Rat Model Of Myocardial Ischemia-reperfusion Injury Research Articles

Network pharmacology, molecular docking technology integrated with pharmacodynamic study to explore the potential targets and mechanism of Xinkeshu tablets against myocardial ischemia reperfusion injury

Xinkeshu tablets (XKST) is a Chinese patent medicine (CPM) against cardiovascular diseases. However, the specific action and mechanism of XKST against Myocardial ischemia reperfusion injury (MIRI) have not been clarified and discussed in detail. This study aims to discover the potential targets and mechanism of XKST in the treatment of MIRI. The therapeutic effects of XKST on MIRI in rats were evaluated by pharmacodynamics indexes, including myocardial enzyme content, cardiac troponin content, myocardial infarction rate, 2,3,5-triphenyltetrazolium chloride (TTC) staining and hematoxylin-eosin (HE) staining. The intersection of compound targets and disease targets were obtained and the coincidence targets were used to construct a compound-target network. We further performed GO and KEGG enrichment analysis on the targets to study its potential molecular mechanism. Differentially expressed genes (DEGs) analysis based on Gene expression omnibus (GEO) database was used to further identify core targets. Finally, molecular docking study was approved for core targets and quality markers of XKST. The pharmacodynamics results indicated that XKST had a good therapeutic effect on MIRI rat model. Additionally, our research showed that the protein targets ALOX12, MMP8 and HSD11B1 may be potential therapeutic core targets of XKST in the treatment of MIRI. Furthermore, it may play therapeutic roles through AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, prostate cancer, TNF signaling pathway and fluid shear stress and atherosclerosis, etc. This study reveals the potential targets and mechanism of XKST in the treatment of MIRI, and provides a reference for subsequent basic research.

Galectin‑1 alleviates myocardial ischemia‑reperfusion injury by reducing the inflammation and apoptosis of cardiomyocytes

Myocardial ischemia-reperfusion injury (MIRI) is one of the leading causes of morbidity and mortality worldwide, for which there is no effective treatment. The present study aimed to assess novel methods of clinical MIRI treatment by studying the effects of galectin-1 (Gal-1) on MIRI. Male 2-month-old Sprague Dawley rats and the rat cardiomyocyte cell line H9c2 were utilized in the present study. A rat model of MIRI was constructed by ligating the left anterior descending coronary artery, which was subsequently treated with Gal-1. Differences in myocardial injury were then assessed by hematoxylin and eosin (H&E) staining. In addition, the levels of inflammation and apoptosis in rat myocardial tissue were determined by immunohistochemistry staining. Hypoxia-reoxygenation was used to construct a model of MIRI in H9c2 cells. The effect of Gal-1 on the apoptosis and viability of H9c2 cells was also verified by flow cytometry and a Cell Counting Kit-8 assay. The results of H&E staining revealed that Gal-1 alleviated MIRI. Echocardiography demonstrated that Gal-1 improved cardiac function in rats following MIRI. In addition, MIRI increased levels of inflammation and apoptosis in rat myocardial tissues, with Gal-1 treatment reversing this effect. In cellular experiments, Gal-1 served anti-inflammatory and anti-apoptotic effects in hypoxic/reoxygenated cardiomyocytes. In conclusion, Gal-1 served a significant protective effect on the myocardial tissue after ischemia-reperfusion by reducing the level of inflammation and apoptosis in cardiomyocytes.

Retraction.

Retraction: "Anti-Apoptotic Effect of MicroRNA-30b in Early Phase of Rat Myocardial Ischemia-Reperfusion Injury Model," Chun-Li Song, Bin Liu, Jin-Peng Wang, Bei-Lin Zhang, Ji-Chang Zhang, Li-Yan Zhao, Yong-Feng Shi, Yang-Xue Li, Guan Wang, Hong-Ying Diao, Qian Li, Xin Xue, Jun-Duo Wu, Jia Liu, Yun-Peng Yu, Dan Cai, and Zhi-Xian Liu, J Cell Biochem. 2015; 2610-2619: The above article, published online on 28 April 2015 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.25208) has been retracted by agreement between the authors, the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed because the authors stated that unintentional errors occurred during the research process, and the experimental results cannot be verified. Thus, the conclusions are considered to be invalid. The authors apologize for the unintentional errors.

