Myocardial Apoptosis In Rats Research Articles

Influence of miR-34a on myocardial apoptosis in rats with acute myocardial infarction through the ERK1/2 pathway.

To study the influence of micro-ribonucleic acid (miR)-34a on myocardial apoptosis in rats with acute myocardial infarction (AMI) through the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. A total of 24 Sprague-Dawley (SD) rats were randomly divided into sham group (n=12) and model group (n=12). The heart was exposed in the sham group, while the AMI model was established in the model group. After sampling, the morphology of myocardial tissues was observed via hematoxylin-eosin (HE) staining, the expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were detected via immunohistochemistry, and the protein expression levels of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) were detected via Western blotting. Moreover, the expression of miR-34a was detected via quantitative Polymerase Chain Reaction (qPCR), the apoptosis was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and the myocardial injury indexes were detected using a fully-automatic biochemical analyzer. The morphology of myocardial tissues was normal with a complete structure in the sham group, while there was damage to myocardial tissues in different degrees in the model group. The immunohistochemical results revealed that the Bax expression was increased and the Bcl-2 expression was decreased in the model group compared with those in the sham group (p<0.05). The results of Western blotting showed that the protein expression levels of both ERK1/2 and p-ERK1/2 were significantly increased in the model group compared with those in the sham group (p<0.05). The qPCR results manifested that the expression of miR-34a in the model group markedly declined compared with that in the sham group (p<0.05). Besides, the TUNEL detection showed that the apoptosis rate in the model group was remarkably increased compared with that in the sham group (p<0.05), and the content of cardiac troponin T and creatine kinase isoenzyme in the model group was significantly higher than that in the sham group ((p<0.05). MiR-34a affects the apoptosis in AMI by regulating the ERK1/2 signaling pathway.

Influence of MiR-154 on myocardial apoptosis in rats with acute myocardial infarction through Wnt/β-catenin signaling pathway.

To explore the influence of micro ribonucleic acid (miR)-154 on myocardial apoptosis in rats with acute myocardial infarction (AMI), and to analyze whether Wnt/β-catenin signaling pathway was involved in the regulation. The Sprague-Dawley (SD) rat model of AMI was established via ligation of left anterior descending artery. Rats were randomly divided into model group (M group, n=12) and ICG-001 intervention group (I group, n=12). At the same time, sham operation group (S group, n=12) was established. In I group, ICG-001 (5 mg/kg) was intraperitoneally injected every day after operation. Meanwhile, an equal amount of normal saline was injected in rats of S group and M group. 21 d after operation, the cardiac function of rats in each group was detected via echocardiography. After that, the rats were immediately executed. MI area in each group was detected via 2,3,5-triphenyltetrazolium chloride (TTC) staining. Myocardial apoptosis level in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Moreover, the changes of apoptotic proteins in rat myocardial cells were detected via Western blotting. Moreover, the expression level of miR-154 in myocardial cells of rats was detected via quantitative polymerase chain reaction (qPCR). Furthermore, the influence of miR-154 on Wnt/β-catenin signaling pathway was detected via Western blotting. Compared with S group, left ventricular ejection fraction (LVEF, %) and left ventricular fractional shortening (LVFS, %) were significantly decreased in M group (p<0.01). However, left ventricular internal diameter at end-diastole (LVIDd) and left ventricular internal diameter at end-systole (LVIDs) were significantly increased (p<0.01). In I group, LVEF (%) and LVFS (%) were significantly higher than those of M group (p<0.05), whereas LVIDs and LVIDd were significantly lower (p<0.05). MI area in M group was remarkably larger than that of S group (p<0.01). Meanwhile, MI area in I group was significantly smaller than that of M group (p<0.01). Compared with S group, the number of apoptotic myocardial cells and the protein expression level of cleaved caspase-3 were significantly increased in M group (p<0.01). However, the expression level of B-cell lymphoma-2/Bcl-2 associated X protein (Bcl-2/Bax) was significantly decreased (p<0.01). The number of apoptotic myocardial cells and the protein expression level of cleaved caspase-3 were significantly declined in I group when compared with those of M group (p<0.01). However, the expression level of Bcl-2/Bax was significantly increased in I group (p<0.01). The expression level of miR-154 in myocardial cells of M group and I group was remarkably increased when compared with that of S group (p<0.01). Furthermore, the expression levels of β-catenin and Cyclin D1 in myocardial cells of M group were remarkably higher than those of S group and I group (p<0.01). AMI significantly increases the expression level of miR-154. Moreover, miR-154 can activate Wnt/β-catenin signaling pathway, eventually promoting myocardial apoptosis.

Effect of miR-21/TLR4/NF-κB pathway on myocardial apoptosis in rats with myocardial ischemia-reperfusion.

