Erxian decoction ameliorates myocardial tissue damage through activating PI3K/AKT signaling pathway in ovariectomized rats.

BACKGROUND: Erxian decoction (EXD) is an empirical formula for treating cardiovascular disease, our previous work has shown that EXD could improve the cardiovascular structure and function in ovariectomized (OVX) rats, but its pharmacological mechanism is still unclear. MATERIALS AND METHODS: Network pharmacology was utilized to assess the key active components and central targets of EXD in treating postmenopausal cardiovascular disease. Then, an OVX rat model was established, HE staining and transmission electron microscope were utilized to observe myocardial tissue morphology, TUNEL staining was utilized to detect cardiomyocyte apoptosis, western blot, and ELISA were used to confirm efficacy and pathway of EXD. RESULTS: The network pharmacology prediction results showed that 129 common targets were identified by intersecting EXD targets and postmenopausal cardiovascular disease targets, including AKT1, TNF, IL-6, IL-1ß, PTGS2 and other core targets, apoptosis, PI3K/AKT, and other signaling pathways may be closely related to postmenopausal cardiovascular disease. After ovariectomy, the myocardial tissue of rats was damaged, the expression level of PI3K/AKT pathway-related molecules in the myocardial tissue were decreased, the apoptosis index of cardiomyocytes was increased, and the levels of inflammatory factors (TNF-α, IL-6, and IL-1ß) were enhanced. EXD intervention could improve myocardial tissue injury, EXD could up-regulate the protein expression of PI3K and p-AKT in myocardial tissue, and thereby prevent myocardial cell apoptosis. At the same time, EXD downregulated the levels of inflammatory factors in serum of ovariectomized rats. CONCLUSION: EXD may prevent myocardial tissue damage through induction of the PI3K/AKT signaling pathway, thereby reducing cardiomyocyte apoptosis and inflammation. EXD may be a potential drug for the treatment of postmenopausal cardiovascular disease.

Evaluation of time-dependent phenotypes of myocardial ischemia-reperfusion in mice.

BACKGROUND: A mouse model of myocardial ischemia-reperfusion (I/R) is widely used to study myocardial ischemia-reperfusion injury (I/RI). However, few studies focus on the direct comparison of the extent of pathological events resulting from variant durations of ischemia and reperfusion process. METHODS: A mouse model of I/RI was established by ligation and perfusion of the left anterior descending coronary artery (LAD), and the dynamic changes were recorded by electrocardiogram at different stages of I/R. Subsequently, reperfusion duration was used as a variable to directly compare the phenotypes of different myocardial injury degrees induced by 3 h, 6 h and 24 h reperfusion from myocardial infarct size, myocardial apoptosis, myocardial enzyme, and inflammatory cytokine levels. RESULTS: All mice subjected to myocardial I/R surgery showed obvious myocardial infarction, extensive myocardial apoptosis, dynamic changes in serum myocardial enzyme and inflammatory cytokines, at least for the first 24 h of reperfusion. The infarct size and apoptosis rates gradually increased with the extension of reperfusion time. The peaks of serum myocardial enzyme and inflammatory cytokines occurred at 6 h and 3 h of reperfusion, respectively. We also established I/R mice models with 30 and 60 mins of ischemia. After 21 days of remodeling, longer periods of ischemia increased the degree of fibrosis and reduced cardiac function. CONCLUSIONS: In summary, we conclude that reperfusion durations of 3 h, 6 h, and 24 h induces different injury phenotypes in ischemia-reperfusion mouse model. At the same time, the ischemia duration before reperfusion also affects the degree of cardiac remodeling.

IL-38 attenuates myocardial ischemia-reperfusion injury by inhibiting macrophage inflammation.

BACKGROUND: Reperfusion therapy is the most effective approach to resolve coronary occlusion, but myocardial injury caused by excessive inflammation during myocardial ischemia-reperfusion will also pose a new threat to health. Our prior study revealed the expression pattern of interleukin-38 (IL-38) in the peripheral blood serum of patients with ischemic cardiomyopathy and the role of IL-38 in acute myocardial infarction in mice. However, its role and potential mechanisms in myocardial ischemia/reperfusion injury (MIRI) remain to be determined. METHODS AND RESULTS: The left anterior descending artery of C57BL/6 mice was transiently ligated to induce the MIRI model. We found that MIRI induced the expression of endogenous IL-38, which was mainly produced by locally infiltrating macrophages. Overexpression of IL-38 in C57BL/6 mice attenuated inflammatory injury and decreased myocardial apoptosis after myocardial ischemia-reperfusion. Furthermore, IL-38 inhibited lipopolysaccharide-induced macrophage inflammation in vitro. Cardiomyocytes cocultured with the supernatant of IL-38- and troponin I-treated macrophages showed a lower rate of apoptosis than controls. CONCLUSIONS: IL-38 attenuates MIRI by inhibiting macrophage inflammation. This inhibitory effect may be partially achieved by inhibiting the activation of NOD-like receptor pyrin domain-related protein 3 inflammasome, resulting in decreased expression of inflammatory factors and reduced cardiomyocyte apoptosis.

