Rat Model Of Myocardial Ischemia-reperfusion Injury Research Articles

Proteomics Profiling of Bilirubin Nanoparticle Treatment against Myocardial Ischemia-Reperfusion Injury.

In myocardial infarction, ischemia-reperfusion injury (IRI) poses a significant challenge due to a lack of effective treatments. Bilirubin, a natural compound known for its anti-inflammatory and antioxidant properties, has been identified as a potential therapeutic agent for IRI. Currently, there are no reports about proteomic studies related to IRI and bilirubin treatment. In this study, we explored the effects of bilirubin nanoparticles in a rat model of myocardial IRI. A total of 3616 protein groups comprising 76,681 distinct peptides were identified using LC-MS/MS, where we distinguished two kinds of protein groups: those showing increased expression in IRI and decreased expression in IRI with bilirubin treatment, and vice versa, accounting for 202 and 35 proteins, respectively. Our proteomic analysis identified significant upregulation in the Wnt and insulin signaling pathways and increased Golgi markers, indicating their role in mediating bilirubin nanoparticle's protective effects. This research contributes to the proteomic understanding of myocardial IRI and suggests bilirubin nanoparticles as a promising strategy for cardiac protection, warranting further investigation in human models.

Targeting delivery of miR-146a via IMTP modified milk exosomes exerted cardioprotective effects by inhibiting NF-κB signaling pathway after myocardial ischemia-reperfusion injury

Reperfusion therapy is critical for saving heart muscle after myocardial infarction, but the process of restoring blood flow can itself exacerbate injury to the myocardium. This phenomenon is known as myocardial ischemia-reperfusion injury (MIRI), which includes oxidative stress, inflammation, and further cell death. microRNA-146a (miR-146a) is known to play a significant role in regulating the immune response and inflammation, and has been studied for its potential impact on the improvement of heart function after myocardial injury. However, the delivery of miR-146a to the heart in a specific and efficient manner remains a challenge as extracellular RNAs are unstable and rapidly degraded. Milk exosomes (MEs) have been proposed as ideal delivery platform for miRNA-based therapy as they can protect miRNAs from RNase degradation. In this study, the effects of miR-146a containing MEs (MEs-miR-146a) on improvement of cardiac function were examined in a rat model of MIRI. To enhance the targeting delivery of MEs-miR-146a to the site of myocardial injury, the ischemic myocardium-targeted peptide IMTP was modified onto the surfaces, and whether the modified MEs-miR-146a could exert a better therapeutic role was examined by echocardiography, myocardial injury indicators and the levels of inflammatory factors. Furthermore, the expressions of miR-146a mediated NF-κB signaling pathway-related proteins were detected by western blotting and qRT-PCR to further elucidate its mechanisms. MiR-146 mimics were successfully loaded into the MEs by electroporation at a square wave 1000 V voltage and 0.1 ms pulse duration. MEs-miR-146a can be up-taken by cardiomyocytes and protected the cells from oxygen glucose deprivation/reperfusion induced damage in vitro. Oral administration of MEs-miR-146a decreased myocardial tissue apoptosis and the expression of inflammatory factors and improved cardiac function after MIRI. The miR-146a level in myocardium tissues was significantly increased after the administration IMTP modified MEs-miR-146a, which was higher than that of the MEs-miR-146a group. In addition, intravenous injection of IMTP modified MEs-miR-146a enhanced the targeting to heart, improved cardiac function, reduced myocardial tissue apoptosis and suppressed inflammation after MIRI, which was more effective than the MEs-miR-146a treatment. Moreover, IMTP modified MEs-miR-146a reduced the protein levels of IRAK1, TRAF6 and p-p65. Therefore, IMTP modified MEs-miR-146a exerted their anti-inflammatory effect by inhibiting the IRAK1/TRAF6/NF-κB signaling pathway. Taken together, our findings suggested miR-146a containing MEs may be a promising strategy for the treatment of MIRI with better outcome after modification with ischemic myocardium-targeted peptide, which was expected to be applied in clinical practice in future.

Identification of New, Translatable ProtectomiRs against Myocardial Ischemia/Reperfusion Injury and Oxidative Stress: The Role of MMP/Biglycan Signaling Pathways.

