Oxygen-glucose Deprivation/reoxygenation Research Articles

Abstract 4134837: Checkpoint Kinase 1 Attenuates Myocardial Ischemia-Reperfusion Injury Through Maintaining SIRT1-Dependent Mitochondrial Homeostasis

Background: Mitochondrial dysfunction is linked to myocardial ischemia-reperfusion (I/R) injury. Checkpoint kinase 1 (CHK1) could facilitate cardiomyocyte proliferation and cardiac repair post myocardial infarction, however, its role on mitochondrial function in I/R injury remains unknown. Research Questions: The purpose of this study is to explore the potential effects and mechanisms of CHK1 on mitochondrial homeostasis during myocardial I/R injury. Methods: To investigate the role of CHK1 on mitochondrial function following I/R injury, cardiomyocyte-specific knockout/knock-in mouse models were generated. Mass spectrometry-proteomics analysis and protein co-immunoprecipitation assays were conducted to dissect the molecular mechanism of CHK1. The interaction between CHK1 and SIRT1 was explored using truncated plasmids. Results: CHK1 was downregulated in myocardium post I/R and neonatal mouse cardiomyocytes (NMCMs) post oxygen-glucose deprivation/re-oxygenation (OGD/R). In vivo , CHK1 overexpression protected against myocardial I/R injury, while heterogenous CHK1 knockout exacerbated cardiomyopathy. In vitro , CHK1 inhibited OGD/R-induced cardiomyocyte apoptosis and bolstered cardiomyocyte survival. Mechanistically, CHK1 attenuated oxidative stress and preserved mitochondrial metabolism in cardiomyocytes under I/R. Moreover, disrupted mitochondrial homeostasis in I/R myocardium was restored by CHK1 through the promotion of mitochondrial biogenesis and mitophagy. Through mass spectrometry analysis following co-immunoprecipitation, SIRT1 was identified as a direct target of CHK1. The 266-390 domain of CHK1 interacted with the 160-583 domain of SIRT1. Importantly, CHK1 phosphorylated SIRT1 at Thr530 residue, thereby inhibiting SMURF2-mediated degradation of SIRT1. CHK1 Δ390 amino acids (aa) mutant functioned similarly to full-length CHK1 in scavenging ROS and maintaining mitochondrial dynamics. Consistently, cardiac-specific SIRT1 knockdown partially attenuated the protective role of CHK1 in I/R injury. Conclusions: Our findings revealed that CHK1 mitigates I/R injury and restores mitochondrial dynamics in cardiomyocytes through a SIRT1-dependent mechanism.

MRPL41, as a target for acupuncture, promotes neuron apoptosis in models of ischemic stroke via activating p53 pathway

Neuronal death is the key cause of ischemic stroke. Acupuncture (Acu) is a recognized method for the treatment and amelioration of cerebral ischemia. However, the molecular mechanism of Acu for treating ischemic stroke has not yet been detailedly elucidated. Based our microarray analysis results, mitochondrial ribosomal protein L41 (MRPL41), which is related to apoptosis, was identified as the target of Acu. MRPL41 expression was increased in middle cerebral artery occlusion/reperfusion (MCAO/R) model and reduced after Acu treatment. Following, MCAO/R model and oxygen and glucose deprivation/reoxygenation (OGD/R) model were established to explore the effect of MRPL41. Knockdown of MRPL41 increased cell viability and ani-apoptotic protein (Bcl-2) expression, and reduced apoptosis intensity and pro-apoptotic protein (Bax and Cleaved caspase-3) of OGD/R neurons. In vivo, MRPL41 silencing decreased neurological severity score, shrank infarct area, reduced encephaledema and neuron apoptosis. In addition, reduction of MRPL41 caused loss of p53. Our data uncover that Acu targets MRPL41, following with inhibiting neuron apoptosis via p53 pathway, thereby ameliorating ischemic stroke.