The aim of the study was to investigate the influences of micro ribonucleic acid (miR)-21 and downstream Toll-like receptor 4 (TLR4)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway on myocardial apoptosis induced by myocardial ischemia-reperfusion (I/R) injury in rats. Recombinant adeno-associated virus rAAV9-ZsGreen-pre-miR-21 and blank control virus were constructed. A total of 48 Sprague-Dawley (SD) rats were randomly divided into S1 group (open chest only), S2 group (transfection with blank virus + open chest), I/R1 group (transfection with blank virus + 6 d of myocardial I/R), and I/R2 group (transfection with miR-21 + 6 d of myocardial I/R). The cardiac function and myocardial infarct size of rats were evaluated in each group. Quantitative Polymerase Chain Reaction (qPCR) was applied to measure the expression level of miR-21 in the myocardium. The level of myocardial apoptosis in each group was detected through terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. Western blotting was performed to determine the protein expression levels of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax), Caspase-3, TLR4, and NF-κB in the myocardium. The content of interleukin-6 (IL-6) and IL-10 was measured using enzyme-linked immunosorbent assay (ELISA) kits. The cardiac function of rats in I/R1 and I/R2 groups was significantly lower than that in S1 and S2 groups (p<0.01). Rats in I/R2 group had better cardiac function than those in I/R1 group (p<0.01). In I/R1 group, the level of myocardial apoptosis of rats was overtly increased compared with that in S1, S2, and I/R2 groups (p<0.01), while the expression level of miR-21 in myocardium was evidently lower than that in S1, S2, and I/R2 groups (p<0.01). Compared with S1, S2, and I/R2 groups, I/R1 group had markedly decreased Bcl-2/Bax expression level and IL-10 content and overtly elevated expression levels of Caspase-3, p-TLR4, p-NF-κB, and IL-6 content in the myocardium (p<0.01). Myocardial I/R injury in rats leads to decreased expression of miR-21. The overexpression of miR-21 is able to effectively inhibit the TLR4/NF-κB pathway and reduce the level of myocardial apoptosis of rats and the release of inflammatory factors.

PL-011 Astaxanthin Reduces High Intensity Training Induced Myocardial Cell Apoptosis Via Activating Nrf2 in Rats

Objective Long-term intensive training may led to ischemia oxygen reaction and increase the ROS. Astaxanthin, as the super antioxidant, was investigated to against anti-oxidative stress. By supplementing the astaxanthin, we wanted to observe if it can mediated Nrf2 reduces myocardial cell oxidative injury in rats after high intensity training of 6 weeks. &#x0D; Methods 7-week SD male rats were divided into 3 groups randomly: control group ( C group，n =10)，high intensity training group ( HT group，n = 15)，astaxanthin and high intensity training group (HTA group，n = 15) . The rats in HTA group were given with astaxanthin 20 mg /kg·d and in HT group were given with oil during the training day．The serum cTnI，myocardial apoptosis index, the expression of myocardial BAX, Bcl2, Nrf2, HO-1, myocardial MDA，SOD and T- AOC activity were measured 24 hours after the last training．&#x0D; Results After 6-week tranning of high intensity, compared with group C, the serum cTNI, myocardial apoptosis index, the expression of BAX and myocardial MDA were significantly higher in group HT(P&lt;0.01).The Bcl2/Bax, the expression of HO-1, SOD and T-AOC activity were significantly declined (P&lt;0.01). After the intervention of 6-week astaxanthin, compared with group HT, the serum cTNI, myocardial MDA, the myocardial apoptosis index, the expression of BAX were significantly lower in HTA group (cTNI(ng/ml): 1.16±0.27 VS 2.47±0.39, P&lt;0.05; myocardial apoptosis index: 164.27±3.98 VS 196.20±9.65, P&lt;0.01; BAX: 58.40±5.95 VS 78.03±3.80, P&lt;0.01 ). Finally, Bcl2/Bax, SOD, T-AOC activity, the expression of Nrf2 and HO-1 were significantly higher (Bcl2/Bax : 1.92±0.10 VS 1.19±0.18, P&lt;0.01; SOD(U/mg): 52.38±6.15 VS 38.32±3.36, P&lt;0.01; T-AOC(U/mg): 30.22±4.07 VS 23.76±3.20, P&lt;0.01; Nrf2: 93.61±8.53 VS 74.26±6.69, P&lt;0.01; HO-1: 84.99±13.78 VS 64.22±11.39, P&lt;0.05). &#x0D; Conclusions The supplement of astaxanthin can mediate Nrf2 signaling pathway, and elevate the expression of Nrf2 and HO-1. Then it can increase the activity of SOD and T-AOC and reduce the myocardial oxidative level and myocardial apoptosis in rats caused by 6-week high intensity training. Finally, the structure and function of heart tissue are back to normal.

Eugenol protects the transplanted heart against ischemia/reperfusion injury in rats by inhibiting the inflammatory response and apoptosis.