PPARγ Regulates Macrophage Polarization by Inhibiting the JAK/STAT Pathway and Attenuates Myocardial Ischemia/Reperfusion Injury In Vivo.

This study aimed to investigate the role of PPARγ and underlying mechanisms in myocardial ischemia/reperfusion injury (IRI). IRI was surgically induced in mice and neonatal rat cardiomyocytes (NRCM) were exposed to oxygen-glucose deprivation and reoxygenation (OGD/R). Quantitative genetic analysis and western blotting were performed to assess mRNA and protein levels, respectively, of PPARγ, as well as of different inflammatory, fibrosis, and apoptosis markers in cells and tissues. PPARγ was overexpressed in the heart of mice and NRCMs by viral transfection. Apoptosis and fibrosis were detected by TUNEL and Masson's trichrome staining, respectively. Enzyme-linked immunosorbent assay was performed to detect M1 and M2 macrophage-related inflammatory factors present in mouse sera. PPARγ overexpression significantly inhibited OGD/R- and IRI-induced cardiomyocyte apoptosis and fibrosis in vitro and in vivo. Moreover, PPARγ overexpression inhibited IRI-induced secretion of M1-related proinflammatory factors, whereas it supported the secretion of M2-related anti-inflammatory factors. Notably, these events were found to be mediated by the JAK/STAT pathway. In conclusion, PPARγ regulates macrophage polarization upon IRI via the JAK/STAT pathway, which will in turn prevent myocardial apoptosis and fibrosis. Hence, PPARγ may represent a valuable target for myocardial IRI treatment.

Sphingosine-1-phosphate Attenuates Endoplasmic Reticulum Stress-induced Cardiomyocyte Apoptosis Through Sphingosine-1-phosphate Receptor 1.

BACKGROUND: Endoplasmic reticulum stress (ER stress) is involved in the development and progression of various forms of heart disease and may lead to myocardial apoptosis. Sphingosine-1-phosphate (S1P) possesses cardioprotective properties, including anti-apoptosis. However, little is known about the link between S1P and ER stress-induced myocardial apoptosis. This study investigated the regulatory role of S1P in ER stress-induced apoptosis in cardiomyocytes. METHODS: ER stress and myocardial apoptosis were induced by transverse aortic constriction (TAC) or tunicamycin in mice, which were then treated with 2-acetyl-5-tetrahydroxybutyl imidazole (THI) or S1P. AC16 cells were treated with tunicamycin or thapsigargin, or pretreated with S1P, sphingosine-1-phosphate receptor (S1PR) subtype antagonists, S1PR1 agonist, and PI3K and MEK inhibitors. Cardiac function, the level of S1P in plasma and heart, ER stress markers, cell viability, and apoptosis were detected. RESULTS: S1P reduced the expression of ER stress-related molecules and ER stress-induced myocardial apoptosis in mice subjected to TAC or an injection of tunicamycin. Furthermore, in AC16 cells exposed to thapsigargin or tunicamycin, S1P decreased the expression of ER stress-related molecules, promoting cell viability and survival. Nevertheless, the S1PR1 antagonist abrogated the protection of S1P. Subsequently, in TAC S1PR1 heterozygous (S1PR1+/-) mice, S1P had no effect on ER stress and apoptosis in cardiomyocytes. Notably, in vitro, the impact of anti-ER stress-induced myocardial apoptosis by the S1PR1 agonist was reversed by PI3K and MEK inhibitors. CONCLUSION: This study is the first to demonstrate that S1P relieves ER stress-induced myocardial apoptosis via S1PR1/AKT and S1PR1/ERK1/2, which are potential therapeutic targets for heart disease.

Resveratrol Pretreatment Inhibits Myocardial Apoptosis in Rats Following Coronary Microembolization via Inducing the PI3K/Akt/GSK-3ß Signaling Cascade.