Salidroside facilitates neuroprotective effects in ischemic stroke by promoting axonal sprouting through promoting autophagy

BackgroundIschemic stroke is a common cerebrovascular disease characterized by high incidence, disability, mortality, and recurrence. The limitations of current pharmacological treatments, which have primarily single neuroprotective action and a narrow therapeutic time window, lead to unsatisfactory therapeutic efficacy. Activation of autophagy can facilitate neural regeneration. ObjectiveTo clarify whether salidroside can promote axonal sprouting through autophagy resulting in protecting neurons. MethodsIn vivo, a Middle Cerebral Artery Occlusion/reperfusion (MCAO/IR) model was used, and in vitro, an Oxygen-Glucose Deprivation/Reoxygenation (OGD/R)-induced primary neuronal cell model was employed to evaluate the neuroprotective effects of salidroside. BDA neurotracer, immunofluorescence, and Western blot (WB) were utilized to determine its impact on axonal sprouting and the levels of related proteins (MAP2, GAP43, and PSD-95). Proteomics, transmission electron microscopy (TEM), and WB were applied to identify the effects on autophagy-related proteins (beclin1, LC3, p62, and LAMP2), autophagosomes and lysosomes. The mechanism of salidroside in promoting axonal sprouting through inducing autophagy was further confirmed by blocking with the autophagy inhibitor 3-MA. ResultsSalidroside reduced neurologic deficits and infarct volume induced by MCAO/IR in vivo and protected OGD/R induced primary neuronal cells in vitro. Both in vivo and in vitro, it increased the number and length of axons and upregulated the expression of key axonal proteins (MAP2, GAP43, and PSD-95) and mediated autophagy-related proteins. Mechanistic studies showed that the promoting effects of salidroside on autophagy and axonal sprouting disappeared after the blockade by 3-MA. ConclusionThis study reports for the first time that the neuroprotective effect of salidroside in ischemic stroke can be executed through mediating autophagy-related protein (beclin1, LC3, p62, and LAMP2), resulting in induced axonal sprouting or mature protein (MAP2, GAP43, and PSD-95).

2-Acetylacteoside improves recovery after ischemic stroke by promoting neurogenesis via the PI3K/Akt pathway

Ischemic stroke induces adult neurogenesis in the subventricular zone (SVZ), even in elderly patients. Harnessing of this neuroregenerative response presents the therapeutic potential for post-stroke recovery. We found that phenylethanoid glycosides (PhGs) derived from Cistanche deserticola aid neural repair after stroke by promoting neurogenesis. Among these, 2-acetylacteoside had the most potent on the proliferation of neural stem cells (NSCs) in vitro. Furthermore, 2-acetylacteoside was shown to alleviate neural dysfunction by increase neurogenesis both in vivo and in vitro. RNA-sequencing analysis highlighted differentially expressed genes within the PI3K/Akt signaling pathway. The candidate target Akt was validated as being regulated by 2-acetylacteoside, which, in turn, enhanced the proliferation and differentiation of cultured NSCs after oxygen-glucose deprivation/reoxygenation (OGD/R), as evidenced by western blot analysis. Subsequent analysis using cultured NSCs from adult subventricular zones (SVZ) confirmed that 2-acetylacteoside enhanced the expression of phosphorylated Akt (p-Akt), and its effect on NSC neurogenesis was shown to be dependent on the PI3K/Akt pathway. In summary, our findings elucidate for the first time the role of 2-acetylacteoside in enhancing neurological recovery, primarily by promoting neurogenesis via Akt activation following ischemic brain injury, which offers a novel strategy for long-term cerebrological recovery in ischemic stroke.

Galangin Regulates Astrocyte Phenotypes to Ameliorate Cerebral Ischemia-reperfusion Injury by Inhibiting the RhoA/ROCK/LIMK Pathway.

This study aimed to explore whether Galangin (Gal) could improve cerebral Ischemia- reperfusion (I/R) injury by regulating astrocytes, and clarify its potential molecular mechanism. An I/R injury model of rats was established using the Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R) method, followed by the administration of Gal (25, 50, 100 mg/kg) via gavage for 14 consecutive days. Besides, astrocytes were isolated from the rats to construct an Oxygen-Glucose Deprivation/Re-oxygenation (OGD/R) cell model, with treatments of Gal or the Ras homolog gene family member A (RhoA)/Rho-associated Coiled-coil containing protein Kinase (ROCK) inhibitor Y-27632. Subsequently, the severity of nerve injury was assessed using the modified Neurological Severity Score (mNSS) test; behavioral disorders in I/R rats were observed through the open field and ladder-climbing tests. Pathological damages and neuron survival in the peri-infarct zone were examined by hematoxylin and eosin staining and NeuN staining, respectively. Additionally, immunofluorescence staining was employed to determine astrocyte polarization and TUNEL staining was carried out to measure the level of cell apoptosis; also, western blot was performed to detect the expression of proteins related to the RhoA/ROCK/LIM domain Kinase (LIMK) pathway. Gal significantly ameliorated the neurological and motor dysfunctions caused by I/R in rats, reduced pathological damage in the peri-infarct zone, and promoted neuronal survival. Additionally, Gal increased the number of A2 astrocytes, while it decreased the number of A1 astrocytes. In vitro experiments revealed that the effect of Gal was consistent with that of Y-27632. Additionally, Gal significantly enhanced the survival of OGD/R cells, increased the number of A2 astrocytes, and inhibited the expression of proteins associated with the RhoA/ROCK pathway. Gal could reduce the level of apoptosis, promote the polarization of A2 astrocytes, and improve cerebral I/R injury, and its mechanism may be related to the inhibition of the RhoA/ROCK pathway.

