Rat Model Of Ischemia-reperfusion Research Articles

#829 Renoprotective effects of laxative linaclotide: inhibition of acute kidney injury and fibrosis in a rat model of renal ischemia-reperfusion

Abstract Background and Aims Acute kidney injury caused by renal ischemia/reperfusion (I/R) is a major clinical problem in many aspects of urology fields such as kidney transplantation and partial nephrectomy, but there is no effective treatment. Linaclotide, a guanylate cyclase C agonist clinically approved as a laxative, has recently been shown to be renoprotective in a chronic kidney disease (CKD) model. Therefore, the present study investigated the possibility that the renoprotective effects of linaclotide in renal I/R may extend beyond its gastrointestinal effects. Method The left renal pelvis of adult male Wistar rats was clamped for 45 minutes to induce renal I/R injury. Kidney tissue, colon, and blood were collected 24 hours and 14 days later. Linaclotide was administered for 14 days preoperatively and until postoperative tissue collection. Tissue sections were stained with standard histologic techniques such as HE, Sirius Red, F4/80, etc. As genes associated with I/R, TGF-β and related fibrosis markers α-SMA, MMP2, TIMP1 and inflammatory cytokines TNF, IL-1β and IL-6 were analyzed by quantitative PCR analysis. Results Serum creatinine levels decreased significantly after linaclotide administration, indicating improved renal function. Histologic examination showed less tubular damage and less collagen deposition on Sirius Red staining. Further analysis showed decreased expression of TGF-β and related fibrosis markers α-SMA, MMP2 and TIMP1, suggesting downregulation of the fibrotic TGF-β pathway by linaclotide. Furthermore, 1 day after I/R injury, linaclotide significantly reduced macrophage infiltration and suppressed key proinflammatory cytokines such as TNF, IL-1β, and IL-6. Conclusion These results suggest that linaclotide, with its established safety profile, could extend its benefits beyond gastrointestinal issues and potentially serve as a therapeutic intervention in many aspects of kidney surgeries. In addition, it could provide immediate and practical insights into the selection of laxatives for the management of patients with AKI or CKD, regardless of cause, and for those receiving dialysis or transplant therapy.

#604 Oral administration of UNI-494 ameliorates acute kidney injury in a rat model of delayed graft function

Abstract Background and Aims There are no FDA approved drugs for the treatment of acute kidney injury (AKI), which affects 10-15% of hospitalized patients and often results in renal transplantation or lifelong dialysis. UNI-494 is a novel nicotinamide ester derivative and selective mitochondrial ATP-sensitive potassium channel activator that may be beneficial for several disease states, including AKI. We present results from a study evaluating the in vivo efficacy of oral (PO) UNI-494 in the unilateral renal ischemia-reperfusion (I/R) rat model of AKI, which is a well-established model of delayed graft function (DGF) (Cavaille-Coll, 2013). Method Rats were anesthetized, the right kidney was removed, and I/R was induced in three groups of rats by clamping the renal vessels in the left kidney for 30 minutes; a fourth group had no surgery (sham group) without clamping the renal vessels. 50 mg/kg or 100 mg/kg UNI-494 were administered PO 30 minutes prior to I/R. One I/R group and the sham group were not treated. After 24 hours of reperfusion in metabolic cages, blood samples were collected for serum creatinine (sCr), and urinary samples were collected for albumin-creatinine ratio (ACR) and the tubular injury marker neutrophil gelatinase-associated lipocalin (NGAL). The clamped left kidney was collected and processed for histology for tubular injury scores. Results In this study, I/R induced significant increases of sCr, ACR, NGAL, and proximal convoluted tubular injury scores in the vehicle treated I/R group vs No I/R sham group. A single PO dose of UNI-494 at 50 mg/kg/PO or 100 mg/kg/PO reduced the kidney functional markers sCr and ACR, the tubular injury marker (NGAL), and proximal tubular injury scores (all p < 0.001, Fig. 1). Conclusion The results indicate that UNI-494 dose dependently prevented AKI markers and proximal tubular injury in a rat model of AKI and therefore is a potential candidate for the prevention of DGF and other AKI clinical conditions.

