Ischemia-reperfusion Group Research Articles

Empagliflozin restores the deregulated cardiac endothelial cell transcriptome in vivo upon myocardial ischemia reperfusion injury and reduces the infiltration of immune cells

Abstract Background/Introduction Empagliflozin (EMPA) is a sodium-glucose co-transporter 2 (SGLT-2) inhibitor with established cardioprotective effects in heart failure but its use in myocardial ischemia-reperfusion (IR) injury is still under investigation. We have shown that chronic administration of EMPA reduces infarct size in normoglycemic mice subjected to IR and that endothelial cells (ECs) are implicated in the protective mechanism. Purpose We aimed to determine EMPA’s distinct effect on three cell populations: cardiac ECs, fibroblasts (FBs) and cardiomyocytes (CMs) in vivo. Methods C57Bl6 male mice were randomized as follows: 1) Control-Sham, 2) Control-IR and 3) EMPA-IR. EMPA group received EMPA 10mg/kg/day for 6 weeks which is a clinically relevant dose and Control groups received vehicle (5% DMSO in normal saline). At the end of the administrations, mice were subjected to 30’I/2hR or sham operation and the ischemic heart was collected to isolate CMs. From the non-CMs populations the cardiac ECs and FBs were sorted. RNA was obtained from the distinct cell populations and 3’ RNA sequencing was applied to investigate the pathways affected by IR and EMPA treatment. An additional cohort of mice was employed to validate the results at mRNA level. In order to examine the effects of EMPA upon prolonged reperfusion, we repeated the experimental protocol, and the mice were sacrificed upon 7 days of R. Based on the RNA sequencing data, the infiltration of the heart with immune cells was examined. Results Comparison between IR and Sham groups revealed that IR alters the transcriptomic profile of ECs and FBs with 576 and 571 genes respectively being significantly deregulated (FDR<0.05). CMs transcriptome was not altered by IR. The comparison between Control-IR and EMPA-IR groups revealed that EMPA does not affect the FBs’ gene expression profile while in CMs, EMPA affects only 3 genes expression (FDR<0.05) related to mitochondrial metabolism. The ECs are mostly affected by EMPA treatment with 211 genes being significantly altered (FDR<0.05). Pathway enrichment analysis in ECs revealed that the deregulated pathways by IR related to extracellular matrix organization, matrix degradation and immune cell adhesion are restored by EMPA treatment. The expression levels of several significant genes of our dataset including Mmp-2, Timp-1, Icam, Vecam, etc was validated in the independent cohort of mice. On the 7th day of R, EMPA reduced the infiltration of neutrophils and inflammatory (Ly6C high) monocytes in the myocardium compared to the Control group indicating that EMPA may affect the remodeling process upon IR. Conclusions We identified ECs and FBs as cell target populations for therapeutic interventions at 2h of R. EMPA pre-treatment reverses the deregulated transcriptome related to extracellular matrix organization, matrix degradation and immune cell adhesion in ECs and eventually reduces the infiltration of immune cells in the ischemic myocardium.

Abstract 12887: Cyclin D2 Overexpressing and Hypoimmunogenic Cardiomyocyte Spheroid Transplantation Induced Endogenous Myocardial Regeneration in the Pig Heart After Myocardial Infarction

Background: hiPSCs derived cardiomyocytes (hiPSC-CMs) have very low proliferative capacity, while patient hiPSC-CMs may have disease phenotypes which compromise therapeutic efficacy and allogenic hiPSC-CMs are expected to induce an immune response during clinical use. In order to solve these problems, we established a hiPSC line by knocking-out two components of the human leukocyte antigen (HLA) and over-expressing myosin heavy chain driven cyclin D2 (CCND2) systems in hiPSCs ( HLA-KO/CCND2-OE hiPSCs). Aim: To determine the therapeutic efficacy and mechanism of the xeno-transplantation of HLA-KO/CCND2-OE hiPSCs derived cardiomyocytes ( HLA-KO/CCND2-OE hiPSC-CMs) in the pig hearts after myocardial ischemia/reperfusion (IR). Methods and Results: HLA-KO/CCND2-OE hiPSC-CM spheroids were differentiated in shaking flasks. Purified HLA-KO/CCND2-OE hiPSC-CM spheroids were intramyocardially injected into pig hearts after IR (the Spheroid Group), while control animals received basal medium injection (the IR Group). Cardiac magnetic resonance imaging (cMRI) showed that pig heart function improved significantly compared to the IR Group at 4 weeks after IR. This finding was associated with a dramatical reduction in the scar size as determined by late gadolinium enhancement measurement. Immunohistology showed that significantly increased porcine CMs expressed cell proliferation markers, such as Ki67, phospho-histone 3 (pH3), and symmetrical Aurora B, and abundant YAP protein expression in porcine CM nuclei in the porcine hearts of the Spheroid Group compared to the IR Group at 1 week after IR. Western Blot analysis of pig heart tissue showed significantly reduced expression of phospho-YAP in the Spheroid Group. snRNAseq showed that number of cycling porcine CMs was > 5 folds in the Spheroid Groups than that in the IR Group. Angiogenic microarray showed that HLA-KO/CCND2-OE hiPSC-CM spheroids produced abundant follistatin which induced YAP protein expression and cytokinesis in CMs. Conclusions: Transplantation of HLA-KO/CCND2-OE hiPSC-CM spheroids effectively recovered porcine heart function, reduced scar size, and induced porcine CM proliferation through modulating YAP protein expression by follistatin secreted by transplanted cells.