VEGFD/VEGFR3 signaling contributes to the dysfunction of the astrocyte IL-3/microglia IL-3Rα cross-talk and drives neuroinflammation in mouse ischemic stroke.

Astrocyte-derived IL-3 activates the corresponding receptor IL-3Rα in microglia. This cross-talk between astrocytes and microglia ameliorates the pathology of Alzheimer's disease in mice. In this study we investigated the role of IL-3/IL-3Rα cross-talk and its regulatory mechanisms in ischemic stroke. Ischemic stroke was induced in mice by intraluminal occlusion of the right middle cerebral artery (MCA) for 60 min followed by reperfusion (I/R). Human astrocytes or microglia subjected to oxygen-glucose deprivation and reoxygenation (OGD/Re) were used as in vitro models of brain ischemia. We showed that both I/R and OGD/Re significantly induced decreases in astrocytic IL-3 and microglial IL-3Rα protein levels, accompanied by pro-inflammatory activation of A1-type astrocytes and M1-type microglia. Importantly, astrocyte-derived VEGFD acting on VEGFR3 of astrocytes and microglia contributed to the cross-talk dysfunction and pro-inflammatory activation of the two glial cells, thereby mediating neuronal cell damage. By using metabolomics and multiple biochemical approaches, we demonstrated that IL-3 supplementation to microglia reversed OGD/Re-induced lipid metabolic reprogramming evidenced by upregulated expression of CPT1A, a rate-limiting enzyme for the mitochondrial β-oxidation, and increased levels of glycerophospholipids, the major components of cellular membranes, causing reduced accumulation of lipid droplets, thus reduced pro-inflammatory activation and necrosis, as well as increased phagocytosis of microglia. Notably, exogenous IL-3 and the VEGFR antagonist axitinib reestablished the cross-talk of IL-3/IL-3Rα, improving microglial lipid metabolic levels via upregulation of CPT1A, restoring microglial phagocytotic function and attenuating microglial pro-inflammatory activation, ultimately contributing to brain recovery from I/R insult. Our results demonstrate that VEGFD/VEGFR3 signaling contributes to the dysfunction of the astrocyte IL-3/microglia IL-3Rα cross-talk and drives pro-inflammatory activation, causing lipid metabolic reprogramming of microglia. These insights suggest VEGFR3 antagonism or restoring IL-3 levels as a potential therapeutic strategy for ischemic stroke.

SRAGE attenuates myocardial ischemia-reperfusion injury by recruiting Tregs through chemokine CCL4

Abstract Background Immune response has recently been shown to play a crucial role in myocardial ischemia-reperfusion (MIR). Our previous studies have confirmed that soluble receptors for advanced glycation end-products (sRAGE) could reduce myocardial ischemia-reperfusion injury (MIRI) by increasing the number of regulatory T cells (Tregs). However, the precise mechanism is still incompletely understood. Purpose The present study aimed to determine which kind of chemokines play a major role in recruiting Tregs during MIRI. The potential mechanism of sRAGE on the chemokine production was further explored. Methods We modeled mice with cardiac-specific overexpression of sRAGE by crossing the floxed sRAGE mouse model with the cardiomyocyte-specific Cre transgenic mouse. Experimental models were established utilizing left anterior descending coronary artery ligation in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Transcriptomics, quantitative real-time polymerase chain reaction, immunohistochemistry, flow cytometry, and western blotting were used to determine the chemokines and related pathways involved in the recruitment of Tregs during MIRI. Results Using transcriptomics, quantitative real-time polymerase chain reaction, and western blotting approach, we identify the chemokine CCL4 plays a crucial role during regulatory T cell recruitment in the heart after MIRI. In vivo studies show that depletion of CCL4 reduces Tregs infiltration into the ischemic myocardium, subsequently deteriorating cardiac injury post-MIR. Mechanistically, sRAGE promotes cardiomyocytes to express and secrete CCL4 via the JNK/AP-1 pathway in vitro. Selective inhibition of the JNK/AP-1 pathway could decrease CCL4 production in sRAGE overexpression cardiomyocytes. Conclusions sRAGE induces Tregs recruitment and alleviates MIRI via the JNK/AP-1/CCL4 pathway, preserving myocardial function and impeding inflammation and apoptosis, providing new perspectives for immunotherapeutic targets in MIRI treatment.

