Myocardial Ischemia-reperfusion Injury Model Research Articles

Effect of "Jiaji" (EX-B2)-electroacupuncture preconditioning on expression of myocardial cytochrome P450 enzyme in rats with acute myocardial ischemia-reperfusion injury

To investigate the effect of "Jiaji"(EX-B2)-electroacupuncture(EA) preconditioning on structural changes of myocardium and expression of cytochrome P450 (CYP450) enzymes in acute myocardial ischemia-reperfusion injury (MIRI) rats. Sixty male Wistar rats were randomly divided into sham operation, model, EX-B2, Neiguan (PC6), Yanglingquan(GB34) and Quchi (LI11) groups (n＝10 in each group). The MIRI model was established by occlusion of the descending anterior branch of the left coronary artery for 30 min, followed by reperfusion for 30 min. EA preconditioning (2 Hz/100 Hz, 1 mA) was respectively applied to EX-B2, PC6, GB34, and LI11 for 30 min, once daily for 7 days. Morphological changes of myocardium were observed by H．E. staining. The ultrastructure of myocardial cells was observed by transmission electron microscope (TEM). The protein expression levels of myocardial CYP450 (CYP1A1, CYP2B2, CYP2C11 and CYP4A2) were detected by Western blot. In MIRI rats, myocardial pathological changes as swollen, disorganized arrangement, and partially ruptured myocardial fibers with unclear limit and necrosis and infiltration of lots of inflammatory cells under microscope, and focal myofilament degeneration, dissolution and necrosis with interstitial edema, vague mitochondria with swelling and vacuolation, ruptured and disorganized arrangement of crests under TEM were found in the ischemic area, which was obviously milder in rats of both EX-B2 and PC6 groups. After modeling, the expression of myocardial CYP1A1 and CYP2B2 protein were significantly up-regulated in the model group relevant to the sham operation group (P<0.05). In EA preconditioning groups, the expression levels of CYP1A1 and CYP4A2 proteins in the EX-B2, PC6 and GB34 groups were obviously down-regulated (P<0.01), and those of CYP2B2 and CYP2C11 proteins in both EX-B2 and PC6 groups were markedly up-regulated relevant to the model group (P<0.01). Compared with the EX-B2 group, the expression levels of CYP1A1, CYP4A2 proteins in the LI11 group were significantly up-regulated (P<0.05), and CYP2B2, CYP2C11 proteins were significantly down-regulated (P<0.05). No significant differences were found between the model and sham operation groups in the expression levels of myocardial CYP2C11 and CYP4A2 proteins, between the LI11 and model groups in the expression of CYP1A1, CYP2B2, CYP2C11 and CYP4A2, between the GB34 and model groups in the expression of CYP2B2, CYP2C11 (all P>0.05)．. EA preconditioning at EX-B2 and PC6 can suppress MIRI induced up-regulation of myocardial CYP450 ω-hydroxylase CYP1A1 and CYP4A2 expression and further up-regulate the expression of myocardial epoxygenase CYP2B2 and CYP2C11 proteins in acute MIRI rats, thus playing a role in myocardial protection.

Serum metabolic profile involving protective effect of "Neiguan"(PC6)-electroacupuncture preconditioning in rats with myocardial ischemia reperfusion injury

To observe the effect of "Neiguan" (PC6)-electroacupuncture (EA) preconditioning on serum metabolites in myocardial ischemia-reperfusion injury (MIRI) rats, so as to reveal its mechanism underlying improvement of ischemic myocardium from metabonomics. A total of 48 male SD rats were randomly divided into control, model, EA "Neiguan"(PC6) and EA "Hegu"(LI4) groups (n＝12 rats/ group). Rats of the control group were just banded on animal boards for 30 min, once daily for 7 days. The MIRI model was established by occlusion of the left anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 1 h, and rats of the model group were also banded as those in the control group. Before modeling, EA (10 Hz/50 Hz, 1 mA) was applied to bilateral "Neiguan"(PC6) and "Hegu"(LI4) for 30 min, once daily for 7 successive days. After the treatment, serum samples were collected to be analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy. The orthogonal partial least squares discriminate analysis (PLS-DA) was employed to distinguish the serum differential metabolic profile of rats in different groups and identify potential biomarkers. After modeling, the ECG of model group and electroacupuncture groups showed T wave towering, and there was no obvious ST segment between R wave and T wave. The T wave decreased more than 0.2 mV after reperfusion, and there was no obvious ST segment. Compared with the control group, MIRI induced significant changes of metabolites in the serum including increase of acetoacetate acid, lectic acid, creatine, glycerol and glucose, and decrease of alanine, glutamine, glycerophosphoryl choline and phosphorylcholine. In comparison with the model group, PC6-EA preconditioning induced significant changes, including an increase of glucose, and a decrease of leucine，isoleucine, valine，3-hydroxybutyric acid，lactate，acetate，acetone，acetoacetate acid，pyruvic acid，glutamine，creatine and glycerol. There is no significant difference in metabolic patterns between "Hegu" group and model group. Metabolic pathway enrichment analysis indicated that the protective effect of PC6-EA pretreatment was realized mainly by regulating pathways of glycolysis, gluconeogenesis, citric acid metabolism, pyruvate metabolism, ketone body metabolism, etc. PC6-EA pretreatment has a role in regulating gluconeogenesis, pyruvate metabolism, amino metabolism, ketone body metabolism and energy metabolism in rats with MIRI, which maybe contribute to its protective effect on ischemic myocardium, but the specific metabolic pathways and mechanisms need being studied further.