Abstract 15334: Laminin Alpha 2 Increases the Protective Effect of Exosomes in Human IPSC-Derived Cardiomyocytes During Ischemia-Reperfusion

Acute myocardial infarction (AMI) is a leading cause of death and disability worldwide. With early treatment, restoration of blood flow to ischemic myocardium can be achieved and therefore reduce mortality risk. Evidence suggests that mesenchymal stromal cell (MSC)-derived exosomes (EXO) promote myocardial repair after AMI due to paracrine activity. Laminin alpha 2 (LAMA2) is a major cardiac extracellular matrix (ECM) component and can be used to produce cardiac-specific EXO. Here we coated MSC-derived EXOs in LAMA2 to enhance their recognition by human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that LAMA2-coated MSC-EXO would confer protective effects on CMs during ischemia-reperfusion. The LAMA2-EXO had a similar yield of particles, with a size range of 130nm as the control (gelatin-EXO). No cytotoxic effect was observed in CMs after 3-day exposure to LAMA2-EXO. CMs were then treated with LAMA2-EXO two hours prior to 4h ischemia (1% O 2 , 5% CO 2 , and glucose-free media). LAMA2-EXO had a 2-fold protective effect compared to non-treatment, and after 1.5h of reperfusion (5% CO 2 , CM-enriched media) CMs treated with LAMA2-EXO recovered faster than the control group. Conversely, due to the laminin 511/411 ECM component of endothelial cells (ECs), we did not see a significant protective effect nor a difference between LAMA2-EXO vs. the control in the EC ischemia-reperfusion group. This is the first report of suggesting the specificity of LAMA2-EXO to treat CM post-exposure to ischemia-reperfusion injury. Overall, these results indicate that LAMA2-EXO can be a promising treatment to target CM survival in ischemic cardiovascular disease.

The effect of Edaravone on lung injury after lower limb ischemiareperfusion in a rat model: Biochemical and histopathological insights

Aim: Lower limb ischemia/reperfusion leads to distant organ dysfunction, notably affecting the lungs, resulting in respiratory failure and significant morbidity and mortality. Edaravone is a new free radical scavenger and has attracted the attention of researchers. This study aims to examine edaravone's influence on lung injury arising from lower limb ischemia-reperfusion. Material and Methods: Forty male Wistar rats were categorized into groups: Sham, Ischemia/Reperfusion (IR), Solvent, and Edaravone. The infrarenal abdominal aorta was clamped for 120 minutes and then reperfused for another 120 minutes. Edaravone was administered 30 minutes before the ischemic event. Then we analyzed serum and lung tissue samples for malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), and nitric oxide (NO) levels. Furthermore, a thorough histopathological assessment was conducted. Results: In the IR group, edaravone notably reduced serum and lung MDA levels. While significant differences in serum SOD and NO levels existed between the IR and edaravone groups, similar differences were not found in the lung tissue samples. There appeared to be no significant impact of edaravone on serum and lung GPx levels. The histopathological investigation revealed that the lung injury score was significantly higher in the IR group compared to the control group, a difference mitigated by edaravone treatment. Conclusion: Our findings suggest that edaravone mitigates lower limb ischemia-reperfusion-induced lung injury, both biochemically and histopathologically. Our findings imply the potential utility of this agent in the context of acute ischemia-reperfusion injury following vascular surgery.