Tilianin suppresses NLRP3 inflammasome activation in myocardial ischemia/reperfusion injury via inhibition of TLR4/NF-κB and NEK7/NLRP3.

Tilianin, a flavonoid compound derived from Dracocephalum moldavica L., is recognized for its diverse biological functionalities, in particular alleviating myocardial ischemia-reperfusion injury (MIRI). There is ample evidence suggesting that the NLRP3 inflammasome has a significant impact on the development of MIRI. In this study, rats undergoing the ligation and subsequent release of the left anterior descending (LAD) coronary artery and H9c2 cardiomyocytes subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used to investigate the effects of tilianin on NLRP3 inflammasome and its anti-MIRI mechanisms. Upon reperfusion, the rats were intraperitoneally injected with tilianin at doses of 3, 10, 30mg/kg. H9c2 cells were treated with tilianin at concentrations of 10, 30, and 50μg/mL. Echocardiography, TTC staining and TUNEL staining demonstrated that tilianin remarkably improved cardiac function and mitigated myocardial damage in MIRI rats. Additionally, notable inflammatory response reduction by tilianin was evidenced by subsequent hematatoxylin-eosin (HE) staining, inflammatory cytokines assay, and quantitative proteomics. Further western blotting analysis and immunofluorescence staining showed tilianin decreased the levels of TLR4, p-NF-κB, NLRP3, and ASC in MIRI rats and H9c2 cells exposed to OGD/R, alongside a significant reduction in cleaved gasdermin D, mature IL-1β and IL-18. Molecular docking, cellular thermal shift assay (CETSA) and co-immunoprecipitation (co-IP) assay revealed that tilianin impeded the interaction between NLRP3 and NEK7. Taken together, tilianin protects cardiomyocytes from MIRI by suppressing NLRP3 inflammasome through the inhibition of the TLR4/NF-κB signaling pathway and the disruption of the NEK7/NLRP3 interface. These findings underscore the potential of tilianin as a promising therapeutic candidate for MIRI.

The deubiquitinase OTUD3 stabilizes IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to ameliorate cerebral ischemia–reperfusion injury

BackgroundIschemic stroke (IS) is known for its high morbidity, disability and mortality rates, and studies designed to explore its pathophysiological mechanisms and identify novel therapeutic strategies are urgently needed. We aimed to probe the effects of the deubiquitinase OTUD3-IRP2-p53/PTGS2 pathway on cerebral ischemia‒reperfusion (I/R) injury and hippocampal neuron ferroptosis.MethodsA cerebral I/R mouse model was established. Furthermore, lentiviral vectors that overexpressed OTUD3 and knocked down IRP2 were constructed, and a series of assays were performed to probe the OTUD3/IRP2/p53/PTGS2 mechanism. An oxygen‒glucose deprivation and reoxygenation (OGD/R) model of mouse hippocampal neurons was constructed. Then, OTUD3 and IRP2 were knocked down and overexpressed, and p53 was overexpressed to explore the mechanism of the OTUD3/IRP2/p53/PTGS2 pathway.ResultsOTUD3 and IRP2 were expressed at low levels in cerebral I/R models. OTUD3 promoted IRP2 expression to protect damaged hippocampal neurons. Moreover, IRP2 affected ferroptosis in hippocampal neurons. In addition, IRP2 inhibited p53. After IRP2 and p53 were overexpressed, IRP2 regulated the p53/PTGS2 pathway and affected ferroptosis in hippocampal neurons. In vivo, after overexpressing OTUD3 and knocking down IRP2, we found that overexpression of OTUD3 promoted IRP2 expression to reduce ferroptosis in hippocampal neurons and improve cerebral I/R injury via the inhibition of the p53/PTGS2 pathway.ConclusionsThe deubiquitinase OTUD3 stabilized IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to subsequently ameliorate cerebral I/R injury.

