, including morphogenesis, motility, endocytosis, and cell death, their role in ferroptosis remains unclear. Here, our study reveals that actin microfilaments undergo remodeling and disassembly during ferroptosis. Interestingly, inhibitors that target actin microfilament remodeling do not affect cell sensitivity to ferroptosis, with the exception of CK-666 and its structural analogue CK-636. Mechanistically, CK-666 attenuates ferroptosis independently of its canonical function in inhibiting the Arp2/3 complex. Further investigation revealed that CK-666 modulates the ferroptotic transcriptome, prevents lipid degradation, and diminishes lipid peroxidation. In addition, CK-666 does not impact the labile iron pool within cells, nor does the inhibition of FSP1 impact its anti-ferroptosis activity. Notably, the results of DPPH assay and liposome leakage assay suggest that CK-666 mitigates ferroptosis by directly eliminating lipid peroxidation. Importantly, CK-666 significantly ameliorated renal ischemia-reperfusion injury and ferroptosis in renal tissue, underscoring its potential therapeutic impact.

Inhibition of Hmbox1 Promotes Cardiomyocyte Survival and Glucose Metabolism Through Gck Activation in Ischemia/Reperfusion Injury.

BACKGROUND: Exercise-induced physiological cardiac growth regulators may protect the heart from ischemia/reperfusion (I/R) injury. Homeobox-containing 1 (Hmbox1), a homeobox family member, has been identified as a putative transcriptional repressor and is downregulated in the exercised heart. However, its roles in exercise-induced physiological cardiac growth and its potential protective effects against cardiac I/R injury remain largely unexplored. METHODS: We studied the function of Hmbox1 in exercise-induced physiological cardiac growth in mice after 4 weeks of swimming exercise. Hmbox1 expression was then evaluated in human heart samples from deceased patients with myocardial infarction and in the animal cardiac I/R injury model. Its role in cardiac I/R injury was examined in mice with adeno-associated virus 9 (AAV9) vector-mediated Hmbox1 knockdown and in those with cardiac myocyte-specific Hmbox1 ablation. We performed RNA sequencing, promoter prediction, and binding assays and identified glucokinase (Gck) as a downstream effector of Hmbox1. The effects of Hmbox1 together with Gck were examined in cardiomyocytes to evaluate their cell size, proliferation, apoptosis, mitochondrial respiration, and glycolysis. The function of upstream regulator of Hmbox1, ETS1, was investigated through ETS1 overexpression in cardiac I/R mice in vivo. RESULTS: We demonstrated that Hmbox1 downregulation was required for exercise-induced physiological cardiac growth. Inhibition of Hmbox1 increased cardiomyocyte size in isolated neonatal rat cardiomyocytes and human embryonic stem cell-derived cardiomyocytes but did not affect cardiomyocyte proliferation. Under pathological conditions, Hmbox1 was upregulated in both human and animal postinfarct cardiac tissues. Furthermore, both cardiac myocyte-specific Hmbox1 knockout and AAV9-mediated Hmbox1 knockdown protected against cardiac I/R injury and heart failure. Therapeutic effects were observed when sh-Hmbox1 AAV9 was administered after I/R injury. Inhibition of Hmbox1 activated the Akt/mTOR/P70S6K pathway and transcriptionally upregulated Gck, leading to reduced apoptosis and improved mitochondrial respiration and glycolysis in cardiomyocytes. ETS1 functioned as an upstream negative regulator of Hmbox1 transcription, and its overexpression was protective against cardiac I/R injury. CONCLUSIONS: Our studies unravel a new role for the transcriptional repressor Hmbox1 in exercise-induced physiological cardiac growth. They also highlight the therapeutic potential of targeting Hmbox1 to improve myocardial survival and glucose metabolism after I/R injury.

Extracellular RIPK3 Acts as a Danger-Associated Molecular Pattern to Exaggerate Cardiac Ischemia/Reperfusion Injury.