PURPOSE: Coronary microembolization (CME) is associated with progressive cardiac dysfunction, myocardial inflammation, and apoptosis. Resveratrol (RES) has a considerable role in cardioprotection. However, the contribution and possible mechanisms of RES in CME have not been clearly understood. METHODS: In the current study, 40 SD rats were randomly selected and categorized into various groups including CME, CME + resveratrol (CME + RES), CME + resveratrol+ LY294002 (CME + RES + LY), and sham groups (10 animals in each group). The inert plastic microspheres (42 µm) were injected into the rats' left ventricle for developing the CME model. Then resveratrol (25 mg/kg/d) was given to the rats in the CME + RES and CME + RES + LY groups for one week before CME induction. Furthermore, LY294002 (10 mg/kg) was intraperitoneally injected into the rats of the CME + RES + LY group 0.5 hours before CME modeling. The cardiac functions, serum levels of myocardial injury biomarkers, myocardial histopathology, and mRNA and proteins associated with myocardial apoptosis were all assessed 12 hours after surgery. RESULTS: The results revealed that resveratrol pretreatment alleviated the CME-induced myocardial damage by improving cardiac dysfunction, and lowering the serum level of myocardial injury biomarkers, myocardial microinfarct size, and cardiomyocyte apoptotic index. Pretreatment with resveratrol reduced the level of proteins and mRNAs associated with the pro-apoptosis in myocardial tissues and increased the levels of proteins and mRNAs associated with the anti-apoptosis. Moreover, the combined treatment of resveratrol and LY294002 reversed the observed protective effects. CONCLUSION: Resveratrol can inhibit cardiomyocyte apoptosis, thus attenuating the CME-induced myocardial injury by triggering the PI3K/Akt/GSK-3ß cascade.

Pericardial Adipose Tissue-Derived Leptin Promotes Myocardial Apoptosis in High-Fat Diet-Induced Obese Rats Through Janus Kinase 2/Reactive Oxygen Species/Na+/K+-ATPase Signaling Pathway.

Background Pathophysiologic mechanisms underlying cardiac structural and functional changes in obesity are complex and linked to adipocytokines released from pericardial adipose tissue (PAT) and cardiomyocyte apoptosis. Although leptin is involved in various pathological conditions, its role in paracrine action of pericardial adipose tissue on myocardial apoptosis remains unknown. This study was designed to investigate the role of PAT-derived leptin on myocardial apoptosis in high-fat diet-induced obese rats. Methods and Results Hearts were isolated from lean or high-fat diet-induced obese Wistar rats for myocardial remodeling studies. Obese rats had abnormal myocardial structure, diastolic dysfunction, greatly elevated cardiac apoptosis, enhanced cardiac fibrosis, and increased oxidative stress level. ELISA detected significantly higher than circulating leptin level in PAT of obese, but not lean, rats. Western blot and immunohistochemical analyses demonstrated increased leptin receptor density in obese hearts. H9c2 cardiomyoblasts, after being exposed to PAT-conditioned medium of obese rats, exhibited pronounced reactive oxygen species-mediated apoptosis, which was partially reversed by leptin antagonist. Moreover, leptin derived from PAT of obese rats inhibited Na+/K+-ATPase activity of H9c2 cells through stimulating reactive oxygen species, thereby activating calcium-dependent apoptosis. Pretreatment with specific inhibitors revealed that Janus kinase 2/signal transducer and activator of transcription 3 and phosphoinositide 3-kinase/protein kinase B signaling pathways were involved in leptin-induced myocardial apoptosis. Conclusions PAT-derived leptin induces myocardial apoptosis in high-fat diet-induced obese rats via activating Janus kinase 2/signal transducer and activator of transcription 3/reactive oxygen species signaling pathway and inhibiting its downstream Na+/K+-ATPase activity.

Guan Xin Dan Shen formulation protects db/db mice against diabetic cardiomyopathy via activation of Nrf2 signaling.

Guan Xin Dan Shen formulation (GXDSF) is a widely used treatment for the management of coronary heart disease in China and is composed of three primary components: Dalbergiae odoriferae Lignum, Salviae miltiorrhizae Radix et Rhizoma and Panax notoginseng Radix et Rhizoma. However, the potential use of GXDSF for the management of diabetic cardiomyopathy (DCM) has not been previously assessed. The present study aimed to assess the effects of GXDSF on DCM, as well as the underlying mechanism. In the present study, db/db mice were used. Following treatment with GXDSF for 10 weeks, fasting blood glucose, insulin sensitivity, serum lipid levels and cardiac enzyme levels were detected. Cardiac pathological alterations and cardiac function were assessed by performing hematoxylin and eosin staining and echocardiograms, respectively. TUNEL assays were conducted to assess cardiomyocyte apoptosis. Additionally, reverse transcription­quantitative PCR and western blotting were performed to evaluate the expression of apoptosis­associated genes and proteins, respectively. In the model group, the db/db mice displayed obesity, hyperlipidemia and hyperglycemia, accompanied by noticeable myocardial hypertrophy and diastolic dysfunction. Following treatment with GXDSF for 10 weeks, serum triglyceride levels were lower and insulin sensitivity was enhanced in db/db mice compared with the model group, which indicated improvement in condition. Cardiac hypertrophy and dysfunction were also improved in db/db mice following treatment with GXDSF, resulting in significantly increased left ventricular ejection fraction and fractional shortening compared with the model group. Following treatment with metformin or GXDSF, model­induced increases in levels of myocardial enzymes were decreased in the moderate and high dose groups. Moreover, the results indicated that, compared with the model group, GXDSF significantly inhibited cardiomyocyte apoptosis in diabetic heart tissues by increasing Bcl­2 expression and decreasing the expression levels of Bax, cleaved caspase­3 and cleaved caspase­9. Mechanistically, GXDSF enhanced Akt phosphorylation, which upregulated antioxidant enzymes mediated by nuclear factor erythroid 2­related factor 2 (Nrf2) signaling. Collectively, the results of the present study indicated that GXDSF attenuated cardiac dysfunction and inhibited cardiomyocyte apoptosis in diabetic mice via activation of Akt/Nrf2 signaling. Therefore, GXDSF may serve as a potential therapeutic agent for the management of DCM.