Protective effect of MitoTEMPO against cardiac dysfunction caused by ischemia–reperfusion: MCAO stroke model study

Purpose Neurological impairments are the leading cause of post-stroke mortality, while stroke-related cardiovascular diseases rank second in significance. This study investigates the potential protective effects of MitoTEMPO (2,2,6,6-tetramethyl-4-[[2-(triphenylphosphonio) acetyl] amino]-1-piperidinyloxy, monochloride, monohydrate), a mitochondria-specific antioxidant, against cardiac and neurological complications following stroke. The objective is to assess whether MitoTEMPO can be utilized as a protective agent for individuals with a high risk of stroke. Materials and methods Seventeen-week-old male Wistar Albino rats were randomly assigned to three groups: SHAM, ischemia–reperfusion and MitoTEMPO + ischemia–reperfusion (MitoTEMPO injection 0.7 mg/kg/day for 14 days). The SHAM group underwent a sham operation, while the ischemia–reperfusion group underwent 1-h middle cerebral artery occlusion followed by three days of reperfusion. Afterwards, noninvasive thoracic electrical bioimpedance and electrocardiography measurements were taken, and sample collection was performed for histological and biochemical examinations. Results Our thoracic electrical bioimpedance and electrocardiography findings demonstrated that MitoTEMPO exhibited a protective effect on most parameters affected by ischemia–reperfusion compared to the SHAM group. Furthermore, our biochemical and histological data revealed a significant protective effect of MitoTEMPO against oxidative damage. Conclusions The findings suggest that both ischemia–reperfusion-induced cardiovascular abnormalities and the protective effect of MitoTEMPO may involve G-protein coupled receptor-mediated signaling mechanisms. This study was conducted with limitations including a single gender, a uniform age group, a specific stroke model limited to middle cerebral artery, and pre-scheduled only one ischemia–reperfusion period. In future studies, addressing these limitations may enable the implementation of preventive measures for individuals at high risk of stroke.

Effects of transcranial direct current stimulation on the glutamatergic pathway in the male rat hippocampus after experimental focal cerebral ischemia.

This study aimed to assess the focal cerebral ischemia-induced changes in learning and memory together with glutamatergic pathway in rats and the effects of treatment of the animals with transcranial Direct Current Stimulation (tDCS). One hundred male rats were divided into five groups as sham, tDCS, Ischemia/Reperfusion (IR), IR + tDCS, and IR + E-tDCS groups. Learning, memory, and locomotor activity functions were evaluated by behavioral experiments in rats. Glutamate and glutamine levels, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR1), N-Methyl-D-Aspartate receptors (NMDAR1 and NMDAR2A), vesicular glutamate transporter-1 (VGLUT-1), and excitatory amino acid transporters (EAAT1-3) mRNA expressions in hippocampus tissues were measured. Ischemic areas were analyzed by TTC staining. The increase was observed in IR + tDCS, and IR + E-tDCS groups compared to the IR group while a significant decrease was observed in IR group compared to the sham in the locomotor activity, learning, and memory tests. While glutamate and glutamine levels, AMPAR1, NMDAR1, NMDAR2A, VGLUT1, and EAAT1 mRNA expressions were significantly higher in IR group compared to the sham group, it was found to be significantly lower in IR + tDCS and IR + E-tDCS groups compared to the IR group. EAAT2 and EAAT3 mRNA expressions were significantly higher in IR + tDCS and IR + E-tDCS groups compared to the IR group. Ischemic areas were significantly decreased in IR + tDCS and IR + E-tDCS groups compared to the IR group. Current results suggest that tDCS application after ischemia improves learning and memory disorders and these effects of tDCS may be provided through transporters that regulate glutamate levels.

Neuroprotective effects of ivermectin against transient cerebral ischemia-reperfusion in rats.

Stroke is a leading cause of disability and death worldwide. Ivermectin is a broad-spectrum anti-parasitic agent with potential anti-bacterial, anti-viral, and anti-cancer effects. However, the effects of ivermectin on the brain are poorly described. This study examined the effects of ivermectin on cerebral ischemia-reperfusion (IR) in rats. A rat model of transient global IR was induced by bilateral carotid artery occlusion for 20min. Rats received ivermectin (2mg/kg/day, ip) one hour after inducing cerebral IR for three consecutive days at 24-h intervals. Next, we examined the effects of ivermectin on brain infarction, histopathology, malondialdehyde levels, myeloperoxidase activity, spatial learning and memory, and phospho-AMPK protein levels. The results showed that ivermectin reduced brain infarct size (P < 0.001) and histopathological changes such as cerebral leukocyte accumulation and edema (P < 0.05) compared to untreated rats with IR. Treatment with ivermectin also decreased myeloperoxidase activity (P < 0.01) and malondialdehyde levels (P < 0.05) while increasing AMPK activity (P < 0.001), memory, and learning compared to the untreated IR group. Overall, we show for the first time that ivermectin conferred neuroprotective effects in a rat model of cerebral IR. Our results indicate that three days of treatment with ivermectin reduced brain infarct size, lipid peroxidation, and myeloperoxidase activity and improved memory and learning in rats with cerebral IR. These effects likely occurred via AMPK-dependent mechanisms.

Segmental and transmural motion of the rat myocardium estimated using quantitative ultrasound with new strategies for infarct detection.

