24-h Reperfusion Research Articles

Kirenol Ameliorates Myocardial Ischemia-Reperfusion Injury by Promoting Mitochondrial Function and Inhibiting Inflammasome Activation.

Macrophage-mediated inflammation plays a crucial role in the pathophysiological process of myocardial ischemia/reperfusion (I/R) injury. Recent studies have highlighted the importance of mitochondrial function and inflammasome activation in the inflammatory process. Kirenol, a well-known natural compound, has been shown to regulate inflammation in various diseases. This study investigated whether Kirenol could exert anti-inflammatory effects on macrophages during myocardial I/R injury. Mouse myocardial I/R models were established by 45min of ischemia followed by 24h of reperfusion. Saline or Kirenol treatment was administered. In vivo assessments included the evaluation of cardiac function, infarcted area, and immune cell infiltration. Subsequently, bone marrow-derived macrophages (BMDMs) were isolated, and mitochondrial function and pyroptosis were assessed. Furthermore, the study compared the cardioprotective effects of Kirenol with a specific NOX1/NOX4 inhibitor, GKT137831. Kirenol gavage improved cardiac function, decreased infarct area, and alleviated inflammatory infiltration in mice subjected to myocardial I/R injury. Mechanistically, Kirenol inhibited NOX1 and NOX4 and enhanced mitochondrial function, ultimately attenuating the pyroptosis of macrophages. The therapeutic effects of Kirenol and GKT137831 were not significantly different. This study demonstrates that Kirenol mitigates myocardial I/R injury by inhibiting NOX1 and NOX4, restoring mitochondrial function, and ameliorating macrophage pyroptosis.

Sevoflurane Affects Myocardial Autophagy Levels After Myocardial Ischemia Reperfusion Injury via the microRNA-542-3p/ADAM9 Axis.

This research focused on investigating the effects of sevoflurane (Sev) on myocardial autophagy levels after myocardial ischemia reperfusion (I/R) injury via the microRNA-542-3p (miR-542-3p)/ADAM9 axis. Mice underwent 30min occlusion of the left anterior descending coronary (LAD) followed by 2h reperfusion. Cardiac infarction was determined by 2,3,5-triphenyltetrazolium chloride triazole (TTC) staining. Cardiac function was examined by echocardiography. Cardiac markers and oxidative stress factors were evaluated by ELISA. Autophagy-associated factors were detected by western blot. Relationship between miR-542-3p and ADAM9 was tested by dual-luciferase reporter gene assay, RT-qPCR, and western blot. Sev treatment ameliorated cardiac dysfunction, myocardial oxidative stress, and histopathological damages, decreased myocardial infarction size and myocardial apoptotic cells after myocardial I/R injury. Sev treatment elevated miR-542-3p expression and decreased ADAM9 expression in myocardial tissues after myocardial I/R injury. miR-542-3p overexpression could enhance the ameliorative effects of Sev on myocardial injury and myocardial autophagy in I/R mice. miR-542-3p targeted and negatively regulated ADAM9 expression. ADAM9 overexpression reversed the ameliorative effects of miR-542-3p up-regulation on myocardial injury and myocardial autophagy in Sev-treated I/R mice. Sev treatment could ameliorate myocardial injury and myocardial autophagy in I/R mice, mediated by mechanisms that include miR-542-3p up-regulation and ADAM9 down-regulation.

Simulated ischaemia/reperfusion impairs trophoblast function through divergent oxidative stress- and MMP-9-dependent mechanisms.