Puerarin pretreatment inhibits myocardial apoptosis and improves cardiac function in rats after acute myocardial infarction through the PI3K/Akt signaling pathway.

BACKGROUND: Puerarin demonstrates a protective effect in many cardiovascular diseases. However, the role of puerarin in acute myocardial infarction (AMI)-induced injury and the exact molecular mechanisms are not fully understood. OBJECTIVES: To investigate whether puerarin pretreatment improves cardiac function and to study the mechanism of action of puerarin. MATERIAL AND METHODS: Thirty rats were grouped into sham group, AMI group and AMI+puerarin (PUE) group at random (n = 10 per group). Except for the sham group, a model of AMI was established via left anterior descending artery ligation. The PUE group received puerarin 120 mg/(kg × day) for 7 days before the operation. Echocardiography was used for evaluation of cardiac function in rats and TUNEL staining for measuring myocardial apoptosis. The expression levels of p-PI3K, t-Akt, p-Akt, Bax, Bcl-2, and cleaved caspase-3 proteins were measured with western blot. RESULTS: Compared to the sham group, the AMI group demonstrated poor cardiac function and decreased p-PI3K, p-Akt and Bcl-2 proteins levels, while Bax, cleaved caspase-3, and myocardial apoptosis levels increased. Compared with the AMI group, the PUE group showed significant improvement in cardiac function and increased protein expression of p-PI3K, p-Akt and Bcl-2, while Bax and cleaved caspase-3 levels decreased and myocardial apoptosis was attenuated. CONCLUSIONS: Puerarin pretreatment in AMI can effectively improve cardiac function by inhibiting myocardial apoptosis. The molecular mechanism of this protective effect may be mediated by activating the PI3K/Akt pathway in cardiomyocytes.

Effects of D-pinitol on myocardial apoptosis and fibrosis in streptozocin-induced aging-accelerated mice.

Diabetic cardiomyopathy (DCM) causes heart failure and increases the mortality in diabetic patients. Myocardial apoptosis and fibrosis are the main features of DCM and aging. The aim is to study the underlying mechanism of D-pinitol (DP) on myocardial apoptosis and fibrosis in an elderly diabetic mouse model. The diabetic model was established by SAMP-8 mice that were injected with streptozotocin daily for five consecutive days. The mice were administrated of DP (150 mg kg-1  day-1 ) by gavage for 10 weeks. The common metabolic disorder indices, cardiac dysfunction, oxidative stress, myocardial apoptosis and fibrosis, and PI3K/Akt/mTOR pathway were investigated. Our findings suggested that DP has a protective effect on DCM, which may be related to regulating oxidative stress, and PI3K/Akt/mTOR pathway involving cardiac fibrosis and apoptosis. DP may be a novel clinical application in fighting against DCM. PRACTICAL APPLICATIONS: D-pinitol (DP) was found in large quantities in soybean and legume foods. DP has a variety of functions, including hypoglycemic, anti-oxidation, anti-inflammatory, cardioprotective, and anti-tumor activity. We used the streptozotocin-induced SAMP8 mice as the diabetic model and treated with DP. We found that DP can improve cardiac dysfunction and inhibits the oxidative stress, myocardial apoptosis and fibrosis. DP has a significant effect on diabetic cardiomyopathy (DCM). The molecular mechanisms are related to regulating oxidative stress, and PI3K/Akt/mTOR pathway involving cardiac fibrosis and apoptosis. DP can prevent and/or delay the onset of DCM.

Overexpression of lncRNA HULC Attenuates Myocardial Ischemia/reperfusion Injury in Rat Models and Apoptosis of Hypoxia/reoxygenation Cardiomyocytes via Targeting miR-377-5p through NLRP3/Caspase­1/IL­1ß Signaling Pathway Inhibition.