BACKGROUND: Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease. Currently, there is no effective therapy for reducing cardiac I/R injury. Damage-associated molecular patterns are endogenous molecules released after cellular damage to exaggerate tissue inflammation and injury. RIPK3 (receptor-interacting protein kinase 3), a well-established intracellular mediator of cell necroptosis and inflammation, serves as a circulating biomarker of multiple diseases. However, whether extracellular RIPK3 also exerts biological functions in cardiac I/R injury remains totally unknown. METHODS: Patients with acute myocardial infarction receiving percutaneous coronary intervention (PCI) were recruited independently in the discovery cohort (103 patients) and validation cohort (334 patients), and major adverse cardiovascular events were recorded. Plasma samples were collected before and after PCI (6 and 24 h) for RIPK3 concentration measurement. Cultured neonatal rat ventricular myocytes, macrophages and endothelial cells, and in vivo mouse models with myocardial injury induced by I/R (or hypoxia/reoxygenation) were used to investigate the role and mechanisms of extracellular RIPK3. Another cohort including patients with acute myocardial infarction receiving PCI and healthy volunteers was recruited to further explore the mechanisms of extracellular RIPK3. RESULTS: In the discovery cohort, elevated plasma RIPK3 levels after PCI are associated with poorer short- and long-term outcomes in patients with acute myocardial infarction, as confirmed in the validation cohort. In both cultured cells and in vivo mouse models, recombinant RIPK3 protein exaggerated myocardial I/R (or hypoxia/reoxygenation) injury, which was alleviated by the RIPK3 antibody. Mechanistically, RIPK3 acted as a damage-associated molecular pattern and bound with RAGE (receptor of advanced glycation end-products), subsequently activating CaMKII (Ca2+/calmodulin-dependent kinase II) to elicit the detrimental effects. The positive correlation between plasma RIPK3 concentrations and CaMKII phosphorylation in human peripheral blood mononuclear cells was confirmed. CONCLUSIONS: We identified the positive relationship between plasma RIPK3 concentrations and the risk of major adverse cardiovascular events in patients with acute myocardial infarction receiving PCI. As a damage-associated molecular pattern, extracellular RIPK3 plays a causal role in multiple pathological conditions during cardiac I/R injury through RAGE/CaMKII signaling. These findings expand our understanding of the physiological and pathological roles of RIPK3, and also provide a promising therapeutic target for myocardial I/R injury and the associated complications.

Liver Transplantation: A Test of Cellular Physiology, Preservation, and Injury.

Liver transplantation has evolved into a mature clinical field, but scarcity of usable organs poses a unique challenge. Expanding the donor pool requires novel approaches for protecting hepatic physiology and cellular homeostasis. Here we define hepatocellular injury during transplantation, with an emphasis on modifiable cell death pathways as future therapeutics.

Mitochondrial Calcium Regulation of Cardiac Metabolism in Health and Disease.

Oxidative phosphorylation is regulated by mitochondrial calcium (Ca2+) in health and disease. In physiological states, Ca2+ enters via the mitochondrial Ca2+ uniporter and rapidly enhances NADH and ATP production. However, maintaining Ca2+ homeostasis is critical: insufficient Ca2+ impairs stress adaptation, and Ca2+ overload can trigger cell death. In this review, we delve into recent insights further defining the relationship between mitochondrial Ca2+ dynamics and oxidative phosphorylation. Our focus is on how such regulation affects cardiac function in health and disease, including heart failure, ischemia-reperfusion, arrhythmias, catecholaminergic polymorphic ventricular tachycardia, mitochondrial cardiomyopathies, Barth syndrome, and Friedreich's ataxia. Several themes emerge from recent data. First, mitochondrial Ca2+ regulation is critical for fuel substrate selection, metabolite import, and matching of ATP supply to demand. Second, mitochondrial Ca2+ regulates both the production and response to reactive oxygen species (ROS), and the balance between its pro- and antioxidant effects is key to how it contributes to physiological and pathological states. Third, Ca2+ exerts localized effects on the electron transport chain (ETC), not through traditional allosteric mechanisms but rather indirectly. These effects hinge on specific transporters, such as the uniporter or the Na+/Ca2+ exchanger, and may not be noticeable acutely, contributing differently to phenotypes depending on whether Ca2+ transporters are acutely or chronically modified. Perturbations in these novel relationships during disease states may either serve as compensatory mechanisms or exacerbate impairments in oxidative phosphorylation. Consequently, targeting mitochondrial Ca2+ holds promise as a therapeutic strategy for a variety of cardiac diseases characterized by contractile failure or arrhythmias.