Penehyclidine hydrochloride alleviates lung ischemia-reperfusion injury by inhibiting pyroptosis

ObjectiveThe aim of this research was to examine how penehyclidine hydrochloride (PHC) impacts the occurrence of pyroptosis in lung tissue cells within a rat model of lung ischemia-reperfusion injury.MethodsTwenty-four Sprague Dawley (SD) rats, weighing 250 g to 270 g, were randomly distributed into three distinct groups as outlined below: a sham operation group (S group), a control group (C group), and a test group (PHC group). Rats in the PHC group received a preliminary intravenous injection of PHC at a dose of 3 mg/kg. At the conclusion of the experiment, lung tissue and blood samples were collected and properly stored for subsequent analysis. The levels of malondialdehyde, superoxide dismutase, and myeloperoxidase in the lung tissue, as well as IL-18 and IL-1β in the blood serum, were assessed using an Elisa kit. Pyroptosis-related proteins, including Caspase1 p20, GSDMD-N, and NLRP3, were detected through the western blot method. Additionally, the dry-to-wet ratio (D/W) of the lung tissue and the findings from the blood gas analysis were also documented.ResultsIn contrast to the control group, the PHC group showed enhancements in oxygenation metrics, reductions in oxidative stress and inflammatory reactions, and a decrease in lung injury. Additionally, the PHC group exhibited lowered levels of pyroptosis-associated proteins, including the N-terminal segment of gasdermin D (GSDMD-N), caspase-1p20, and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3).ConclusionPre-administration of PHC has the potential to mitigate lung ischemia-reperfusion injuries by suppressing the pyroptosis of lung tissue cells, diminishing inflammatory reactions, and enhancing lung function. The primary mechanism behind anti-pyroptotic effect of PHC appears to involve the inhibition of oxidative stress.

Intrathecal Anesthesia Prevents Ventricular Arrhythmias in Rats with Myocardial Ischemia/Reperfusion

Introduction: Ventricular arrhythmia is commonly provoked by acute cardiac ischemia through sympathetic exaggeration and is often resistant to anti-arrhythmic therapies. Thoracic epidural anesthesia has been reported to terminate fatal ventricular arrhythmia; however, its underlying mechanism is unknown. Methods: Rats were randomly divided into four groups: sham, sham plus bupivacaine, ischemia/reperfusion (IR), and IR plus bupivacaine groups. Bupivacaine (1 mg/mL, 0.05 mL/100 g body weight) was injected intrathecally into the L5–L6 intervertebral space prior to establishing a myocardial IR rat model. Thereafter, cardiac arrhythmia, cardiac function, myocardial injury, and electrical activities of the heart and spinal cord were evaluated. Results: Intrathecal bupivacaine inhibited spinal neural activity, improved heart rate variability, reduced ventricular arrhythmia score, and ameliorated cardiac dysfunction in IR rats. Furthermore, intrathecal bupivacaine attenuated cardiac injury and myocardial apoptosis and regulated cardiomyocyte autophagy and connexin-43 distribution during myocardial IR. Conclusion: Our results indicate that intrathecal bupivacaine blunts spinal neural activity to prevent cardiac arrhythmia and dysfunction induced by IR and that this anti-arrhythmic activity may be associated with regulation of autonomic balance, myocardial apoptosis and autophagy, and cardiac gap junction function.

Rebaudioside B Attenuates Lung Ischemia-reperfusion Injury-associated Apoptosis and Inflammation.

At present, no proven effective treatment is available for Lung Ischemiareperfusion Injury (LIRI). Natural compounds offer promising prospects for developing new drugs to address various diseases. This study sought to explore the potential of Rebaudioside B (Reb B) as a treatment compound for LIRI, both in vivo and in vitro. This study involved utilizing the human pulmonary alveolar cell line A549, consisting of epithelial type II cells, subjected to Oxygen-glucose Deprivation/recovery (OGD/R) for highthroughput in vitro cell viability screening. The aim was to identify the most promising candidate compounds. Additionally, an in vivo rat model of lung ischemia-reperfusion was employed to evaluate the potential protective effects of Reb B. Through high-throughput screening, Reb B emerged as the most promising natural compound among those tested. In the A549 OGD/R models, Reb B exhibited a capacity to enhance cell viability by mitigating apoptosis. In the in vivo LIRI model, pre-treatment with Reb B notably decreased apoptotic cells, perivascular edema, and neutrophil infiltration within lung tissues. Furthermore, Reb B demonstrated its ability to attenuate lung inflammation associated with LIRI primarily by elevating IL-10 levels while reducing levels of IL-6, IL-8, and TNF-α. The comprehensive outcomes strongly suggest Reb B's potential as a protective agent against LIRI. This effect is attributed to its inhibition of the mitochondrial apoptotic pathway and its ability to mitigate the inflammatory response.