OBJECTIVE: Acute myocardial infarction (AMI) is characterized by myocardial tissue necrosis and activation of inflammatory response. This study aims to elucidate the potential mechanism underlying the protective effects of long non-coding RNA (lncRNA) highly up-regulated in liver cancer (HULC) against myocardial ischemia/reperfusion (I/R) injury in rat models and apoptosis of cardiomyocytes. METHODS: We firstly established rat models of myocardial I/R injury and rat cardiomyocyte (H9c2 cells) models of hypoxia/reoxygenation (H/R) injury. Sprague-Dawley (SD) neonatal rats were randomized into four groups: sham, I/R, I/R+ microRNA (miR) -377-5p mimic, and I/R+ miR-377-5p antagomir, respectively. Then, histopathological examination was applied. Apoptosis was evaluated by transferase-mediated dUTP nick end labeling (TUNEL) staining. Cell vitality was measured using MTT assay. The concentrations of creatine kinase MB (CK-MB), cardiac troponin I (cTnI), interleukin (IL) -6 (IL-6), and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The expression of Cleaved-Caspase-3, Caspase-3, NOD-like receptor P3 (NLRP3), Caspase-1, and IL-1ß was analyzed by immunohistochemical (IHC) or Western blot analysis. RESULTS: We found that HULC was downregulated and miR-377-5p was upregulated in IR-injured myocardial tissue and the H/R-induced H9c2 cell. Overexpression of miR-377-5p increased myocardial dysfunction and apoptosis and activated formation and secretion of IL-6 and TNF-α. The preprocessing of miR-377-5p silencing emerged opposite results. Strikingly, dual luciferase reporter assay showed that HULC was a sponge of miR-377-5p. Subsequently, mechanism experiments revealed that NLRP3/Caspase­1/IL­1ß was a target axis of miR-377-5p. In vitro, the protective effect of HULC overexpression on H9c2 cell viability and inflammation was offset by miR-377-5p silencing. Finally, rescue assay suggested that HULC-miR-377-5p -NLRP3/Caspase­1/IL­1ß axis regulated the apoptosis and inflammation of H/R-induced H9c2 cells. CONCLUSIONS: Overall, these results indicate that the protective effect of HULC against myocardial I/R injury and H/R cardiomyocyte apoptosis partially relies on the inhibition of NLRP3/Caspase­1/IL­1ß signaling pathway.

Mitochondrial Ca2+ regulation in the etiology of heart failure: physiological and pathophysiological implications.

Heart failure (HF) represents one of the leading causes of cardiovascular diseases with high rates of hospitalization, morbidity and mortality worldwide. Ample evidence has consolidated a crucial role for mitochondrial injury in the progression of HF. It is well established that mitochondrial Ca2+ participates in the regulation of a wide variety of biological processes, including oxidative phosphorylation, ATP synthesis, reactive oxygen species (ROS) generation, mitochondrial dynamics and mitophagy. Nonetheless, mitochondrial Ca2+ overload stimulates mitochondrial permeability transition pore (mPTP) opening and mitochondrial swelling, resulting in mitochondrial injury, apoptosis, cardiac remodeling, and ultimately development of HF. Moreover, mitochondria possess a series of Ca2+ transport influx and efflux channels, to buffer Ca2+ in the cytoplasm. Interaction at mitochondria-associated endoplasmic reticulum membranes (MAMs) may also participate in the regulation of mitochondrial Ca2+ homeostasis and plays an essential role in the progression of HF. Here, we provide an overview of regulation of mitochondrial Ca2+ homeostasis in maintenance of cardiac function, in an effort to identify novel therapeutic strategies for the management of HF.

Composition of Ophiopogon Polysaccharide, Notoginseng Total Saponins and Rhizoma Coptidis Alkaloids Inhibits the Myocardial Apoptosis on Diabetic Atherosclerosis Rabbit.