MiR-129 Attenuates Myocardial Ischemia Reperfusion Injury by Regulating the Expression of PTEN in Rats.

PTEN/AKT signaling plays pivotal role in myocardial ischemia reperfusion injury (MIRI), and miRNAs are involved in the regulation of AKT signaling. This study was designed to investigate the interaction between miR-129 and PTEN in MIRI. A MIRI rat model and a hypoxia reoxygenation (H/R) H9C2 cell model were constructed to simulate myocardial infarction clinically. TTC staining, creatine kinase (CK) activity, TUNEL/Hoechst double staining, Hoechst staining and flow cytometer were used for evaluating myocardial infarction or cell apoptosis. miR-129 mimic transfection experiment and luciferase reporter gene assay were conducted for investigating the function of miR-129 and the interaction between miR-129 and PTEN, respectively. Real-time PCR and western blotting were performed to analyze the gene expression. Compared to the control, MIRI rats presented obvious myocardial infarction, higher CK activity, increased expression of caspase-3 and PTEN, decreased expression of miR-129, and insufficient AKT phosphorylation. Consistently, H/R significantly increased the apoptosis of H9C2 cells, concomitant with the downregulation of miR-129, upregulation of PTEN and caspase-3, and insufficient phosphorylation of AKT, while miR-129 mimic obviously inhibited the expression of PTEN and caspase-3, increased the AKT phosphorylation, and decreased the cell apoptosis. Additionally, miR-129 mimic obviously decreased the relative luciferase activity in H9C2 cells. To our best knowledge, this study firstly found that the low expression of miR-129 accelerates the myocardial cell apoptosis by directly targeting 3′UTR of PTEN. miR-129 is an important biomarker for MIRI, as well as a potential therapy target.

HOTAIR/miR-17-5p Axis is Involved in the Propofol-Mediated Cardioprotection Against Ischemia/Reperfusion Injury.

BackgroundPropofol (PPF) ameliorates ischemia/reperfusion (I/R) injury in multiple organs by reducing apoptosis and release of pro-inflammatory cytokines. This study aims to explore the mechanism of PPF in attenuating myocardial ischemia-reperfusion injury (MIRI).Materials and MethodsRat MIRI model was established, and PPF pre-treatment was performed 10 min before I/R. H9c2 cardiomyocytes treated with hypoxia/reoxygenation (H/R) were used to establish an in vitro model. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate HOTAIR and miR-17-5p expression levels. Flow cytometry was employed to detect the apoptosis of H9c2 cells. The interaction between HOTAIR and miR-17-5p was determined by bioinformatics analysis, luciferase reporter gene analysis, and RNA immunoprecipitation experiments. STAT3 and p-STAT3 expressions were detected by Western blot.ResultsPPF pre-treatment significantly reduced creatine kinase isoenzyme (CK-MB) and serum lactate dehydrogenase (LDH) levels in the serum of the rats with MIRI. PPF pre-treatment remarkably up-regulated HOTAIR expression and down-regulated miR-17-5p expression in both in vivo and in vitro models. HOTAIR adsorbed miR-17-5p to repress the expression of miR-17-5p. PPF pre-treatment markedly inhibited cardiomyocyte apoptosis induced by I/R or H/R. HOTAIR knockdown could partially reverse the protective effects of PPF on MIRI. HOTAIR could activate STAT3 signaling via repressing miR-17-5p expression.ConclusionPPF protects the MIRI by modulating the HOTAIR/miR-17-5p/STAT3 axis.

Sufentanil preconditioning protects against myocardial ischemia/reperfusion injury via miR-125a/DRAM2 axis

ABSTRACT This project aimed to investigate the protective mechanism of sufentanil pretreatment on myocardial ischemia-reperfusion injury (IRI). An in vivo rat model of myocardial IRI and an in vitro cultured cardiomyocyte model of hypoxia-reoxygenation (H/R) were used to confirm the anti-oxidation and anti-autophagy effects of sufentanil. The interaction between miR-125a and damage-regulated autophagy regulator 2 (DRAM2) was verified by luciferase reporter assay. We showed that pretreatment with sufentanil suppressed myocardial damage caused by IRI in rats by inhibiting oxidative stress and mitochondrial autophagy. Furthermore, the cardioprotective mechanism of sufentanil was mediated by miR-125a. MiR-125a targeted DRAM2 to ameliorate cardiomyocyte autophagy and oxidative injury following H/R treatment. In conclusion, our results demonstrated that sufentanil pretreatment produced a protective effect against myocardial IRI via regulating miR-125a/DRAM2 signaling axis.