Early-onset pre-eclampsia is believed to arise from defective placentation in the 1st trimester, leading to placental ischaemia/reperfusion (I/R) and oxidative stress. However, our current understanding of the effects of I/R and oxidative stress on trophoblast function is ambiguous in part due to studies exposing trophoblasts to hypoxia instead of I/R, and which report conflicting results. Here we present a model of simulated ischaemia/reperfusion (SI/R) to recapitulate the pathophysiological events of early-onset PE, by exposing 1st trimester cytotrophoblast HTR-8/SVneo cells to a simulated ischaemia buffer followed by reperfusion. We examined different ischaemia and reperfusion times and observed that 1h ischaemia and 24h reperfusion induced an increase in reactive oxygen species (ROS) production (p < 0.0001) and oxygen consumption rate (p < 0.01). SI/R-exposed trophoblast cells exhibited deficits in migration, proliferation and invasion (p < 0.01). While the deficits in migration and proliferation were rescued by antioxidants, suggesting a ROS-dependent mechanism, the loss of invasion was not affected by antioxidants, which suggests a divergent ROS-independent pathway. In line with this, we observed a decrease in MMP-9, the key regulatory enzyme necessary for trophoblast invasion (p < 0.01), which was similarly unaffected by antioxidants, and pharmacological inhibition of MMP-9 replicated the phenotype of deficient invasion (p < 0.01). Collectively, these data demonstrate that I/R impairs trophoblast migration and proliferation via a ROS-dependent mechanism, and invasion via a ROS-independent loss of MMP-9, disambiguating the role of oxidative stress and providing insights into the response of trophoblasts to I/R in the context of early-onset PE.

Pharmacokinetics and cardioprotective efficacy of intravenous miR-125b* microRNA mimic in a mouse model of acute myocardial infarction.

MicroRNA (miRNA) therapy is a promising approach to induce cardioprotection. We have previously identified cardiac microRNA-125b* (microRNA-125b-2-3p; miR-125b*) as a potential cardioprotective miRNA, termed ProtectomiR. We aimed to characterize the pharmacokinetics and pharmacodynamics, and the effect of miR-125b* mimic on infarct size using an in vivo mouse model. To characterize the pharmacokinetics properties of miR-125b* mimic, a single injection of 10-μg miR-125b* mimic or its scramble miRNA control, or vehicle i.v. was given to C57BL/6 mice. MiR-125b* expression was measured from plasma, heart, kidney and liver samples. Effect of miR-125b* on area at risk and infarct size was assessed after 45-min coronary occlusion, followed by 24-h reperfusion; 10-μg miR-125b* mimic or 10-μg non-targeting miRNA mimic control or vehicle were administered via the right jugular vein at 10th mins of coronary occlusion. To assess molecular mechanism involved in cardioprotection, expression of mRNA targets of miR-125b* were measured from ventricular myocardium at 1, 2, 4, 8 or 24 hpost-treatment using quantitative real time polymerase chain reaction. MiR-125b* expression was markedly increased in plasma and myocardium 1h, and in the liver 2h after treatment. Infarct size was significantly reduced after miR-125b* mimic treatment when compared to the vehicle. The expression of Ccna2, Eef2k and Cacnb2 target mRNAs was significantly reduced 8h after injection of miR-125b* mimic. This is the first demonstration of pharmacokinetic and molecular pharmacodynamic properties as well as the cardioprotective effect of miR-125b* mimic in vivo.

Nephroprotective role of resveratrol in renal ischemia-reperfusion injury: a preclinical study in Sprague-Dawley rats.

Renal ischemia-reperfusion injury (IRI) is a significant contributor to renal dysfunction, acute kidney injury (AKI), and associated morbidity and mortality. Resveratrol, a polyphenol and phytoalexin, is known for its anti-inflammatory, antioxidant, and anti-cancer properties. This study investigates the nephroprotective potential of resveratrol in a rat model of renal IRI. Twenty-eight male Sprague-Dawley rats were divided into four groups: Sham, IRI, DMSO, and Resveratrol. The Sham group underwent identical procedures without renal pedicle clamping, while the IRI group experienced 30min of ischemia followed by 2h of reperfusion. The DMSO group received dimethyl sulfoxide (DMSO) intraperitoneally 30min before ischemia, and the Resveratrol group received 30mg/kg resveratrol intraperitoneally 30min before ischemia. Biochemical parameters (Urea, creatinine, IL-1β, NF-κβ, SOD, GSH, Bcl-2, and caspase-3) and histopathological changes were assessed. IRI caused a substantial increase in serum creatinine, Urea, IL-1β, NF-κβ, and caspase-3 levels, while simultaneously decreasing SOD, GSH, and Bcl-2 levels. Resveratrol treatment mitigated these effects by lowering inflammatory and apoptotic markers, enhancing antioxidant defenses, and improving histological outcomes. Resveratrol demonstrates significant nephroprotective effects in renal IRI, primarily through its antioxidant, anti-inflammatory, and anti-apoptotic properties.