OBJECTIVE: To investigate the effects of Composition of Ophiopogon polysaccharide, Notoginseng total saponins and Rhizoma Coptidis alkaloids (CONR) on myocardial apoptosis of diabetic atherosclerosis (DA) rabbits METHODS: Sixty male New Zealand white rabbits were randomly divided into 6 groups [control group, model group, CONR high-dose group (450 mg/kg), CONR medium-dose group (150 mg/kg), CONR low-dose group (50 mg/kg), and simvastatin group] by using a completely random method, 10 in each group. DA model was established by intravenously injected alloxan combined with high-fat diet and abdominal aortic balloon injury. After mediation for 10 weeks, fasting blood glucose (FBG), glycosylated hemoglobin (GHB), glycosylated serum protein (GSP), fructoseamine (FRA), aldose reductase (AR), advanced glycation end products (AGEs) in serum were measured by enzyme linked immunosorbent assay (ELISA) method; the expression of receptor of AGEs (RAGE) in myocardial tissue were observed by immunohistochemical method; and p-Jun N-terminal kinase (p-JNK), caspase-3, B-cell lymphoma-2 (bcl-2) protein expression in myocardial tissue were measured by Western blotting. The myocardial apoptosis was detected by TdT-mediated dUTPnick-end labeling (TUNEL) method, and apoptosis index (AI) was calculated. RESULTS: Compared with the control group, serum FBG, GHB, GSP, FRA, AR, AGEs and the expression of myocardium RAGE, p-JNK, caspase-3 proteins, as well as apoptosis index (AI) were significantly increased and bcl-2 protein was significantly decreased in the model group (P<0.01). Compared with the model group, the levels of serum FBG, GHB, GSP, FRA and AR showed a significant decline in CONR high- and medium-dose groups (P<0.01). FBG and GHB showed a significant decline in CONR low-dose group (P<0.01). Compared with the model group, the expression of serum AGEs and myocardium RAGE, p-JNK and caspase-3 protein as well as AI were significantly decreased and bcl-2 protein was significantly up-regulated in all treatment groups (P<0.01); high-dose CONR had the most significant effect on abovementioned indices compared with other treatment groups (P<0.01). Middle-dose CONR had better effect on serum AGEs compared with the low-dose group (P<0.01); middle-dose CONR and simvastatin groups had better effect on the expression of caspase-3, bcl-2 protein, myocardium apoptosis compared with the CONR low-dose group (P<0.01). CONCLUSION: CONR may effectively inhibit myocardial apoptosis on DA rabbits by intervening AGEs-RAGE and JNK, caspase-3, and bcl-2 protein expressions.

Qiliqiangxin Attenuates Oxidative Stress-Induced Mitochondrion-Dependent Apoptosis in Cardiomyocytes via PI3K/AKT/GSK3ß Signaling Pathway.

Qiliqiangxin capsule (QLQX) is a well-known traditional Chinese medicine that exhibits cardioprotective effects in heart failure patients. However, it remains unclear whether and by which mechanism QLQX attenuates oxidative stress-induced mitochondria-dependent myocardial apoptosis. In vivo, Sprague Dawley (SD) rats received left anterior descending coronary artery ligation for 4 weeks to establish a model of heart failure after acute myocardial infarction, and then were treated with QLQX for another 4 weeks. We evaluated cardiac function, oxidative stress injury, as well as the expressions of mitochondria-dependent apoptosis and its signaling factors. The results indicated that QLQX protected cardiac function and attenuated oxidative stress-induced myocardial apoptosis. Meanwhile, QLQX elevated the Bcl-2 expression, declined the expressions of Bax, cytochrome c, apoptotic protease activating factor-1 (Apaf-1), cleaved-caspase 9 and cleaved-caspase 3, and up-regulated the ratios of phospho-AKT/AKT and phospho-glycogen synthase kinase-3ß (GSK3ß)/GSK3ß. In vitro, H9c2 cardiomyocytes were pretreated with QLQX, then exposed to H2O2 for 24 h. QLQX promoted the proliferation of H9c2 cardiomyocytes induced by H2O2 and reversed oxidative stress damage. Moreover, QLQX inhibited the apoptosis rate and the pro-apoptosis protein expressions, but improved the Bcl-2 expression as well as the ratios of phospho-AKT/AKT and phospho-GSK3ß/GSK3ß. Meanwhile, it further ameliorated mitochondrion-related apoptosis by inhibiting the mitochondrial fission, mitochondrial permeability transition pore (MPTP) opening, and mitochondrial membrane potential (MMP) decline in H9c2 cardiomyocytes induced by H2O2. In addition, all the effects of QLQX on H2O2-induced mitochondria-dependent apoptosis could be blocked by the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. We conclude that QLQX may ameliorate oxidative stress-induced mitochondria-dependent apoptosis in cardiomyocytes through PI3K/AKT/GSK3ß signaling pathway.

Stomatin-like protein-2 relieve myocardial ischemia/reperfusion injury by adenosine 5'-monophosphate-activated protein kinase signal pathway.

Previous studies have shown that stomatin-like protein-2 (SLP-2) could regulate mitochondrial biogenesis and function. The study was designed to explore the contribution of SLP-2 to the myocardial ischemia and reperfusion (I/R) injury. Anesthetized rats were treated with SLP-2 and subjected to ischemia for 30 minutes before 3 hours of reperfusion. An oxygen-glucose deprivation/reoxygenation model of I/R was established in H9C2 cells. In vivo, SLP-2 significantly improved cardiac function recovery of myocardial I/R injury rats by increasing fractional shortening and ejection fraction. SLP-2 pretreatment alleviated infarct area and myocardial apoptosis, which was paralleled by decreasing the level of cleaved caspase-3 and the ratio of Bax/Bcl-2, increasing the content of superoxide dismutase and reducing oxidative stress damage in serum. In addition, SLP-2 increased the level of ATP and stabilized mitochondrial potential (Ψm). The present in vitro study revealed that overexpression with SLP-2 reduced H9C2 cells apoptosis, accompanied by an increased level of ATP, the ratio of mitochondrial DNA/nuclear DNA, activities of complex II and V, and decreased the production of mitochondrial reactive oxygen species. Simultaneously, SLP-2 activated the adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway in myocardial I/R injury rats and H9C2 cells. This study revealed that SLP-2 mediates the cardioprotective effect against I/R injury by regulating AMPK signaling pathway.