Ischemic conditionings (ICon) were intensively investigated and several protective signaling pathways were identified. Previously, we have shown the role of matrix metalloproteinases (MMP) in myocardial ischemia/reperfusion injury (MIRI) and the cardioprotective role of biglycan (BGN), a small leucine-rich proteoglycan in vitro. Here, we hypothesized that cardiac MMP and BGN signaling are involved in the protective effects of ICon. A reverse target-microRNA prediction was performed by using the miRNAtarget™ 2.0 software to identify human microRNAs with a possible regulatory effect on MMP and BGN, such as on related genes. To validate the identified 1289 miRNAs in the predicted network, we compared them to two cardioprotective miRNA omics datasets derived from pig and rat models of MIRI in the presence of ICons. Among the experimentally measured miRNAs, we found 100% sequence identity to human predicted regulatory miRNAs in the case of 37 porcine and 24 rat miRNAs. Upon further analysis, 42 miRNAs were identified as MIRI-associated miRNAs, from which 24 miRNAs were counter-regulated due to ICons. Our findings highlight 24 miRNAs that potentially regulate cardioprotective therapeutic targets associated with MMPs and BGN in a highly translatable porcine model of acute myocardial infarction.

DECIPHERING THE NOVEL ROLE OF ERDOSTEINE IN PREVENTION OF MYOCARDIAL ISCHEMIA-REPERFUSION INJURY IN RAT

Objective: Myocardial ischemia-reperfusion injury (MIRI) is caused by thrombotic obstruction of the coronary artery subsequently leading to heart failure. Despite the availability of improved prognosis, MIRI is a foremost cause of morbidity and mortality worldwide. In this study, we investigated the therapeutic potential of Erdosteine (a mucoactive antioxidant molecule, with multifaceted pharmacological properties), to attenuate myocardial infarction in rat model of MIRI. Design and method: This study was conducted in adult male Wistar rats (n=40), randomized into five groups: Sham, IR-C (disease control), Erdosteine (80 mg/kg; s.c.) + IR, Telmisartan (10 mg/kg; orally) +IR and Erdosteine (80 mg/kg) perse groups. Erdosteine and telmisartan were administered for 28 days as a pre-treatment. On the 29th day, animals were subjected to LAD coronary artery occlusion for 1 h followed by reperfusion for 45 minutes. The cardioprotective potential was assessed through measurement of hemodynamic parameters, biochemical markers (MDA, GSH, SOD), cardiac injury markers (CK-MB and LDH), inflammatory markers (IL-6, TNF), histopathological evaluation, TUNEL assay and immunohistochemistry analysis (Bax, caspase 3, Bcl2). Further, the underlying mechanism was evaluated using immune-blotting of MAPK, Nrf-2/HO-1, and Akt/p-Akt. The statistics analysis was performed by one-way ANOVA followed by Turkey-Kramer post hoc tests. Results: Erdosteine and telmisartan pretreatment improves hemodynamic parameters by preserving MAP, ±LVdP/dt, and HR as compared to the IR-C group. Pretreatment markedly restored the levels of antioxidants (GSH and SOD) and decreased peroxidation marker (MDA) compared to the IR-C group. Also, the pretreatment significantly prevented the damage following IR injury as compared to the IR-C group when investigated through cardiac injury markers and histopathological evaluation. Erdosteine profoundly reversed the expressions of Bcl2/BAX ratio, in comparison to the IR-C group indicating its anti-apoptotic effect when reconfirmed through TUNEL assay. Erdosteine pretreatment preserved the levels of NF-kB, TNF, and IL-6 following IR challenge. Erdosteine pretreatment restores FABP, Nrf-2, and Hsp27. Conclusions: Erdosteine protects the myocardial cell membrane from peroxidation and inhibits oxidative burst and inflammation through NF-kB pathway activation. Erdosteine attenuates MIRI injury in rats preserving the MAPK and Nrf-2 signaling cascade.

Inhibition of circ_0073932 attenuates myocardial ischemia‒reperfusion injury via miR-493-3p/FAF1/JNK.

Oxidative stress and apoptosis play crucial roles in myocardial ischemia‒reperfusion injury (MIRI). In this study, we investigated the role of circ_0073932 in MIRI as well as its molecular mechanism. A hypoxia/reoxygenation (H/R) cardiomyocyte model was established with H9C2 cardiomyocytes, and RT-qPCR was used to measure gene expression. We observed that circ_0073932 expression was abnormally increased in the H/R cardiomyocyte model and in blood samples from MIRI patients. Inhibition of circ_0073932 suppressed H/R-induced cell apoptosis, oxidative stress (ROS, LDH and MDA), and p-JNK expression. Dual luciferase reporter assays showed that circ_0073932 targeted the downregulation of miR-493-3p, and miR-493-3p targeted the downregulation of FAF1. Furthermore, si-circ_0073932, an miR-493-3p inhibitor, oe-FAF1, or si-FAF1 were transfected into H9C2 cardiomyocytes to investigate the roles of these factors in MIRI. Our results showed that compared with the H/R group, si-circ_0073932 inhibited H/R-induced cell apoptosis, oxidative stress (ROS, LDH and MDA), and p-JNK expression. These results were reversed by the miR-493-3p inhibitor or oe-FAF1. Finally, a rat model of MIRI was established, and si-circ_0073932 was administered. Inhibition of circ_0073932 reduced the area of myocardial infarction and decreased the levels of apoptosis and oxidative stress by inhibiting the JNK signaling pathway. Our study indicated that circ_0073932 mediates MIRI via miR-493-3p/FAF1/JNK in vivo and in vitro, revealing novel insights into the pathogenesis of MIRI and providing a new target for the clinical treatment of MIRI.