Empagliflozin treatment pre- and post- acute myocardial infarction reduces no-reflow, inflammatory cell infiltration and infarct size while preserving cardiac function.

Abstract Background and aims Empagliflozin (EMPA) and other members of the family of sodium glucose co-transporter 2 inhibitors have demonstrated clinical benefit in terms of survival and rehospitalizations in heart failure. However, their effect on acute myocardial infarction (AMI) is still under investigation. Microvascular injury (MVI) and no-reflow are major determinants of myocardial infarct size (IS) and prognosis. We investigated EMPA’s cardioprotective potential in AMI in terms of no-reflow, MVI and IS emphasizing on a translational regimen with EMPA treatment after AMI. Methods In a first series of experiments, C57Bl6 male mice (n=10 per group) were randomized into 1) Sham, 2) Control-AMI and 3) EMPA-Pre-AMI (10mg/kg/day orally for 6 weeks) groups. Mice underwent 30 min ischemia (I) and 2h reperfusion (R) or sham operation. Cell sorting and 3’ mRNA sequencing were applied to identify the cell types that are transcriptionally affected by EMPA pretreatment in mice with AMI. In a second series of experiments, the protocol was repeated, and an additional group was added, EMPA-Post-AMI (n=8-11 mice per group) in which oral treatment was performed 1h after R. Assessment of no-reflow via thioflavin S staining and electron microscopy was performed at 48 h. The infiltration of inflammatory cells in the ischemic heart was examined via flow cytometry. IS and myocardial function were determined by cardiac magnetic resonance (CMR). Type-2 diabetes mellitus patients received insulin (n=18) or EMPA (n=24) within 2 months post-AMI. Endothelial glycocalyx and endothelial-related circulating markers were examined at 4 months and 12 months post-AMI. Results EMPA pretreatment reversed AMI-induced alterations in cardiac endothelial cells’ (ECs) transcriptome. As such, it promoted the expression of survival genes, reduced gene expression related to adhesion molecules and ECs matrix degradation. Conversely, EMPA pretreatment exerted minimal effects on cardiomyocyte and fibroblast transcriptome. In support of the effect of EMPA on ECs, EMPA-Pre-AMI and EMPA-Post-AMI groups significantly reduced no-reflow and MVI as indicated by electron microscopy and histology at 48h of R. Moreover, both EMPA treatment pre-AMI and post-AMI reduced the infiltration of inflammatory monocytes and neutrophils in the infarcted myocardium suggesting enhanced vascular integrity. Both EMPA-Pre-AMI and EMPA-Post-AMI reduced IS and preserved cardiac function defined by CMR. Patients receiving EMPA presented with improved endothelial glycocalyx thickness, % global longitudinal strain and preserved pulse wave velocity. Conclusions EMPA treatment either before or after AMI protects against MVI, reduces no-reflow and IS and preserves cardiac function. Our data support the use of EMPA after recanalization in patients with AMI to confer improvement of cardiovascular function.

Retinal ischemia-reperfusion injury and pretreatment with Lycium barbarum glycopeptide.

To investigate the antioxidant protective effect of Lycium barbarum glycopeptide (LbGP) pretreatment on retinal ischemia-reperfusion (I/R) injury (RIRI) in rats. RIRI was induced in Sprague Dawley rats through anterior chamber perfusion, and pretreatment involved administering LbGP via gavage for 7d. After 24h of reperfusion, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CREA) levels, retinal structure, expression of Caspase-3 and Caspase-8, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) in the retina were measured. The pretreatment with LbGP effectively protected the retina and retinal tissue from edema and inflammation in the ganglion cell layer (GCL) and nerve fiber layer (NFL) of rats subjected to RIRI, as shown by light microscopy and optical coherence tomography (OCT). Serum AST was higher in the model group than in the blank group (P=0.042), but no difference was found in ALT, AST, and CREA across the LbGP groups and model group. Caspase-3 expression was higher in the model group than in the blank group (P=0.006), but no difference was found among LbGP groups and the model group. Caspase-8 expression was higher in the model group than in the blank group (P=0.000), and lower in the 400 mg/kg LbGP group than in the model group (P=0.016). SOD activity was lower in the model group than in the blank group (P=0.001), and the decrease was slower in the 400 mg/kg LbGP group than in the model group (P=0.003). MDA content was higher in the model group than in the blank group (P=0.001), and lower in the 400 mg/kg LbGP group than in the model group (P=0.016). The pretreatment with LbGP did not result in any observed liver or renal toxicity in the model. LbGP pretreatment exhibits dose-dependent anti-inflammatory, and antioxidative effects by reducing Caspase-8 expression, preventing declines of SOD activity, and decreasing MDA content in the RIRI rat model.