The possible protective effect of l-cysteine in a rat model of sciatic nerve ischemia-reperfusion: A possible role for NRF1 and Caspase 3; Biochemical, Histological, and Immunohistochemical study

Organ damage brought on by ischemia is exacerbated by the reperfusion process. L-cysteine is a semi-essential amino acid that acts as a substrate for cystathionine-β-synthase in the central nervous system. The aim of this study was to investigate the possible protective effects of L- cysteine against the structural and biochemical changes that occur in the rat sciatic nerve after ischemia reperfusion (I/R) and to address some of the underlying mechanisms of these effects. Rats were divided into 4 groups: sham, l-cysteine, I/R, and l-cysteine- I/R groups. Specimens of sciatic nerve were processed for biochemical, histological, and immunohistochemical assessment. The results showed in I/R group, a significant increase in malondialdehyde with a significant decrease in both Nuclear respiratory factor-1 (NRF1) and superoxide dismutase levels. Moreover, with histological alteration. There was a significant increase in the mean surface area fraction of anti-caspase immunopositive cells as well as a significantdecrease in mean surface area fraction of anti-CD 34 immunopositive cells. In contrast, the l-cysteine- I/R group showed amelioration of these biochemical, structural, and immunohistochemical changes. To the best of our knowledge, this is the first study showed the protective effects of l-cysteine in sciatic nerve I/R via NRF1and caspase 3 modulation as well as telocyte activation.

Renoprotective effects of laxative linaclotide: Inhibition of acute kidney injury and fibrosis in a rat model of renal ischemia-reperfusion

Ischemia-reperfusion (I/R) leads to tissue damage in transplanted kidneys, resulting in acute kidney injury (AKI) and chronic graft dysfunction, which critically compromises transplant outcomes, such as graft loss. Linaclotide, a guanylate cyclase C agonist clinically approved as a laxative, has recently been identified to exhibit renoprotective effects in a chronic kidney disease (CKD) model. This study evaluates the therapeutic effects of linaclotide on AKI triggered by I/R in a rat model with an initial comparison with other laxatives. Here, we show that linaclotide administration resulted in substantial reduction in serum creatinine levels, reflective of enhanced renal function. Histological examination revealed diminished tubular damage, and Sirius Red staining confirmed less collagen deposition, collectively indicating preserved structural integrity and mitigation of fibrosis. Further analysis demonstrated lowered expression of TGF-β and associated fibrotic markers, α-SMA, MMP2, and TIMP1, implicating the downregulation of the fibrogenic TGF-β pathway by linaclotide. Furthermore, one day after I/R insult, linaclotide profoundly diminished macrophage infiltration and suppressed critical pro-inflammatory cytokines such as TNF, IL-1β, and IL-6, signifying its potential to disrupt initial inflammatory mechanisms integral to AKI pathology. These findings suggest that linaclotide, with its established safety profile, could extend its benefits beyond gastrointestinal issues and potentially serve as a therapeutic intervention for organ transplantation. Additionally, it could provide immediate and practical insights into selecting laxatives for managing patients with AKI or CKD, regardless of the cause, and for those receiving dialysis or transplant therapy.

A novel 14-amino acid peptide from yak alleviates kidney damage in the rat model of myocardial ischemia-reperfusion

Myocardial ischemia-reperfusion (MI/R) not only causes cardiac damage, but also causes severe renal damage. T8 is the 8th peptide identified by peptiomics in digested yak milk dregs and our previous studies showed that T8 had strong antioxidant activity. This study evaluated the protective effects and molecular mechanisms of MI/R-induced kidney injury in rats. Our results indicated that peptide T8 could increase ejection fraction (EF) and shortened fraction (FS), and degraded ST segment elevation, which ameliorated cardiac function in the MI/R rats. Peptide T8 could increase activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and decreased the contents of malondialdehyde (MDA), serum creatinine (Scr) and blood urea nitrogen (BUN), which could ameliorate renal insufficiency. Peptide T8 and kidney injury-related targets in the MI/R-damaged rats were obtained from network pharmacology analysis. KEGG analysis revealed that T8 might affect 54 signaling pathways and 13 key targets were obtained by PPI network analysis. The binding affinity of peptide T8 to Keap1 was found to be the strongest by molecular docking analysis. In the H2O2-induced HEK293 cell model, peptide T8 decreased reactive oxygen species (ROS) content and changed the expression ratio of Bcl-2 and Bax, thereby inhibiting mitochondria-dependent apoptosis. Further studies indicated that T8 could regulate Nrf2 pathway and downstream target genes such as NQO1, which could reduce oxidative stress-induced damage. These results suggest that peptide T 8 can exert renal protection via regulating Nrf2 pathway and apoptosis-related genes.