Bioinformatics integration reveals key genes associated with mitophagy in myocardial ischemia-reperfusion injury

BackgroundMyocardial ischemia is a prevalent cardiovascular disorder associated with significant morbidity and mortality. While prompt restoration of blood flow is essential for improving patient outcomes, the subsequent reperfusion process can result in myocardial ischemia–reperfusion injury (MIRI). Mitophagy, a specialized autophagic mechanism, has consistently been implicated in various cardiovascular disorders. However, the specific connection between ischemia–reperfusion and mitophagy remains elusive. This study aims to elucidate and validate central mitophagy-related genes associated with MIRI through comprehensive bioinformatics analysis.MethodsWe acquired the microarray expression profile dataset (GSE108940) from the Gene Expression Omnibus (GEO) and identified differentially expressed genes (DEGs) using GEO2R. Subsequently, these DEGs were cross-referenced with the mitophagy database, and differential nucleotide sequence analysis was performed through enrichment analysis. Protein–protein interaction (PPI) network analysis was employed to identify hub genes, followed by clustering of these hub genes using cytoHubba and MCODE within Cytoscape software. Gene set enrichment analysis (GSEA) was conducted on central genes. Additionally, Western blotting, immunofluorescence, and quantitative polymerase chain reaction (qPCR) analyses were conducted to validate the expression patterns of pivotal genes in MIRI rat model and H9C2 cardiomyocytes.ResultsA total of 2719 DEGs and 61 mitophagy-DEGs were identified, followed by enrichment analyses and the construction of a PPI network. HSP90AA1, RPS27A, EEF2, EIF4A1, EIF2S1, HIF-1α, and BNIP3 emerged as the seven hub genes identified by cytoHubba and MCODE of Cytoscape software. Functional clustering analysis of HIF-1α and BNIP3 yielded a score of 9.647, as determined by Cytoscape (MCODE). In our MIRI rat model, Western blot and immunofluorescence analyses confirmed a significant elevation in the expression of HIF-1α and BNIP3, accompanied by a notable increase in the ratio of LC3II to LC3I. Subsequently, qPCR confirmed a significant upregulation of HIF-1α, BNIP3, and LC3 mRNA in the MIRI group. Activation of the HIF-1α/BNIP3 pathway mediates the regulation of the degree of Mitophagy, thereby effectively reducing apoptosis in rat H9C2 cardiomyocytes.ConclusionsThis study has identified seven central genes among mitophagy-related DEGs that may play a pivotal role in MIRI, suggesting a correlation between the HIF-1α/BNIP3 pathway of mitophagy and the pathogenesis of MIRI. The findings highlight the potential importance of mitophagy in MIRI and provide valuable insights into underlying mechanisms and potential therapeutic targets for further exploration in future studies.

Chaihu Sanshen Capsules protect rats from myocardial ischemia reperfusion injury via PKCβⅡ/NOX2/ROS signaling pathway

This study aims to explore the pathogenesis of myocardial ischaemia reperfusion injury(MIRI) based on oxidative stress-mediated programmed cell death and the mechanism and targets of Chaihu Sanshen Capsules in treating MIRI via the protein kinase Cβ(PKCβⅡ)/NADPH oxidase 2(NOX2)/reactive oxygen species(ROS) signaling pathway. The rat model of MIRI was established by the ligation of the left anterior descending branch. Rats were randomized into 6 groups: sham group, model group, clinically equivalent-, high-dose Chaihu Sanshen Capsules groups, N-acetylcysteine group, and CGP53353 group. After drug administration for 7 consecutive days, the area of myocardial infarction in each group was measured. The pathological morphology of the myocardial tissue was observed by hematoxylin-eosin(HE) staining. The apoptosis in the myocardial tissue was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL). Enzyme-linked immunosorbent assay(ELISA) was employed to measure the le-vels of indicators of myocardial injury and oxidative stress. The level of ROS was detected by flow cytometry. The protein and mRNA levels of the related proteins in the myocardial tissue were determined by Western blot and real-time quantitative PCR(RT-qPCR), respectively. Compared with the sham group, the model group showed obvious myocardial infarction, myocardial structural disorders, interstitial edema and hemorrhage, presence of a large number of vacuoles, elevated levels of myocardial injury markers, myocardial apoptosis, ROS, and malondialdehyde(MDA), lowered superoxide dismutase(SOD) level, and up-regulated protein and mRNA le-vels of PKCβⅡ, NOX2, cysteinyl aspartate specific proteinase-3(caspase-3), and acyl-CoA synthetase long-chain family member 4(ACSL4) in the myocardial tissue. Compared with the model group, Chaihu Sanshen Capsules reduced the area of myocardial infarction, alleviated the pathological changes in the myocardial tissue, lowered the levels of myocardial injury and oxidative stress indicators and apoptosis, and down-regulated the mRNA and protein levels of PKCβⅡ, NOX2, caspase-3, and ACSL4 in the myocardial tissue. Chaihu Sanshen Capsules can inhibit oxidative stress and programmed cell death(apoptosis, ferroptosis) by regulating the PKCβⅡ/NOX2/ROS signaling pathway, thus mitigating myocardial ischemia reperfusion injury.