SGLT1 contributes to glucose-mediated exacerbation of ischemia-reperfusion injury in ex vivo rat heart.

Hyperglycaemia is common during acute coronary syndromes (ACS) irrespective of diabetic status and portends excess infarct size and mortality, but the mechanisms underlying this effect are poorly understood. We hypothesized that sodium/glucose linked transporter-1 (SGLT1) might contribute to the effect of high-glucose during ACS and examined this using an ex-vivo rodent heart model of ischaemia-reperfusion injury. Langendorff-perfused rat hearts were subjected to 35min ischemia and 2h reperfusion, with variable glucose and reciprocal mannitol given during reperfusion in the presence of pharmacological inhibitors of SGLT1. Myocardial SGLT1 expression was determined in rat by rtPCR, RNAscope and immunohistochemistry, as well as in human by single-cell transcriptomic analysis. High glucose in non-diabetic rat heart exacerbated reperfusion injury, significantly increasing infarct size from 45 ± 3 to 65 ± 4% at 11-22mmol/L glucose, respectively (p < 0.01), an association absent in diabetic heart (32 ± 1-37 ± 5%, p = NS). Rat heart expressed SGLT1 RNA and protein in vascular endothelium and cardiomyocytes, with similar expression found in human myocardium by single-nucleus RNA-sequencing. Rat SGLT1 expression was significantly reduced in diabetic versus non-diabetic heart (0.608 ± 0.08 compared with 1.116 ± 0.13 probe/nuclei, p < 0.01). Pharmacological inhibitors phlorizin, canagliflozin or mizagliflozoin in non-diabetic heart revealed that blockade of SGLT1 but not SGLT2, abrogated glucose-mediated excess reperfusion injury. Elevated glucose is injurious to the rat heart during reperfusion, exacerbating myocardial infarction in non-diabetic heart, whereas the diabetic heart is resistant to raised glucose, a finding which may be explained by lower myocardial SGLT1 expression. SGLT1 is expressed in vascular endothelium and cardiomyocytes and inhibiting SGLT1 abrogates excess glucose-mediated infarction. These data highlight SGLT1 as a potential clinical translational target to improve morbidity/mortality outcomes in hyperglycemic ACS patients.

Electroacupuncture alleviates myocardial ischemia-reperfusion injury by inhibiting hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla.

Accumulating evidence suggests that electroacupuncture (EA) has obvious therapeutic effects and unique advantages in alleviating myocardial ischemia-reperfusion injury (MIRI), while the underlying neuromolecular mechanisms of EA intervention for MIRI have not been fully elucidated. The aim of the study is to investigate the role of the neural pathway of hypothalamic paraventricular nucleus (PVN) neurons projecting to the rostral ventrolateral medulla (RVLM) in the alleviation of MIRI rats by EA preconditioning. MIRI models were established by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for 2h. Electrocardiogram recording, chemogenetics, enzyme-linked immunosorbent assay, multichannel physiology recording and haematoxylin-eosin and immunofluorescence staining methods were conducted to demonstrate that the firing frequencies of neurons in the PVN and the expression of c-Fos decreased by EA pretreatment. Meanwhile, EA preconditioning significantly reduced the levels of creatine kinase isoenzymes (CK-MB), cardiac troponin I (cTnI) and lactic dehydrogenase (LDH). Virus tracing showed a projection connection between PVN and RVLM. The inhibition of the PVN-RVLM neural pathway could replicate the protective effect of EA pretreatment on MIRI rats. However, the activation of the pathway weakened the effect of EA preconditioning. EA pretreatment alleviated MIRI by regulating PVN neurons projecting to RVLM. This work provides novel evidence of EA pretreatment for alleviating MIRI.