Introduction: The estimation of myocardial motion abnormalities has great potential for the early diagnosis of myocardial infarction (MI). This study aims to quantitatively analyze the segmental and transmural myocardial motion in MI rats by incorporating two novel strategies of algorithm parameter optimization and transmural motion index (TMI) calculation. Methods: Twenty-one rats were randomly divided into three groups (n = 7 per group): sham, MI, and ischemia-reperfusion (IR) groups. Ultrasound radio-frequency (RF) signals were acquired from each rat heart at 1 day and 28 days after animal model establishment; thus, a total of six datasets were represented as Sham1, Sham28, MI1, MI28, IR1, and IR28. The systolic cumulative displacement was calculated using our previously proposed vectorized normalized cross-correlation (VNCC) method. A semiautomatic regional and layer-specific myocardium segmentation framework was proposed for transmural and segmental myocardial motion estimation. Two novel strategies were proposed: the displacement-compensated cross-correlation coefficient (DCCCC) for algorithm parameter optimization and the transmural motion index (TMI) for quantitative estimation of the cross-wall transmural motion gradient. Results: The results showed that an overlap value of 80% used in VNCC guaranteed a more accurate displacement calculation. Compared to the Sham1 group, the systolic myocardial motion reductions were significantly detected (p < 0.05) in the middle anteroseptal (M-ANT-SEP), basal anteroseptal (B-ANT-SEP), apical lateral (A-LAT), middle inferolateral (M-INF-LAT), and basal inferolateral (B-INF-LAT) walls as well as a significant TMI drop (p < 0.05) in the M-ANT-SEP wall in the MI1 rats; significant motion reductions (p < 0.05) were also detected in the B-ANT-SEP and A-LAT walls in the IR1 group. The motion improvements (p < 0.05) were detected in the M-INF-LAT wall in the MI28 group and the apical septal (A-SEP) wall in the IR28 group compared to the MI1 and IR1 groups, respectively. Discussion: Our results show that the MI-induced reductions and reperfusion-induced recovery in systolic myocardial contractility could be successfully evaluated using our method, and most post-MI myocardial segments could recover systolic function to various extents in the remodeling phase. In conclusion, the ultrasound-based quantitative estimation framework for estimating segmental and transmural motion of the myocardium proposed in our study has great potential for non-invasive, novel, and early MI detection.

Potential nephroprotective effects of angiotensin II type 2 receptor agonist Compound 21 in renal ischemia-reperfusion injury.

This study examined the reno-protective potential of Compound 21 during renal ischemia-reperfusion injury by regulating the PI3K expression. 20 adult male Swiss-albino mice, aged 8-12 weeks and weighing 20-30g, were randomly assigned to four equal groups: sham, control, vehicle, and Compound 21. Serum urea, creatinine, inflammatory mediators, tissue 8-isoprostane, and myeloperoxidase were quantified using ELISA. Compared to the sham group, blood levels of urea, creatinine, TNF-α, IL-6, and IL-10 were significantly higher in the ischemia-reperfusion group than in the sham group (p<0.05). However, these indicators were significantly lower in the Compound 21 group (p<0.05). Histological analysis revealed significant renal tissue damage in the ischemia-reperfusion group (p<0.05), which was significantly reduced in the Compound 21 group (p<0.05). PCR results showed that PI3K expression was significantly lower (p<0.05) in the control group compared to the sham group but significantly higher in the Compound 21 group (p<0.05). Furthermore, P-AKT expression levels in the control group were considerably lower than in the sham group (p<0.05). On the other hand, the level of P-AKT expression in the Compound 21 group was significantly upregulated compared to the control group (p<0.05). The findings revealed that Compound 21 could mitigate renal dysfunction induced by ischemia-reperfusion injury in male mice through modulation of the PI3K/AKT signaling pathway, resulting in decreased levels of pro-inflammatory cytokines and renal oxidative stress markers.

Investigation of the protective effect of anzer propolis in cerebral ischemia-reperfusion injury.

Globally, stroke is the leading cause of disability and death. With the use of thrombolytic therapy, reperfusion injury, and its consequences came to the fore. We aimed to find out how anzer propolis, which can only be obtained in Turkey's Eastern Black Sea region, affected ischemia-reperfusion injury using biochemical and histological techniques. 32 female Wistar albino rats were divided into 4 groups, including a control group. Three of the groups underwent 30 minutes of induced ischemia via clamping of the common carotid artery, followed by ischemia-reperfusion injury through the release of the clamp. One group received no treatment, another received oral administration of 100 mg/kg of anzer propolis one hour before surgery, and the third group received oral administration of 40 mg/kg of acetylsalicylic acid just before surgery. Histopathological examination assessed apoptosis and tissue necrosis, while serum and brain tissue were evaluated for levels of nerve growth factor (NGF), Interlokin 1β (IL-1β), Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), total antioxidant capacity (TAS), and total oxidant capacity (TOS). Anzer propolis and acetylsalicylic acid significantly reduced hyperemia in vessels, vacuolization in neurons, glial cell infiltration, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity. The anzer propolis group had the highest NGF levels. The anzer propolis and acetylsalicylic acid groups had lower levels of TNF-a and IL-6 in the brain tissue than the ischemia-reperfusion group, while TAS levels were higher. The findings obtained in this study suggest that anzer propolis has a neuroprotective effect against ischemia-reperfusion injury and will have beneficial effects on neurodegeneration. We believe our findings will contribute to the clinical treatment of ischemia-reperfusion injury.