The aim of the present study was to investigate the protective effect of eugenol on the transplanted heart and explore its mechanisms of action. Male Sprague-Dawley rats were randomly divided into a sham group (n=10), a eugenol group (n=10 pairs, donors and recipients) and a control group (n=10 pairs, donors and recipients). The recipients in the eugenol group received an intraperitoneal injection of eugenol (20 mg/kg/day). The sham group and the control group received equal volumes of physiological saline by intraperitoneal injection. After 15 days the recipients in the control and eugenol groups underwent abdominal heterotopic heart transplantation, while the sham group received only a coeliotomy. The orthotopic hearts in the sham group and the heterotopic hearts in the eugenol and control groups, as well as the peripheral blood samples from all three groups were taken 3 h post operation for biochemical, histopathological, molecular and apoptosis analyses. Compared with the control group, the eugenol treatment significantly reduced the myocardial malondialdehyde content, serum cardiac troponin I, creatine kinase-MB, tumor necresis factor-α and interleukin-6 levels (P<0.05) and significantly alleviated myocardial injury. Western blot analysis demonstrated that the protein expression of cleaved Poly (ADP-ribose) polymerase 1, BAX and active caspase-3 in the eugenol group were significantly decreased, while B-cell lymphoma 2 expression was significantly increased compared with the control group (P<0.05). The myocardial apoptosis rate of the eugenol group was significantly decreased compared with the control group (P<0.05). In conclusion eugenol treatment significantly reduced myocardial injury and demonstrated protective effects for the transplanted heart.

The effect of neuregulin-1 on myocardial ischemia reperfusion injury in rats

Objective To investigate the effect and mechanism of neuregulin-1 (NRG-1) on myocardial ischemia reperfusion injury in rats. Methods The model of myocardial ischemia and reperfusion in rats were randomly divided into the model group and the NGR-1 group. At the same time, set up the sham operation group, and the left anterior descending artery of the sham operation group was not ligation. NRG-1 group rats were injected with NRG-1 before modeling. Three phenyl tetrazolium chloride (TTC) method was used to detect the myocardial infarct size in rat heart. Isolated rat myocardial cells, flow cytometry was used to detect cell apoptosis. Detection of B cell lymphoma/leukemia 2 (bcl-2), bcl-2 related X protein (bax), signal transduction and transcription factor 3 (STAT3), phosphorylated STAT3 (p-STAT3) protein and reactive oxygen species (ROS) level, SOD activity and malondialdehyde (MDA) content in cardiac tissue. Results The myocardial infarction area of model group was higher than that of sham operation group (P=0.000), and the infarct size of NRG-1 group was lower than that of model group (P=0.000). In model group, myocardial apoptosis was significantly more than that in sham operation group (P=0.000), the rate of myocardial apoptosis in NRG-1 group was significantly lower than that in model group (P=0.000). The myocardial tissue of bcl-2 and p-STAT3 levels in the model rats were significantly lower than the sham operation group (P=0.000), bcl-2 and p-STAT3 level of myocardial tissue in NRG-1 rats was higher than in the model group (P=0.000). The level of bax in myocardial tissue of rats in the model group was higher than in the sham operation group (P=0.000), the bax level of myocardial tissue in NRG-1 rats was lower than the model group (P=0.000). The levels of ROS and MDA in the model group were significantly higher than those in the sham operation group (P=0.000). The levels of ROS and MDA in NRG-1 group were significantly lower than those in model group (P=0.000). In model group, the activity of SOD was significantly lower than that of sham operation group (P=0.000). The activity of SOD in NRG-1 group was significantly higher than that in model group (P=0.000). Conclusion NRG-1 can effectively improve the myocardial ischemia reperfusion injury, and the mechanism of action is related to the regulation of myocardial apoptosis and antioxidation. Key words: Myocardial ischemia reperfusion; Neuregulin-1; Apoptosis; Oxygen free radical

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.

Icariin influences cardiac remodeling following myocardial infarction by regulating the CD147/MMP-9 pathway.

ObjectiveWe investigated the protective effect of icariin on myocardial infarction-induced cardiac remodeling.MethodsA cardiac remodeling model was constructed by ligating rats’ coronary artery. Different icariin and CD147 concentrations were administered in the model group, and echocardiography was used to detect systolic function, screening out ideal experimental concentrations. The ventricular systolic function, myocardial apoptosis rate, and expression of collagen type I (Col I), Col III, CD147, matrix metalloproteinase 9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) were detected by hematoxylin–eosin staining, TUNEL assay, and western blot. MMP-9 activity was evaluated by gelatin zymography.ResultsThe expression of Col I, Col III, CD147, and MMP-9 was higher, the expression of TIMP-1 was lower, and the maximal rates of left ventricular pressure rise and fall (+dp/dtmax and −dp/dtmax, respectively) were lower in model than control rats. The expression of CD147, MMP-9, Col I, and Col III was lower, the expression of TIMP-1 was higher, and the +dp/dtmax and −dp/dtmax were higher in the icariin than model group. The apoptosis rate was lower in the icariin and icariin + CD147 groups than control group.ConclusionIcariin attenuated myocardial apoptosis following myocardial infarction by apoptosis rate reduction and CD147/MMP-9 pathway inhibition.