Stroke triggers dynamic m6A reprogramming of cerebral circular RNAs

We previously showed that stroke alters circular RNA (circRNA) expression profiles. Many circRNAs undergo epitranscriptomic modifications, particularly methylation of adenosine to form N6-methyladenosine (m6A). This modification significantly influences the circRNA metabolism and functionality. Hence, we currently evaluated if transient focal ischemia in adult C57BL/6J mice alters the m6A methylation of circRNAs. Changes in m6A were profiled in the peri-infarct cortex following immunoprecipitation coupled with microarrays. Correlation and gene ontology analyses were performed to understand the association of m6A changes with circRNA regulation and functional implications after stroke. Many circRNAs showed differential regulation (up or down) after stroke, and this change was highest at 24h of reperfusion. Notably, most circRNAs differentially regulated after stroke also exhibited temporal changes in m6A modification patterns. The majority of circRNAs that showed post-stroke differential m6A modifications were derived from protein-coding genes. Hyper-than hypomethylation of circRNAs was most prevalent after stroke. Gene ontology analysis of the host genes suggested that m6A-modified circRNAs might regulate functions such as synapse-related processes, indicating that m6A epitranscriptomic modification in circRNAs could potentially influence post-stroke synaptic pathophysiology.

The relationship between ischemic penumbra progression and the oxygen content of cortex microcirculation in acute ischemic stroke

The precise oxygen content thresholds of ischemic deep parenchymal (OCIDP) and that in cortical microcirculation (OCCM), which leads to ischemic penumbra converting into the infarcted core, remain uncertain. This study employed an invasive fiber-optic oxygen meter and a newly developed oxygen-responsive probe called RuA3-Cy5-rtPA (RC-rtPA) based on recombinant tissue-type plasminogen activator (rtPA) to examine the oxygen content thresholds. A mouse model of middle cerebral artery occlusion was generated and animals were randomly divided into a sham, 24-h reperfusion after 3-h ischemia (IR 3-h), and IR 6-h groups, all of which were sacrificed following reperfusion. Stroke severity was evaluated based on the infarction area, neurological symptoms, microcirculation perfusion, and microemboli in microcirculation. OCIDP was characterized based on its extent and distribution, whereas OCCM was measured using RC-rtPA. During ischemia, stroke severity escalation manifested as increasing infarction area, severe neurologic symptoms, and poorer microcirculation perfusion with more microthrombi depositions. OCIDP presented rapid decline following artery occlusion along with a gradual increase in the hypoxic area. Within 3 ​h following ischemia induction, the ischemic tissue that experienced hypoxia could be rescued, and this reversibility would disappear after 6 ​h. Within 6 ​h, OCCM continued to decrease. A significant decrease in oxygen content in cortical venules and cortical parenchyma was observed. These findings assist in establishing the extent of the ischemic penumbra at the microcirculation level and offer a foundation for assessing the ischemic penumbra that could respond positively to reperfusion therapy beyond the typical time window.

Effects of edaravone on testicular torsion-detorsion injury in rats.

This study aimed to assess the protective ability of edaravone on testicular torsion-detorsion injury in rats. Eighteen adult male Sprague-Dawley rats were randomly divided into three groups: Sham group (control, n=6); testicular torsion/detorsion (T/D group, n=6) and T/D+edaravone (T/D+E group, n=6). The spermatic cords of rats of the T/D group and the T/D+E group were rotated 720° in a clockwise direction and maintained for 120min in this torsion position. Around 90min after the torsion, edaravone at a dose of 10mg/kg dissolved in saline was administered IP to the T/D+E group. The testicle was counter-rotated to its normal position to allow reperfusion for 4h. Left testes of each animal were excised 240min after beginning of reperfusion. Oxidative stress markers (TAS, TOS, SOD, and MDA) and apoptotic pathways (Caspase 3, Caspase 8, Caspase 9, Bcl-2, and Bax,) were assessed by ELISA methods. Also, testicles were subjected to the histopathologic and ultrasound examinations. Ultrasound imaging showed that edaravone reduced the surface area and increased vascularization in testicles with T/D (p<0.0001, p<0.05, respectively). Edaravone pretreatment markedly decreased the levels of MDA, TOS, Bcl-2, Bax, Caspase 3, Caspase 8, and Caspase 9 (p<0.0001). Also, it increased significantly TAS levels (p<0.0001) and reduced insignificantly SOD activity. Histopathologic examinations demonstrated that edaravone significantly attenuated the histological damage caused by T/D in testicles. Taken together, the findings indicate that pretreatment of edaravone has protective effect against testicular T/D injury.

