Targeting Pin1 Protects Mouse Cardiomyocytes from High-Dose Alcohol-Induced Apoptosis.
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ABSTRACT:
SUBMITTER: Wang Y
PROVIDER: S-EPMC4678095 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2? mRNA and eIF2? phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.
| S-EPMC4546295 | biostudies-literature
Project description:MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, has been implicated as critical regulatory molecules in many cardiovascular diseases, including ischemia/reperfusion induced cardiac injury. Here, we report microRNA-145, a tumor suppressor miRNA, can protect cardiomyocytes from hydrogen peroxide H?O?-induced apoptosis through targeting the mitochondrial pathway. Quantitative real-time PCR (qPCR) demonstrated that the expression of miR-145 in either ischemia/reperfused mice myocardial tissues or H?O?-treated neonatal rat ventricle myocytes (NRVMs) was markedly down-regulated. Over-expression of miR-145 significantly inhibited the H?O?-induced cellular apoptosis, ROS production, mitochondrial structure disruption as well as the activation of key signaling proteins in mitochondrial apoptotic pathway. These protective effects of miR-145 were abrogated by over-expression of Bnip3, an initiation factor of the mitochondrial apoptotic pathway in cardiomyocytes. Finally, we utilized both luciferase reporter assay and western blot analysis to identify Bnip3 as a direct target of miR-145. Our results suggest miR-145 plays an important role in regulating mitochondrial apoptotic pathway in heart challenged with oxidative stress. MiR-145 may represent a potential therapeutic target for treatment of oxidative stress-associated cardiovascular diseases, such as myocardial ischemia/reperfusion injury.
| S-EPMC3445575 | biostudies-literature
Project description:AimThe aim of the present study was to investigate the role of Lin28a in protecting against hypoxia/reoxygenation (H/R)-induced cardiomyocytes apoptosis under high glucose/high fat (HG/HF) conditions.MethodsPrimary cardiomyocytes which were isolated from neonatal mouse were randomized to be treated with lentivirus carrying Lin28a siRNA, Lin28acDNA 72 h before H/R (9 h/2 h). Cardiomyocytes biomarkers release (LDH and CK), cardiomyocytes apoptosis, mitochondria biogenesis and morphology, intracellular reactive oxygen species (ROS) production, ATP content and inflammatory cytokines levels after H/R injury in high glucose/high fat conditions were compared between groups. The target proteins of Lin28a were examined by western blot analysis.ResultsOur results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions. Lin28a siRNA transfection (knockdown) rendered the cardiomyocytes more susceptible to H/R injury as evidenced by increased apoptotic index, impaired mitochondrial biogenesis, decreased ATP production and increased ROS level. Interestingly, these effects of Lin28a were blocked by pretreatment with the PI3K inhibitor wortmannin. Lin28a overexpression increased, while Lin28a knockdown inhibited IGF1R, Nrf-1, Tfam, p-IRS-1, p-Akt, p-mTOR, p-p70s6k, p-AMPK expression levels after H/R injury in HG/HF conditions. Moreover, pretreatment with wortmannin abolished the effects of Lin28a on the expression levels of p-AKT, p-mTOR, p-p70s6k, p-AMPK.ConclusionsThe present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions. The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.
| S-EPMC4196990 | biostudies-literature
Project description:Activation of apoptosis in cardiomyocytes by saturated palmitic acids contributes to cardiac dysfunction in diabetic cardiomyopathy. Beta-catenin (b-catenin) is a transcriptional regulator of several genes involved in survival/anti-apoptosis. However, its role in palmitate-induced cardiomyocyte apoptosis remains unclear. Glucagon-like peptide 1 (GLP1) has been shown to exhibit potential cardioprotective properties. This study was designed to evaluate the role of b-catenin signalling in palmitate-induced cardiomyocyte apoptosis and the molecular mechanism underlying the protective effects of GLP1 on palmitate-stressed cardiomyocytes. Exposure of neonatal rat cardiomyocytes to palmitate increased the fatty acid transporter CD36-mediated intracellular lipid accumulation and cardiomyocyte apoptosis, decreased accumulation and nuclear translocation of active b-catenin, and reduced expression of b-catenin target protein survivin and BCL2. These detrimental effects of palmitate were significantly attenuated by GLP1 co-treatment. However, the anti-apoptotic effects of GLP1 were markedly abolished when b-catenin was silenced with a specific short hairpin RNA. Furthermore, analysis of the upstream molecules and mechanisms responsible for GLP1-associated cardiac protection revealed that GLP1 restored the decreased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3b (GSK3b) in palmitate-stimulated cardiomyocytes. In contrast, inhibition of Akt with an Akt-specific inhibitor MK2206 or blockade of GLP1 receptor (GLP1R) with a competitive antagonist exendin-(9-39) significantly abrogated the GLP1-mediated activation of GSK3b/b-catenin signalling, leading to increased apoptosis in palmitate-stressed cardiomyocytes. Collectively, our results demonstrated for the first time that the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis, while GLP1 can protect cardiomyocytes from palmitate-induced apoptosis through activation of GLP1R/Akt/GSK3b-mediated b-catenin signalling.