MicroRNA-29b Regulates the Mitochondria-Dependent Apoptotic Pathway by Targeting Bax in Doxorubicin Cardiotoxicity.

BACKGROUND/AIMS: Myocardial apoptosis plays an important role in doxorubicin (Dox) cardiotoxicity. MicroRNA-29 (miR-29) is suggested to function as an anti-fibrotic factor with potential therapeutic effects on cardiac fibrosis. However, it has not been shown whether there is an association between miR-29b and myocardial apoptosis. METHODS: Male Wistar rats were transfected with miR-29b agomir by local delivery to the myocardium prior to Dox treatment. Rat cardiomyocytes were pretreated with miR-29b mimics or inhibitor followed by Dox incubation in vitro. Cardiac function and underlying mechanisms were evaluated by echocardiography, immunofluorescence, flow cytometry, real-time PCR, and western blotting. RESULTS: Our results revealed that miR-29b is the only member of the miR-29 family that was significantly downregulated in myocardium from Dox-treated rats. Delivery of miR-29b agomir to myocardium resulted in a marked improvement of cardiac function. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining showed that rescue of miR-29b expression inhibited Dox-induced myocardial apoptosis, concomitantly with increased Bcl-2 expression and decreased Bax expression and caspase-3 activity. In vitro, miR-29b overexpression mitigated, whereas inhibition of miR-29b promoted, Dox-induced cardiomyocyte apoptosis. Mechanistically, miR-29b negatively regulated Bax expression by directly targeting the 3' untranslated region of Bax. In Dox-treated cardiomyocytes, upregulation of miR-29b resulted in a significant decrease in Bax expression, with an increase in Bcl-2 expression, accompanied by inhibition of mitochondrial membrane depolarization, cytochrome c release, and caspase activation. However, inhibition of miR-29b produced the opposite effects by further augmenting the effects of Dox. CONCLUSIONS: These data demonstrate that miR-29b prevents Dox-induced myocardial apoptosis through inhibition of the mitochondria-dependent pathway by directly targeting Bax, suggesting that miR-29b is a potential novel therapeutic target for the treatment of Dox cardiotoxicity.

[Effect of fracture of lower limbs with hemorrhage on myocardial injury and its mechanism in rats].

OBJECTIVES: To test whether myocardial apoptosis can be induced by traumatic fracture of lower limbs with hemorrhage, in order to lay a foundation of myocardial injury after traumatic fracture for the follow-up study. METHODS: Twenty SD rats were randomly divided into two groups, i. e. control group and trauma group(n=10). A rat model of traumatic hemorrhage was establish, and a traumatic model of the original generation of myocardial cell culture was constructed in vitro. The level of interleukin-2(IL-2),IL-6,IL-10 and tumor necrosis factor-α(TNF-α) in rat serum was detected by ELISA at 0, 1, 2, 4, 8, 12, 16, 24 and 48 hour to find the most significant point. The pathological cardiac injury in rats was observed by HE staining under a microscope, and the apoptosis of cultured cardiomyocyte in vitro was detected by TUNEL methods. The expressions of apoptosis gene,(Bcl-2) and Bax, in myocardium of rat and cultured cardiomyocyte in vitro were detected by Western blot and RT-PCR. RESULTS: At the 4th hour after trauma, IL-6 and IL-10 in the serum of rats reached its highest, IL-2 reached its lowest at the 8th hour after trauma, and TNF-αreached its highest at 1 hour after trauma, then all recovered to their normol level gradually. Myocardial HE staining indicated that cardiomyocyte was swelling, disordered derangement, inflammatory cell infiltrated; a large number of myocardial cell nuclei was dyedbrown in TUNEL test which proved that the apoptosis index increased (P<0.05). Western blot and RT-PCR results showed that the expression of pro-apoptotic gene Bax was up-regulated (P<0. 05), while expression of anti apoptosis gene Bcl-2 down-regulated (P<0.05). CONCLUSIONS: The myocardial apoptosis can be induced by traumatic fracture of lower limbs with hemorrhage in rats, and then lead to myocardial injury.