Neuropeptide W protects against ovarian oxidative injury and reinforces ovarian steroidogenic activity via the upregulation of ERα expression.

The aim of this study was to investigate the protective effect of neuropeptide W (NPW) on ovarian ischemia-reperfusion-induced oxidative injury and ovarian steroid metabolism. Rats were randomly divided into control and ischemia groups that received either saline or NPW (0.1 or 5 μg/kg/day). Bilateral ovarian ischemia was performed for 3 h followed by a 72-h reperfusion. Blood, ovary, and uterus samples were collected for biochemical and histological assessments. Treatment with either dose of NPW alleviated oxidative injury of the ovaries with a significant suppression in free radical formation and decreased histopathological injury in both the ovarian and uterine tissues, along with reduced lipid peroxidation and neutrophil accumulation in the uterus. Moreover, NPW treatment reversed the decrease in aromatase expression with a concomitant reduction in the expression of the inactivity enzyme estrogen sulfotransferase. Also, downregulation of estrogen receptor-α (ERα) expression in the injured ovarian tissue was abolished by NPW treatment, which implicates that the protective effect of NPW on the female reproductive system may involve the upregulation of the ERα-mediated signaling pathway. Our study demonstrated for the first time that NPW protects against ovarian oxidative injury and reinforces ovarian steroidogenic activity, which is accompanied by the upregulation of ERα expression in the ovaries.

Aloperine Alleviates Myocardial Injury Induced by Myocardial Ischemia and Reperfusion by Activating the ERK1/2/β-catenin Signaling Pathway.

Myocardial ischemia/reperfusion (I/R) injury can cause severe cardiac damage. Aloperine is a quinolizidine alkaloid found in the leaves and seeds of Sophora alopecuroides L. It has been recognized that aloperine has organ-protective properties; however, its role in cardioprotection is poorly characterized. This study aimed to evaluate the cardioprotective effects of aloperine against myocardial I/R injury in vivo. Adult male Sprague‒Dawley rats were randomly divided into sham-operated, control, and aloperine groups. All rats except for the sham-operated rats were subjected to 45min of myocardial ischemia (by left anterior descending ligation) followed by 3h of reperfusion. Aloperine (10mg/kg) was given intravenously at the onset of reperfusion. The cardioprotective effects of aloperine were evaluated by determining infarct size, hemodynamics, histological changes, cardiac biomarkers, and cardiac apoptosis. Aloperine limited infarct size; improved hemodynamics; attenuated myocardial I/R-induced histological deterioration; decreased serum LDH, CK-MB, and α-HBDH levels; and inhibited apoptosis after myocardial I/R injury. Moreover, aloperine stimulated the phosphorylation of ventricular ERK1/2, which is a major module of MAPK signaling pathways. Furthermore, aloperine increased the ventricular expression levels of β-catenin. Pharmacological inhibition of ERK1/2 diminished aloperine-induced cardioprotection and blocked ERK1/2/β-catenin signaling. These data support the cardioprotective effect of aloperine against myocardial I/R injury, which is mediated, at least in part, by the ERK1/2/β-catenin signaling pathway.

Potential protective effects of L-carnitine against myocardial ischemia/reperfusion injury in a rat model.