Protective action of the ginsenoside Rh3 in a rat myocardial ischemia-reperfusion injury model by inhibition of apoptosis induced via p38 mitogen-activated protein kinase/caspase-3 signaling.

ObjectiveTo investigate the protective effects of the ginsenoside Rh3 on rats subjected to myocardial ischemia-reperfusion (MIR) via its impact on caspase-3 and the p38 mitogen-activated protein kinase (MAPK) pathway.MethodsFifteen male Sprague-Dawley rats were randomly categorized into the MIR group (MY group, n = 5), sham surgery group (SS group, n = 5), and ginsenoside Rh3 group (GR group, n = 5).ResultsThe MY group exhibited the largest myocardial infarctions compared with the GR and SS groups. The GR group exhibited significantly higher cell viability of cardiomyocytes and significantly decreased apoptosis compared with the MY group. Fibrils of infarcted tissue in the GR group were disordered but less swollen, with a more organized fibril orientation than those in the MY group. The GR group showed reduced p-p38 MAPK protein and caspase-3 mRNA expression levels compared with the MY and SS groups.ConclusionsRh3 significantly improved myocardial necrosis and caspase-3 levels in myocardial tissues by suppressing the p38 MAPK pathway, thereby inhibiting caspase-3 involvement in apoptosis. Thus, Rh3 was effective in inhibiting the escalated apoptotic pathway in myocardial infarction and can potentially serve as a useful therapeutic agent to rescue myocardial infarction.

Effects of Promethazine on Proliferation and Apoptosis of Myocardial Cells in Rats with Myocardial Ischemia-Reperfusion Injury Through PI3K/Akt Signaling Pathway

Objective: To assess promethazine's effect on myocardial cells in rats with myocardial ischemiareperfusion injury (MIRI). Methods: The rat MIRI model was established and treated as the ischemia group. MIRI rats were treated with promethazine and included as the drug group. Rats only undergoing thoracotomy were enrolled as the control group. The physiological function of heart was assessed using the ultrasound cardiotachograph, and the apoptosis and proliferation of myocardial cells were detected using TUNEL assay and Ki67 staining, respectively. Moreover, the expressions of Caspase-3, Bcl-2, PI3K, GSK-3, PDK-1 and PKB were determined via Western blotting and qPCR. Results: There were significant differences in cardiac function indexes [left ventricular enddiastolic diameter (LVEDd), left ventricular end systolic diameter (LVESd), ejection fraction (EF) and fractional shortening (FS)] among the three groups (p= 0 002, 0.004, 0.025 and 0.012), and ischemia group had the highest LVEDd [(8.73± 0.31) mm] and LVESd [(7.98± 0.37) mm] and lowest EF [(42± 3.8)%] and FS [(40.3± 2.8)%]. The number of apoptotic myocardial cells was significant higher in ischemia group than control ( p< 0 05), while it was significantly declined after treatment with promethazine ( p< 0 05). Caspase-3 was significantly upregulated and Bcl-2 was downregulated in ischemia group which were all significantly reversed in drug group. Besides, Ki67 level was significantly reduced in ischemia group compared to control and higher in drug group than ischemia group, indicating that drug treatment increased cell proliferation ability. The levels of PI3K, GSK-3 and PKB in myocardial tissues were significantly declined in ischemia group and elevated after the treatment with promethazine without difference of PDK-1 level in myocardial tissues among the three groups. Conclusion: Promethazine inhibits apoptosis and promotes proliferation of myocardial cells in MIRI rats through PI3K/Akt signaling pathway.

MiR-346 Inhibited Apoptosis Against Myocardial Ischemia-Reperfusion Injury via Targeting Bax in Rats.