METTL3 mediates m6A modification of lncRNA CRNDE to promote ATG10 expression and improve brain ischemia/reperfusion injury through YTHDC1

BackgroundIschemia/reperfusion (I/R) injury is a severe brain disorder with currently limited effective treatments. This study aims to explore the role of N6-methyladenosine (m6A) modification and associated regulatory factors in I/R to identify potential therapeutic targets.MethodsWe utilized a middle cerebral artery occlusion (MCAO) rat model and SH-SY5Y cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to assess m6A levels and investigate the impact of METTL3 overexpression on long non-coding RNA (lncRNA) CRNDE expression. The effects of silencing lncRNA CRNDE on the interaction between YTHDC1 and ATG10 mRNA, as well as the stability of ATG10 mRNA, were evaluated. Additionally, apoptosis rates, pro-inflammatory and anti-inflammatory factor levels, ATG10 expression, and autophagic activity were analyzed to determine the effects of METTL3. The reverse effects of YTHDC1 overexpression were also examined.ResultsMCAO rats and OGD/R-treated SH-SY5Y cells exhibited reduced m6A levels. METTL3 overexpression significantly inhibited lncRNA CRNDE expression. Silencing lncRNA CRNDE mitigated OGD/R-induced apoptosis and inflammation in SH-SY5Y cells, while enhancing autophagy and stabilizing ATG10 mRNA. METTL3 overexpression decreased cell apoptosis, reduced the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and increased IL-10 secretion. Furthermore, METTL3 overexpression upregulated ATG10 expression and promoted autophagy. Conversely, lncRNA CRNDE overexpression negated these effects.ConclusionThe inhibition of lncRNA CRNDE affects the interaction between YTHDC1 and ATG10 mRNA and stabilizes ATG10 mRNA, mediated by METTL3 overexpression. These findings suggest that targeting lncRNA CRNDE to reduce apoptosis, inhibit inflammation, increase ATG10 expression, and enhance autophagy could offer new therapeutic strategies for I/R injury.

Tetrahydroxy Stilbene Glucoside Promotes Mitophagy and Ameliorates Neuronal Injury after Cerebral Ischemia Reperfusion via Promoting USP10-Mediated YBX1 Stability.

Tetrahydroxy stilbene glucoside (TSG) from Polygonum multiflorum exerts neuroprotective effects after ischemic stroke. We explored whether TSG improved ischemic stroke injury via PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy. Oxygen glucose deprivation/reoxygenation (OGD/R) in vitro model and middle cerebral artery occlusion (MCAO) rat model were established. Cerebral injury was assessed by neurological score, hematoxylin and eosin staining, 2,3,5-triphenyltetrazolium chloride staining, and brain water content. Apoptosis, cell viability, and mitochondrial membrane potential were assessed by flow cytometry, cell counting kit-8, and JC-1 staining, respectively. Colocalization of LC3-labeled autophagosomes with lysosome-associated membrane glycoprotein 2-labeled lysosomes or translocase of outer mitochondrial membrane 20-labeled mitochondria was observed with fluorescence microscopy. The ubiquitination level was determined using ubiquitination assay. The interaction between molecules was validated by coimmunoprecipitation and glutathione S-transferase pull-down. We found that TSG promoted mitophagy and improved cerebral ischemia/reperfusion damage in MCAO rats. In OGD/R-subjected neurons, TSG promoted mitophagy, repressed neuronal apoptosis, upregulated Y-box binding protein-1 (YBX1), and activated PINK1/Parkin signaling. TSG upregulated ubiquitin-specific peptidase 10 (USP10) to elevate YBX1 protein. Furthermore, USP10 inhibited ubiquitination-dependent YBX1 degradation. USP10 overexpression activated PINK1/Parkin signaling and promoted mitophagy, which were reversed by YBX1 knockdown. Moreover, TSG upregulated USP10 to promote mitophagy and inhibited neuronal apoptosis. Collectively, TSG facilitated PINK1/Parkin pathway-mediated mitophagy by upregulating USP10/YBX1 axis to ameliorate ischemic stroke.