Amplified P2X3 pathway activity in muscle afferent dorsal root ganglion neurons and exercise pressor reflex regulation in hindlimb ischaemia-reperfusion.

Hindlimb ischaemia-reperfusion (IR) is among the most prominent pathophysiological conditions observed in peripheral artery disease (PAD). An exaggerated arterial blood pressure (BP) response during exercise is associated with an elevated risk of cardiovascular events in individuals with PAD. However, the precise mechanisms leading to this exaggerated BP response are poorly elucidated. The P2X3 signalling pathway, which plays a key role in modifying the exercise pressor reflex (EPR), is the focus of the present study. We determined the regulatory role of P2X3 on the EPR in a rat model of hindlimb IR. In vivo and in vitro approaches were used to determine the expression and functions of P2X3 in muscle afferent nerves and EPR in IR rats. We found that in IR rats there was (1) upregulation of P2X3 protein expression in the L4-6 dorsal root ganglia (DRG); (2) amplified P2X currents in isolated isolectin B4 (IB4)-positive muscle DRG neurons; and (3) amplification of the P2X-mediated BP response. We further verified that both A-317491 and siRNA knockdown of P2X3 significantly decreased the activity of P2X currents in isolated muscle DRG neurons. Moreover, inhibition of muscle afferents' P2X3 receptor using A-317491 was observed to alleviate the exaggerated BP response induced by static muscle contraction and P2X-induced BP response by α,β-methylene ATP injection. P2X3 signalling pathway activity is amplified in muscle afferent DRG neurons in regulating the EPR following hindlimb IR.

Effect of sugammadex, rocuronium and sevoflurane on oxidative stress and apoptosis in cerebral ischemia reperfusion model in rats.

Cerebral ischemia-reperfusion (I/R) injury causes neurological dysfunction and cell death. Sugammadex, as a large molecule, is normally difficult to pass through the blood-brain barrier (BBB). In ischemia, molecules can pass into the brain tissue. In this study, we aimed to evaluate the effect of sugammadex in the presence of cerebral I/R damage in rats with a general anesthesia model with sevoflurane and rocuronium. Rats were divided into 7 groups; Group 1 (Control), Group 2 (Sham), Group 3 (Sevoflurane), Group 4 (Sugammadex), Group 5 (Sevoflurane + Rocuronium), Group 6 (Sevoflurane + Sugammadex), Group 7 (Sevoflurane + Rocuronium + Sugammadex). Brain tissues of rats with cerebral I/R damage with bilateral carotid occlusion were removed. Tissue Malondialdehyde (MDA), Myeloperoxidase (MPO), and Superoxide dismutase (SOD) levels were examined with ELISA and apoptosis was examined by Caspase-3. The number of caspase-3 positive cells decreased the most in Group 4 compared to the other groups. Group 4's mean MDA and MPO levels were lower than Group 2. There was no significant difference in terms of SOD levels. The apoptotic effect of sugammadex was lowest compared to other agent groups, and it did not increase oxidative damage as much as the other groups.

The Anti-Apoptotic Effect of Ranolazine on Cerebral Protection during Cardiopulmonary Bypass and Carotid Artery Surgery.

We aimed to determine the usability of ranolazine (Rn) as a neuroprotective during cardiac surgeries and carotid artery interventions where cerebral blood flow is interrupted. Female Wistar albino rats were used. The rats were divided into 4 groups of 8 rats each. The first group (Group 1) was the control group. Group 2 underwent ischemia induction but was not treated with Rn. Group 3 received 25 mg/kg/day and Group 4 50 mg/kg/day Rn intraperitoneally, starting 3 days before ischemia induction. Bilateral carotid arteries were explored and clamped simultaneously. Ischemia was induced for 15 minutes. After 72 hours, the experimental animals were sacrificed. Superoxide dismutase, alkaline phosphatase, and interleukin 6 levels were similar among the 4 groups. Acetylcholine esterase (Group 3: p = 0.007, Group 4: p = 0.002), tumor necrosis factor-alpha (Group 4: p = 0.01), and annexin V (Group 3: p = 0.001) levels were statistically significantly lower in the Rn-treated groups. Malondialdehyde (Group 3: p = 0.003, Group 4: p = 0.009), reduced glutathione (Group 4: p = 0.04), acid phosphatase (Group 3: p = 0.04), noradrenaline (Group 3: p = 0.01), and Bcl-2 (Group 4: p = 0.004) levels were significantly higher in the Rn-treated groups. The results of this study demonstrated the antiapoptotic effect of Rn in a brain ischemia-reperfusion model of rats receiving Rn before the procedure.