PurposeMyocardial ischemia-reperfusion injury (MIRI) is a common pathophysiological process after occlusion of the blood vessels to restore blood supply. Apoptosis is one of the ways of myocardial cell death in this process. MicroRNAs (miRNAs), a class of short and noncoding RNAs, are involved in multiple biological processes by post-transcriptionally targeting their downstream effectors. To date, whether miRNAs exert biological effects in myocardial ischemia-reperfusion (I/R) injury remains to be further studied.MethodsIn this study, we induced MIRI model by ligating rat left anterior descending artery (LAD) for 30 mins and reperfusion for 2 hrs. The differential expression profile of miRNAs in rat models of MIRI was analyzed by miRNAs sequencing.ResultsWe found that miRNAs sequencing analysis showed the expressions of 15 types of miRNAs, including miR-346, were downregulated and 29 types of miRNAs were elevated in the MIRI rat model. We observed the key regulator of apoptosis Bax was a predicted downstream target of miR-346 using online software TargetScan. And luciferase reporter assay was utilized to certify this prediction. Over-expression of miR-346 can attenuate myocardial injury and narrow infarct area by inhibiting myocardial cell apoptosis in rat models.ConclusionThis study revealed a novel pathway, miR-346/Bax axis, in the regulation of apoptosis in MIRI and which might be a new molecular mechanism and therapeutic target.

MiR-25 exerts cardioprotective effect in a rat model of myocardial ischemia-reperfusion injury by targeting high-mobility group box 1.

We previously confirmed the targeting of high-mobility group box 1 (HMGB1) by miR-25. This project aims to further investigate whether miR-25 improves myocardial ischemia-reperfusion injury (IRI) in vivo by targeting HMGB1. A rat model of myocardial IRI was established by the ligation of the left anterior descending coronary artery for 45 minutes followed by 2, 4, or 6 hours reperfusion. The expression of miR-25, HMGB1, and apoptosis-related proteins in the myocardium was determined by quantitative real-time polymerase chain reaction (PCR) and western blotting. The activities of myocardial enzymes and the release of inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay. Evans blue/triphenyltetrazolium chloride double staining was performed to assess infarct size. Myocardial apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. MiR-25 expression was significantly downregulated, while HMGB1 was highly expressed at the mRNA and protein levels in myocardial tissues after induction of the IRI model. MiR-25 agomir administration suppressed the expression of HMGB1 in myocardial tissues. Furthermore, administration of both miR-25 agomir and lentivirus-mediated short hairpin RNA (shRNA) interference targeting HMGB1 sh-HMGB1 resulted in reduced serum myocardial enzyme activities, cytokine secretion, and myocardial apoptosis during myocardial IRI. MiR-25 mitigated myocardial IRI-induced damage by targeting HMGB1.

Combination of the ginsenosides Rb3 and Rb2 exerts protective effects against myocardial ischemia reperfusion injury in rats.

Ginsenoside Rb3 (G-Rb3) has been demonstrated to alleviate myocardial ischemia reperfusion injury (MIRI); however, it is difficult to separate G-Rb2 from its isomer G-Rb3. The current study aimed to compare the cardioprotective effects of G-Rb3 and the concomitant use of G-Rb3 and G-Rb2 (G-Rb3/Rb2) on MIRI in rats. A rat model of MIRI was established by ligation of the left anterior descending coronary artery and the rats were randomly divided into five groups. Prior to MIRI, G-Rb3/Rb2 (20 mg/kg), G-Rb3 (20 mg/kg) and diltiazem (DLZ; 20 mg/kg, as a positive control) were orally administered to the rats once a day for 3 consecutive days. After 30 min of ischemia and 120 min of reperfusion, cardiac function, infarct size, cardiac marker enzymes, antioxidative parameters, inflammatory factors, histopathological changes, cardiomyocyte apoptosis, and B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein and caspase-3 expression were determined using a multi-channel physiological recording system, nitrotetrazolium blue chloride, biochemical kits, radioimmunoassay kits, hematoxylin and eosin, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay, immunohistochemistry and reverse transcription-quantitative PCR, respectively. The results indicated that treatment with G-Rb3/Rb2 significantly protected rats against MIRI, as shown by improved cardiac function, reduced myocardial ischemic area, decreased serum activities of aspartate aminotransferase, lactate dehydrogenase and creatine kinase MB, decreased serum concentrations of interleukin-6 and tumor necrosis factor-α, decreased malondialdehyde concentration in myocardial tissues, increased activities of superoxide dismutase, glutathione peroxidase and catalase in myocardial tissues, reduced histopathological changes in myocardial tissues, reduced number of apoptotic cardiomyocytes, and changes in the expression levels of caspase-3, Bcl-2 and Bax. In addition, the effects of treatment with G-Rb3/Rb2, G-Rb3 or DLZ were equivalent. The protective effects of G-Rb3/Rb2 on MIRI were similar to those of G-Rb3 in terms of oxidative stress, inflammatory factors and inhibition of cardiomyocyte apoptosis. Therefore, G-Rb3/Rb2 may be developed as a concomitant treatment for MIRI.