Shuangshen ningxin formula attenuates cardiac microvascular ischemia/reperfusion injury through improving mitochondrial function

Ethnopharmacological relevanceShuangshen Ningxin Formula (SSNX) is a traditional Chinese medicine formula used to treat myocardial ischemia–reperfusion injury (MIRI). A randomized controlled trial previously showed that SSNX reduced cardiovascular events, and experiments have also verified that SSNX attenuated ischemia–reperfusion (I/R) injury. However, the mechanism of SSNX in the treatment of microvascular I/R injury is still unclear. Aim of the studyTo determine whether SSNX protects the microvasculature by regulating I/R induction in rats and whether this effect depends on the regulation of NR4A1/Mff/Drp1 pathway. MethodsThe anterior descending coronary artery was ligated to establish a rat MIRI model with 45 min of ischemia and 24 h of reperfusion. The rats were subjected to a 7-day pretreatment with SSNX and nicorandil, after which their cardiac function and microvascular functional morphology were evaluated through diverse methods, including hematoxylin and eosin (HE) staining, wheat germ agglutinin (WGA) staining, and transmission electron microscopy. Cell apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Additionally, serum levels of ET-1 and eNOS were determined through an enzyme-linked immunosorbent assay (ELISA). The expression levels of NR4A1, Mff, and proteins related to mitochondrial fission were examined by Western blot (WB). Cardiac microcirculation endothelial cells (CMECs) were cultured and the oxygen–glucose deprivation/reoxygenation (OGD/R) model was duplicated. Following treatment with SSNX and DIM-C-pPhOH, an NR4A1 inhibitor, cell viability was assessed. Fluorescence was used to evaluate mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (MPTP) opening. Moreover, vascular endothelial function was evaluated through transendothelial electrical resistance (TEER), Transwell assays and tube formation assays. ResultsThe results showed that SSNX reduced the infarction area and no-flow area, improved cardiac function, mitigated pathological alterations, increased endothelial nitric oxide synthase expression, protected endothelial function, and attenuated microvascular damage after I/R injury. I/R triggered mitochondrial fission and apoptotic signaling in CMECs, while SSNX restored mitochondrial fission to normal levels and inhibited mitochondrial apoptosis. A study using CMECs revealed that SSNX protected endothelial function after OGD/R, attenuating the increase in NR4A1/Mff/Drp1 protein and inactivating VDAC1, HK2, cytochrome c (cyt-c) and caspase-9. Research also shows that SSNX can affect CMEC cell migration and angiogenesis, reduce mitochondrial membrane potential damage, and inhibit membrane opening. Moreover, DIM-C-pPhOH, an NR4A1 inhibitor, partially imitated the effect of SSNX. ConclusionSSNX has a protective effect on the cardiac microvasculature by inhibiting the NR4A1/Mff/Drp1 pathway both in vivo and in vitro.

Ticagrelor alleviates pyroptosis of myocardial ischemia reperfusion-induced acute lung injury in rats: a preliminary study.