Identification of 3-methyl-1-(3-methylpyridin-2-yl)-1H-pyrazol-5-ol as promising neuroprotective agent

Pyrazolol derivatives are gaining significant attention for their diverse pharmacological effects, such as analgesic, anti-inflammatory, antioxidant, and anticancer activities. In this study, 20 pyrazolol derivatives were designed and synthesized to develop an anti-ischemic stroke formulation with free radical scavenging activity. Most of these synthesized compounds demonstrated antioxidant capabilities in DPPH, ABTS radical scavenging, and ORACFL assays. The methyl-substituted compound Y12, in particular, showed exceptional antioxidant capacity. Additionally, these compounds showed excellent neurocytoprotective effects in the SH-SY5Y cell injury model subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Notably, Y12 exhibited significant metal chelating activity with Cu2+. In vivo studies confirmed that compound Y12 has neuroprotective effects and can significantly reduce the infarct area in a mouse model of focal cerebral ischemia induced by transient middle cerebral artery occlusion (tMCAO).

Mechanism of ginsenoside Rb3 against OGD/R damage based on metabonomic and PCR array analyses.

In order to study the mechanisms of ginsenoside Rb3 (G-Rb3) against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in HT22 cells based on metabolomics and PCR array, HT22 cells were randomly divided into control group, model group, G-Rb3 high-dose group (10 µmol/l) and G-Rb3 low-dose group (5 µmol/l). Except for the control group, which was left untreated, the remaining groups were incubated with 10 mmol/l Na2S2O4 in sugar-free DMEM medium for 2 h and then replaced with serum-free high-sugar DMEM medium for 2 h in order to replicate in vitro OGD/R model. Trypan blue staining was used to detect the cell viability; flow cytometry was used to detect apoptosis; western blotting was used to detect the protein expression levels of Bax, Bcl-2 and caspase-3. The metabolomics were used to analyze the differential metabolites of G-Rb3 affecting OGD/R in order to find the relevant metabolic pathways. PCR array assay was performed to identify the expression of the differential genes. G-Rb3 could inhibit HT22 apoptosis according to the result of cell morphology, trypan blue staining and flow cytometry. The levels of Bax and caspase-3 protein expression were decreased, whereas the level of Bcl-2 protein expression was increased after the treatment of G-Rb3. Metabolomics results showed that a total of 31 differential metabolites between OGD/R group and G-Rb3 group, such as guanosine level, was downregulated, the levels of enalaprilat and sorbitol were upregulated, affecting ABC transporters, galactose metabolism, citrate cycle and other related metabolic pathways; according to the result of PCR array, it was observed that G-Rb3 significantly downregulated Trp63, Trp73, Dapk1, Casp14 and Cd70 pro-apoptotic genes. In conclusion, G-Rb3 has a significant protective effect on the OGD/R model simulated in vitro, and the mechanism may be related to the inhibition of apoptosis by affecting metabolites.

Glycyrrhizic Acid Inhibits Hippocampal Neuron Apoptosis by Activating the PI3K/ AKT Signaling Pathway.

Glycyrrhizic acid (GA), one of the main active substances in Glycyrrhiza, has anti-inflammatory, anti-viral, and neuroprotective effects. GA can significantly reduce cerebral infarction size in middle cerebral artery occlusion (MCAO) rats and suppress inflammatory responses. However, the underlying mechanism by which GA protects the neuronal system remains poorly understood. Cell proliferation and viability were tested using CCK-8 and Edu assays. The effects of GA on apoptosis were detected using flow cytometry and Tunel assays. Western blotting was performed to assess protein expression. Behavioral experiments were conducted using the Morris water maze and rotation tests. Infarct size was observed using TTC staining. We report that GA protects neurons by inhibiting apoptosis, mainly through the PI3K/AKT pathway in oxygen-glucose deprivation/reoxygenation (OGD/R) and MCAO rat models. GA increases the viability and proliferation of oxygen- and glucose-deprived hippocampal neurons. Hippocampal neuron apoptosis decreased after GA treatment in vitro and in vivo. Furthermore, we determined that GA treatment increased the active state of PI3K and its downstream protein p-AKT, whereas when using a specific inhibitor of PI3K, Y294002, the levels of p-PI3K and p-AKT decreased. Finally, we showed that GA treatment improved spatial memory and motor coordination in MCAO rats, while TTC staining showed that GA decreased cerebral infarct size in MCAO rats. We reveal that GA protects hippocampal neurons by inhibiting their apoptosis, mainly through the PI3K/AKT signaling pathway.

Quercetin Promotes the Repair of Mitochondrial Function in H9c2 Cells Through the miR-92a-3p/Mfn1 Axis.