Analysis of lipid metabolites derived from gut microbiota in ischemia-reperfusion model.

Disruption of intestinal barrier caused by intestinal ischemia due to hemorrhagic shock is associated with the pathogenesis of multiple organ dysfunction (MOD) after severe trauma. Mesenteric lymph (ML) plays an important role as a route for transporting inflammatory mediators, including lipids. Postbiotics, such as 10-hydroxy-cis-12-octadecenoic acid (HYA), have received much attention as a treatment option. However, the relationship between postbiotics and MOD has yet to be clarified. The aim of the present study was to analyze lipid metabolites derived from gut microbiota in the intestinal ischemia-reperfusion (IR) rat model. Male Sprague-Dawley rats underwent laparotomy, and their ML duct and superior mesenteric artery were exposed. The superior mesenteric artery was clamped for 60 minutes, followed by 120 minutes of reperfusion. The ML and the plasma were collected before and after intestinal IR. Lipids were extracted from plasma and ML, and liquid chromatography-tandem mass spectrometry was performed. The concentration of linoleic acid in plasma samples was not different before and after IR; however, the linoleic acid concentration in the ML samples increased after intestinal IR. Eicosapentaenoic acids and docosahexaenoic related to linoleic acids showed similar changes with IR-induced increase in the ML. The concentration of HYA, a linoleic acid-derived bioactive metabolite produced by gut bacteria, was high in ML samples, while that in plasma samples was low. The relative increase rate of HYA in ML samples after IR was higher than that of the plasma samples (the ML samples: relative increase, 3.23 ± 1.36; the plasma samples: relative increase, 0.95 ± 0.35; n = 3, p = 0.048). The present study demonstrated increased linoleic acids and high concentrations of HYA, lipid metabolites derived from gut bacteria in the ML after intestinal IR. These findings may contribute to clarifying the relation between gut microbiota and MOD after severe trauma.

Resveratrol Inhibited Nanoparticles Stromal Interaction Molecule 2 in Regulating miR-20b-5p Signaling Pathway to Improve Mitochondrial Function During Myocardial Ischemia-Reperfusion Injury

Myocardial ischemia-reperfusion (IR) in diabetes can cause severe myocardial damages. In this study, resveratrol (RES) nanoparticles were used in diabetic myocardial IR rat model injury to assess its effect on mitochondria function. Rat models were assigned into sham group, IR group, IR+RES group, IR+RES+mir-NC group, and IR+RES+miR-20b-5p inhibitor group. Myocardial infarction area was measured by TTC in 5 rats from each group, and ultrasound was used to detect left ventricular end-systolic internal diameters (LVIDs) and end-diastolic internal diameters (LVIDd), along with analysis of cardiomyopathy by HE staining. miR-20b-5p and Stromal interaction molecule 2 (STIM2) expressions, cardiomyocyte proliferation, apoptosis, cell viability, mitochondrial function, and relationship between miR-20b-5p and STIM2 were also analyzed. Resveratrol (RES) nanoparticles were prepared successfully. Myocardial infarct size, LVIDd and LVIDs of rats in IR+RES group decreased (vs. IR group), but were higher than sham group. miR-20b-5p expression also increased in the IR+RES group (vs. IR group), and the above indicators were decreased by the miR-20b-5p inhibitor (vs. IR+RES group, P <0.05). The myocardial changes in rats from the IR+RES+miR-20b-5p antagomir group were smaller (vs. IR group), while STIM2 expression was lower than in the IR group after using the RES nanoparticles (P < 0.05). RES nanoparticles can thus enhance mitochondrial function and cell viability of cardiomyocytes, increasing cell proliferation rate and decreasing apoptosis rate (vs. IR group,P <0.05). After using the RES nanoparticles to interfere with myocardial IR in the diabetic rats, they were found to inhibit STIM2 and improve mitochondria by regulating miR-20b-5p signaling pathway.