Denervation aggravates renal ischemia reperfusion injury via BMAL1-mediated Nrf2/ARE pathway

AimTo explore the change of clock gene rhythm under renal denervation (RDN) and its effect on renal function and oxidative stress during renal ischemia-reperfusion (IR) injury. MethodC57/BL6 mice were randomly divided into 4 groups at daytime 7 A M (zeitgeber time [ZT] 0) or at nighttime 7 P M (ZT12) in respectively: Sham (S) group, RDN group, IR group and RDN + IR (DIR) group. Renal pathological and functional changes were assessed by H&E staining, and serum creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin levels. Renal oxidative stress was detected by SOD and MDA levels, and renal inflammation was measured by IL-6, IL-17 A F and TNF-ɑ levels. BMAL1, CLOCK, Nrf2 and HO-1 mRNA and protein expressions were tested by qPCR and Western Blot. ResultCompared with S groups, the rhythm of BMAL1, CLOCK and Nrf2 genes in the kidney were disordered in RDN groups, while renal pathological and functional indexes did not change significantly. Compared with IR groups, renal pathological and functional indexes were significantly higher in the DIR groups, as well as oxidative stress and inflammation in renal tissues. The nocturnal IR injury in the RDN kidney was the worst while the BMAL1, Nrf2 and HO-1 expressions were the highest. In DIR groups, renal injury was aggravated after the Brusatol treatment, but there was no significant improvement after the t-BHQ treatment at night, which might be consistent with the changes of Nrf2 and HO-1 protein expressions. ConclusionRDN lead to the disruption of BMAL1-mediated Nrf2 rhythm accumulation in the kidney, which reduced the renal ability to resist oxidative stress and inflammation, due to the impaired effect of activating Nrf2/ARE pathway in renal IR injury at nighttime.

Gallic acid treatment protects intestinal tissue against ischaemia-reperfusion.

This study aimed to investigate the protective effects of gallic acid (GA) in the rat intestine against ischaemia-reperfusion (IR) injury. Thirty male Wistar albino rats with a mean weight of 200-250 g were used. Animals were categorized into the sham, IR, and IR+GA groups. Ischaemia of the intestine was induced for 3 h by occluding the superior mesenteric artery (SMA) and then left for 3 h of reperfusion. In the IR+GA group, after ischaemia induction, 50 mg/kg GA was orally administered to the animals. Blood samples were collected for biochemical assays. Intestinal tissues were excised for histopathologic and immunohistochemical processing. Malondialdehyde (MDA) levels were increased, and catalase (CAT) and glutathione (GSH) levels were decreased in the IR group compared to the sham group. After GA treatment, MDA levels decreased and CAT and GSH levels increased in the GA-treated group compared to the IR group. In the sham group, normal intestinal histology was observed. In the IR group, the villi structures were completely degenerated. In the IR+GA group, histology was improved after GA treatment. In the sham group, the caspase-3 reaction was generally negative in the epithelium and glands. In the IR group, the caspase-3 reaction increased in apoptotic bodies and inflammatory cells. The caspase-3 reaction was negative in goblet cells and the epithelium. A moderate caspase-3 reaction was observed in the IR+GA group. The beclin-1 reaction was negative in epithelial cells and goblet cells in villi in the sham group. In the IR group, the beclin-1 reaction was positive in the degenerated villi. An intense beclin-1 reaction was also observed in some inflammatory cells. After GA treatment, the beclin-1 reaction was positive in a few cells. In general, moderate beclin-1 positivity was observed. Gallic acid, with its antioxidative effect, inhibited the apoptotic pathway (caspase-3) through beclin-1 regulation.

Neuroprotective effect of Potentilla reptans L. root in the rat brain global ischemia/reperfusion model.