Myocardial ischemia/reperfusion (I/R) injury is a growing concern for global public health. This study seeks to explore the potential protective effects of L-carnitine (LC) against heart ischemia-reperfusion injury in rats. To induce I/R injury, the rat hearts underwent a 30-min ligation of the left anterior descending coronary artery, followed by 24h of reperfusion. We evaluated cardiac function through electrocardiography and heart rate variability (HRV) and conducted pathological examinations of myocardial structure. Additionally, the study investigated the influence of LC on myocardial apoptosis, inflammation, and oxidative stress in the context of I/R injury. The results show that pretreatment with LC led to improvements in the observed alterations in ECG waveforms and HRV parameters in the nontreated ischemic reperfusion model group, althoughmost of these changes did not reach statistical significance. Similarly, although without a significant difference, LC reduced the levels of proinflammatory cytokines when compared to the values in the nontreated ischemic rat group. Furthermore, LC restored the reduced expressions of SOD1, SOD2, and SOD3. Additionally, LC significantly reduced the elevated Bax expressions and showed a nonsignificant increase in Bcl-2 expression, resulting in a favorable adjustment of the Bcl-2/Bax ratio. We also observed a significant enhancement in the histological appearance of cardiac muscles, a substantial reduction in myocardial fibrosis, and suppressed CD3 + cell proliferation in the ischemic myocardium. This small-scale, experimental, in vivo study indicates that LC was associated with enhancements in the pathological findings in the ischemic myocardium in the context of ischemia/reperfusion injury in this rat model. Although statistical significance was not achieved, LC exhibits potential and beneficial protective effects against I/R injury. It does so by modulating the expression of antioxidative and antiapoptotic genes, inhibiting the inflammatory response, and enhancing autonomic balance, particularly by increasing vagal tone in the heart. Further studies are necessary to confirm and elaborate on these findings.

Abstract TP302: Circular RNAs Methylation Patterns Positively Correlated With Their Expression Level in the Post-Stroke Brain

Circular RNAs (circRNAs), a novel subgroup of noncoding RNAs derived from reverse splicing of various genes, have emerged as potential regulators of transcription and translation. Recent findings indicate that methylation of adenosine (m 6 A; N6-methyladenosine) in various RNA types might govern their levels by modulating their degradation as well as functional roles. This study investigated the spatiotemporal correlation of methylation patterns of circRNAs with their abundance in the post-stroke brain. Adult male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and m 6 A methylation in circRNAs was profiled using immunoprecipitated methylated RNAs with microarrays in the peri-infarct cortex at 6h, 12h, and 24h of post-reperfusion. Computational analysis was employed to identify the change in m 6 A levels and evaluate the correlation between expression and methylation patterns. We noted that most circRNAs that alter their levels following ischemic stroke are transcribed from the exonic regions of all chromosomes. While most genes generate a single circRNA, a unique situation occurs with the gene Rn45s, giving rise to a minimum of 30 circRNAs within the brain. Interestingly, following the stroke, the overall m 6 A levels of circRNAs displayed notable alterations. Moreover, the percentage shift in circRNA m 6 A levels increased from 6h to 24h of reperfusion, with some circRNAs, including mmu_circRNA_32433, demonstrating an increase of &gt;40% at the 24h reperfusion. More significantly, many cerebral circRNAs that displayed increased expression also showed elevated m 6 A methylation following stroke. The increased m 6 A methylation suggests potential stabilization and altered function of circRNAs in the post-stroke brain.

Abstract TMP113: RNA Sequencing Reveals Novel Pericytic MiR-15a/16-1 Targets in Post-Stroke Cerebral Angiogenesis