Antagonism of cortistatin against cyclosporine-induced apoptosis in rat myocardial cells and its effect on myocardial apoptosis gene expression.

To investigate the role of cortistatin (CST) on cyclosporine A (CsA)-induced myocardial apoptosis in rats and determine its effect on the expressions of myocardial apoptosis genes. H9C2 cells were treated with different concentrations of CsA solution (0.04, 0.2, 1 and 5 μM) for 24, 48 and 72 h, respectively. The cell viability was detected via methyl thiazolyl tetrazolium (MTT) assay, and the appropriate dose and time were compared and determined. At the same time, CST in different concentrations (0.08, 0.04, 0.2, 1, 5 and 25 μM) was added into cell culture, and the appropriate dose was identified using MTT assay. The cellular morphology in each group was observed, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed for the detection of cell apoptosis. Moreover, in molecular mechanism research, the apoptosis-associated factors, factor associated suicide (Fas), Fas ligand (FasL) and B-cell lymphoma-2-associated X protein (Bax), were detected via quantitive Real-time polymerase chain reaction (qPCR). Finally, the levels of a protein related to myocardial apoptosis in rats were investigated via Western blotting. The treatment with 1 μM CsA for 48 h caused significant apoptosis. The results of TUNEL staining showed the inhibitory role of CST on the myocardial apoptosis in rats induced by CsA. The detection of apoptosis factors via Real-time PCR revealed that after the induction of CsA, the expressions of Fas, FasL and Bax mRNA in cells were significantly higher than those in control group, but were significantly decreased after administration of CST. Western blotting showed that the protein expressions of Caspase 3 and Caspase 9 were remarkably elevated in cells after the use of CsA, but were significantly reduced after administration of CST (p < 0.01). CST contributes to antagonistic function against the CsA-induced apoptosis of rat myocardial cells, and its effect is related to the down-regulation of expressions of apoptotic factors, Fas, FasL, Bax, Caspase 3, and Caspase 9.

Melatonin ameliorates myocardial apoptosis by suppressing endoplasmic reticulum stress in rats with long‑term diabetic cardiomyopathy.

The effects of melatonin (MLT), which exerts cardioprotective effects against myocardial apoptosis, in long‑term diabetic cardiomyopathy are not currently well defined. The present study aimed to investigate how MLT protects the heart through modulating myocardial apoptosis in rats with type 2 diabetes mellitus (DM). In total, 36 rats were randomly divided into three groups, including control (n=12), DM (n=12) and DM+MLT (n=12) groups. The results demonstrated that, in DM rats, a significant increase was observed in the serum fasting blood glucose and lipid levels, in addition to insulin resistance and cardiac dysfunction, which were attenuated in DM rats treated with MLT. Additionally, cellular apoptosis in rats with DM was increased, and the expression of Bcl‑2 was downregulated, while levels of Bcl‑2‑associated X and caspase‑3 were upregulated, and these observations were reversed by MLT, as determined by TUNEL and western blot analysis, respectively. As increased endoplasmic reticulum (ER) stress induced by hyperglycemia is reported to be a factor for apoptosis, the present study also determined the expression of proteins associated with ER stress in cardiac tissues following MLT treatment by western blotting. The results further indicated that MLT decreased the expression of ER stress hallmarks, including CCAAT/enhancer‑binding protein homologous protein, glucose‑regulated protein 78, protein kinase RNA‑like endoplasmic reticulum kinase (PERK) and activating transcription factor 6α in cardiac tissues. In conclusion, the results of the present study indicate that MLT may protect heart by ameliorating cardiac ER stress‑induced apoptosis in diabetic cardiomyopathy.

Renal sympathetic denervation improves myocardial apoptosis in rats with isoproterenol-induced heart failure by downregulation of tumor necrosis factor-α and nuclear factor-κB.

Chronic congestive heart failure (CHF) is the end outcome of organic heart diseases and one of the major diseases harmful to human health. Renal sympathetic denervation (RSD) is the anatomical basis of transcatheter renal sympathetic nerve ablation within the renal artery. To date, the roles of norepinephrine and angiotensin II (Ang II) in myocardial apoptosis and their underlying mechanisms have not been well explored. The aim of the present study was to verify the hypothesis that RSD is likely to inhibit myocardial apoptosis by inhibiting the release of norepinephrine and Ang II. An isoproterenol-induced CHF rat model was established, and the effects of RSD on myocardial apoptosis were examined using flow cytometry and TUNEL staining. The expression of factors associated with myocardial apoptosis, including p53, tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), caspase-2 and −3, were measured using quantitative polymerase chain reaction and western blot analysis. The results indicated that the mRNA levels of p53, TNF-α, NF-κB, caspase-2 and −3 were significantly reduced in the myocardial tissues of rats in the CHF+RSD group when compared with the levels in the CHF+sham group (P<0.01 for all). In addition, the protein levels of p53, TNF-α, NF-κB and caspases-2 and −3 were decreased by 42.6, 41.3, 46.7, 30.0 and 35.8%, respectively, in myocardial tissues of rats in the CHF+RSD group in comparison with the CHF+sham group (P<0.01 for all). Furthermore, myocardial apoptosis was improved in rats in the CHF+RSD group compared with that in the CHF+sham group (P<0.01). In conclusion, the present study provides a theoretical basis for application of RSD in the treatment of CHF.