| S-EPMC4621609 | biostudies-literature
Project description:microRNA-3568 (miR-3568) has been reported to be associated with atherosclerosis. Only few data describe the expression and underlying mechanism of miR-3568 in regulating cardiac ischemia-reperfusion (I/R) injury such as apoptosis. In this study, we therefore sought to investigate the potential function of miR-3568 in simulated I/R-induced apoptosis in H9C2 cardiomyocytes and related signaling pathways involved. Flow cytometry was performed to examine the cell apoptosis. The expression of miR-3568, Survivin, Bcl-2, ERK, JNK, p38, AKT, and STAT3 was measured by western blot and quantitative real-time PCR. The correlation between TRIM62 and p-STAT3 was measured by co-immunoprecipitation and ubiquitination. We found that miR-3568 expression in simulated I/R-induced H9C2 cardiomyocytes was increased in a time-dependent manner. miR-3568 mimic transfection in H9C2 cardiomyocytes significantly enhanced cell apoptosis, decreased the expression of Bcl-2 and Survivin, and activated STAT3 signaling, which were reversed by miR-3568 inhibitor. The direct interaction between miR-3568 and the 3'-untranslated region (UTR) of TRIM62 mRNA was confirmed by dual-luciferase reporter assay. TRIM62 overexpression or AG490, a selective inhibitor of JAK2/STAT3 significantly, significantly inhibited I/R and miR-3568 mimic induced cell apoptosis and STAT3 activation. TRIM62 was found to interact with and induce ubiquitination of p-STAT3. The facilitating role of miR-3568 in I/R injury was also observed in our in vivo rat models. In conclusion, our study suggests that miR-3568 promotes simulated I/R-induced apoptosis in H9C2 cardiomyocytes through targeting TRIM62.
| S-EPMC7031202 | biostudies-literature
Project description:Plumbagin (PLB) has been previously reported to alleviate myocardial ischemia/reperfusion injury in vivo. In the present study, the potential of plumbagin to protect against hydrogen peroxide-induced injury in cardiomyocytes was analyzed. Specifically, the cytoprotective effects of PLB were evaluated in H9c2 cardiomyocytes, in which oxidative stress was induced by tertiary butyl hydrogen peroxide (TBHP; 75 µM) treatment. After the cardiomyocytes were treated with different concentrations of PLB, cell viability, creatine kinase (CK) activity and lactate dehydrogenase (LDH) release were determined. The apoptosis rate and reactive oxygen species (ROS) levels were evaluated by flow cytometry. Western blot analyses of cleaved caspase-3, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme 4 (NOX4), and phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK) were performed. PLB pretreatment (5, 10 or 20 µM) restored TBHP-treated H9c2 cell viability (P<0.01). Additionally, PLB significantly decreased CK (P<0.01) and LDH activity (P<0.01). TBHP induced apoptosis and oxidative stress in cardiomyocytes, whereas PLB pretreatment significantly reduced the TBHP-induced apoptosis rate (P<0.01) and ROS levels (P<0.01). Furthermore, PLB resulted in a decrease in the expression of cleaved caspase-3, NOX4, and p-p38 MAPK in TBHP-treated H9c2 cells. The active marker of autophagosomes, LC3-II/LC3-I, was increased following treatment with PLB, indicating the induction of autophagy. The present study revealed the protective role of PLB against TBHP-induced cardiomyocyte injury via the alleviation of ROS-mediated apoptosis and induction of autophagy.
| S-EPMC9257763 | biostudies-literature
Project description:Myocardial ischemia/reperfusion (MIR) injury, which occurs following acute myocardial infarction, can cause secondary damage to the heart. Tripartite interaction motif (TRIM) proteins, a class of E3 ubiquitin ligases, have been recognized as critical regulators in MIR injury. Zenglv Fumai Granule (ZFG) is a clinical prescription for the treatment of sick sinus syndrome, a disease that is associated with MIR injury. The present study aimed to investigate the effect of ZFG on MIR injury and to determine whether ZFG exerts its effects via regulation of TRIM proteins. In order to establish an in vitro MIR model, human cardiomyocyte cell line AC16 was cultured under hypoxia for 5 h and then under normal conditions for 1 h. Following hypoxia/reoxygenation (H/R) treatment, these cells were cultured with different ZFG concentrations. ZFG notably inhibited H/R-induced cardiomyocyte apoptosis. The expression levels of four TRIM proteins, TRIM7, TRIM14, TRIM22 and TRIM28, were also detected. These four proteins were significantly upregulated in H/R-injured cardiomyocytes, whereas their expression was inhibited following ZFG treatment. Moreover, TRIM28 knockdown inhibited H/R-induced cardiomyocyte apoptosis, whereas TRIM28 overexpression promoted apoptosis and generation of reactive oxygen species (ROS) in cardiomyocytes. However, the effects of TRIM28 overexpression were limited by the action of ROS inhibitor N-acetyl-L-cysteine. In addition, the mRNA and protein levels of antioxidant enzyme glutathione peroxidase (GPX)1 were significantly downregulated in H/R-injured cardiomyocytes. TRIM28 knockdown restored GPX1 protein levels but had no effect on mRNA expression levels. Co-immunoprecipitation and ubiquitination assays demonstrated that TRIM28 negatively regulated GPX1 via ubiquitination. In sum, the present study revealed that ZFG attenuated H/R-induced cardiomyocyte apoptosis by regulating the TRIM28/GPX1/ROS pathway. ZFG and TRIM28 offer potential therapeutic options for the treatment of MIR injury.