Effect of hyperoxic reperfusion on final infarct size after ST elevation myocardial infarction (STEMI) in old Wistar rats

Elderly people above 65 years old account for more than 80% of patients with ischemic heart disease [1] . Most of animal models of myocardial ischemia-reperfusion injury (IRI) involve young animals, with the assumption that findings are applicable to older animals and thus applicable to humans at highest risk, the elderly. Current studies failed to analyse the effect of hyperoxia during myocardial infarction (MI) reperfusion in old rodent model. Assess the final myocardial infarct size after normoxic and hyperoxic MI reperfusion in old Wistar rats. STEMI was induced in old male Wistar rats (35–40 weeks old) by ligation of the left anterior descending (LAD) artery and maintained for 60 minutes. Animals were randomised either in the hyperoxic reperfusion (HR) group (receiving 100% oxygen) or the normoxic reperfusion (NR) group (21% of oxygen) for a 3-hour reperfusion. At the end of reperfusion, Evans Blue dye solution was injected to delineate the ischemic area from the non-ischemic area. The heart was cut into transverse slices and immerged in TTC solution. Myocardial infarct size was assessed in two ways; first troponin T concentration was measured after reperfusion, secondly ischemic zone and area at risk of the myocardium slices were measured by planimetry. A total of 8 rats underwent HR vs 11 NR. No difference was observed in the final myocardial infarct size between the two groups in both methods of myocardial infarct size assessment. These very preliminary results corroborate the observations made in young animals’ studies [2] , [3] , however they should be taken cautiously since they are based on a very small cohort. There is a need to understand the effect of hyperoxic MI reperfusion as animal studies contradict the findings in human studies [4] .

Comparative regenerative properties of mesenchymal stem cells isolated from MRL (Murphy Roths Large) versus C57Bl6 mice

Adult Mammals are unable to regenerate their injuried myocardium by contrast to Zebrafish or MRL mice. While myocardial repair has been shown after cryoinjury in MRL mice, it failed after coronary ligation leading to ischemic injury. This underlies the impact of the inflammatory environment on the intrinsic regenerative potential of MRL stem cells. Mesenchymal stem cells (MSC) administered as cardiac therapy have emerged as breakthrough immunomodulative and cytoprotective treatments for myocardial infarction. to compare the intrinsic regenerative properties of MSC derived from C57BL/6 (MSC-BL6) and MRL (MSC-MRL) mice in an ex vivo myocardial ischemia-reperfusion (IR) injury model and decipher the underlying mechanisms. FACS analysis allowed characteristic phenotyping of MSC isolated from C57BL/6 (MSC-B6) or MRL (MSC-MRL) murine bone marrow. Their differentiation potential into chondrocytes, osteoblasts and adipocytes upon inductive conditions was studied. Their migration capacities were evaluated using the in vitro scratch wound healing assay. Infarct size was measured in mouse hearts subjected to ex vivo 30 min-ischemia and 1 hour-reperfusion and treated by MSCs. In vitro, MSC-MRL showed a significantly higher migration potential than MSC-B6 in accordance with reduced expression levels of E-cadherin and cadherin 11 adhesion molecules. Using MRL-MSC conditioned medium, we demonstrated an increased migration capacity of MSC-B6 suggesting that the MRL-MSC release a soluble factor responsible for their potent homing potential to the wound site. Injected during reperfusion, MSC-MRL were able to protect the injured myocardium more efficiently than MSC-B6 in the ex vivo IR model (MSC-MRL: 47.18 ± 0.66%, n = 3 vs MSC-B6: 56.36 ± 3.17%, n = 3). MSC-MRL have superior cardioprotective effects for the treatment of IR injury compared to MSC-B6 probably due to higher migration capacities and specific regenerative secretome.

Pretreatment with Tilianin improves mitochondrial energy metabolism and oxidative stress in rats with myocardial ischemia/reperfusion injury via AMPK/SIRT1/PGC-1 alpha signaling pathway

Mitochondrial energy metabolism and oxidative stress play a crucial role in ameliorating myocardial ischemia/reperfusion injury (MIRI). Tilianin has been reported to have a significant protection for mitochondrion in MIRI. However, the underlying mechanisms remain unknown. This study investigated whether Tilianin regulates mitochondrial energy metabolism and oxidative stress in MIRI via AMPK/SIRT1/PGC-1 alpha signaling pathway. The MIRI model was established by 30 min of coronary occlusion followed by 2 h of reperfusion in rats. The results revealed that Tilianin significantly reduced myocardial infarction, improved the pathological morphology of myocardium, markedly increased the contents of ATP and NAD+, decreased ADP and AMP contents and the ratio of AMP/ATP, reduced the level of ROS and MDA, enhanced SOD activity, evidently increased the levels of AMPK, SIRT1 and PGC-1 alpha mRNA, up-regulated the expressions of AMPK, pAMPK, SIRT1, PGC-1alpha, NRF1, TFAM and FOXO1 proteins. However, these effects were respectively abolished by Compound C (a specific AMPK inhibitor) and EX-527 (a specific SIRT1 inhibitor). Taken together, this study found that Tilianin could attenuate MIRI by improving mitochondrial energy metabolism and reducing oxidative stress via AMPK/SIRT1/PGC-1 alpha signaling pathway.