Introduction: Angiogenesis actively contributes to post-stroke functional recovery and improves long-term survival in stroke patients. Previously, we demonstrated that endothelium-targeted deletion of miR-15a/16-1 promotes post-stroke angiogenesis by enhancing classic pro-angiogenic factors and their receptors, and genetic deletion of miR-15a/16-1 in pericytes likewise promotes post-stroke functional recovery by stimulating cerebral angiogenesis. Here, we further investigate the underlying mechanisms and downstream targets of pericytic miR-15a/16-1 in cerebral angiogenesis after ischemic stroke. Methods: Pericyte-miR15a/16-1 conditional knockout mice (Pericyte-miR-15a/16-1 cKO) and wild-type (WT) controls were subjected to 1h MCAO followed by 7d reperfusion. Cerebral microvessels were extracted and subjected to RNA-seq to identify the significant differentially expressed genes (DEGs) that related to miR-15a/16-1 and angiogenesis among experimental groups. TargetScan and miRDB were used to predict potential miR-15a/16-1 downstream targets. Primary mouse brain vascular pericytes (mBVPs) were treated with lentivirus to achieve loss- or gain-of-miR-15a/16-1 function, and subjected to OGD 8h and reperfusion 24h. Pro-angiogenic factors were further verified by dual-luciferase reporter assay, qPCR, and western blotting. Results: RNA-seq analysis showed 289 upregulated genes (P &lt; 0.05 &amp; at least one CPM &gt; 1) and 399 upregulated genes from 100 positive enriched gene sets (GESA) in cerebral microvessels of pericyte-miR-15a/16-1 cKO mice compared to WT controls 7d after MCAO. By comparing to miR-15a/16-1 potential target database from TargetScan and miRDB, 9 angiogenic genes were identified. Further biochemical analysis revealed that Pappa2, Fgf9, Islr, and Ccr2 mRNA and protein expression are significantly increased in cerebral microvessels of cKO mice and mBVPs with loss-of-miR-15a/16-1 function. Furthermore, dual-luciferase assay results verified that loss- or gain-of-miR-15a/16-1 function significantly increased or decreased luciferase reporter activity of these four genes, respectively. Conclusion: Our findings reveal novel pericytic miR-15a/16-1 mediated angiogenic targets in ischemic stroke.

The crucial role of SETDB1 in structural and functional transformation of epithelial cells during regeneration after intestinal ischemia reperfusion injury.

Su (var) 3-9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75min of ischemia followed by 3, 24, and 48h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.

Theaflavin pretreatment ameliorates renal ischemia/reperfusion injury by attenuating apoptosis and oxidative stress in vivo and in vitro

Oxidative stress-induced apoptosis is an important pathological process in renal ischemia/reperfusion injury (RIRI). Theaflavin (TF) is the main active pigment and polyphenol in black tea. It has been widely reported because of its biological activity that can reduce oxidative stress and protect against many diseases. Here, we explored the role of theaflavin in the pathological process of RIRI. In the present study, the RIRI model of 45 min ischemia and 24 h reperfusion was established in C57BL/6 J male mice, and theaflavin was used as an intervention. Compared with the RIRI group, the renal filtration function, renal tissue damage and antioxidant capacity of the theaflavin intervention group were significantly improved, while the level of apoptosis was reduced. TCMK-1 cells were incubated under hypoxia for 48 h and then reoxygenated for 6 h to simulate RIRI in vitro. The application of theaflavin significantly promoted the translocation of p53 from cytoplasm to nucleus, upregulated the expression of glutathione peroxidase 1 (GPx-1) in cells, and inhibited oxidative stress damage and apoptosis. Transfection with p53 siRNA can partially inhibit the effect of theaflavin. Thus, theaflavin exerted a protective effect against RIRI by inhibiting apoptosis and oxidative stress via regulating the p53/GPx-1 pathway. We conclude that theaflavin has the potential to become a candidate drug for the prevention and treatment of RIRI.

The mechanism of intestinal stem cells differentiation after ischemia-reperfusion injury in a rat model.

Notch and Wnt/β-catenin signaling are responsible for regulation of intestinal stem cells (ISCs) proliferation and differentiation. The purpose of the study was to evaluate Wnt/β-catenin and Notch signaling roles in regulation of ISC differentiation following ischemia-reperfusion (IR) injury in a rat. Rats were assigned into two groups: Sham rats underwent laparotomy without vascular intervention and IR rats underwent occlusion of SMA and portal vein for 20min followed by 48h of reperfusion. Wnt/β-catenin and Notch-related gene expression were determined using Real-Time PCR. Enterocyte proliferation, differentiation and Wnt-related proteins were determined by immunohistochemistry. IR rats demonstrated a significant decrease in β-catenin gene expression, a decrease in cyclin D1 and β-catenin positive cells in jejunum and ileum compared to Sham rats. IR rats demonstrated a significant increase in Notch-related gene expression in jejunum and ileum compared to Sham rats. The number of secretory cells was higher mainly in the jejunum and number of absorptive cells was significantly lower in jejunum and lower in ileum in IR rats compared to Sham rats. Intestinal stem-cell differentiation is toward secretory cells 48h after IR injury; however, Wnt/β-catenin pathway inhibition and Notch-related gene expression stimulation suggest crosstalk between pathways.