MiR-487b mitigates chronic heart failure through inhibition of the IL-33/ST2 signaling pathway.

We investigated the effects of microRNA-587b (miR-487b) in a rat model of chronic heart failure (CHF). Wistar rats were assigned to 10 groups (n=8 per group). Expression of interleukin-33 (IL-33), somatostatin 2 (ST2), IL-6, and TNF-α was higher in the CHF group than the control group. In the CHF, negative control (NC) for si-IL-33, NC for miR-487b mimic, NC for miR-487b inhibitor, and miR-487b inhibitor + si IL-33 groups, as compared to the blank and sham groups: steroid binding protein (SBP), D binding protein (DBP), left ventricular systolic pressure (LVSP), ± dp/dtmax, and superoxide dismutase (SOD) were all lower; myocardial fibrosis, MDA, left ventricular end-diastolic pressure (LVEDP), myocardial apoptosis rate, IL-6, and TNF-α were all higher; levels of IL-33 and ST2 mRNA and protein were higher; and levels of miR-487b were lower. Levels of IL-33 and ST2 mRNA and protein were lower, and SBP, DBP, LVSP, ± dp/dtmax, and SOD were higher in the miR-487b mimic and si-IL-33 groups than the CHF group. Expression of miR-487b was increased in the miR-487b mimic group, and expression of IL-33 and ST2 were increased and expression of miR-487b was decreased in the miR-487b inhibitor group. MiR-487b reduces apoptosis, inflammatory responses, and fibrosis in CHF by suppressing IL-33 through inhibition the IL-33/ST2 signaling pathway.

Crude terpene glycoside component from Radix paeoniae rubra protects against isoproterenol-induced myocardial ischemic injury via activation of the PI3K/AKT/mTOR signaling pathway

Ethnopharmacological relevanceRadix paeoniae rubra, also known as chishao (CS), is a frequently used traditional Chinese medicine that can promote blood circulation to remove blood stasis. It has been widely used for the prevention and treatment of cardiovascular diseases in China. Although terpene glycoside (TG), the major component in CS, has been shown to possess cardioprotective properties, the mechanism underlying CS-TG's preventive effect against myocardial ischemia injury is unknown. This study was conducted to explore the protective and curative effects of CS-TG against isoproterenol (ISO)-induced myocardial ischemic injury in rats and investigate the underlying myocardial protective mechanisms. Materials and methodsA rat model of ISO-induced myocardial ischemia was established to evaluate the protective effect of CS-TG in ameliorating heart injury. Myocardial ischemia was induced by administering ISO (40mg/kg/d) subcutaneously for 2 days. Serum was collected and analyzed for the levels of different cardiac biomarkers, and heart tissues were isolated and prepared for ATP analysis, glycogen content determination, histopathology assay, and ultrastructure observation. The regulatory effects of CS-TG on myocardial apoptosis in rats were studied by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and the levels of cleaved caspase-3, Bax, and Bcl-2 were detected by western blotting. Furthermore, in vitro experiments were conducted to examine whether the CS-TG's cardioprotective effects were linked to the inhibition of apoptosis via activation of the phosphoinositide-3-kinase/serine-threonine kinase AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. ResultsCS-TG (300mg/kg/d) significantly decreased serum levels of creatine kinase and lactate dehydrogenase in ISO-induced myocardial ischemic rats. Analysis of ATP and glycogen contents, myocardial ultrastructure, and pathological examination showed that CS-TG (300mg/kg/d) significantly improved energy metabolism and alleviated myocardial injury in vivo. In addition, the expression of p-AKT and p-mTOR in rats subjected to CS-TG significantly elevated, while the levels of caspase-3 and Bax/Bcl-2 dramatically reduced. Moreover, treatment with LY294002, a PI3K inhibitor, abrogated CS-TG (200μg/mL) induced down-regulation of cleaved caspase-3, Bax/Bcl-2 in the serum. ConclusionsCS-TG protects the heart from ISO-induced myocardial ischemia, potentially by improving cardiac energy metabolism and inhibiting cardiomyocyte apoptosis via activation of the PI3K/AKT/mTOR signaling pathway. Thus, CS -TG might be a potential therapeutic candidate for the prevention and treatment of myocardial ischemia.

Protective effect of active perfusion in porcine models of acute myocardial ischemia.