Ginkgo biloba extract-761 protects myocardium by regulating Akt/Nrf2 signal pathway.

ObjectiveThe aim of this study was to investigate the protective effect and mechanism of Ginkgo biloba extract-761 (EGb 761) in the rat with myocardial ischemia–reperfusion injury (MIRI).Materials and methodsForty Sprague Dawley rats were randomly divided into following four groups: sham group, I/R group and EGb 761 groups (20 and 40 mg/kg). MIRI model was established after 14 days of administration. The myocardial infarct size and myocardial histology were measured and compared. Meanwhile, the levels of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), troponin T (TnT), TNF-α, IL-6, IL-1β, superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were evaluated. Western blot was used to detect the expression of Caspase-3, Bax, Bcl-2, HO-1, Nrf2, Akt, p-Akt and nuclear protein Nrf2.ResultsThe levels of infarct size, CK-MB, LDH, TnT, TNF-α, IL-6 and IL-1β in the EGb 761 groups were significantly lower than those in the ischemia/reperfusion (I/R) group. The content of MDA was lower in the myocardium, whereas the activities of SOD and GSH-Px were higher than those in the I/R group. The expressions of Caspase-3 and Bax in the EGb 761 groups were significantly lower than those in the I/R group, whereas the expressions of Bcl-2, p-Akt and HO-1 and nuclear protein Nrf2 in the EGb 761 groups were higher than those in the I/R group.ConclusionEGb 761 might inhibit the apoptosis of myocardial cells and protect the myocardium by activating the Akt/Nrf2 pathway, increasing the expression of HO-1, decreasing oxidative stress and repressing inflammatory reaction.

Effect of isoflurane on myocardial ischemia-reperfusion injury through the p38 MAPK signaling pathway.

To investigate the effect of isoflurane on myocardial ischemia-reperfusion injury through the p38 mitogen-activated protein kinase (MAPK) signaling pathway. A total of 36 specific-pathogen-free (SPF) Sprague-Dawley rats were randomly divided into sham group (n=12), model group (n=12) and isoflurane group (n=12). In model group and isoflurane group, the myocardial ischemia-reperfusion injury model was established via the ligation of left anterior descending coronary artery (ischemia for 30 min and reperfusion for 3 h). In sham group, the left anterior descending coronary artery was not ligated, but the chest was opened and threaded using the same method. After ischemia, the rats in isoflurane group were inhaled with isoflurane. The cardiac function of rats in each group was detected before ischemia (T0) and once every 2 h after reperfusion (T1-T4) for a total of 5 times, and the cardiac function indexes included ejection fraction (EF), fractional shortening (FS), left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP). After the rats were executed, the myocardial infarction tissues were taken for hematoxylin-eosin (HE) staining and 2,3,5-triphenyltetrazolium chloride (TTC) staining to observe the morphological changes in tissues and the degrees of myocardial ischemia and infarction. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in myocardial cells in the infarction site in each group were detected using the MDA and SOD kits. Moreover, the expression levels of related proteins in the p38 MAPK signaling pathway in myocardial cells in the infarction site were detected via Western blotting. In model group, the cardiac function was significantly damaged (p<0.01), there was significant pathological damage in the myocardium, the area of myocardial infarction was significantly increased (p<0.01), the MDA content was significantly increased (p<0.01), the SOD activity declined obviously (p<0.01), and the expression levels of p-p38 and p-tau protein were significantly increased (p<0.01) compared with those in control group. After intervention with isoflurane, the cardiac function of rats was significantly improved (p<0.01), the pathological damage in myocardial tissues was alleviated, the area of myocardial infarction was reduced (p<0.01), the MDA content declined (p<0.01), the SOD activity was increased (p<0.01), and the expression levels of p-p38 and p-tau protein were decreased (p<0.01). Isoflurane can, through inhibiting the p38 MAPK signaling pathway, effectively protect the cardiac function of rats from myocardial ischemia-reperfusion injury, reduce the area of myocardial infarction, alleviate the pathological damage in myocardial cells and reduce the oxidative stress response.

Electroacupuncture and moxibustion pretreatments reduce cardiomyocyte apoptosis and autophagy in rats with myocardial ischemia/reperfusion injury