Multipotent mesenchymal stromal cells of various origins reduce reactive gliosis in the hippocampal CA1 area during acute ischemia-reperfusion of the rat brain

Neuroprotective therapy in acute cerebrovascular disorders is directly aimed at preserving neurons in the penumbra zone, but it also emphasizes the importance of gliogenesis in the affected area. Glial cells exhibit rapid reactivity and are highly sensitive to cerebral ischemic damage. Recent experimental studies have demonstrated the successful application of multipotent mesenchymal stromal cells (MMSCs) in stroke to modulate microglial activation. The aim of the study was to investigate the impact of MMSCs of different origins, MMSC lysate, and citicoline on glial components in a model of cerebral ischemia-reperfusion in rats. Materials and methods. The experiments were conducted on 190 male Wistar rats aged 4 months, weighing 160-190 g. After modeling cerebral ischemia-reperfusion through bilateral 20-minute occlusion of the internal carotid arteries, rats were intravenously administered MMSCs derived from human Wharton’s jelly, human adipose tissue, and rat Wharton’s jelly at a dose of 106 cells per animal. Other groups received fetal rat fibroblasts (106 cells/animal in 0.2 ml of physiological solution) and lysate from human Wharton’s jelly at a dose of 0.2 ml per animal. Control animals received 0.2 ml of physiological solution intravenously. The last group of rats received a single dose of the reference drug citicoline at a dose of 250 mg/kg. On the 7th and 14th days, the area and fluorescence intensity of cells expressing markers of astrocytes (GFAP), microglia (Iba1), and oligodendrocytes (Rip) were quantitatively assessed in CA1 hippocampal region slices using immunohistochemical examination and confocal microscopy. Results. On the 7th and 14th days after cerebral ischemia-reperfusion in rats, the intensity of fluorescence of GFAP-positive astrocytes and Iba1-positive microglial cells increased, indicating pronounced reactive astrogliosis and microglial activation in the CA1 region of the hippocampus. Meanwhile, ischemia-reperfusion had a significant impact on the content of Rip-positive oligodendrocytes in brain slices. The application of all treatments (transplantation of MMSCs of different origins, their lysate, or the reference drug citicoline) had a cytoprotective effect, reducing reactive astrogliosis and microgliosis both on the 7th and 14th days after injury. The best result was demonstrated with the treatment using MSCs from human Wharton’s jelly.s. Conclusion. Cerebral ischemia-reperfusion induces reactive gliosis through the activation of GFAP- and Iba1-positive glial cells in all layers of the hippocampus. The application of MSCs from human Wharton’s jelly and fetal rat fibroblasts significantly reduces its intensity on both the 7th and 14th days after injury modeling.

Temporal proteomic and phosphoproteomic profiling of the left ventricle in a novel rat model of ischemia/reperfusion with or without ischemic preconditioning

Abstract Background The relationship between ischemic preconditioning (IPC) and myocardial stunning is incompletely understood. Preconditioning has been shown to protect against ischemic injury, whereas myocardial stunning, i.e. transient contractile dysfunction in the setting of ischemia, can lead to severe heart failure and has been considered a deleterious process. To understand this relationship, we recently developed a novel rat model of ischemia-reperfusion (I/R) injury in the presence or absence of IPC. In the presence of IPC (2 cycles of 5 min of I/R followed by 13.5 min of ischemia), we observe a profound myocardial stunning at 4h of reperfusion which completely resolves at 48h. In the absence of IPC, we observe necrosis at 4h of reperfusion and sustained injury at 48h. Despite extensive studies on IPC and myocardial stunning in the setting of I/R, the molecular basis of the relationship between these phenomena are still unknown. Purpose To investigate alterations in protein expression after IPC and myocardial stunning in a novel rat model of I/R injury. Methods We utilized tandem mass tags and mass spectrometry to identify temporal changes in the proteomic- and phosphoproteomic-profiles in left ventricular (LV) tissue from rat hearts following 13.5 min of ischemia and 4h of reperfusion in the presence of absence of IPC. Results Proteomics analysis of the LV tissues collected at baseline showed that 115 proteins were differentially expressed between IPC and non-IPC groups. We observed abundant changes in sarcoplasmic proteins, sarcoplasmic reticulum-related proteins, and actin-based cell projection proteins. Most of these proteins are implicated in arylesterase activity, haemoglobin binding, and nitrogen metabolism. At 4h, differentially expressed proteins were predominantly related to wound healing, coagulation, and lipase activity. Phosphoproteomic analysis at baseline showed that 686 phosphosites were differentially regulated between IPC and non-IPC groups. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins, Z-disc, I-band, contractile fiber associated with actin binding, and the thyroid hormone signalling pathway. At 4h, 520 phosphosites differed between IPC and non-IPC rats; these phosphosites were abundant in contractile fiber proteins, myofibril proteins, and sarcomere proteins associated with calcium signalling and cardiomyopathies. Conclusions This study demonstrates marked changes in proteins and phosphosites related to lipase activity, haemoglobin binding, calcium signalling, and the cardiac contractile apparatus in IPC versus non-IPC groups. This extensive catalogue of novel proteins and phosphosites associated with IPC and myocardial stunning in the setting of I/R injury sets the stage for future manipulation of myocardial stunning and preconditioning in both experimental and clinical settings.