Stroke is the most common cause of death among neurological diseases. The protective effects of Potentilla reptans L. include antioxidative, anti-inflammatory, and antiapoptotic effects. In this study, the brain protection and beta-amyloid effects of P. reptans root extract were investigated in the rat brain ischemia/reperfusion (IR) model. Forty male Wistar rats were randomly divided into five groups (n = 8), including IR, sham, and three groups receiving P. reptans with concentrations of 0.025, 0.05, and 0.1 (g/kg/b.w.), which were injected daily for 7 days. For the IR model, the common carotid artery was occluded bilaterally for 8 min. All injections were intraperitoneal (IP). The shuttle box test was used to measure passive avoidance memory. Then the brain tissue was extracted for the histological examination of neuron counts and β-amyloid plaques using a morphometric technique, and finally, Statistical Package for the Social Sciences software was used for statistical analysis of the data. Pretreatment with P. reptans improved memory impairment. Also, by examining the tissues of the CA1, CA3, and dentate gyrus areas of the hippocampus, it was observed that the number of plaques in the groups receiving P. reptans extract was reduced compared to the IR group, especially at the concentration of 0.05 g/kg/b.w. Also, P. reptans improved the number of neurons at all concentrations, in which the concentration of 0.05 g/kg/b.w. showed more effective therapeutic results. Taken together, we found that P. reptans root extract has beneficial effects on memory impairment, neuronal loss, and β-amyloid accumulation.

Effects of Coriandrum sativum on distant organ inflammation and apoptosis due to liver ischemia/reperfusion injury.

Liver ischemia/reperfusion (IR) often affects distant organs, such as small intestine, kidney, and lung. Coriandrum sativum (CS) has an antioxidant and anti-inflammatory effect on liver damage. The aim of this study was to investigate the anti-inflammatory and antiapoptotic effects of CS extract on small intestine, lung, and kidney after the liver IR injury. Small intestine, lung, and kidney tissues were evaluated and scored in terms of cell degeneration, inflammation, and congestion, as well as caspase-3 (Cas-3) and cluster of differentiation 31 (CD31) immunostainings were carried out. Renal enzymes, creatinine and urea levels were measured biochemically in serum. After IR, a decrease in villi size, diffuse degeneration, epithelial cell shedding and extensive congestion in the capillaries were observed. Meanwhile, the number of degenerated villi and congestion decreased in the IR+CS group. Due to IR, increased congestion was detected in the interalveolar septum of the lungs and in the capillaries between the kidney tubules. It was also observed that the positively stained cells with Cas-3 and CD31 were increased in the lung, kidney, and small intestine tissues of the IR group, and decreased in the IR+CS group. Kidney enzymes, urea and creatinine levels were significantly increased in the IR group and decreased in the IR+CS group. In conclusion, it was observed that liver IR caused changes in distant organs, especially in the small intestine, lung, and kidneys. Damaging effects of IR as well as apoptosis and inflammation were found to be decreased in the groups treated with CS.

The Role of the JAK-STAT Signaling Pathway in the Protective Effects of Hepatic Ischemia Post-conditioning Against the Injury Induced by Ischemia/Reperfusion in the Rat Liver.

Hepatic ischemic post-conditioning (IPOC) is shown to protect the liver from injury induced by ischemia/reperfusion (IR). However, the mechanism underlying this protection has remained elusive. The present study aimed to investigate the role of the interleukin 6-Janus kinase-signal transducers and activators of transcription (IL-6-JAK-STAT) pathway in the protective effect of hepatic IPOC against the IR-induced injury in the liver. Twenty-five rats were randomly divided into 5 groups of (1) sham-operated, (2) IR, (3) IR+hepatic IPOC, (4) IR+tofacitinib (TOFA), and (5) IR+TOFA+hepatic IPOC. The changes induced by IR and the effects of different treatments were assessed by enzyme release, histopathological observations, the serum level of IL-6, and the occurrence of apoptosis detected via the expression of the Bax/Bcl-2 ratio. The hepatic IPOC improved the liver injury induced by IR as shown by histological changes, reduction of IL-6 level, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) compared to the IR group (P<0.001, P<0.05, P<0.05, respectively). There was also downregulation of the Bax/Bcl2 ratio in the rats exposed to IR+hepatic IPOC compared with those in the IR group (P<0.05). However, TOFA, an inhibitor of JAK-STAT activity, inhibited the protective effect of hepatic IPOC. It suggests that the protective effect of hepatic IPOC against IR-induced injury may be mediated by activating the IL-6-JAK-STAT pathway.