The involvement of Nox4 in fine particulate matter exposure-induced cardiac injury in mice.

Epidemiological studies have confirmed that ambient fine particulate matter (PM2.5) exposure is associated with cardiovascular disease (CVD). However, the underlying mechanisms in PM2.5 exposure-induced heart injury are largely unknown. It has been acknowledged that NADPH oxidase (Nox) 4 plays a critical role in CVD development. To investigate the acute effects of PM2.5 on the mouse heart and the role of Nox4 in PM2.5 exposure-induced cardiac injury, C57BL/6J mice were instilled with saline or 1.5, 3.0, 6.0 mg/kg BW PM2.5 suspension for two weeks (five days per week). The levels of malondialdehyde (MDA), super oxide dismutase (SOD), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß in heart supernatants were determined using related kits. The expression of Nox4, p67phox, p47phox and p22phox in heart tissue was evaluated by immunofluorescence staining or Western blotting, respectively. Protein levels of p53, Bax, Bcl-2 and Caspase-3 in the heart were examined using immunohistochemical staining and Western blotting. TUNEL assay was used to measure myocardial apoptosis. PM2.5 exposure leads to obvious cardiac injury. PM2.5 exposure increases MDA level and iNOS activity, and decreases activity of SOD in heart supernatants of mice. High levels of TNF-α and IL-1ß in heart supernatants of mice with PM2.5 instillation were determined. Nox4 and Nox-associated subunits such as p67phox, p47phox and p22phox expression levels were increased in heart tissue of mice after PM2.5 exposure. Additionally, PM2.5 exposure causes myocardial apoptosis in the mouse heart. This study suggested that Nox4 is involved in PM2.5 exposure-induced cardiac injury in mice.

[Effects of Moxibustion on Ventricular Mass Index and Expression of Apoptosis Related Proteins in Myocardium of Rats with Chronic Heart Failure].

OBJECTIVE: To observe the effect of moxibustion on cardiac function and the expression of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X (Bax), Fas, Fas ligand (FasL) in cardiomyocytes of chronic heart failure (CHF) rats, so as to explore its underlying mechanisms in preventing and treating CHF. METHODS: SD rats were randomly divided into normal control, model, moxibustion, Captopril and moxibustion ＋ Captopril (M＋C) groups (n＝12 rats/group). The CHF model was established by intraperitoneal injection of Adriamycin (ADR, from 1 to 4 mg/kg, once every other day for 15 days). Mild moxibustion was applied to bilateral"Feishu"(BL 13) and "Xinshu"(BL 15). Rats of the Captopril group was treated by gavage of Captopril suspension (5 mg/mL, 25 mL/kg), and those of the M＋C group treated by the combined two methods. All the treatments were given once a day for 3 weeks. The general conditions and behaviors of rats were observed. The left ventricular mass index (LVMI) and right ventricular mass index (RVMI) were detected for assessing the cardiac performance. Morphological changes of myocardium were observed by HE staining. Enzyme linked immunosorbent assay (ELISA) was used to detect the concentrations of B-type natriuretic peptide (BNP) and precursor N-terminal pro-brain natriuretic peptide (NT-pro BNP) in the serum. The expression levels of Bcl-2, Bax, Fas and FasL of the left ventricle of heart were detected by Western blot. RESULTS: After modeling, the pathological changes of myocardium (as myocardial cell swelling with vacuoles, myocardial fibre breakage, etc.) were obvious, the LVMI, RVMI, serum BNP and NT-pro BNP concentrations, and myocardial Bax, Fas and FasL protein expression levels were significantly increased in the model group compared with the normal group (P<0.01), while the expression level of Bcl-2 was significantly down-regulated (P<0.01). Following the interventions, the myocardial injury was reduced, both LVMI and RVMI, serum BNP concentration and Bax, Fas and FasL expression levels in the three treatment groups, and serum NT-pro BNP concentration in the moxibustion and M＋C groups were significantly decreased (P<0.05, P<0.01), while the myocardial Bcl-2 protein levels in the three treatment groups were significantly increased relevant to the model group (P<0.01). Comparison among the three treatment groups showed that the effects of moxibustion ＋ Captopril were significantly superior to those of simple moxibustion and simple Captopril in suppressing CHF-induced increased expression of myocardial Bax, Fas and FasL, and in lessening CHF-induced decrease of Bcl-2 level (P<0.05, P<0.01). No significant differences were found among the three treatment groups in down-regulating LVMI and RVMI, and serum BNP content (P>0.05)．. CONCLUSION: Moxibustion can reduce myocardial injury and improve cardiac function in CHF rats, which may be related to its effects in down-regulating the expression of myocardial Bax, Fas and FasL proteins, and up-regulating the expression of Bcl-2 protein to inhibit cardiomyocyte apoptosis.