Pulmonary infection is highly prevalent in patients with acute myocardial infarction undergoing percutaneous coronary intervention. However, the potential mechanism is not well characterized. Myocardial ischemia-reperfusion injury (MIRI) induces acute lung injury (ALI) related to pulmonary infection and inflammation. Recent studies have shown that pyroptosis mediates ALI in several human respiratory diseases. It is not known whether MIRI induces pyroptosis in the lungs. Furthermore, ticagrelor is a clinically approved anti-platelet drug that reduces ALI and inhibits the expression levels of several pyroptosis-associated proteins, but the effects of ticagrelor on MIRI-induced ALI have not been reported. Therefore, we investigated whether ticagrelor alleviated ALI in the rat MIRI model, and its effects on pyroptosis in the lungs. Sprague-Dawley rats were randomly divided into four groups: control, MIRI, MIRI plus low ticagrelor (30 mg/kg), and MIRI plus high ticagrelor (100 mg/kg). Hematoxylin and Eosin (HE) staining was performed on the lung sections, and the HE scores were calculated to determine the extent of lung pathology. The wet-to-dry ratio of the lung tissues were also determined. The expression levels of pyroptosis-related proteins such as NLRP3, ASC, and Cleaved caspase-1 were estimated in the lung tissues using the western blot. ELISA was used to estimate the IL-1β levels in the lungs. Immunohistochemistry was performed to determine the levels of MPO-positive neutrophils as well as the total NLRP3-positive and Cleaved caspase-1-positive areas in the lung tissues. The lung tissues from the MIRI group rats showed significantly higher HE score, wet-to-dry ratio, and the MPO-positive area compared to the control group, but these effects were attenuated by pre-treatment with ticagrelor. Furthermore, lung tissues of the MIRI group rats showed significantly higher expression levels of pyroptosis-associated proteins, including NLRP3 (2.1-fold, P < 0.05), ASC (3.0-fold, P < 0.01), and Cleaved caspase-1 (9.0-fold, P < 0.01). Pre-treatment with the high-dose of ticagrelor suppressed MIRI-induced upregulation of NLRP3 (0.46-fold, P < 0.05), ASC (0.64-fold, P < 0.01), and Cleaved caspase-1 (0.80-fold, P < 0.01). Immunohistochemistry results also confirmed that pre-treatment with ticagrelor suppressed MIRI-induced upregulation of pyroptosis in the lungs. In summary, our data demonstrated that MIRI induced ALI and upregulated pyroptosis in the rat lung tissues. Pre-treatment with ticagrelor attenuated these effects.

Platelet membrane-derived biomimetic microbubbles with enhanced targeting ability for the early detection of myocardial ischemia-reperfusion injury

Myocardial ischemia-reperfusion injury (MIRI) is a widely recognized cardiovascular disease that significantly impacts the prognosis of patients undergoing myocardial infarction recanalization. This condition can be fatal and involves complex pathophysiological mechanisms. Early diagnosis of MIRI is crucial to minimize myocardial damage and reducing mortality. Based on the inherent relationship between platelets and MIRI, we developed biomimetic microbubbles coated with platelet membrane (MB-pla) for early identification of MIRI. The MB-pla were prepared through a recombination process involving platelet membrane obtained from rat whole blood and phospholipids, blended in appropriate proportions. By coating the microbubbles with platelet membrane, MB-pla acquired various adhesion molecules, thereby gaining the capability to selectively adhere to damaged endothelial cells in the context of MIRI. In vitro experiments demonstrated that MB-pla exhibited remarkable targeting characteristics, particularly toward type IV collagen and human umbilical vein endothelial cells that had been injured through hypoxia/reoxygenation procedures. In a rat model of MIRI, the signal intensity produced by MB-pla was notably higher than that of control microbubbles. These findings were consistent with results obtained from fluorescence imaging of isolated hearts and immunofluorescence staining of tissue sections. In conclusion, MB-pla has great potential as a non-invasive early detection method for MIRI. Furthermore, this approach can potentially find application in other conditions involving endothelial injury in the future.

Mechanism of DYRK1a in myocardial ischemia-reperfusion injury by regulating ferroptosis of cardiomyocytes.

This study aimed to explore the role and mechanism of DYRK1a regulating ferroptosis of cardiomyocytes during myocardial ischemia-reperfusion injury (MIRI). H9c2 cells treated with oxygen-glucose deprivation/reoxygenation (OGD/R) were used as MIRI cell models and transfected with sh-DYRK1a or/and erastin. Cell viability, apoptosis, and DYRK1a mRNA/protein expression were measured accordingly. The levels of reactive oxygen species (ROS), iron, malondialdehyde (MDA), and glutathione (GSH) were determined. The expression of ferroptosis-related proteins (GPX4, SLC7A11, ACSL4, and TFR1) was detected using western blotting. The MIRI rat model was established to explore the possible role of DYRK1a suppression in cell injury and ferroptosis. OGD/R cells showed elevated mRNA and protein expression for DYRK1a. OGD/R cells transfected with sh-DYRK1a showed elevated cell viability, GSH content, increased GPX4 and SLC7A11 expression, suppressed iron content, MDA, ROS, ACSL4, and TFR1 expression, and reduced apoptosis rate, whereas co-transfection of sh-DYRK1a with erastin reversed the attenuation of sh-DYRK1a on MIRI. The suppressive effect of sh-DYRK1a on MI/R injury was confirmed in an MIRI rat model. DYRK1a mediates ferroptosis of cardiomyocytes to deteriorate MIRI progression.

Salvianolic Acid B Inhibits Ferroptosis and Apoptosis during Myocardial Ischemia/Reperfusion Injury via Decreasing the Ubiquitin-Proteasome Degradation of GPX4 and the ROS-JNK/MAPK Pathways.