MSC-Derived Small Extracellular Vesicles Exert Cardioprotective Effect Through Reducing VLCFAs and Apoptosis in Human Cardiac Organoid IRI Model.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs' ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

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.

Mid1 aggravates hepatic ischemia-reperfusion injury by inducing immune cell infiltration.

Hepatic ischemia-reperfusion injury (HIRI) represents a major risk factor in liver transplantation and resection surgeries. Kupffer cells (KCs) produce proinflammatory cytokines and lead to hepatic neutrophil infiltration in the liver, which is one of the leading causes of HIRI. Mid1 is involved in immune infiltration, but the role of Mid1 remains poorly understood. Herin, our study aimed to investigate the effect of Mid1 on HIRI progression. Male C57BL/6 mice aged 6 weeks were used for the HIRI model established. The function of Mid1 on liver injury and hepatic inflammation was evaluated. In vitro, KCs were used to investigate the function and mechanism of Mid1 in modulating KC inflammation upon lipopolysaccharide (LPS) stimulation. We found that Mid1 expression was up-regulated upon HIRI. Mid1 inhibition alleviated liver damage, as evidenced by neutrophil infiltration, intrahepatic inflammation, and hepatocyte apoptosis. In vitro experiments further revealed that Mid1 knockdown reduced the secretion of proinflammatory cytokines and chemokines in KCs. Moreover, silenced-Mid1 suppressed proinflammatory responses by the inhibition of NF-κB, JNK, and p38 signaling pathways. Taken together, Mid1 contributes to HIRI via regulating the proinflammatory response of KCs and inducing neutrophil infiltration. Targeting Mid1 may be a promising strategy to protect against HIRI.

Histone deacetylase 6 deficiency protects the liver against ischemia/reperfusion injury by activating PI3K/AKT/mTOR signaling.

Liver transplantation (LT) is the only effective method to treat end-stage liver disease. Hepatic ischemia-reperfusion injury (IRI) continues to limit the prognosis of patients receiving LT. Histone deacetylase 6 (HDAC6) is a unique HDAC member involved in inflammation and apoptosis. However, its role and mechanism in hepatic IRI have not yet been reported. We examined HDAC6 levels in liver tissue from LT patients, mice challenged with liver IRI, and hepatocytes subjected to hypoxia/reoxygenation (H/R). In addition, HDAC6 global-knockout (HDAC6-KO) mice, adeno-associated virus-mediated liver-specific HDAC6 overexpressing (HDAC6-LTG) mice, and their corresponding controls were used to construct hepatic IRI models. Hepatic histology, inflammatory responses, and apoptosis were detected to assess liver injury. The molecular mechanisms of HDAC6 in hepatic IRI were explored in vivo and in vitro. Moreover, the HDAC6-selective inhibitor tubastatin A was used to detect the therapeutic effect of HDAC6 on liver IRI. Together, our results showed that HDAC6 expression was significantly upregulated in liver tissue from LT patients, mice subjected to hepatic I/R surgery, and hepatocytes challenged by hypoxia/reoxygenation (H/R) treatment. Compared with control mice, HDAC6 deficiency mitigated liver IRI by inhibiting inflammatory responses and apoptosis, whereas HDAC6-LTG mice displayed the opposite phenotype. Further molecular experiments show that HDAC6 bound to and deacetylated AKT and HDAC6 deficiency improved liver IRI by activating PI3K/AKT/mTOR signaling. In conclusion, HDAC6 is a key mediator of hepatic IRI that functions to promote inflammation and apoptosis via PI3K/AKT/mTOR signaling. Targeting hepatic HDAC6 inhibition may be a promising approach to attenuate liver IRI.