Quantitative proteomics reveal the alterations in the spinalcord after myocardial ischemia‑reperfusion injury in rats.

There is now substantial evidence that myocardial ischemia-reperfusion (IR) injury affects the spinal cord and brain, and that interactions may exist between these two systems. In the present study, the spinal cord proteomes were systematically analyzed after myocardial IR injury, in an attempt to identify the proteins involved in the processes. The myocardial IR injury rat model was first established by cross clamping the left anterior descending coronary artery for 30-min ischemia, followed by reperfusion for 2 h, which resulted in a significant histopathological and functional myocardial injury. Then using the stable isotope dimethyl labeling quantitative proteomics strategy, a total of 2,362 shared proteins with a good distribution and correlation were successfully quantified. Among these proteins, 33 were identified which were upregulated and 57 were downregulated in the spinal cord after myocardial IR injury, which were involved in various biological processes, molecular function and cellular components. Based on these proteins, the spinal cord protein interaction network regulated by IR injury, including apop-tosis, microtubule dynamics, stress-activated signaling and cellular metabolism was established. These heart-spinal cord interactions help explain the apparent randomness of cardiac events and provide new insights into future novel therapies to prevent myocardial I/R injury.

Protective effects of troxerutin preconditioning on ischemia-reperfusion myocardial in rats

Objective To investigate the protective effects of troxerutin preconditioning on ischemia-reperfusion myocardial in rats. Methods 24 male Wistar rats were divided into sham group, ischemia/reperfusion group (I/R group) and troxerutin preconditioning group (I/R+ TRX group) randomly. The myocardial ischemia-reperfusion injury rat model was created with troxerutin preconditioning. Myocardial infarct size was evaluated by the Evans blue-triphenyltetrazolium chloride (TTC) method. Cell apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) method. Hemodynamic parameters and cardiac function were monitored. Serum cardiac troponin I (cTnI), creatine kinase (CK), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α) and interleukin (IL)-10 were measured. Results Myocardial infarct size in I/R+ TRX group was significantly decreased than that in the I/R group (P 0.05). Serum cTnI, CK, LDH, TNF-α and IL-10 concentration in I/R group were significantly higher than those in sham group and I/R+ TRX group (P 0.05). Conclusion Troxerutin preconditioning may inhibition of the inflammatory response after myocardial ischemia-reperfusion, attenuates myocardial cell apoptosis, and protected against myocardial ischemia-reperfusion injury in rats. Key words: Troxerutin; Ischemia-reperfusion injury; Myocardial

Research on cardioprotective effect of irbesartan in rats with myocardial ischemia-reperfusion injury through MAPK-ERK signaling pathway.