Myocardial ischemia/reperfusion injury (MIRI) is related to ferroptosis and apoptosis elicited by reactive oxygen species (ROS). In this research, we investigated the protective effect of salvianolic acid B (SAB) as a natural antioxidant on ferroptosis and apoptosis in the MIRI process, and discussed the protective mechanism inhibiting ubiquitin-proteasome degradation of glutathione peroxidase 4 (GPX4) and the c-Jun N-terminal kinases (JNK) apoptosis signal pathway. We observed that ferroptosis and apoptosis occurred in the MIRI rat model in vivo and the H9c2 cardiomyocyte hypoxia/reoxygenation (H/R) damage model in vitro. SAB can alleviate tissue damage related to ROS, ferroptosis and apoptosis. Ubiquitin-proteasome degradation of GPX4 occurred in H/R models, and SAB reduced the ubiquitin-proteasome degradation of GPX4. SAB downregulates JNK phosphorylation and the expression of BCL2-Associated X (Bax)/B-cell lymphoma-2 (Bcl-2) and Caspase-3 to inhibit apoptosis. The role of GPX4 in the cardioprotection of SAB was further verified by the elimination effect of the GPX4 inhibitor RAS-selective lethal 3 (RSL3). This research shows that SAB may be used as a myocardial protective agent against oxidative stress, ferroptosis and apoptosis, and has potential clinical application prospects.

Angelica sinensis polysaccharide ameliorates myocardial ischemia-reperfusion injury in rats by inhibiting TLR4/NF-κB

Purpose: To evaluate the ameliorative effect of Angelica sinensis polysaccharide (ASP) in myocardial ischemia-reperfusion injury (MIRI) rats through Toll-like receptor 4 (TLR4)/Nuclear factor κB (NF-κB) pathway. &#x0D; Methods: The MIRI rat model was established. Sprague-Dawley rats were randomized to sham, MIRI, and ASP low-dose (50 mg/kg) and high-dose (100 mg/kg) groups, and a model of hypoxia-reoxygenation (H/R) injury was established. In in vitro studies, H/R cardiomycetes (derived from neonatal cell culture) were randomized to control, H/R, H/R + ASP low dose (5 μg/mL), H/R + ASP high dose (10 μg/mL), and ASP high dose + TLR4 activator groups. &#x0D; Results: After intragastric administration of ASP for 4 consecutive weeks, creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) were reduced after ASP treatment in MIRI rats (p &lt; 0.05). Both in vivo and in vitro, ASP reduced TNF-α, IL-6, and IL-1β expressions (p &lt; 0.05), and alleviated inflammatory response. Apoptosis was inhibited by ASP, which also increased Bcl2, reduced Bax and cleaved caspase-3 expressions, while TLR-4, p-IκBα, and p-p65 were decreased. &#x0D; Conclusion: The cardioprotective effect of ASP on MIRI results in the inhibition of TLR4/NF-κB pathway. Thus, this study broadens the current body of knowledge on the pharmacological prevention of MIRI and the therapeutic potential of ASP.

CircHECTD1 promoted MIRI-associated inflammation via inhibiting miR-138-5p and upregulating ROCK2.

Myocardial ischemia-reperfusion injury (MIRI) was often observed after surgeries, causing a lot of suffering to patients. Inflammation and apoptosis were critical determinants during MIRI. We conveyed experiments to reveal the regulatory functions of circHECTD1 in MIRI development. The Rat MIRI model was established and determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. We analyzed cell apoptosis using TUNEL and flow cytometry. Proteins expression was evaluated by western blot. The RNA level was determined by qRT-PCR. Secreted inflammatory factors were analyzed by ELISA assay. To predict the interaction sequences on circHECTD1, miR-138-5p, and ROCK2, bioinformatics analysis was performed. Dual-luciferase assay was used to confirm these interaction sequences. CircHECTD1 and ROCK2 were upregulated in the rat MIRI model, while miR-138-5p was decreased. CircHECTD1 knockdown alleviated H/R-induced inflammation in H9c2 cells. Direct interaction and regulation of circHECTD1/miR-138-5p and miR-138-5p/ROCK2 were confirmed by dual-luciferase assay. CircHECTD1 promoted H/R-induced inflammation and cell apoptosis by inhibiting miR-138-5p. miR-138-5p alleviated H/R-induced inflammation, while ectopic ROCK2 antagonized such effect of miR-138-5p. Our research suggested that the circHECTD1-modulated miR-138-5p suppressing is responsible for ROCK2 activation during H/R-induced inflammatory response, providing a novel insight into MIRI-associated inflammation.

