Su Ying Wen

Diallyl trisulfide protects against high glucose-induced cardiac apoptosis by stimulating the production of cystathionine gamma-lyase-derived hydrogen sulfide☆

BACKGROUND Cystathionine-γ-lyase (CSE)-derived hydrogen sulfide (H2S) is a potent cardioprotective agent. We investigated the effects of diallyl trisulfide (DATS) on CSE expression and H2S generation in myocardium and examined whether DATS-mediated H2S generation effectively protects rat heart from diabetes-induced cardiac damage. METHODS The correlations between the effects of hyperglycemia and diabetes on CSE expression and the effects of DATS and H2S on hyperglycemia and diabetes were examined in vitro in the cardiomyocyte cell line H9c2 and in vivo in hearts from rats with streptozotocin-induced diabetes mellitus (DM). RESULTS Expression of CSE, a catalyst of H2S production, was suppressed in H9c2 cells treated with high glucose (33 mM) and in DM rat hearts. CSE suppression also correlated with a decrease in the activation of the pro-survival protein kinase Akt. Treatment of H9c2 cells with DATS resulted in increased CSE expression and a reduction in apoptosis via a mechanism involving IGF1R/pAkt signaling and by modulating the expression of reactive oxygen species-related enzymes. The role CSE plays in the cardioprotective effects of DATS was further confirmed by CSE inhibition assays including inhibitors and siRNA. CONCLUSION DATS produces H2S as efficiently as NaSH and DATS-derived H2S provides effective cardioprotection. Further, our data indicate that H2S plays a major role in the protective effect of DATS against apoptosis of cardiomyocytes.

Deep sea minerals prolong life span of streptozotocin-induced diabetic rats by compensatory augmentation of the IGF-I-survival signaling and inhibition of apoptosis.

Consumption of deep sea minerals (DSM), such as magnesium, calcium, and potassium, is known to reduce hypercholesterolemia‐induced myocardial hypertrophy and cardiac‐apoptosis and provide protection against cardiovascular diseases. Heart diseases develop as a lethal complication among diabetic patients usually due to hyperglycemia‐induced cardiac‐apoptosis that causes severe cardiac‐damages, heart failure, and reduced life expectancy. In this study, we investigated the potential of DSM and its related cardio‐protection to increase the life expectancy in diabetic rats. In this study, a heart failure rat model was developed by using streptozotocin (65 mg kg−1) IP injection. Different doses of DSM‐1× (37 mg kg−1 day−1), 2× (74 mg kg−1 day−1) and 3× (111 mg kg−1 day−1), were administered to the rats through gavages for 4 weeks. The positive effects of DSM on the survival rate of diabetes rats were determined with respect to the corresponding effects of MgSO4. Further, to understand the mechanism by which DSM enhances the survival of diabetic rats, their potential to regulate cardiac‐apoptosis and control cardiac‐dysfunction were examined. Echocardiogram, tissue staining, TUNEL assay, and Western blotting assay were used to investigate modulations in the myocardial contractile function and related signaling protein expression. The results showed that DSM regulate apoptosis and complement the cardiomyocyte proliferation by enhancing survival mechanisms. Moreover DSM significantly reduced the mortality rate and enhanced the survival rate of diabetic rats. Experimental results show that DSM administration can be an effective strategy to improve the life expectancy of diabetic subjects by improving cardiac‐cell proliferation and by controlling cardiac‐apoptosis and associated cardiac‐dysfunction.

Diallyl trisulfide suppresses doxorubicin-induced cardiomyocyte apoptosis by inhibiting MAPK/NF-κB signaling through attenuation of ROS generation

Doxorubicin (Dox) is an effective anticancer agent. However, its effectiveness is limited by its cardiotoxic effects. It has also been reported that the mitogen‐activated protein kinase family and NF‐κB can be activated by Dox treatment. DATS has been shown to be a potent antioxidant with cardioprotective effects. We investigate whether Dox induces cardiac apoptosis through JNK‐ and ERK‐dependent NF‐κB upregulation that can be reduced by DATS treatment.

E2/ER β enhances calcineurin protein degradation and PI3k/Akt/MDM2 signal transduction to inhibit ISO-induced myocardial cell apoptosis

Secretion of multifunctional estrogen and its receptor has been widely considered as the reason for markedly higher frequency of heart disease in men than in women. 17β-Estradiol (E2), for instance, has been reported to prevent development of cardiac apoptosis via activation of estrogen receptors (ERs). In addition, protein phosphatase such as protein phosphatase 1 (PP1) and calcineurin (PP2B) are also involved in cardiac hypertrophy and cell apoptosis signaling. However, the mechanism by which E2/ERβ suppresses apoptosis is not fully understood, and the role of protein phosphatase in E2/ERβ action also needs further investigation. In this study, we observed that E2/ERβ inhibited isoproterenol (ISO)-induced myocardial cell apoptosis, cytochrome c release and downstream apoptotic markers. Moreover, we found that E2/ERβ blocks ISO-induced apoptosis in H9c2 cells through the enhancement of calcineurin protein degradation through PI3K/Akt/MDM2 signaling pathway. Our results suggest that supplementation with estrogen and/or overexpression of estrogen receptor β gene may prove to be effective means to treat stress-induced myocardial damage.