Abstract 12634: Amplified P2x 3 Pathway Activity in IB4 Positive Muscle DRG Neurons and Exercise Pressor Reflex Regulation in Hindlimb Ischemia-Reperfusion

Background: Hindlimb ischemia-reperfusion (IR) represents one of the most prominent pathological conditions observed in peripheral artery disease (PAD). Arterial blood pressure response (BP) during exercise, partly driven by the exaggerated exercise pressor reflex (EPR), is associated with an elevated risk of cardiovascular events in PAD patients. However, the precise underlying mechanisms leading to this exaggerated BP response are poorly elucidated. The purinergic P2X 3 signaling pathway, which plays a key role in modifying the EPR, was the focus of the present study. Objectives: To examine the regulatory role of P2X 3 on the EPR in a rat model of hindlimb IR, which was induced by 6 hours’ ischemia followed by 18 hours’ reperfusion (referred to as IR rats). Results: We found that, in IR rats, there were: 1) upregulation of P2X 3 protein expression in the L4-6 dorsal root ganglion (DRG) (1.48±0.16 in IR rats/n=8 vs. 1.00±0.41 in sham rats/n=9; P<0.05), which was localized in IB4 positive DRG neurons by the method of immunohistochemistry, 2) amplified P2X currents in isolated muscle DRG neurons (P2X transient currents: 1281.64±776.74 pA in IR rats/n=25, vs. 717.91±556.2 pA in sham rats/n=9, P<0.05; and sustain currents: 466.94±168.68 pA in IR rats/n=11, vs. 271.49±200.90 pA in sham rats/n=8, P<0.05); 3) amplification of P2X-mediated BP response (29±8 mmHg in IR rats/n=12 vs. 21±4 mmHg in sham rats/n=7; P<0.05); and 4) alleviation of the exaggerated BP response induced by static muscle contraction (18±8 mmHg in IR +A317491/n=8, vs. 29±11 mmHg in IR/n=7, P<0.05) and P2X-mediated BP response by α, β-me-ATP injection (15±4 mmHg in IR+A317491 /n=6 vs. 23±7 mmHg in IR/n=4, P<0.05) following the inhibition of muscle afferents’ P2X 3 receptor by A-317491. We further verified that both A-317491 and siRNA knockdown of P2X 3 significantly decreased the activity of P2X currents in the isolated muscle DRG neurons (all P<0.05 vs. their respective controls). Conclusion: With the above evidence from multiple approaches, we concluded that the P2X 3 signaling pathway activity is amplified following hindlimb IR via the enhancing expression and activity of P2X 3 receptors in IB4 positive muscle DRG afferents. This contributes to the exacerbated EPR responses in PAD.

Protective Effects of Diospyros Kaki Leaves Extract against Oxidative Damage and Inflammation in the Testicular Ischemia-Reperfusion Model in Rats

Protective Effects of Diospyros Kaki Leaves Extract against Oxidative Damage and Inflammation in the Testicular Ischemia-Reperfusion Model in Rats

Overexpression of miR-124 in astrocyte improves neurological deficits in rat with ischemic stroke via DLL4 modulation