Mortality rates associated with off-pump coronary artery bypass (CAB) are relatively high, as the majority of patients requiring CAB are at a high risk for cardiac events. The present study aimed to establish porcine models of acute myocardial ischemia, and evaluate the protective role of shunt and active perfusion. A total of 30 pigs were randomly assigned to five groups, as follows: i) Sham (control); ii) A1 (shunt; stenosis rate, 55%); iii) A2 (shunt; stenosis rate, 75%); iv) B1 (active perfusion; stenosis rate, 55%); and v) B2 (active perfusion; stenosis rate, 75%) groups. Aortic pressure (P0), left anterior descending coronary pressure (P1), and coronary effective perfusion pressure (P1/P0) were measured. The expression levels of tumor necrosis factor-α (TNF-α), cardiac troponin (cTnI), creatine kinase-myocardial band (CK-MB), interleukin (IL)-6, IL-10, B-cell lymphoma 2 (Bcl-2), and caspase-3 were detected using enzyme-linked immunosorbent assay or western blotting. The myocardial apoptosis rate was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Ischemia models with stenosis rates of 55 and 75% were successfully constructed following suturing of the descending artery. Compared with the control, the 55 and 75% stenosis groups demonstrated significantly decreased P1/P0, increased expression levels of TNF-α, cTnI, CK-MB, IL-6, IL-10 and caspase-3, an increased rate of myocardial apoptosis, and a decreased expression level of anti-apoptotic protein, Bcl-2. At 30 min following successful establishment of the model (ST segment elevation to 1 mm), group B demonstrated significantly increased P1/P0, decreased expression levels of TNF-α, cTnI, CK-MB, IL-6, IL-10 and caspase-3, a decreased rate of myocardial apoptosis, and an increased expression level of anti-apoptotic protein, Bcl-2. Furthermore, the current study indicated that active perfusion was more efficacious in maintaining myocardial perfusion and alleviating ischemic injury when compared with traditional shunt perfusion.

Maternal exposure to ambient PM2.5 exaggerates fetal cardiovascular maldevelopment induced by homocysteine in rats.

Maternal exposure to airborne particulate matter with aerodynamic diameter <2.5 µm (PM2.5 ) during pregnancy and lactation periods is associated with filial congenital cardiovascular diseases. This study aimed to investigate the toxic effects of maternal exposure to ambient levels of PM2.5 on filial cardiovascular maldevelopment induced by homocysteine. Using a 2 × 2 factorial design, rats were randomized into four groups and were exposed to ambient PM2.5 or filtered air (FA) throughout the pregnancy and lactation periods coupled with the administration of either homocysteine (HCY) or normal saline (NS) daily from gestation days 8-10. Morphological changes in the heart, myocardial apoptosis, expressions of cardiac progenitor transcriptional factors, and levels of cytokines were investigated in the offspring. The apoptosis-like changes in the myocardium were seen in the FA plus HCY-treated group and more obviously in the PM2.5 plus HCY-treated group, which was in accordance with an increased myocardial apoptosis rate in the two groups. PM2.5 exposure resulted in significantly decreased Nkx2-5 protein level and GATA4 and Nkx2-5 mRNA expressions, and significantly increased TNF-α and IL-1β levels. There were significant interactions between PM2.5 exposure and HCY-treatment that PM2.5 exposure reduced Nkx2-5 protein levels and GATA4 and Nkx2-5 mRNA expressions in the HCY-treated groups. These results suggest that maternal exposure to PM2.5 , even at the ambient levels in urban regions in China, exaggerates filial cardiovascular maldevelopment induced by HCY in a murine model, exacerbating structural abnormalities in the filial cardiac tissue, which is possibly associated with oxidative stress and reduced GATA4 and Nkx2-5 transcription factor expressions. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 877-889, 2017.

Pretreatment with low-dose gadolinium chloride attenuates myocardial ischemia/reperfusion injury in rats.

We have shown that low-dose gadolinium chloride (GdCl3) abolishes arachidonic acid (AA)-induced increase of cytoplasmic Ca(2+), which is known to play a crucial role in myocardial ischemia/reperfusion (I/R) injury. The present study sought to determine whether low-dose GdCl3 pretreatment protected rat myocardium against I/R injury in vitro and in vivo. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with GdCl3 or nifedipine, followed by exposure to anoxia/reoxygenation (A/R). Cell apoptosis was detected; the levels of related signaling molecules were assessed. SD rats were intravenously injected with GdCl3 or nifedipine. Thirty min after the administration the rats were subjected to LAD coronary artery ligation followed by reperfusion. Infarction size, the release of serum myocardial injury markers and AA were measured; cell apoptosis and related molecules were assessed. In A/R-treated NRVMs, pretreatment with GdCl3 (2.5, 5, 10 μmol/L) dose-dependently inhibited caspase-3 activation, death receptor-related molecules DR5/Fas/FADD/caspase-8 expression, cytochrome c release, AA release and sustained cytoplasmic Ca(2+) increases induced by exogenous AA. In I/R-treated rats, pre-administration of GdCl3 (10 mg/kg) significantly reduced the infarct size, and the serum levels of CK-MB, cardiac troponin-I, LDH and AA. Pre-administration of GdCl3 also significantly decreased the number of apoptotic cells, caspase-3 activity, death receptor-related molecules (DR5/Fas/FADD) expression and cytochrome c release in heart tissues. The positive control drug nifedipine produced comparable cardioprotective effects in vitro and in vivo. Pretreatment with low-dose GdCl3 significantly attenuates I/R-induced myocardial apoptosis in rats by suppressing activation of both death receptor and mitochondria-mediated pathways.