Cardio Protective Effects of Lumbrokinase and Dilong on Second-Hand Smoke-Induced Apoptotic Signaling in the Heart of a Rat Model

Exposure to second-hand tobacco smoke (SHS) has been epidemiologically linked to heart disease among non-smokers. However, the molecular mechanism behind SHS-induced cardiac disease is not well known. This study found that SD rats exposed to cigarette smoke at a dose of 10 cigarettes for 30 min twice a day for 1 month had a reduced left ventricle-to-tibia length ratio (mg/mm), increased cardiomyocyte apoptosis by TUNEL assay and a wider interstitial space by H&E staining. However, lumbrokinase and dilong both reversed the effects of SHS. Western blotting demonstrated significantly increased expression of the pro-apoptotic protein caspase-3 in the hearts of the rats exposed to SHS. Elevated protein expression levels of Fas, FADD and the apoptotic initiator activated caspase-8, a molecule in the death-receptor-dependent pathway, coupled with increased t-Bid and apoptotic initiator activated caspase-9 were found. Molecules in the mitochondria-dependent pathway, which disrupts mitochondrial membrane potential, were also found in rats exposed to SHS. These factors indicate myocardial apoptosis. However, treatment with lumbrokinase and dilong inhibited SHS-induced apoptosis. Regarding regulation of the survival pathway, we found in western blot analysis that cardiac protein expression of pAkt, Bcl2, and Bcl-xL was significantly down-regulated in rats exposed to SHS. These effects were reversed with lumbrokinase and dilong treatment. The effects of SHS on cardiomyocytes were also found to be mediated by the Fas death receptor-dependent apoptotic pathway, an unbalanced mitochondria membrane potential and decreased survival signaling. However, treatment with both lumbrokinase and dilong inhibited the effects of SHS. Our data suggest that lumbrokinase and dilong may prevent heart disease in SHS-exposed non-smokers.

Early fluid resuscitation by lactated ringer's solution alleviate the cardiac apoptosis in rats with trauma-hemorrhagic shock

Cardiac trauma has been recognized as a complication associated with blunt chest trauma involving coronary artery injury, myocardium contusion and myocardial rupture. Secondary cardiac injuries after trauma supposed to be a critical factor in trauma patients, but the mechanism is not fully explored. Overproduction of TNF-alpha had been reported in multiple trauma animals, this induces oxidative stress resulting in cardiac apoptosis. Apoptosis gradually increases after trauma and reaches to a maximum level in 12 h time. TNF-alpha increases the expression of NFkB, and induces the expression of caspase-3 and resulted in cell apoptosis. The effect can be attenuated by non-selective caspase inhibitor and IL10. Fas induced cardiac apoptosis and hypertrophy in ischemic heart disease. In this study, we demonstrated a trauma-hemorrhagic shock (THS) model in rats and resuscitated rats by lactated Ringer’s (L/R) solution after shock in different hours (0 hour, 4 hours, 8 hours). NFkB gradually increased after the first 8 hours of shock, and can be reduced by fluid resuscitation. NFkB is known as a downstream pathway of Fas related apoptosis, we found Fas ligand, caspase-8 levels elevate after shock, and can be reduced by resuscitation. In addition, resuscitation can activate insulin-like growth factor (IGF-1)/Akt pathway, at the same time. It can block mitochondrial damage by decrease the effect of tBid. In conclusion, THS can induce secondary cardiac injury. Fas showed to be an important element in caspase cascade induced myocardium apoptosis. By L/R fluid resuscitation, the suppression of caspase cascade and activation of IGF-I/Akt pathway showed antiapoptotic effects in traumatic heart of rats.

Fenofibrate induced PPAR alpha expression was attenuated by oestrogen receptor alpha overexpression in Hep3B cells

The physiological regulation of Oestrogen receptor α (ERα) and peroxisome proliferator‐activated receptor alpha (PPARα) in Hepatocellular carcinoma (HCC) remains unknown. The present study we first treat the cells with fenofibrate and further investigated the possible mechanisms of 17β‐estradiol (E2) and/or ERα on regulating PPARα expression. We also found higher PPARα expression in the tumor area than adjacent areas and subsequently compared PPARα expression in four different hepatic cancer cell lines. Hep3B cells were found to express more PPARα than the other cell lines. Using the PPARα agonist fenofibrate, we found that fenofibrate increased Hep3B cell proliferation efficiency by increasing cell cycle proteins, such as cyclin D1 and PCNA, and inhibiting p27 and caspase 3 expressions. Next, we performed transient transfections and immuno‐precipitation studies using the pTRE2/ERα plasmid to evaluate the interaction between ERα and PPARα. ERα interacted directly with PPARα and negatively regulated its function. Moreover, in Tet‐on ERα over‐expressed Hep3B cells, E2 treatment inhibited PPARα, its downstream gene acyl‐CoA oxidase (ACO), cyclin D1 and PCNA expression and further increased p27 and caspase 3 expressions. However, over‐expressed ERα plus 17‐β‐estradiol (10−8 M) reversed the fenofibrate effect and induced apoptosis, which was blocked in ICI/melatonin/fenofibrate‐treated cells. This study illustrates that PPARα expression and function were negatively regulated by ERα expression in Hep3B cells.