Cardiocerebrovascular disease is a severe threat to human health. Quercetin has a wide range of pharmacological effects such as antitumor and antioxidant. In this study, we aimed to determine how quercetin regulates mitochondrial function in H9c2 cells. An H9c2 cell oxygen glucose deprivation/reoxygenation (OGD/R) model was constructed. The expression of miR-92a-3p and mitofusin 1 (Mfn1) mRNA in the cells was detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Changes in the mitochondrial membrane potential of cells were examined by JC-1 staining. ATP production in the cells was detected using a biochemical assay. Mitochondrial morphological changes were observed using transmission electron microscopy. Detection of miR-92a-3p binding to Mfn1 was done using dual luciferase. Western blotting was used to detect the protein expression of Mfn1 in the cells. miR-92a-3p is essential in regulating cell viability, apoptosis, and tumor cell metastasis. OGD/R induced miR-92a-3p expression, decreased mitochondrial membrane potential and mitochondrial ATP production, and increased mitochondrial damage. Mitochondria are the most critical site for ATP production. Continued opening of the mitochondrial permeability transition pore results in an abnormal mitochondrial transmembrane potential. Both quercetin and inhibition of miR-29a-3p were able to downregulate miR-29a-3p levels, increase cell viability, mitochondrial membrane potential, and ATP levels, and improve mitochondrial damage morphology. Furthermore, we found that downregulation of miR-29a-3p upregulated the protein expression of Mfn1 in cells. Additionally, miR-92a-3p was found to bind to Mfn1 in a luciferase assay. miR- 29a-3p overexpression significantly inhibited the protein expression level of Mfn1. Quercetin treatment partially reversed the effects of miR-29a-3p overexpression in H9c2 cells. Quercetin promoted the recovery of mitochondrial damage in H9c2 cells through the miR-92a-3p/Mfn1 axis.

Zinc sulfide nanoparticles serve as gas slow-release bioreactors for H2S therapy of ischemic stroke

Stroke is one of the leading causes of death and disability in the world. Ischemic stroke causes overproduction of reactive oxygen/nitrogen species (RONS) after reperfusion, triggering inflammatory responses that further leads to cell damage. In order to develop novel neuroprotective materials, we synthesized zinc sulfide nanoparticles (ZnS NPs) to function as gas slow-release bioreactors, showcasing stable and sustained H2S release while effectively removing RONS. In cultured cells, ZnS NPs can reduce the oxidative damage caused by oxygen-glucose deprivation and reoxygenation (OGD/R), promote the expression of p-AMPK, enhance microglia M2 polarization, decrease inflammatory factors and reduce neuronal apoptosis. Additionally, it increases the proliferation and migration of endothelial cells, promoting the formation of new neurovascular units by regulating the protein of p-AKT. In mice with ischemic stroke induced by middle cerebral artery occlusion/reperfusion (MCAO/R), ZnS NPs significantly reduce the infarct area and restore the mobility of mice owing to the slow release of H2S. In summary, our results indicate that ZnS NPs can be used as H2S slow-release bioreactors, offering a potentially innovative approach to treat ischemia-reperfusion injury caused by stroke.

Roflumilast Attenuates Microglial Senescence and Retinal Inflammatory Neurodegeneration Post Retinal Ischemia Reperfusion Injury Through Inhibiting NLRP3 Inflammasome.

Retinal ischemia-reperfusion (RIR) injury is implicated in various retinal diseases, leading to retinal ganglion cells (RGCs) degeneration. Microglial senescence exacerbates inflammation, contributing to neurodegeneration. This study aimed to investigate the potential therapeutic role of Roflumilast (Roflu) in ameliorating microglial senescence and neuroinflammation following RIR injury. C57BL/6J mice underwent RIR surgery, and Roflu treatment was administered intraperitoneally. BV2 microglial cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) to simulate ischemic conditions in vitro. SA-β-gal staining was used to detect cellular senescence. Quantitative PCR and ELISA were used to examine the levels of senescence-associated secretory phenotype (SASP) factors. Hematoxylin and eosin (H&E) staining was performed on retinal sections to assess retinal morphology and thickness. Surviving RGCs were labeled and quantified in retinal whole-mounts using immunofluorescence (IF). Furthermore, Western blot and IF staining were used to quantify the proteins associated with the cell cycle and NLRP3 inflammasomes. Roflu treatment reduced microglial senescence, ROS production, and secretion of pro-inflammatory cytokines in OGD/R-exposed BV2 cells. It also restored cell proliferation capacity and reversed OGD/R-induced cell cycle arrest. In vivo, Roflu alleviated retinal senescence, preserved retinal thickness, and protected against RGCs death in the RIR mouse model. Mechanistically, Roflu inhibited the NLRP3 inflammasome activation and suppressed DNA damage signaling pathway in microglia. Roflu exerts neuroprotective effects by mitigating microglial senescence and inflammation via inhibition of the NLRP3 inflammasome in RIR injury. These findings suggest that Roflu may serve as a promising therapeutic strategy for retinal diseases associated with ischemic injury by targeting microglial senescence.