The aim of this study was to explore whether the mechanism of Irbesartan (IRB) in the treatment of rats with myocardial ischemia-reperfusion injury (MIRI) was related to the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) signaling pathway. The rat model of MIRI was first successfully established. All rats were randomly divided into 5 groups, including the Sham group (sham operation control group, ligation only), Model group (MIRI rat model group), IRB12.5 group [low-dose IRB (12.5 mg/kg/d) group], IRB50.0 group [medium-dose IRB (50.0 mg/kg/d) group] and IRB200.0 group [high-dose IRB (200.0 mg/kg/d) group]. After treatment of IRB in MIRI rats, the activities of four myocardial enzyme indexes, including creatine kinase-MB (CK-MB), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and cardiac troponin T (cTnT), were detected via enzyme-linked immunosorbent assay (ELISA). The effect of IRB on myocardial apoptosis in MIRI rats was detected via Annexin V-fluorescein isothiocyanate/Propidium Iodide (FITC/PI) double staining. Meanwhile, the messenger ribonucleic acid (mRNA) levels of ERK, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in myocardial cells after treatment of IRB were detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Furthermore, the protein levels of ERK and p-ERK were detected via Western blotting. Different concentrations of IRB could protect myocardium from MIRI. IRB at doses of 50.0 mg/kg/d and 200.0 mg/kg/d could significantly downregulate myocardial enzyme indexes in MIRI (p<0.01). Meanwhile, both the doses could markedly inhibit myocardial apoptosis in MIRI rat model by regulating the expressions of apoptosis-related genes (Bcl-2 and Bax) (p<0.01), eventually improving myocardial pathological damage. At the same time, it could also significantly decrease the mRNA and protein levels of ERK in the MAPK-ERK signaling pathway (p<0.05). The cardioprotective mechanism of IRB in MIRI rats may be related to the inhibition of the activation of the MAPK-ERK signaling pathway.

Effect of ulinastatin on myocardial ischemia reperfusion injury through ERK signaling pathway.

To study the effect of ulinastatin (UTI) on myocardial ischemia-reperfusion injury (MIRI) through the extracellular signal-regulated kinase (ERK) signaling pathway. A total of 24 Sprague-Dawley rats were randomly divided into sham group (n=8), I/R group (n=8), and UTI group (n=8), and the rat model of MIRI was established. The changes in the content of serum biochemical indexes, including superoxide dismutase (SOD) and malondialdehyde (MDA), were detected using the kits, and the changes in the expressions of serum inflammatory factors, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were detected using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) kits. Moreover, the ERK phosphorylation level in myocardial tissues was detected using the immunofluorescence method, and the ERK phosphorylation level and cleaved caspase-3 expression were detected via qRT-PCR and Western blotting. Compared with those in sham group, the serum SOD content significantly declined, while the MDA content was significantly increased in I/R group, and they were significantly improved in UTI group (p<0.01). The results of detection using qRT-PCR and ELISA kits revealed that the inflammatory factors (IL-6 and TNF-α) in UTI group were significantly improved (p<0.01). The immunofluorescence results showed that the ERK phosphorylation level in myocardial tissues was significantly increased in UTI group. The results of qRT-PCR and Western blotting manifested that both ERK phosphorylation level and cleaved caspase-3 expression were significantly improved in UTI group (p<0.01). UTI can play a protective role in MIRI through up-regulating the ERK signaling pathway.

The role of high-mobility group box protein 1 in the signaling pathways of myocardial ischemia-reperfusion injury in rats

Objective: To investigate the role of high-mobility group box protein 1 (HMGB1) in the signaling pathways of myocardial ischemia-reperfusion injury (MIRI) in rats. Methods: Forty male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 4 groups (n=10) using a random number table: sham operation group (group sham), MIRI group (group IR-C), anti-HMGB1 antibody group (group IR-H-Ig), contrast antibody control group (group IR-Ig). The rat model of MIRI was established by 30 min occlusion of left anterior descending branch (LAD) of coronary artery followed by 180 min reperfusion. In sham group, no blocking of LAD was adopted after thoracotomy. Anti-HMGB1 antibody and contrast antibody immunoglobulin G (IgG) (2 mg/kg) were injected respectively at 30 min of reperfusion in IR-H-Ig and IR-Ig groups. Blood samples were collected from the femoral vein for determination of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), HMGB1, creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) concentrations at 180 min of reperfusion. The rats were then sacrificed after blood samples were taken and the pathological changes of myocardial tissue were observed. The mRNA and protein expressions of HMGB1, toll-like receptor 4(TLR4) and nuclear factor (NF)-κB in myocardial tissues were detected by Western blot and real-time quantitative PCR respectively. Results: Compared with the Sham group, the plasma level of IL-6, TNF-α, HMGB1 increased significantly and HMGB1, TLR4 and NF-κB mRNA and protein levels of myocardial tissues up-regulated in IR-C and IR-Ig groups (all P<0.05). The plasma level of CK-MB and cTnI increased significantly in IR-C, IR-H-Ig, IR-Ig group (all P<0.05). Compared with the IR-C group, the levels of the plasma HMGB1, the cytokines mentioned above, CK-MB and cTnI were significantly decreased, and mRNA and protein expressions of HMGB1, TLR4 and NF-κB of myocardial tissues down-regulated in IR-H-Ig group (all P<0.05). Inflammatory cell infiltration in MIRI groups increased significantly, while it was significantly reduced in IR-H-Ig group. Conclusion: Blocking the combination of HMGB1 and TLR4 can effectively alleviate the tissue damage caused by myocardial ischemia-reperfusion in rats.