To observe the effect of "Neiguan" (PC6)-electroacupuncture (EA) or moxibustion (Moxi) pretreatment on myocardial apoptosis and expression of autophagy related proteins light chain (LC) 3-Ⅰ and LC3-Ⅱ in rats with myocardial ischemia/reperfusion injury (MIRI), so as to explore their mechanisms underlying improvement of MIRI. Forty SD rats (half male and half female) were randomly divided into sham operation, model, ischemic preconditioning (IP), EA and Moxi groups (n＝8 in each group). EA (10 Hz/50 Hz, 1 mA) or Moxi (ignited moxa stick) was respectively applied to bilateral "Neiguan" (PC6) for 20 min, once daily for 7 days. The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 60 min. The ultrastructural changes and autophagy of myocardial cells were observed by electron microscopy (EM), and the myocardial cellular apoptosis [apoptotic index ＝ (number of apoptotic cells/total number of cardiomyocytes)×100%] was detected by the terminal deoxyribonucleotidyl transferase mediated dUTP nick end labelling (TUNEL) method. The expressions of LC3-Ⅰ and LC3-Ⅱ proteins (markers for autophagy) in myocardial tissue were detected by Western blot. Following MI, EM observation revealed a vague structure of cardiomyocytes and muscular horizontal grain, dissolution of myofibers, mitochondrial swelling, some autophagic vacuoles and autophagic lysosomes at different degrees and surrounded by a double membrane in the model group, these situations were apparently milder in the EA and Moxi groups. The apoptosis index, myocardial LC3-Ⅰ and LC3-Ⅱ protein expression levels, and the ratio of LC3-Ⅱ/Ⅰ were significantly increased in the model group relevant to the sham operation group (P<0.05). After the treatment, the apoptosis index, the expression level of myocardial LC3-Ⅱ protein and the ratio of LC3-Ⅱ/Ⅰ were considerably down-regulated in the IP, EA and Moxi groups in comparison with those in the model group (P<0.05). The effect of EA was obviously superior to those of IP and Moxi in down-regulating the apoptosis index (P<0.05), but obviously inferior to those of IP and Moxi in down-regulating the levels of LC3-Ⅱ and LC3-Ⅱ/Ⅰ (P<0.05). No significant changes were found in the expression of LC3-Ⅰ after IP, EA and Moxi interventions in comparison with the model group (P>0.05), and no significant differences were observed in the apoptosis index and levels of LC3-Ⅱ and LC3-Ⅱ/Ⅰ between the IP and Moxi groups (P>0.05)．. Both EA and moxibustion pretreatments, similar to IP, have a positive role in reducing myocardiocyte apoptosis and regulating autophagy-related protein expression in MIRI rats, which maybe contribute to their protective effects on ischemic myocardium.

Downregulated microRNA-330 suppresses left ventricular remodeling via the TGF-β1/Smad3 signaling pathway by targeting SRY in mice with myocardial ischemia-reperfusion injury.

microRNAs (miRs) are essential in the development of heart failure. The aim of this study is to investigate the effect of microRNA-330 (miR-330) on left ventricular remodeling via the TGF-β1/Smad3 signaling pathway by targeting the sex-determining region Y (SRY) in mice with myocardial ischemia-reperfusion injury (MIRI). Differentially expressed gene (DEG) in myocardial ischemia-reperfusion (IR) was screened out and the miR that targeted the DEG was also predicted and verified. A model of MIRI was established to detect the expression of miR-330, SRY, transforming growth factor-β (TGF-β1), and Sekelsky mothers against dpp3 (Smad3). To further investigate the role of miR-330 in MIRI with the involvement of SRY and TGF-β1/Smad3 signaling pathway, the modeled mice were treated with different mimic, inhibitor, or small interfering RNA (siRNA) to observe the changes of the related gene expression, as well as the myocardial infarction size and volume of myocardial collagen. SRY was screened out and verified as a target gene of miR-330. The MIRI mice showed enlarged myocardial infarction size, increased volume of myocardial collagen, increased expression of miR-330, TGF-β1 and Smad3, while decreased the expression of SRY. The MIRI mice treated with miR-330 inhibitor showed decreased myocardial infarction size, the volume of myocardial collagen, and expression of TGF-β1 and Smad3 but promoted expression of SRY. Our findings demonstrated that downregulated miR-330 could suppress left ventricular remodeling to inhibit the activation of the TGF-β1/Smad3 signaling pathway via negatively targeting of SRY in mice with MIRI. This can be a potential target in the strategy to attenuate patient suffering.

Notch1 provides myocardial protection by improving mitochondrial quality control.

Mitochondrial quality control is a new target for myocardial protection. Notch signaling plays an important role in heart development, maturation, and repair. However, the role of Notch in the myocardial mitochondrial quality control remains elusive. In this study, we isolated myocardial cells from rats and established myocardial ischemia reperfusion injury (IRI) model. We modulated Notch1 expression level in myocardial cells via infection with recombinant adenoviruses Ad-N1ICD and Ad-shN1ICD. We found that IR reduced myocardial cells viability, but Notch1 overexpression increased the viability of myocardial cells exposed to IRI. In addition, Notch1 overexpression improved ATP production, increased mitochondrial fusion and decreased mitochondrial fission, and inhibited mitophagy in myocardial cells exposed to IRI. However, N1ICD knockdown led to opposite effects. The myocardial protection role of Notch1 was related to the inhibition of Pink1 expression and Mfn2 and Parkin phosphorylation. In conclusion, Notch1 exerts myocardial protection and this is correlated with the maintenance of mitochondrial quality control and the inhibition of Pink1/Mfn2/Parkin signaling.

Influences of remifentanil on myocardial ischemia-reperfusion injury and the expressions of Bax and Bcl-2 in rats.