| S-EPMC7821356 | biostudies-literature
Project description:Uncoupling protein 2 (UCP2) has a cardioprotective role under septic conditions, but the underlying mechanism remains unclear. This study aimed at investigating the effects of UCP2 on the oxidative stress and apoptosis of cardiomyocytes induced by lipopolysaccharide (LPS). First, LPS increased UCP2 expression in cardiomyocytes in a time-dependent manner. LPS increased the production of lactate dehydrogenase (LDH), reactive oxygen species (ROS), and malondialdehyde (MDA) and decreased the level of superoxide dismutase (SOD). However, UCP2 knockdown increased the LPS-induced cardiac injury and oxidative stress. In addition, LPS damaged the mitochondrial ultrastructure and led to the disruption of mitochondrial membrane potential (MMP), as well as the release of mitochondrial cytochrome c. UCP2 knockdown aggravated mitochondrial injury and the release of mitochondrial cytochrome c. LPS increased the protein levels of Bax and cleaved-caspase-3, decreased the protein level of Bcl-2, and upregulated the protein level of mitogen-activated protein kinase. However, upon UCP2 knockdown, the protein levels of Bax and cleaved-caspase-3 increased even further, and the protein level of Bcl-2 was further decreased. The protein level of phosphorylated p38 was also further enhanced. Thus, UCP2 protects against LPS-induced oxidative stress and apoptosis in cardiomyocytes.
| S-EPMC6512061 | biostudies-literature
Project description:Alcoholic liver disease (ALD) is a worldwide health problem and hepatocyte apoptosis has been associated with the development/progression of ALD. However, no definite effective pharmacotherapy for ALD is currently available. Cilostazol, a selective type III phosphodiesterase inhibitor has been shown to protect hepatocytes from ethanol-induced apoptosis. In the present study, the underlying mechanisms for the protective effects of cilostazol were examined. Primary rat hepatocytes were treated with ethanol in the presence or absence of cilostazol. Cell viability and intracellular cAMP were measured. Apoptosis was detected by Hoechst staining, TUNEL assay, and caspase-3 activity assay. The roles of cAMP and AMP-activated protein kinase (AMPK) pathways in the action of CTZ were explored using pharmacological inhibitors and siRNAs. Liver from mice received ethanol (5 g/kg body weight) by oral gavage following cilostazol treatment intraperitoneally was obtained for measurement of apoptosis and activation of AMPK pathway. Cilostazol inhibited ethanol-induced hepatocyte apoptosis and potentiated the increases in cAMP level induced by forskolin. However, the anti-apoptotic effect of cilostazol was not reversed by an inhibitor of adenylyl cyclase. Interestingly, cilostazol activated AMPK and increased the level of LC3-II, a marker of autophagy. The inhibition of AMPK abolished the effects of cilostazol on LC3-II expression and apoptosis. Moreover, the inhibition of LKB1 and CaMKK2, upstream kinases of AMPK, dampened cilostazol-inhibited apoptosis as well as AMPK activation. In conclusion, cilostazol protected hepatocytes from apoptosis induced by ethanol mainly via AMPK pathway which is regulated by both LKB1 and CaMKK2. Our results suggest that cilostazol may have potential as a promising therapeutic drug for treatment of ALD.
| S-EPMC6350983 | biostudies-literature
Project description:ObjectivesArecoline is the main bioactive substance extracted from Areca catechu L, which has cell, neural and genetic toxicity. The function of arecoline in reproductive system has not been well explored.Materials and methodsTo investigate the toxic effects of arecoline on oocyte development, immunofluorescence staining, qPCR, Western blotting, sperm binding assays and in vitro fertilization were performed to evaluate oocyte meiosis competence and embryo development.ResultsOur data revealed that arecoline exposure disrupts actin filament dynamics, spindle assembly and kinetochore-microtubule attachment stability in mouse oocytes, leading to aneuploidy and oocyte meiosis arrest. In addition, arecoline treatment disturbs the distribution of mitochondria, reduces ATP production and increases the level of oxidative stress, which ultimately induces oocyte apoptosis. Supplementation with metformin, a medicine for type 2 diabetes in the clinic, partially alleviates these damages.ConclusionsMetformin has a protective effect on arecoline-induced mouse oocytes apoptosis.
| S-EPMC7377942 | biostudies-literature