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

BackgroundIntestinal 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. PurposeTo 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. MethodsThe 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. ResultsOur 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. ConclusionThis 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.

Curcumin ameliorates ischemic stroke injury by downregulating GMFB expression: An in vitro study.

Ischemic stroke (IS) is a cerebrovascular sickness, and cerebral ischemia-reperfusion (I/R) damage often occurs, but there is still a lack of drugs that can significantly alleviate it. Curcumin (Cur) exerts pharmacological effects such as antioxidative stress, anti-inflammation, and the promotion of apoptosis through regulating various pathways, but its efficacy and specific mechanism of action in IS have not been fully clarified. The purpose of this paper is to study the influence of Cur on IS. Brain microvascular endothelial cells (BMECs) were used to create an oxygen-glucose deprivation/reoxygenation (OGD/R) model to simulate I/R damage. The cell viability was assessed using an MTT assay. The LDH level and ROS positive rate were measured using commercial kits. The cell invasion was examined using a transwell assay. The apoptosis was assessed by flow cytometry. The contents of GMFB, Bax, and Bcl2 were measured using western blot. We confirmed that in the OGD/R-induced IS cell model, the abundance of GMFB was enhanced in the OGD/R group versus the control group. GMFB overexpression promoted OGD/R-induced cell viability diminution, increased LDH and ROS levels, lessened cell invasion ability, enhanced cell apoptosis, enhanced Bax levels, and decreased Bcl2 levels. Silencing GMFB ameliorated OGD/R-induced cell damage. Cur ameliorated OGD/R-induced cell damage. Cur curbed OGD/R-induced cell damage by downregulating GMFB expression. In conclusion, Cur cured ischemic stroke-induced cell damage by downregulating GMFB expression.

NRIP1 is a downstream target of YY1 in promoting OGD/R-induced H9c2 cardiomyocyte injury and mitochondrial dysfunction.

From a clinical standpoint, myocardial ischemia/reperfusion injury (MIRI) has always been an enormous challenge for the treatment of acute myocardial infarction (AMI). Molecular targeting therapy may help overcome this challenge. The present work aimed to elucidate the possible involvement of Yin-Yang 1 (YY1)/nuclear receptor-interacting protein 1 (NRIP1) and discover the molecular mechanism of MIRI. Herein, a cardiomyocyte ischemia/reperfusion (I/R) model was established via oxygen-glucose deprivation/re-oxygenation (OGD/R) damage in H9c2 cardiomyocytes. Reverse transcription-quantitative PCR and western blotting were conducted to measure the levels of YY1 and NRIP1 at the RNA and protein levels, respectively. H9c2 cell viability and apoptosis were assayed using the Cell Counting Kit-8, flow cytometry, and western blotting. In addition, superoxide dismutase, glutathione peroxidase, and malondialdehyde levels were analyzed as markers of oxidative stress. Additionally, mitochondrial membrane potential, which was measured via JC-1 staining, ATP content, Complex I activity, mitochondrial DNA copy number, and mitochondrial permeability transition pore (mPTP) opening rate were analyzed to evaluate mitochondrial activity. Moreover, luciferase reporter and chromatin immunoprecipitation assays experimentally validated the predicted affinity of YY1 with the NRIP1 promoter according to the HumanTFDB online tool. YY1/NRIP1 were both highly expressed in OGD/R-injured H9c2 cardiomyocytes. Downregulation of NRIP1 improved cell viability, whereas it inhibited cell apoptosis and oxidative stress, and suppressed mitochondrial dysfunction in OGD/R-injured H9c2 cardiomyocytes. Importantly, it was verified that YY1 could bind to the NRIP1 promoter to positively regulate NRIP1 expression. The protective effects of NRIP1 knockdown against cardiomyocyte damage and mitochondrial dysfunction in OGD/R-injured H9c2 cardiomyocytes were partly abolished through overexpression of YY1. NRIP1 emerged as a downstream target of YY1 in promoting OGD/R-induced H9c2 cardiomyocyte injury and mitochondrial dysfunction, providing novel ideas for targeted treatments to alleviate MIRI.