BackgroundAstrocytes have been demonstrated to undergo conversion into functional neurons, presenting a promising approach for stroke treatment. However, the development of small molecules capable of effectively inducing this cellular reprogramming remains a critical challenge. MethodsInitially, we introduced a glial cell marker gene, GFaABC1D, as the promoter within an adeno-associated virus vector overexpressing miR-124 into the motor cortex of an ischemia-reperfusion model in rats. Additionally, we administered NeuroD1 as a positive control. Lentiviral vectors overexpressing miR-124 were constructed and transfected into primary rat astrocytes. We assessed the cellular distribution of GFAP, DCX, and NeuN on days 7, 14, and 28, respectively. ResultsIn rats with ischemic stroke, miR-124-transduced glial cells exhibited positive staining for the immature neuron marker doublecortin (DCX) and the mature neuron marker NeuN after 4 weeks. In contrast, NeuroD1-overexpressing model rats only expressed NeuN, and the positive percentage was higher in co-transfection with miR-124 and NeuroD1. Overexpression of miR-124 effectively ameliorated neurological deficits and motor functional impairment in the model rats. In primary rat astrocytes transduced with miR-124, DCX was not observed after 7 days of transfection, but it appeared at 14 days, with the percentage further increasing to 44.6% at 28 days. Simultaneously, 15.1% of miR-124-transduced cells exhibited NeuN positivity, which was not detected at 7 and 14 days. In vitro, double fluorescence assays revealed that miR-124 targeted Dll4, and in vivo experiments confirmed that miR-124 inhibited the expression of Notch1 and DLL4. ConclusionsThe overexpression of miR-124 in astrocytes demonstrates significant potential for improving neurological deficits following ischemic stroke by inhibiting DLL4 expression, and it may facilitate astrocyte-to-neuronal transformation.

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis

Background: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. Methods: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and β-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. Results: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and β-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. Discussion: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Study on the anti-mitochondrial apoptosis mechanism of Erigeron breviscapus injection based on UPLC-Q-TOF-MS metabolomics and molecular docking in rats with cerebral ischemia-reperfusion injury

Ethnopharmacological relevanceErigeron breviscapus is a common medicine of eight ethnic minorities, including Miao, Naxi, and Yi. As early as the Ming Dynasty (AD 1368–1644), Lanmao's Materia Medica of Southern Yunnan (AD 1436) recorded that the medicine is used for the treatment of “Zuo tan you huan.” In modern pharmacological research, Erigeron breviscapus injection is the most commonly used preparation in the treatment of ischemic stroke caused by acute cerebral infarction, but its mechanism of action in the treatment of ischemic stroke is not well understood. Aim of the studyIn this study, a metabonomics study based on ultraperformance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) was used in investigating the effect of a traditional Chinese medicine preparation Erigeron breviscapus injection on the rat model of focal cerebral ischemia-reperfusion and the affinity of its main components with the targets of mitochondrial apoptotic pathways. Materials and methodsThis study used molecular docking technology to verify the effective binding ability of main effective components of Erigeron breviscapus injection to target proteins related to mitochondrial apoptosis pathway. This study developed a metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of focal cerebral ischemia-reperfusion model group and healthy control group was achieved. ResultsErigeron breviscapus injection can significantly reduce the area of cerebral infarction, improve tissue morphological lesion in rats, and can increase the number of Nissl bodies. It may be a promoting factor by inhibiting hippocampal nerve cell apoptosis and Bax protein expression and by exerting effects against ischemia reperfusion after the induction of apoptosis. Thus, it plays a role in brain protection. Moreover, it can considerably promote the recovery of neurological deficiency signs in advance. Meanwhile, Erigeron breviscapus decreased malondialdehyde content and T-NOS activity. Its curative effect from strong to weak order: low dose > high dose > medium dose. The representative components of Erigeron breviscapus have good affinity with the active sites of mitochondrial apoptosis-related proteins. Metabolomics found that the potential biomarkers regulated by breviscapine are kynurequinolinic acid, succinylornithine, and leucine proline. It is speculated that it may participate in TRP–kynurequinolinic acid and succinylornithine–urea cycle–NO metabolic pathways. ConclusionsThis paper revealed the potential biomarkers and metabolic pathways regulated by Erigeron breviscapus. It was speculated that the mechanism is related to its inhibition of mitochondrion-mediated apoptosis. Erigeron breviscapus could restore the metabolic profiles of the model animals to normal animal levels. The mechanism may be related to the potential biomarkers of quinolinic acid, succinylornithine, and leucine proline and the metabolic pathways involved. However, the exact mechanism by which Erigeron breviscapus inhibits mitochondrion-mediated apoptosis remains to be further explored.