To investigate the influences of remifentanil on myocardial ischemia-reperfusion injury in rats and the expressions of b-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and other apoptosis-related proteins. A total of 60 Sprague-Dawley (SD) rats were randomly divided into sham operation (S) group, model (M) group, low-dose remifentanil (L) group and high-dose remifentanil (H) group, with 15 rats in each group. The rat model of myocardial ischemia-reperfusion injury was established by the ligation of the left anterior descending branch (LAD). After ischemia for 30 min and reperfusion for 24 h, the cardiac function of rats in each group was measured by an ultrasonic instrument. Triphenyl tetrazolium chloride (TTC) staining was used to detect the myocardial infarction area of rats in each group. The activity of myocardial enzymes in the serum of rats in each group was detected. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was adopted to examine the apoptosis level of rat cardiomyocytes in each group. Quantitative polymerase chain reaction (qPCR) and Western blotting were applied to detect the expression levels of apoptosis-related proteins and messenger ribonucleic acids (mRNAs) in rat cardiomyocytes in each group. Compared with those in S group, left ventricular internal dimension in systole (LVIDs) and left ventricular internal dimension in diastole (LVIDd) were markedly increased (p<0.01), while left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were notably decreased in M group (p<0.01). LVIDs and LVIDd in L group and H group were lower than those in M group (p<0.05, p<0.01), whereas LVEF and LVFS were higher than those in M group (p<0.05, p<0.01). The myocardial infarction area in M group was significantly larger than that in S group (p<0.01), and those in L group and H group were remarkably smaller than that in M group (p<0.05, p<0.01). The activities of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase-muscle/brain (CK-MB) in the serum of rats in M group were evidently higher than those in S group (p<0.01), and compared with those in M group, those in L group and H group were significantly decreased (p<0.05, p<0.01). The apoptosis level of myocardial cells in M group was significantly higher than that in S group (p<0.01), while those in L group and H group were markedly lower than that in M group (p<0.05, p<0.01). Compared with those in S group, the expression levels of cleaved caspase-3 and its mRNAs in myocardial cells in M group were remarkably increased (p<0.01), while those of Bcl-2/Bax and it mRNAs were significantly decreased (p<0.01). The expression levels of cleaved caspase-3 and its mRNAs in myocardial cells in L group and H group were significantly lower than those in M group (p<0.05, p<0.01), but those of Bcl-2/Bax and its mRNAs were significantly higher than those in M group (p<0.05, p<0.01). Remifentanil can effectively reduce myocardial cell injury caused by myocardial ischemia-reperfusion in rats, improve cardiac function, reduce the myocardial infarction area, decrease cleaved caspase-3 in myocardial cells, and increase Bcl-2/Bax.

Pretreatment with Total Flavonoid Extract from Dracocephalum Moldavica L. Attenuates Ischemia Reperfusion-induced Apoptosis

We previously demonstrated the cardio-protection mediated by the total flavonoid extracted from Dracocephalum moldavica L. (TFDM) following myocardial ischemia reperfusion injury (MIRI). The present study assessed the presence and mechanism of TFDM-related cardio-protection on MIRI-induced apoptosis in vivo. Male Sprague-Dawley rats experienced 45-min ischemia with 12 h of reperfusion. Rats pretreated with TFDM (3, 10 or 30 mg/kg/day) were compared with Sham (no MIRI and no TFDM), MIRI (no TFDM), and Positive (trapidil tablets, 13.5 mg/kg/day) groups. In MIRI-treated rats, high dose-TFDM (H-TFDM) pre-treatment with apparently reduced release of LDH, CK-MB and MDA, enhanced the concentration of SOD in plasma, and greatly reduced the infarct size, apoptotic index and mitochondrial injury. H-TFDM pretreatment markedly promoted the phosphorylation of PI3K, Akt, GSK-3β and ERK1/2 in comparison with the MIRI model group. Western blot analysis after reperfusion also showed that H-TFDM decreased release of Bax, cleaved caspase-3, caspase-7 and caspase-9, and increased expression of Bcl-2 as evident by the higher Bcl-2/Bax ratio. TFDM cardio-protection was influenced by LY294002 (PI3K inhibitor) and PD98059 (ERK1/2 inhibitor). Taken together, these results provide convincing evidence of the benefit of TFDM pretreatment due to inhibited myocardial apoptosis as mediated by the PI3K/Akt/GSK-3β and ERK1/2 signaling pathways.

Hydroxytyrosol protects against myocardial ischemia reperfusion injury by inhibiting mitochondrial permeability transition pore opening.