HIF-1α induced FGF18 alleviates renal ischemia/reperfusion injury via YAP.

Acute kidney injury (AKI) is a devastating clinical condition characterized by an abrupt loss of renal function. The pathophysiology of AKI involves diverse processes and elements, of which survival and regeneration have been established to be significant hallmarks. And early studies have confirmed the fundamental role of FGFs in the regulation of AKI pathology, although the association between FGF18 and AKI still remains elusive. Our study demonstrates a substantial up-regulation of FGF18 in the renal tubules of mice subjected to ischemia. Notably, targeted overexpression of FGF18 effectively mitigates the impairment of kidney function induced by AKI. Mechanistically, FGF18 facilitates cell proliferation and anti-apoptosis in RTECs by enhancing the expression of YAP and facilitating its translocation to the nucleus. Aside from that, we also discovered that the substantial expression of FGF18 under ischemic conditions is HIF-1α dependent. This study aims to uncover the inherent mechanism behind the beneficial effects of FGF18 in attenuating AKI. By doing so, it aims to offer novel insights into the development of therapeutic strategies for AKI.

Tolerogenic CD11c+dendritic cells regulate CD4+Tregs in replacing delayed ischemic preconditioning to alleviate ischemia-reperfusion acute kidney injury.

Ischemia-reperfusion injury (IRI) is one of the primary clinical causes of acute kidney injury (AKI). The key to IRI lies in immune-inflammatory damage, where dendritic cells (DCs) play a central role in eliciting immune responses within the context of inflammation induced by ischemia-reperfusion. Our previous study has confirmed that delayed ischemic preconditioning (DIPC) can reduce the kidney injury by mediating DCs to regulate T-cells. However, the clinical feasibility of DIPC is limited, as pre-clamping of the renal artery is not applicable for the prevention and treatment of ischemia-reperfusion acute kidney injury (I/R-AKI) in clinical patients. Therefore, the infusion of DCs as a substitute for DIPC presents a more viable strategy for preventing renal IRI. In this study, we further evaluated the impact and mechanism of infused tolerogenic CD11c+DCs on the kidneys following IRI by isolating bone marrow-derived dendritic cells and establishing an I/R-AKI model after pre-infusion of DCs. Renal function was significantly improved in the I/R-AKI mouse model after pre-infused with CD11c+DCs. The pro-inflammatory response and oxidative damage were reduced, and the levels of T helper 2 (Th2) cells and related anti-inflammatory cytokines were increased, which was associated with the reduction of autologous DCs maturation mediated by CD11c+DCs and the increase of regulatory T-cells (Tregs). Next, knocking out CD11c+DCs, we found that the reduced immune protection of tolerogenic CD11c+DCs reinfusion was related to the absence of own DCs. Together, pre-infusion of tolerogenic CD11c+DCs can replace the regulatory of DIPC on DCs and T-cells to alleviate I/R-AKI. DC vaccine is expected to be a novel avenue to prevent and treat I/R-AKI.

Ghrelin alleviates intestinal ischemia-reperfusion injury by activating the GHSR-1α/Sirt1/FOXO1 pathway.