Protective effect of erythropoietin on myocardial apoptosis in rats exposed to carbon monoxide

AimsCardiac complications are common in carbon monoxide (CO) poisoning and associated with high morbidity and mortality. We have previously shown that erythropoietin (EPO) could reduce CO-induced cardiac ischemia in rat. In the current study, the anti-apoptotic effect of EPO during CO cardiotoxicity was investigated in order to elucidate the mechanism of EPO anti-ischemic action. Main methodsWistar rats were exposed to CO (250, 1000 and 3000ppm). EPO (5000IU/kg) was administered to all groups by intraperitoneal injection at the end of CO exposure period. TUNEL and caspase-3 activity levels were assessed to investigate the effects of CO exposure and subsequent EPO administration on myocardial apoptosis. The changes of mitochondrial membrane potential (MMP) were also assessed with sensitive lipophilic dye JC-1 by flow cytometry. The roles of Bcl2 and Bax in EPO protective effect were investigated by Western blotting. Key findingsMyocardial apoptosis was observed following CO exposure. Moreover, mitochondrial membrane depolarization and significant reduction in Bcl2/Bax ratio were shown following CO poisoning especially at 3000ppm. On the other hand, EPO administration could effectively suppress apoptosis in myocardial cells. Also, EPO significantly prevented the CO-induced depolarization of MMP (p<0.001) and preserved Bcl2/Bax ratio (p<0.01). SignificanceEPO reduces myocardial injury due to CO intoxication. Thus EPO could be suggested as a possible candidate for the management of CO cardiotoxicity with clinical applications.

Antagonistic effect and mechanism of Rosuvastatin on myocardial apoptosis in rats with acute myocardial infarction

To investigate the protective effect of Rosuvastatin on myocardial cells in rats with acute myocardial infarction and its possible mechanism. Rats were randomly assigned to four groups: Control group, Sham group, AMI and Rosuvastatin group. The levels of lactate dehydrogenase (LDH) and creatine jubase (CK), the vitality of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were detected by assay kits and the levels of C-reactive protein (CRP), tumor necrosis factor (TNF) alpha and interleukin (IL)-6 expression were detected by enzyme linked immunosorbent assay (ELISA). TTC/Evans blue staining was used to determine the relative myocardial infarction area, HE staining was used to detect pathologic changes and myocardial apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). What's more, Western blot was used to detect the protein expression of B-cell lymphoma-2 (Bcl-2), Bax, cleaved-Caspase-3, Rock1, Rock2, I-κB and NF-κBp65. The model of acute myocardial infarction rats was established. Compared with Sham group, the myocardial pathological changes were more severe, and the apoptosis number, the production of inflammatory factors and oxidative damage were significantly increased in AMI group. Compared with AMI group, the relative area of infarction myocardium (43% ± 4% vs 31% ± 8%, P=0.004 3) was dramatically reduced, the levels of LDH (2 545.45 ± 613.67 U/L vs 1 573.43 ± 373.72 U/L, P=0.02) and CK (7.49 ± 1.75 U/ml vs 4.42 ± 1.28 U/ml, P=0.04) in serum were significantly lower (P<0.01), the myocardial pathological damage degree was relieved, the apoptosis number (41% ± 8% vs 23% ± 6%, P=0.014 7) was significantly decreased, the expression of Bax (1.17 ± 0.10 vs 0.57 ± 0.08, P=0.003) and cleaved-Caspase-3 (1.31 ± 0.07 vs 0.70 ± 0.01, P=0.004) were dramatically reduced, and the expression of Bcl-2 (0.19 ± 0.01 vs 0.32 ± 0.01, P=0.003) was enhanced in Rosuvastatin group. Furthermore, the production of inflammatory factors and oxidative damage were effectively alleviated (P<0.05), the protein expression of Rock1, Rock2 and NF-κBp65 were declined in Rosuvastatin group (P<0.01). Rosuvastatin can effectively alleviate the cardiomyocyte apoptosis induced by acute ischemia, and its main mechanism may be the inhibition on RhoA/ROCK activation and the oxidative stress and inflammation reaction mediated by it.