Protective effect of HDL on NADPH oxidase-derived super oxide anion mediates hypoxia-induced cardiomyocyte apoptosis

Cardiovascular diseases are the leading cause of death of death in Taiwan. Atherosclerosis can lead to serious problems, including heart attack, stroke, or even death. Coronary heart disease (CHD) occurs when plaque builds up in the coronary arteries to cause the ischemic heart disease which will enhance myocardial remodeling and also induce myocardial hypoxia. High density lipoprotein (HDL) has been proposed to have cardio-protective effects. Under hypoxic conditions (1%O2 for 24hr), in H9c2 cells, reactive oxygen species (ROS) is induced which leads to cardiomyocyte apoptosis and cardiac dysfunction. Therefore, the present study described the protective effect of HDL on hypoxia-induced cardiomyocyte damage. We investigated the NADPH oxidase-produced ROS-related signaling pathways and apoptosis in cardiomyocytes under hypoxia conditions. Results showed that the ROS mediated cardiac damage might occur via AT1 and PKC activation. Furthermore, hypoxia downregulated the survival protein (p-AKTser473) and anti-apoptotic protein (BCL2), whereas pro-apoptotic protein, Bax and caspase 3 were upregulated. These detrimental effects by ROS and apoptosis were prevented by HDL pretreatment. Our findings revealed the underlying molecular mechanism by which HDL suppresses the hypoxia-induced cardiomyocyte dysfunction. Further, we elucidated the role of HDL on preventing hypoxia induced cardiomyocyte apoptosis is mediated through the inhibition of NADPH oxidase-derived ROS.

High density lipoprotein (HDL) reverses palmitic acid induced energy metabolism imbalance by switching CD36 and GLUT4 signaling pathways in cardiomyocyte

In our previous study palmitic acid (PA) induced lipotoxicity and switches energy metabolism from CD36 to GLUT4 in H9c2 cells. Low level of high density lipoprotein (HDL) is an independent risk factor for cardiac hypertrophy. Therefore, we in the present study investigated whether HDL can reverse PA induced lipotoxicity in H9c2 cardiomyoblast cells. In this study, we treated H9c2 cells with PA to create a hyperlipidemia model in vitro and analyzed for CD36 and GLUT4 metabolic pathway proteins. CD36 metabolic pathway proteins (phospho‐AMPK, SIRT1, PGC1α, PPARα, CPT1β, and CD36) were decreased by high PA (150 and 200 μg/μl) concentration. Interestingly, expression of GLUT4 metabolic pathway proteins (p‐PI3K and pAKT) were increased at low concentration (50 μg/μl) and decreased at high PA concentration. Whereas, phospho‐PKCζ, GLUT4 and PDH proteins expression was increased in a dose dependent manner. PA treated H9c2 cells were treated with HDL and analyzed for cell viability. Results showed that HDL treatment induced cell proliferation efficiency in PA treated cells. In addition, HDL reversed the metabolic effects of PA: CD36 translocation was increased and reduced GLUT4 translocation, but HDL treatment significantly increased CD36 metabolic pathway proteins and reduced GLUT4 pathway proteins. Rat neonatal cardiomyocytes showed similar results. In conclusion, HDL reversed palmatic acid‐induced lipotoxicity and energy metabolism imbalance in H9c2 cardiomyoblast cells and in neonatal rat cardiomyocyte cells.

Nrf2 Activation as a Protective Feedback to Limit Cell Death in High Glucose-Exposed Cardiomyocytes.

Hyperglycemia leads to excess reactive oxygen species (ROS) generation, which causes many diabetic complications, such as cardiomyopathy. Nuclear factor erythroid 2‐related factor 2 (Nrf2), a redox‐sensing transcription factor, can up‐regulate its downstream antioxidant gene expressions in response to oxidative stress. However, the regulatory signal pathway in which high glucose (HG) induces Nrf2 activation is still unclear. Our results demonstrated that HG (33 mM) can indeed stimulate Nrf2 protein expression and translocation into the nucleus in cardiomyocytes, enhancing the downstream antioxidant protein levels. Using siRNAs, p38, JNK, PKCα, and PKCδ, as well as ROS scavengers, it was observed that the dependence of PKCα/PKCδ on ROS production to enhance JNK and p38 phosphorylation mediated HG‐induced cardiac Nrf2 expression and activation. Knockdown of Nrf2 by siRNA transfection increased cleaved‐caspase3, reduced Bcl2 in the cellular protein level and further exacerbated HG‐induced apoptosis. In addition, all of these proteins induced by HG in vitro were also increased in STZ‐induced diabetic rat ventricles in vivo. Our study demonstrated that HG‐induced cardiac Nrf2 activation occurs through PKCα/PKCδ—ROS—JNK/p38 signaling. These findings may provide a therapeutic target to counteract the oxidative stress associated with diabetic cardiomyopathy. J. Cell. Biochem. 118: 1659–1669, 2017.