Hydroxytyrosol (HT), a phenolic compound extracted from olive oil, is reported to protect against myocardial ischemia reperfusion injury (MIRI), but its mechanism has not been fully elucidated. The mitochondria permeability transition pore (MPTP) is an important therapeutic target for MIRI. The present study aimed to investigate the role of MPTP in the cardioprotection of HT. Isolated rat hearts were mounted on a Langendorff apparatus and subjected to 30 min of ischemia followed by 120 min of reperfusion to mimic a MIRI model. Isolated hearts were pretreated with different doses of HT (10, 100 and 1,000 µM) for 10 min prior to ischemia. Myocardial infarct size was detected using TTC staining. Changes in myocardial cell structure were observed using hematoxylin and eosin staining. MPTP opening was detected spectrophotometrically. Myocardial cell apoptosis was observed with terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assays. The expression of apoptosis-associated proteins was measured by western blot analysis. The data revealed that HT (100 and 1,000 µM) treatment significantly alleviated pathological damage in ischemic myocardium and reduced myocardial infarct size compared with the untreated control. However, no significant difference was observed in the 10 µM HT treatment group compared with the untreated control. It was further revealed that HT decreased the B cell lymphoma-2 (Bcl-2)-like protein 4 (Bax)/Bcl-2 ratio, suppressed MPTP opening and subsequently decreased the expression of cytochrome c, cleaved caspase-9 and −3, thereby inhibiting apoptosis. Additionally, the beneficial effects of HT on MIRI were reversed by atractyloside, which induces MPTP opening. In conclusion, the present study demonstrated that HT inhibited MPTP opening, partially via modulation of Bax and Bcl-2, thereby protecting against MIRI and thereby providing a pharmacological basis for future research and treatment of MIRI.

Effects of electroacupuncture at different time during reperfusion on the expression of Bcl-2 and Beclin1 in myocardial tissue in rats with myocardial ischemia reperfusion injury

To compare the effects of electroacupuncture (EA) at different time during reperfusion on the expression of autophagy-related protein Bcl-2 and Beclin1 in myocardial tissue in rats with myocardial ischemia reperfusion injury (MIRI), and to explore the autophagy-related mechanism of EA on protecting MIRI. A total of 72 SD rats were randomly divided into a sham operation group, a model group, a RA group, a RB group, a RC group and a RD group, 12 rats in each group. Except the sham operation group, the rats in the remaining groups were treated with ligating the left anterior descending artery (LAD) for 30 minutes followed by reperfusion to establish the model of MIRI. The rats in the sham operation group were treated with crossing a line through the LAD. The rats in the model group did not receive treatment. The rats in the RA group, RB group, RC group and RD group were treated with EA at "Neiguan" (PC 6) for 20 min, starting at 0 h, 0.5 h, 1 h, 2 h after reperfusion. Evans Blue-TTC double-staining was employed to evaluate myocardial infarct size; the serum CK-MB was detected by ELISA and the expression of Bcl-2 and Beclin1 protein in myocardial tissue were detected by Western blot. Compared with the model group, the percentage of myocardial infarct size in the RB group, RC group and RD group was decreased significantly (all P<0.05), and the reduction in the RB group was more significant than that in the RC group and RD group (both P<0.05). Compared with the sham operation group, the expression of CK-MB and Beclin1 in the model group was significantly increased, but the expression of Bcl-2 was significantly decreased (all P<0.01). Compared with the model group, the expression of CK-MB and Beclin1 was decreased significantly in RA group, RB group, RC group and RD group (all P<0.05), but the expression of Bcl-2 was significantly increased (all P<0.05). Compared with the RA group, the expression of CK-MB was decreased in the RB group and RC group (both P<0.05) but the expression of Bcl-2 was increased (both P<0.01); the expression was not significantly different from that in the RD group (P>0.05); the increasing of Bcl-2 in the RB group was more significant than that in RC group (P<0.05). The expression of Beclin 1 in the RB group was significantly lower than that in the RA group (P<0.05), but there was no significant difference among other EA groups (all P>0.05). EA at different time during reperfusion could reduce myocardial infarct size in rats with MIRI, and EA at 0.5 h after reperfusion has best efficacy; this protective effect may be achieved by increasing Bcl-2 expression and reducing Beclin1 expression to inhibit overautophagy during reperfusion.

Ginsenoside Rb3 protects cardiomyocytes against hypoxia/reoxygenation injury via activating the antioxidation signaling pathway of PERK/Nrf2/HMOX1

ObjectivesThis study aimed to investigate the pharmacological function and underlying regulation mechanisms of Ginsenoside-Rb3 (G-Rb3) in cardioprotection. MethodsCultured H9C2 cells were pre-treated with gradient concentrations of G-Rb3, and subsequently challenged with hypoxia/reoxygenation (H/R) treatment. The generation of intracellular reactive oxygen species (ROS) and cellular antioxidatant capacity were quantified. Cell apoptosis was measured by flow cytometry. Myocardial ischemia reperfusion injury (MIRI) rat models constructed by coronary artery ligation surgery were orally administrated with G-Rb3 for 5 consecutive days, and then infarction area, apoptosis ratio and total antioxidant capacity (T-AOC) of myocardial tissues were measured. PERK phosphorylation inhibitor GSK2656157 and Nrf2 translocation inhibitor ML385 were co-treated with G-Rb3 to further verify the signaling pathway mediated by G-Rb3. ResultsH/R treatment induced prominent ROS deposition and elevated cell apoptosis ratio in H9C2 cells. G-Rb3 pretreatment suppressed intracellular ROS accumulation and enhanced T-AOC, partially rescuing cardiomyocytes from oxidative stress and apoptosis induced by H/R. In vivo, the cardiac infarction area of MIRI model rats was reduced by G-Rb3 treatment via improved total antioxidant levels. In the further functional and mechanistic studies, G-Rb3 was found to induce PERK phosphorylation and nuclear translocation of transcriptional factor Nrf2, promoting the expression of antioxidative genes such as HMOX1. Inhibitors GSK2656157 and ML385 reversed the effects of G-Rb3. ConclusionOur studies revealed a novel mechanism of G-Rb3 to attenuates oxidative stress via activating the antioxidation signaling pathway of PERK/Nrf2/HMOX1 in vivo and in vitro, which may help us to enrich the theoretical knewledge of Ginsenoside-Rb3 in cardiopretection

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.