Ischemia-reperfusion (IR) injury is primarily characterized by the restoration of blood flow perfusion and oxygen supply to ischemic tissue and organs, but it paradoxically leads to tissue injury aggravation. IR injury is a challenging pathophysiological process that is difficult to avoid clinically and frequently occurs during organ transplantation, surgery, shock resuscitation, and other processes. The major causes of IR injury include increased levels of free radicals, calcium overload, oxidative stress, and excessive inflammatory response. Ghrelin is a newly discovered brain-intestinal peptide with anti-inflammatory and antiapoptotic effects that improve blood supply. The role and mechanism of ghrelin in intestinal ischemia-reperfusion (IIR) injury remain unclear. We hypothesized that ghrelin could attenuate IIR-induced oxidative stress and apoptosis. To investigate this, we established IIR by using a non-invasive arterial clip to clamp the root of the superior mesenteric artery (SMA) in mice. Ghrelin was injected intraperitoneally at a dose of 50 µg/kg 20 min before IIR surgery, and [D-Lys3]-GHRP-6 was injected intraperitoneally at a dose of 12 nmol/kg 20 min before ghrelin injection. We mimicked the IIR process with hypoxia-reoxygenation (HR) in Caco-2 cells, which are similar to intestinal epithelial cells in structure and biochemistry. Our results showed that ghrelin inhibited IIR/HR-induced oxidative stress and apoptosis by activating GHSR-1α. Moreover, it was found that ghrelin activated the GHSR-1α/Sirt1/FOXO1 signaling pathway. We further inhibited Sirt1 and found that Sirt1 was critical for ghrelin-mediated mitigation of IIR/HR injury. Overall, our data suggest that pretreatment with ghrelin reduces oxidative stress and apoptosis to attenuate IIR/HR injury by binding with GHSR-1α to further activate Sirt1.

Scutellarin alleviates renal ischemia-reperfusion injury by inhibiting the MAPK pathway and pro-inflammatory macrophage polarization.

Renal ischemia-reperfusion injury (IRI) is an integral process in renal transplantation, which results in compromised graft survival. Macrophages play an important role in both the early inflammatory period and late fibrotic period in response to IRI. In this study, we investigated whether scutellarin (SCU) could protect against renal IRI by regulating macrophage polarization. Mice were given SCU (5-50 mg/kg) by gavage 1 h earlier, followed by a unilateral renal IRI. Renal function and pathological injury were assessed 24 h after reperfusion. The results showed that administration of 50 mg/kg SCU significantly improved renal function and renal pathology in IRI mice. In addition, SCU alleviated IRI-induced apoptosis. Meanwhile, it reduced macrophage infiltration and inhibited pro-inflammatory macrophage polarization. Moreover, in RAW 264.7 cells and primary bone marrow-derived macrophages (BMDMs) exposed to SCU, we found that 150 µM SCU inhibited these cells to polarize to an inflammatory phenotype induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). However, SCU has no influence on anti-inflammatory macrophage polarization in vivo and in vitro induced by in interleukin-4 (IL-4). Finally, we explored the effect of SCU on the activation of the mitogen-activated protein kinase (MAPK) pathway both in vivo and in vitro. We found that SCU suppressed the activation of the MAPK pathway, including the extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38. Our results demonstrated that SCU protects the kidney against IRI by inhibiting macrophage infiltration and polarization toward pro-inflammatory phenotype via the MAPK pathway, suggesting that SCU may be therapeutically important in treatment of IRI.

Preconditioning with ß-hydroxybutyrate attenuates lung ischemia-reperfusion injury by suppressing alveolar macrophage pyroptosis through the SIRT1-FOXO3 signaling pathway.

The complex pathogenesis of lung ischemia-reperfusion injury (LIRI) was examined in a murine model, focusing on the role of pyroptosis and its exacerbation of lung injury. We specifically examined the levels and cellular localization of pyroptosis within the lung, which revealed alveolar macrophages as the primary site. The inhibition of pyroptosis by VX-765 reduced the severity of lung injury, underscoring its significant role in LIRI. Furthermore, the therapeutic potential of ß-hydroxybutyrate (ß-OHB) in ameliorating LIRI was examined. Modulation of ß-OHB levels was evaluated by ketone ester supplementation and 3-hydroxybutyrate dehydrogenase 1 (BDH-1) gene knockout, along with the manipulation of the SIRT1-FOXO3 signaling pathway using EX-527 and pCMV-SIRT1 plasmid transfection. This revealed that ß-OHB exerts lung-protective and anti-pyroptotic effects, which were mediated through the upregulation of SIRT1 and the enhancement of FOXO3 deacetylation, leading to decreased pyroptosis markers and lung injury. In addition, ß-OHB treatment of MH-S cells in vitro showed a concentration-dependent improvement in pyroptosis, linking its therapeutic benefits to specific cell mechanisms. Overall, this study highlights the significance of alveolar macrophage pyroptosis in the exacerbation of LIRI and indicates the potential of ß-OHB in mitigating injury by modulating the SIRT1-FOXO3 signaling pathway.