Effect of icariin on hypoxia/reoxygenation injury in neonatal rat cardiomyocytes

To investigate the effect of icariin on myocardial hypoxia reoxygenation injury and the possible mechanism. Neonatal Sprague-Dawley rat cardiomyocytes in primary culture were treated with different concentrations of icariin for 24 h prior to hypoxia/reoxygenation injury. Cardiomyocyte apoptosis was evaluated with Tunel staining. The expression levels of apoptosis proteins were detected by Western blotting. The nuclear translocation of p65 was evaluated by immunofluorescence. The p65 signaling pathway was also detected by Western blotting. Myocardial apoptosis rate significantly increased after hypoxia/reoxygenation (control: 1.5% ± 0.1%; 23.4% ± 1.3%, P<0.05). While icariin significantly reduced cardiomyocyte apoptosis induced by hypoxia/reoxygenation (1 µmol/L icariin: 7.2% ± 0.9%; 10 µmol/L icariin: 3.9% ± 0.8%, both P<0.05). Western blot showed that the expression levels of pro-apoptotic protein, Bax, increased significantly (control: 0.19 ± 0.05; 0.41 ± 0.03, P<0.05), while the expression of anti-apoptotic protein, B-Cell CLL/Lymphoma 2 (BCL-2), was significantly reduced (control: 0.15 ± 0.02; 0.03 ± 0.01, P<0.05) after hypoxia/reoxygenation. Notably, icariin reduced the expression of Bax (1 µmol/L icariin: 0.29 ± 0.01; 10 µmol/L icariin: 0.33 ± 0.03, both P<0.05) and increased expression of BCL-2 (1 µmol/L icariin: 0.10 ± 0.03; 10 µmol/L icariin: 0.11 ± 0.02, both P<0.05). Immunofluorescence showed that NFκB-p65 nuclear translocation in cardiomyocytes was increased after hypoxia/reoxygenation (control: 3.6% ± 0.5%; 89.5% ± 4.8%, P<0.05), while icariin reduced the nuclear translocation of p65 (1 µmol/L icariin: 32.6% ± 2.3%; 10 µmol/L icariin: 10.6% ± 1.0%, both P<0.05). Moreover, icariin reduced the activation of p65 and phosphorylation of IKBα induced by hypoxia/reoxygenation in cardiomyocytes. Icariin can protect cardiomyocytes against hypoxia reoxygenation injury, which may be via blocking p65 signaling pathway.

Mechanism of QSYQ on anti-apoptosis mediated by different subtypes of cyclooxygenase in AMI induced heart failure rats.

BackgroundQi-shen-yi-qi (QSYQ), one of the most well-known traditional Chinese medicine (TCM) formulas, has been shown to have cardioprotective effects in rats with heart failure (HF) induced by acute myocardial infarction (AMI). However, the mechanisms of its therapeutic effects remain unclear. In this study, we aim to explore the mechanisms of QSYQ in preventing left ventricular remodelling in rats with HF. The anti-apoptosis an anti-inflammation effects of QSYQ were investigated.MethodsSprague–Dawley (SD) rats were randomly divided into 4 groups: sham group, model group, QSYQ treatment group and aspirin group. Heart failure model was induced by ligation of left anterior descending (LAD) coronary artery. 28 days after surgery, hemodynamics were detected. Echocardiography was adopted to evaluate heart function. TUNEL assay was applied to assess myocardial apoptosis rates. Protein expressions of cyclooxygenase1 and 2 (COX1and COX2), Fas ligand (FasL), P53 and MDM2 were measured by western-blot. RT-PCR was applied to detect expressions of our subtype receptors of PGE2 (EP1, 2, 3, and 4).ResultsUltrasonography showed that EF and FS values decreased significantly and abnormal hemodynamic alterations were observed in model group compared to sham group. These indications illustrated that HF models were successfully induced. Levels of inflammatory cytokines (TNF-α and IL-6) in myocardial tissue were up-regulated in the model group as compared to those in sham group. Western-blot analysis showed that cyclooxygenase 2, which is highly inducible by inflammatory cytokines, increased significantly. Moreover, RT-PCR showed that expressions of EP2 and EP4, which are the receptors of PGE2, were also up-regulated. Increased expressions of apoptotic pathway factors, including P53 and FasL, might be induced by the binding of PGE2 with EP2/4. MDM2, the inhibitor of P53, decreased in model group. TUNEL results manifested that apoptosis rates of myocardial cells increased in the model group. After treatment with QSYQ, expressions of inflammatory factors, including TNF-α, IL-6 and COX2, were reduced. Expressions of EP2 and EP4 receptors also decreased, suggesting that PGE2-mediated apoptosis was inhibited by QSYQ. MDM2 was up-regulated and P53 and FasL in the apoptotic pathway were down-regulated. Apoptosis rates in myocardial tissue in the QSYQ group decreased compared with those in the model group.ConclusionsQSYQ exerts cardiac protective efficacy mainly through inhibiting the inflammatory response and down-regulating apoptosis. The anti-inflammatory and anti-apoptosis efficacies of QSYQ are probably achieved by inhibition of COXs-induced P53/FasL pathway. These findings provide experimental evidence for the beneficial effects of QSYQ in the clinical application for treating patients with HF.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0869-z) contains supplementary material, which is available to authorized users.