Etanercept Mitigated Renal Injury in Male Rats Undergoing Global Renal Ischemia-Reperfusion

The kidneys are vulnerable to injury from ischemia-reperfusion (IR), a process that triggers inflammation and apoptosis, primarily mediated by tumor necrosis factor (TNF)-alpha. Numerous studies have investigated renal damage in this context. Etanercept, a soluble receptor for TNF-alpha, has demonstrated anti-inflammatory and anti-apoptotic properties. This study aims to assess the potential of etanercept in mitigating experimental renal IR injury and its capacity to protect against widespread renal ischemia/reperfusion injury. Methods: Male Sprague-Dawley (SD) rats were classified into four groups: sham, DMSO-treated, etanercept-treated, DMSO-treated IR, and etanercept-treated IR groups. After 24 hours following IR injury, renal levels of TNF-alpha and TLRs (Toll-like receptors) were assessed using ELISA and IHC methods, respectively. Histopathological analysis was employed to quantify the extent of renal cell injury. Results: Etanercept treatment significantly lowered tissue levels of TNF-alpha and TLRs in IR-damaged rats compared to DMSO-treated IR rats. Kidneys of DMSO-treated IR rats exhibited substantially elevated levels of TNF-alpha and TLRs when compared to DMSO-treated sham rats. Conversely, etanercept-treated IR rats displayed significantly reduced levels of TNF-alpha and TLRs compared to DMSO-treated IR rats. Pre-treatment with etanercept significantly alleviated the extent of damage in IR-injured rats compared to the control and DMSO groups. Etanercept further promoted the downregulation of TLRs and TNF-alpha, thereby enhancing resistance to renal damage during IR. Conclusion: In conclusion, etanercept shows promise in providing protection against renal ischemia-reperfusion injury by mitigating inflammation and apoptosis, as evidenced by reductions in TNF-alpha and TLR levels. This suggests its potential as a therapeutic intervention to mitigate renal damage resulting from ischemia-reperfusion injury.

Maresin1 inhibits the NF-κB/caspase-3/GSDME signaling pathway to alleviate hepatic ischemia-reperfusion injury

Objective: To investigate the role of Maresin1 (MaR1) in hepatic ischemia-reperfusion injury (HIRI). Methods: The HIRI model was established and randomly divided into a sham operation group (Sham group), an ischemia-reperfusion group (IR group), and a MaR1 ischemia-reperfusion group (MaR1+IR group). MaR1 80ng was intravenously injected into each mouse's tail veins 0.5h before anesthesia. The left and middle hepatic lobe arteries and portal veins were opened and clamped. The blood supply was restored after 1h of ischemia. After 6h of reperfusion, the mice were sacrificed to collect blood and liver tissue samples. The Sham's group abdominal wall was only opened and closed. RAW267.4 macrophages were administered with MaR1 50ng/ml 0.5h before hypoxia, followed by hypoxia for 8h and reoxygenation for 2h, and were divided into the control group, the hypoxia-reoxygenation group (HR group), the MaR1 hypoxia-reoxygenation group (MaR1 + HR group), the Z-DEVD-FMK hypoxia-reoxygenation group (HR+Z group), the MaR1 + Z-DEVD-FMK hypoxia-reoxygenation group (MaR1 + HR + Z group), and the Con group without any treatment. Cells and the supernatant above them were collected. One-way analysis of variance was used for inter-group comparisons, and the LSD-t test was used for pairwise comparisons. Results: Compared with the Sham group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1β, and IL-18 in the IR group were significantly higher (P < 0.05), with remarkable pathological changes, while the level in the MaR1 + IR group was lower than before (P < 0.05), and the pathological changes were alleviated. Compared with the Con group, the HR group had higher levels of IL-1β and IL-18 (P < 0.05), while the MaR1 + HR group had lower levels of IL-1β and IL-18 (P < 0.05). Western blot showed that the expressions of caspase-3, GSDME, and GSDME-N were significantly higher in the HR group and IR group than in the other groups; however, the expression was lower following MaR1 pretreatment. The Z-DEVD-FMK exploration mechanism was inhibited by the expression of caspase-3 in HIRI when using MaR1. Compared with the HR group, the IL-1β and IL-18 levels and the expressions of caspase-3, GSDME, and GSDME-N in the HR + Z group were decreased (P < 0.05), while the expression of nuclear factor κB was increased, but following MaR1 pretreatment, nuclear factor κB was decreased. There was no significant difference in the results between the MaR1 + H/R group and the MaR1 + H/R + Z group (P > 0.05). Conclusion: MaR1 alleviates HIRI by inhibiting NF-κB activation and caspase-3/GSDME-mediated inflammatory responses.

Renal vascular responses to angiotensin II infusion in two kidneys-one clip hypertensive rats under partial ischemia/reperfusion with and without ischemia preconditioning: the roles of AT1R blockade and co-blockades of AT1R and MasR.