Effect of Autophagy Inhibition on the Protection of Ischemia Preconditioning against Myocardial Ischemia/Reperfusion Injury in Diabetic Rats.

Background:Ischemia preconditioning (IPC) remains the most powerful intervention of protection against myocardial ischemia/reperfusion injury (IRI), but diabetes can weaken or eliminate its cardioprotective effect and detailed mechanisms remain unclear. In this study, we aimed to explore whether changes of autophagy in the diabetic condition are attributable to the decreased cardioprotective effect of IPC.Methods:Sixty diabetic male Sprague-Dawley rats were randomly divided into the control (C), IRI, rapamycin (R), wortmannin (W), rapamycin + IPC (R + IPC), and wortmannin + IPC (W + IPC) groups. The in vivo rat model of myocardial IRI was established by ligaturing and opening the left anterior descending coronary artery via the left thoracotomy. Durations of ischemia and reperfusion are 30 min and 120 min, respectively. Blood samples were taken at 120 min of reperfusion for measuring serum concentrations of troponin I (TnI) and creatine kinase isoenzyme MB (CK-MB) using the enzyme-linked immunosorbent assay. The infarct size was assessed by Evans blue and triphenyltetrazolium chloride staining. The expressions of LC3-II, beclin-1, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), and P-Akt/Akt ratio in the ischemic myocardium were assessed by Western blotting.Results:Compared to the IRI group, infarct size (56.1% ± 6.1% vs. 75.4 ± 7.1%, P < 0.05), serum cTnI (0.61 ± 0.21 vs. 0.95 ± 0.26 ng/ml, P < 0.05), and CK-MB levels (6.70 ± 1.25 vs. 11.51 ± 2.35 ng/ml, P < 0.05) obviously decreased in the W + IPC group. Compared with the C group, myocardial expressions of LC3-II (0.46 ± 0.04 and 0.56 ± 0.04 vs. 0.36 ± 0.04, P < 0.05) and beclin-1 (0.34 ± 0.08 and 0.38 ± 0.07 vs. 0.24 ± 0.03, P < 0.05) evidently increased, and myocardial expressions of mTOR (0.26 ± 0.08 and 0.25 ± 0.07 vs. 0.38 ± 0.06, P < 0.05), PI3K (0.29 ± 0.04 and 0.30 ± 0.03 vs. 0.38 ± 0.02, P < 0.05), and P-Akt/Akt ratio (0.49 ± 0.10 and 0.48 ± 0.06 vs. 0.72 ± 0.07, P < 0.05) markedly decreased in the IRI and R groups, indicating an increased autophagy. Compared with the IRI group, myocardial expression of beclin-1 (0.26 ± 0.03 vs. 0.34 ± 0.08, P < 0.05) significantly decreased, and myocardial expressions of mTOR (0.36 ± 0.04 vs. 0.26 ± 0.08, P < 0.05), PI3K (0.37 ± 0.03 vs. 0.29 ± 0.04, P < 0.05), and P-Akt/Akt ratio (0.68 ± 0.05 vs. 0.49 ± 0.10, P < 0.05) increased obviously in the W + IPC group, indicating a decreased autophagy.Conclusions:Increased autophagy in the diabetic myocardium is attributable to decreased cardioprotection of IPC, and autophagy inhibited by activating the PI3K-Akt-mTOR signaling pathway can result in an improved protection of IPC against diabetic myocardial IRI.