JMJD3 activation contributes to renal protection and regeneration following acute kidney injury in mice.

We have recently demonstrated that Jumonji domain-containing protein D3 (JMJD3), a histone demethylase of histone H3 on lysine 27 (H3K27me3), is protective against renal fibrosis, but its role in acute kidney injury (AKI) remains unexplored. Here, we report that JMJD3 activity is required for renal protection and regeneration in murine models of AKI induced by ischemia/reperfusion (I/R) and folic acid (FA). Injury to the kidney upregulated JMJD3 expression and induced expression of H3K27me3, which was coincident with renal dysfunction, renal tubular cell injury/apoptosis, and proliferation. Blocking JMJD3 activity by GSKJ4 led to worsening renal dysfunction and pathological changes by aggravating tubular epithelial cell injury and apoptosis in both murine models of AKI. JMJD3 inhibition by GSKJ4 also reduced renal tubular cell proliferation and suppressed expression of cyclin E and phosphorylation of CDK2, but increased p21 expression in the injured kidney. Furthermore, inactivation of JMJD3 enhanced I/R- or FA-induced expression of TGF-ß1, vimentin, and Snail, phosphorylation of Smad3, STAT3, and NF-κB, and increased renal infiltration by F4/80 (+) macrophages. Finally, GSKJ4 treatment caused further downregulation of Klotho, BMP-7, Smad7, and E-cadherin, all of which are associated with renal protection and have anti-fibrotic effects. Therefore, these data provide strong evidence that JMJD3 activation contributes to renal tubular epithelial cell survival and regeneration after AKI.

Interdependent Nuclear Co-Trafficking of ASPP1 and p53 Aggravates Cardiac Ischemia/Reperfusion Injury.

OBJECTIVE: ASPP1 (apoptosis stimulating of p53 protein 1) is critical in regulating cell apoptosis as a cofactor of p53 to promote its transcriptional activity in the nucleus. However, whether cytoplasmic ASPP1 affects p53 nuclear trafficking and its role in cardiac diseases remains unknown. This study aims to explore the mechanism by which ASPP1 modulates p53 nuclear trafficking and the subsequent contribution to cardiac ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: The immunofluorescent staining showed that under normal condition ASPP1 and p53 colocalized in the cytoplasm of neonatal mouse ventricular cardiomyocytes, while they were both upregulated and translocated to the nuclei upon hypoxia/reoxygenation treatment. The nuclear translocation of ASPP1 and p53 was interdependent, as knockdown of either ASPP1 or p53 attenuated nuclear translocation of the other one. Inhibition of importin-ß1 resulted in the cytoplasmic sequestration of both p53 and ASPP1 in neonatal mouse ventricular cardiomyocytes with hypoxia/reoxygenation stimulation. Overexpression of ASPP1 potentiated, whereas knockdown of ASPP1 inhibited the expression of Bax (Bcl2-associated X), PUMA (p53 upregulated modulator of apoptosis), and Noxa, direct apoptosis-associated targets of p53. ASPP1 was also increased in the I/R myocardium. Cardiomyocyte-specific transgenic overexpression of ASPP1 aggravated I/R injury as indicated by increased infarct size and impaired cardiac function. Conversely, knockout of ASPP1 mitigated cardiac I/R injury. The same qualitative data were observed in neonatal mouse ventricular cardiomyocytes exposed to hypoxia/reoxygenation injury. Furthermore, inhibition of p53 significantly blunted the proapoptotic activity and detrimental effects of ASPP1 both in vitro and in vivo. CONCLUSIONS: Binding of ASPP1 to p53 triggers their nuclear cotranslocation via importin-ß1 that eventually exacerbates cardiac I/R injury. The findings imply that interfering the expression of ASPP1 or the interaction between ASPP1 and p53 to block their nuclear trafficking represents an important therapeutic strategy for cardiac I/R injury.