The renin-angiotensin system activation, partial ischemia/reperfusion (IR) injury, and hypertension contribute to the development of acute kidney injury. The study aims to look at the vascular responses of angiotensin II (Ang II) during Ang II type 1 receptor (AT1R) blockade (losartan) or co-blockades of AT1R and Mas receptor (A779) in two kidneys one clip (2K1C) hypertensive rats which subjected to partial IR injury with and without ischemia preconditioning (IPC). Thirty-three 2K1C male Wistar rats with systolic blood pressure ≥ 150 mmHg were divided into three groups of sham, IR, and IPC + IR divided into two sub-groups receiving losartan or losartan + A779. The IR group had 45 min partial kidney ischemia, while the IPC + IR group had two 5 min cycles of partial ischemia followed by 10 min of reperfusion and then 45 min of partial kidney ischemia followed by reperfusion. The sham group was subjected to similar surgical procedures except for IR or IPC. Ang II increased mean arterial pressure in all the groups, but there were no significant differences between the sub-groups. A significant difference was observed in the renal blood flow response to Ang II between two sub-groups of sham and IR groups treated with AT1R blockade alone or co-blockades of AT1R + A779. These findings demonstrated the significance of AT1R and Mas receptor following partial renal IR in the renal blood flow responses to Ang II in 2K1C hypertensive rats.

Cardiopreventive effect of Klotho protein on oxidative stress, inflammation and apoptosis during cardiac ischemia/reperfusion injury

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre, Poland. Background The injury of the myocardium during ischemia/reperfusion (IRI) involves metabolic, morphological and contractile disorders [1]. Klotho is a membrane or soluble anti-aging protein. Recent studies have proven the correlation of Klotho deficiency and the occurrence and development of cardiovascular diseases [2]. Purpose This study aimed to evaluate the effect of Klotho protein supply on oxidative stress, inflammation and apoptosis in hearts subjected to IRI. Methods Isolated Wistar rat hearts perfused with the Langendorff method were subjected to ischemia, followed by reperfusion, in the presence or absence of Klotho protein. Hemodynamic parameters, and the levels of nitrate/nitrite (NOX)-, glutathione peroxidase (GPx), superoxide dismutase (SOD), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-Κb), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), interleukin-6 (IL-6), matrix metalloproteinase 2 (MMP-2), cytochrome c (cyt c) and caspase-9 were evaluated. Results Infusion supply of Klotho during IRI resulted in a recovery of contractile function (p=0.031) and heart rate (p=0.02). Klotho protein contributed to increase in the production of GPx (p=0.045) and SOD (p=0.006). The levels of (NOX)- (p=0.022), NF-Κb (p=0.006), NFATc1 (p=0.027), IL-6 (p=0.002), MMP-2 (p=0.008), cyt c (p=0.0002) and caspase-9 (p=0.009) were decreased in the IRI+Klotho group, as compared to the IRI group. Conclusions Klotho protein preserved heart mechanical function, enhanced antioxidant capacity, and reduced inflammation and apoptosis in the injured heart. Thus, Klotho can be recognized as a novel cardiopreventive/cardioprotective agent in ischemic damage.

Severe muscle damage after a short period of ischemia and reperfusion in an animal model

BackgroundSkeletal muscle ischemia-reperfusion injuries result in a loss of contractile function, leading to limb disability or amputation. Ischemia causes hypoxia and cellular energy failure, which is aggravated by reperfusion due to the inflammatory response and oxidative stress. The consequences of the injury vary according to the duration of the period of ischemia and reperfusion. Therefore, the present work aims to evaluate ischemia-reperfusion injuries induced in the skeletal muscles of Wistar rats submitted to 3 different application periods based on morphological and biochemical parameters. MethodsFor this, a tourniquet was applied to the root of the animals’ hind limbs, occluding arterial and venous blood flow, and it was followed by reperfusion—the removal of the tourniquet. The groups were: control (without tourniquet); I30'/R60' (30 minutes of ischemia and 1 hour of reperfusion); I120'/R120' (2 hours and 2 hours); and I180'/R180' (3 hours and 3 hours). ResultsAll ischemia-reperfusion groups showed characteristics of muscle injury. Microscopic analyses of the extensor digitorum longus, soleus, tibialis anterior, and gastrocnemius muscles showed a significant increase in the number of injured muscle fibers in the ischemia-reperfusion groups compared to the control group. There were also significant differences between the ischemia-reperfusion groups in all muscles, showing a progressive increase in the degree of injury. The quantification of the number of injured muscle fibers between the muscles revealed that at I30'/R60', the soleus muscles had a higher number of injuries in relation to the other muscles, with statistical significance. In the I120'/R120' group, the gastrocnemius muscles presented a significantly greater number of injured fibers. There were no significant differences in the I180'/R180' group. The serum levels of creatine kinase in the I180'/R180' group were significantly higher than in the control and I30'/R60' groups. ConclusionsTherefore, it was evident that the 3 ischemia-reperfusion models used were capable of causing cell damage, with these findings being more pronounced in the I180'/R180' group.