Shengmai injection reduces apoptosis and enhances angiogenesis after myocardial ischaemia and reperfusion injury in rats

ObjectivesTo investigate whether Shengmai injection (SMI) helps to improve cardiac function and enhances angiogenesis after myocardial ischaemia reperfusion injury (MIRI). A rat model of MIRI was created via occlusion of the left anterior descending coronary artery for 30 min, followed by 3 days or 7 days of reperfusion (n = 6 each group). BackgroundsSMI is widely used for the treatment of myocardial infarction. The mechanism underlying the effect on cardiac function is not known and whether SMI has any effects on angiogenesis during treatment of MIRI is not clear. Results and conclusionEchocardiography showed that SMI improved the left ventricular ejection fraction (LVEF) in the rat model of MIRI. TUNEL staining indicated that SMI decreased the myocardial apoptosis rate after MIRI. This result may be related to the increase of Bcl-2 expression in the SMI group and a reduction in Bax and caspase 3 expression, as determined by immunohistochemical staining. Small vessels (<60 μm in diameter) of the heart of rats in the group treated with SMI were denser (more numerous) than those in the heart of rats in the other groups. Real-time PCR indicated that the SMI-driven reduction in apoptosis was associated with a change in the ratio of Bcl-2 to Bax expression, and treatment-induced angiogenesis was associated with enhanced vascular endothelial growth factor A (VEGF) expression. We elucidated that SMI promotes angiogenesis, which is important for the development of cardiac remodelling after MIRI.

Association of the PPARγ/PI3K/Akt pathway with the cardioprotective effects of tacrolimus in myocardial ischemic/reperfusion injury.

Myocardial ischemia/reperfusion injury (MIRI) induces severe arrhythmias and has a high risk of mortality. The aim of the present study was to investigate the effect of tacrolimus on arrhythmias, cardiac function, oxidative stress and myocardium apoptosis induced by MIRI, and to elucidate the underlying mechanism. The effects of MIRI and tacrolimus on arrhythmias, cardiac function parameters, myocardial oxidative stress and apoptosis were investigated in a rat model of MIRI. The phosphorylation of peroxisome proliferator‑activated receptor γ (PPARγ) and protein kinase B (Akt) was investigated via western blotting. After rats were treated with inhibitors of PPARγ/phosphoinositide 3‑kinase (PI3K)/Akt, cardiac function parameters were measured. The results demonstrated that the MIRI procedure induced arrhythmias and significant impairment of cardiac function, oxidative stress and apoptosis in cardiomyocytes (P<0.05). Tacrolimus significantly alleviated the arrhythmias and impairment of cardiac function and inhibited the oxidative stress and apoptosis in cardiomyocytes (P<0.05). The phosphorylation of PPARγ and Akt was significantly activated by tacrolimus, whereas inhibitors of PPARγ/PI3K/Akt significantly abolished the effects of tacrolimus (P<0.05). Together, these results suggest that tacrolimus may protect rats from MIRI through activation of the PPARγ/PI3K/Akt pathway.

Prdx1 alleviates cardiomyocyte apoptosis through ROS-activated MAPK pathway during myocardial ischemia/reperfusion injury

Apoptosis induced by oxidative stress blocks the recovery of heart function in myocardial ischemia reperfusion injury (MIRI). Peroxiredoxin 1 (Prdx1) inhibits oxidative stress. However, the expression and function of Prdx1 in MIRI are unclear. In present study, Prdx1 protein level increased in rat MIRI model, associated with cardiomyocyte apoptosis. Cultured rat embryonic ventricular myocardial H9c2 cells with hypoxia/reoxygenation (H/R) treatment was utilized to mimic MIRI in vitro, showing that H/R treatment increased the ratio of p-p38/p38, p-JNK/JNK and apoptosis index. But Prdx1 ameliorate the up-regulation of p-p38/p38 ratio and p-JNK/JNK ratio, as well as decreased H9c2 cell apoptosis. SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) inhibited H9c2 cell apoptosis, and at the same time Prdx1 down-regulated the activation of p38 MAPK and JNK during H/R treatment. In addition, a ROS scavenger N-acetyl-l-cysteine (NAC) down-regulated the protein level of p-p38, p-JNK and Prdx1, and H9c2 cell apoptosis. In summary, these findings indicated that Prdx1 inhibited MAPK pathway induced cells apoptosis, and ROS is the upstream regulator of H/R induced apoptosis.