Suxiao Jiuxin Pill alleviates myocardial ischemia–reperfusion injury through the ALKBH5/GSK3β/mTOR pathway

BackgroundMany studies have shown effective protection from myocardial ischemia–reperfusion injury (MIRI) in animal models, but few, if any, treatments have yielded a substantial reduction in clinical. Several studies showed significant therapeutic effects for the Chinese patent medicine Suxiao Jiuxin Pill (SJP) in MIRI, although the specific molecular mechanisms remain undefined. Recently, increasing evidence indicates an important role for m6A modification in autophagy regulation in MIRI, and SJP has not been investigated in this regard.MethodsIn vivo experiments were performed in a Wistar rat MIRI model. In vitro assays were conducted in hypoxia/reoxygenation (H/R)-treated H9c2 cells. H9c2 cells with ALKBH5 and GSK3β silencing were constructed by lentivirus transfection. TUNEL and Annexin V/PI assays were carried out for apoptosis detection. Then, m6A modification was detected with the EpiQuik m6A RNA methylation quantification kit, and GFP-RFP-LC3B was used to observe dynamic changes in autophagy. The autophagosome structure was assessed by Transmission electron microscopy. qPCR and immunoblot were performed for mRNA and protein analyses, receptively.ResultsSJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3β/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3β/mTOR pathway. Additionally, SJP enhanced ALKBH5 expression in H/R cardiomyocytes, which is important in impaired m6A modification. Interestingly, ALKBH5 knockdown enhanced autophagy and apoptosis in H/R-induced cells, whereas SJP reversed these effects. Further experiments showed that autophagic activity and apoptosis enhanced by ALKBH5 deficiency are GSK3β/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3β/mTOR pathway in H/R-induced cardiomyocytes. These effects of SJP were common to its two main constituents, including tetra-methylpyrazine (TMP) and borneol (BOR).ConclusionSJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3β/mTOR pathway, thanks to its two major constituents TMP and BOR.

Cardioprotective efficacy of ultrasound-targeted nitrofatty acid microbubbles in rat myocardial ischemia-reperfusion injury model

Over 1 million Americans suffer from acute myocardial infarction (AMI) annually. Although mortality from AMI has decreased, post-AMI congestive heart failure is increasing due to microvascular obstruction (MVO). MVO comprises mechanical obstruction, along with oxidate stress and inflammation. Currently available treatments for MVO are not consistently effective. Hence, we have been developing ultrasound (US)-targeted microbubble cavitation (UTMC) as a potential treatment for MVO. Nitro-fatty acids (NFA) are pleiotropic signaling molecules with broad anti-inflammatory actions, potentially beneficial for the treatment of MVO. NFA are amphipathic thus can seamlessly integrate into the phospholipid shell of MBs. Thus, we have constructed microbubbles with NFA (NFABs) to treat ischemia-reperfusion injury (IRI) with UTMC in the rat myocardial model. Left anterior descending (LAD) coronary artery was ligated for 30 min, allowing for IRI. After 15 min of reperfusion, UTMC + NFABs therapy was administered. Echocardiography measurements were recorded at baseline, during ligation and post-treatment. Left ventricular fractional shortening (%) was calculated and NFA concentration in cardiac tissue was determined. UTMC with NFABs exhibited promising efficacy in improving fractional shortening post IRI, and in targeted myocardial delivery of NFA. Studies assessing ejection fraction, myocardial area at risk, histopathology, inflammatory burden, oxidative stress and cytoprotective biomarkers are underway.

Dapagliflozin alleviates myocardial ischemia/reperfusion injury by reducing ferroptosis via MAPK signaling inhibition.

Reperfusion is essential for ischemic myocardium but paradoxically leads to myocardial damage that worsens cardiac functions. Ferroptosis often occurs in cardiomyocytes during ischemia/reperfusion (I/R). The SGLT2 inhibitor dapagliflozin (DAPA) exerts cardioprotective effects independent of hypoglycemia. Here, we investigated the effect and potential mechanism of DAPA against myocardial ischemia/reperfusion injury (MIRI)-related ferroptosis using the MIRI rat model and hypoxia/reoxygenation (H/R)-induced H9C2 cardiomyocytes. Our results show that DAPA significantly ameliorated myocardial injury, reperfusion arrhythmia, and cardiac function, as evidenced by alleviated ST-segment elevation, ameliorated cardiac injury biomarkers including cTnT and BNP and pathological features, prevented H/R-triggered cell viability loss in vitro. In vitro and in vivo experiments showed that DAPA inhibited ferroptosis by upregulating the SLC7A11/GPX4 axis and FTH and inhibiting ACSL4. DAPA notably mitigated oxidative stress, lipid peroxidation, ferrous iron overload, and reduced ferroptosis. Subsequently, network pharmacology and bioinformatics analysis suggested that the MAPK signaling pathway was a potential target of DAPA and a common mechanism of MIRI and ferroptosis. DAPA treatment significantly reduced MAPK phosphorylation in vitro and in vivo, suggesting that DAPA might protect against MIRI by reducing ferroptosis through the MAPK signaling pathway.