An autophagic mechanism study on effect of electroacupuncture at different times pretreating myocardial ischemia-reperfusion injury

To investigate the difference of protective effect of electroacupuncture (EA) at different times pretreating myocardial ischemia-reperfusion injury (MIRI) and the protein expressions of ULK1 and Beclin-1 in myocardial tissue, so as to explore the relation of the difference and autophagic mechanism. Sixty-three male SD rats were randomized into 7 groups, a sham group, a model group, an EA pretreating for 1 d (EA-1d) group, an EA pretreating for 2 d (EA-2d) group, an EA pretreating for 3 d (EA-3d) group, an EA pretreating for 4 d (EA-4d) group, an EA pretreating for 5 d (EA-5d) group, 9 rats in each group. All the rats in the pretreating groups were treated with EA 1-5 days before MIRI surgery. EA (2 Hz/100 Hz, 2 mA) was used at bilateral "Neiguan (PC 6)" for 20 min. All the rats except for those in the sham group was underwent left thoracotomy and ligation of the left anterior descending (LAD) coronary artery for 30 minutes followed by 4 hours of reperfusion to establish the MIRI model. The same operation was performed in the sham group except for the ligation of the LAD. Throughout the experiment, electrocardiogram was continuously monitored. The myocardial infarct sizes were assessed by Evans blue and triphenyltetrazolium chloride (TTC) staining. The serum concentrations of cardiac troponinⅠ(cTnⅠ) was detected by ELISA. The expressions of ULK1 and Beclin-1 in the heart tissues were analyze by Western blot. Compared with the sham group, the concentrations of cTnⅠ and the protein expressions of ULK1 and Beclin-1 increased in the model group (all P<0.05). Compared with the model group, the infarct sizes decreased in the EA-1d, EA-2d, EA-3d, EA-4d and EA-5d groups (all P<0.05), with less risk sizes in the EA-3d, EA-4d and EA-5d groups (all P<0.05). The levels of cTnⅠin the EA-4d and EA-5d groups decreased ( both P<0.05); the expressions of ULK1 protein decreased in the EA-1d, EA-2d, EA-3d, EA-4d and EA-5d groups (all P<0.05); the expressions of Beclin-1 protein decreased in the EA-2d, EA-3d, EA-4d and EA-5d groups (all P<0.05). The infarct sizes in the EA-4d and EA-5d groups were lower than that in the EA-1d group (both P<0.05). The cTnⅠconcentration in the EA-4d group was less than that in the EA-1d group (P<0.05). Pretreatment with EA for 1-5 days can improve the infarct size in MIRI, with better effect of the pretreatment for 4-5 days. The cardioprotective effect may be related to the inhibition of autophagy. But the difference of the protective effects is not related to the protein expressions of ULK1 and Beclin-1.

Feasibility Analysis of Oxygen-Glucose Deprivation-Nutrition Resumption on H9c2 Cells In vitro Models of Myocardial Ischemia-Reperfusion Injury.

Background:Oxygen-glucose deprivation-nutrition resumption (OGD-NR) models on H9c2 cells are commonly used in vitro models of simulated myocardial ischemia-reperfusion injury (MIRI), but no study has assessed whether these methods for establishing in vitro models can effectively imitate the characteristics of MIRI in vivo. This experiment was designed to analyze the feasibility of six OGD-NR models of MIRI.Methods:By searching the PubMed database using the keywords “myocardial reperfusion injury H9c2 cells,” we obtained six commonly used OGD-NR in vitro models of MIRI performed on H9c2 cells from more than 400 published papers before January 30, 2017. For each model, control (C), simulated ischemia (SI), and simulated ischemia-reperfusion (SIR) groups were assigned, and cell morphology, lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) levels, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory cytokines were examined to evaluate the characteristics of cell injury. Subsequently, a coculture system of cardiomyocyte-endothelial-macrophage was constructed. The coculture system was dealt with SI and SIR treatments to test the effect on cardiomyocytes survival.Results:For models 1, 2, 3, 4, 5, and 6, SI treatment caused morphological damage to cells, and subsequent SIR treatment did not cause further morphological damage. In the models 1, 2, 3, 4, 5 and 6, LDH release was significantly higher in the SI groups than that in the C group (P < 0.05), and was significantly lower in the SIR groups than that in the SI groups (P < 0.05), except for no significant differences in the LDH release between C, SI and SIR groups in model 6 receiving a 3-h SI treatment. In models 1, 2, 3, 4, 5, and 6, compared with the C group, ATP levels of the SI groups significantly decreased (P < 0.05), ROS levels increased (P < 0.05), and MMP levels decreased (P < 0.05). Compared with the SI group, ATP level of the SIR groups was significantly increased (P < 0.05), and there was no significant ROS production, MMP collapse, and over inflammatory response in the SIR groups. In a coculture system of H9c2 cells-endothelial cells-macrophages, the proportion of viable H9c2 cells in the SIR groups was not reduced compared with the SI groups.Conclusion:All the six OGD-NR models on H9c2 cells in this experiment can not imitate the characteristics of MIRI in vivo and are not suitable for MIRI-related study.