Dexmedetomidine postconditioning attenuates myocardial ischemia/reperfusion injury by activating the Nrf2/Sirt3/SOD2 signaling pathway in the rats

ABSTRACTObjectivesTo observe the protective effects of dexmedetomidine (Dex) postconditioning on myocardial ischemia/reperfusion injury (IRI) and to explore its potential molecular mechanisms.MethodsOne-hundred forty-seven male Sprague-Dawley rats were randomly divided into five groups receiving the different treatments: Sham, ischemia/reperfusion (I/R), Dex, Brusatol, Dex + Brusatol. By the in vivo rat model of myocardial IRI, cardioprotective effects of Dex postconditioning were evaluated by assessing serum CK-MB and cTnI levels, myocardial HE and Tunel staining and infarct size. Furthermore, the oxidative stress-related markers including intracellular ROS level, myocardial tissue MDA level, SOD and GSH-PX activities were determined.ResultsDex postconditioning significantly alleviated myocardial IRI, decreased intracellular ROS and myocardial tissue MDA level, increased SOD and GSH-PX activities. Dex postconditioning significantly up-regulated myocardial expression of Bcl-2, down-regulated Bax and cleaved caspase-3 and decreased cardiomyocyte apoptosis rate. furthermores, Dex postconditioning promoted Nrf2 nuclear translocation, increased myocardial expression of Sirt3 and SOD2 and decreased Ac-SOD2. However, brusatol reversed cardioprotective benefits of Dex postconditioning, significantly decreased Dex-induced Nrf2 nuclear translocation and reduced myocardial expression of Sirt3 and SOD2.ConclusionsDex postconditioning can alleviate myocardial IRI by suppressing oxidative stress and apoptosis, and these beneficial effects are at least partly mediated by activating the Nrf2/Sirt3/SOD2 signaling pathway.

Er-xian ameliorates myocardial ischemia-reperfusion injury in rats through RISK pathway involving estrogen receptors

Curculigo orchioides (CUR) and Epimedium (EPI) are traditional Chinese medicines with estrogen-like biological activity, called Xianmao and Xianlingpi (Er-xian) in Chinese. However, whether Er-xian exerts protective effects on myocardial ischemia-reperfusion injury (MIRI) is unknown. This study aimed to investigate the cardioprotective effects of Er-xian preconditioning against MIRI and the underlying mechanisms. CUR or EPI was administered intragastrically to aged female rats as a monotherapy or combination therapy. 2 weeks later, a rat MIRI model was established. Myocardial infarction size, myocardial morphology, cTnT, cell apoptosis rate, intracellular calcium concentration, mitochondrial permeability transition pore (MPTP) opening and reperfusion injury salvage kinase (RISK) signaling pathway molecules were observed after the surgery. To evaluate the mechanisms of Er-xian, estrogen receptors antagonists ICI 182780 and G15 were used. In this study, Er-xian notably alleviated myocardial tissue damage, maintained mitochondrial morphology, reduced infarct size and cardiac markers, and increased sera levels of E2. Moreover, Er-xian inhibited calcium overload and mPTP opening, and decreased cardiomyocyte apoptosis. We found that the dual therapy of CUR and EPI elicited more noticeable results than CUR or EPI monotherapy. The significant protective effects of Er-xian on ischemia-reperfusion myocardium were attributed to the up-regulation of AKT, ERK1/2 and GSK-3β phosphorylation levels. The cardioprotective effects of Er-xian were significantly reduced after estrogen receptor blockade, especially GPER30. These results indicate that Er-xian attenuates MIRI through RISK signaling pathway and estrogen receptors are the critical mediators.

Effects of Clopidogrel Rehabilitation on Cardiac Protein Kinase C, Cardiac Heat Shock Protein 70, and CI in MIRI Rat Model.

In order to investigate the effects of clopidogrel rehabilitation on the levels of cardiac protein kinase C (PKC), cardiac heat shock protein 70 (HSP70), and cardiac index (CI) in rats with myocardial ischemia-reperfusion injury (MIRI), sixty Wistar rats are randomly divided into three groups (sham operation group, model group, and clopidogrel group), with 20 rats in each group. The clopidogrel group is given clopidogrel by gavage, and the sham operation group and the model group are given the same amount of normal saline by gavage. The experimental results show that compared with the model group, the clopidogrel group has clear horizontal lines and cell edema. The myocardial infarction rate, creatine kinase-MB (CK-MB), and malondialdehyde (MDA) of the model group and clopidogrel group in the control sham operation group significantly increase.