Time-dependent Protective Effect of Electroacupuncture on Ischemic Myocardium and Changes of Myocardial Autophagy and Apoptosis Related Protein Expression in Rats

To observe the effect of electroacupuncture (EA) at different time-points on the injured myocar-dium and expression of myocardial Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ proteins in acute myocardial ischemia-reperfusion injury (MIRI) rats so as to explore its mechanisms underlying myocardial protective effect via reducing cardiomyocyte autophagy and apoptosis. A total of 66 adult SD rats were randomly divided into sham operation (sham), model, EA-R 0min(R＝ reperfusion), EA-R 30min, EA-R 60min, and EA-R 120min groups, with 6 rats being in the sham group and 12 rats being in each of the other 5 groups. The MIRI model was prepared by ligating the anterior descending branch (ADB) of the left coronary artery for 30 min followed by reperfusion for 4 h. In the sham group, the ADB was only threaded without ligation. EA was applied to bilateral "Neiguan" (PC 6) for 20 min at 0, 30, 60, and 120 min when reperfusion. Evans Blue-triphenyltetrazolium chloride (TTC) double staining was performed to determine the myocardial infarction area (MIA) and the ratio of the infarct size of the area at risk (IS/AAR). ELISA was performed to measure serum cardiac troponin 1 (cTn-Ⅰ) content, and Western blot was used to measure the expression of apoptosis-related proteins Bax and Bcl-2 and autophagy related proteins Lc 3 Ⅱ and Lc 3 Ⅰ in the left cardiac ventricle tissue. Compared with the model group, the percentages of MIA in the EA-R 30min, EA-R 60min, and EA-R 120min groups, and the IS/AAR in the EA-R 0min, EA-R 30min, EA-R 60min and EA-R 120min groups were significantly reduced (P<0.05, P<0.01). Comparison among the 4 EA groups showed that the percentages of MIA and the IS/AAR were considerably lower in the EA-R 30min, EA-R 60min, and EA-R 120min groups than in the EA-R 0min group (P<0.05, except IS/AAR in the EA-R 120min group), but significantly higher in the EA-R 60min and EA-R 120min groups than in the EA-R 30min group (P<0.05, P<0.01), suggesting a better therapeutic effect of EA intervention at 30 min of MIRI in improving MI. In comparison with the sham group, myocardial cTn-Ⅰ content and Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ levels in the model group were significantly increased (P<0.01). After EA intervention, the increased cTn-Ⅰ content and Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ levels in the EA-R 0min, EA-R 30min, EA-R 60min, and EA-R 120min groups were significantly reduced (P<0.05, P<0.01). The cTn-Ⅰ content was obviously lower at EA-R 30 min, but markedly increased at EA-R 120min than at EA-R 0min (P<0.05). The expression of Bcl-2 was obviously higher at EA-R 30min than at EA-R 0min (P<0.05). No significant differences were found among the 4 EA intervention time-points in the levels of Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ (P>0.05). EA intervention can reduce MIA in MIRI rats, which is possibly closely related to its effects in reducing apoptosis and autophagy. The best intervention time is at 30 min after MI reperfusion, but the difference of effects of EA at different time-points is independent of Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ expression.

Vaspin protects rats against myocardial ischemia/reperfusion injury (MIRI) through the TLR4/NF-κB signaling pathway

The purpose of our study was to investigate the effect of vaspin on myocardial ischemia reperfusion injury (MIRI) and explore the underlying mechanism. The MIRI model was induced with 30 min of left anterior descending (LAD) occlusion followed by 24 h of reperfusion. In vivo, the rats were randomly divided into five groups: (1) Sham, (2) MIRI, (3) MIRI + vaspin (10 mg/kg), (4) MIRI + vaspin (20 mg/kg) and (5) MIRI + vaspin (40 mg/kg). In vitro, H9C2 cells were assigned to five groups: (1) control, (2) hypoxia-re-oxygenation (H/R), (3) H/R + vaspin (1 μg/ml), (4) H/R + vaspin (2 μg/ml) and (5) H/R + vaspin (4 μg/ml). As a result, vaspin ameliorated MIRI and H/R in a dose-dependent manner, as evidenced by triphenyl tetrazolium chloride (TTC) staining, TUNEL Assay and MTT assay, respectively, meanwhile vaspin decreased the levels of creatine phosphokinase-isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in rat serum, moreover, vaspin could reduce the contents of interleukin-1β (IL-1β), IL-18 and tumor necrosis factor alpha (TNF-α) in serum of rats and supernatant of H9C2 cells. Furthermore, vaspin down-regulated the expression of toll-like receptor 4 (TLR4) and the phosphorylation of nuclear factor κB (NF-κB) in MIRI rats and H/R-induced H9C2 cells. In addition, patients with acute myocardial infarction (AMI) had lower levels of vaspin than patients without. In conclusion, vaspin might be a useful predictive biomarker in patients with AMI; furthermore, vaspin exhibits cardioprotective effects on MIRI which might act through inhibiting TLR4/NF-κB signaling pathway in vivo and in vitro.