Ohwia caudata inhibits doxorubicin-induced cardiotoxicity by regulating mitochondrial dynamics via the IGF-IIR/p-Drp1/PARP signaling pathway.

The most effective drug, doxorubicin (DOX), is widely used worldwide for clinical application as an anticancer drug. DOX-induced cytotoxicity is characterized by mitochondrial dysfunction. There is no alternative treatment against DOX-induced cardiac damage despite intensive research in the present decades. Ohwia caudata has emerged as a potential herbal remedy that prevents from DOX-induced cytotoxicity owing to its pharmacological action of sustaining mitochondrial dynamics by attenuating oxidative stress and inducing cellular longevity. However, its underlying mechanisms are unknown. The novel treatment provided here depends on new evidence from DOX-treated H9c2 cells, which significantly enhanced insulin-like growth factor (IGF) II receptor (IGF-IIR) pathways that activated calcineurin and phosphorylated dynamin-related protein 1 (p-Drp1) at ser616 (p-Drp1[ser616]); cells undergo apoptosis due to these factors, which translocate to mitochondria and disrupt their function and integrity, and in terms of herbal medicine treatment, which significantly blocked these phenomena. Thus, our findings indicate that maintaining integrity of mitochondria is an essential element in lowering DOX-induced cytotoxicity, which further emphasizes that our herbal medicine can successfully block IGF-IIR pathways and could potentially act as an alternative mechanism in terms of cardioprotective against doxorubicin.

Regulation of PM2.5 on mitochondrial damage in H9c2 cells through miR-421/SIRT3 pathway and protective effect of miR-421 inhibitor and resveratrol.

Fine particulate matter (PM2.5) exposure is associated with cardiovascular disease (CVD) morbidity and mortality. Mitochondria are sensitive targets of PM2.5, and mitochondrial dysfunction is closely related to the occurrence of CVD. The epigenetic mechanism of PM2.5-triggered mitochondrial injury of cardiomyocytes is unclear. This study focused on the miR-421/SIRT3 signaling pathway to investigate the regulatory mechanism in cardiac mitochondrial dynamics imbalance in rat H9c2 cells induced by PM2.5. Results illustrated that PM2.5 impaired mitochondrial function and caused dynamics homeostasis imbalance. Besides, PM2.5 up-regulated miR-421 and down-regulated SIRT3 gene expression, along with decreasing p-FOXO3a (SIRT3 downstream target gene) and p-Parkin expression and triggering abnormal expression of fusion gene OPA1 and fission gene Drp1. Further, miR-421 inhibitor (miR-421i) and resveratrol significantly elevated the SIRT3 levels in H9c2 cells after PM2.5 exposure and mediated the expression of SOD2, OPA1 and Drp1, restoring the mitochondrial morphology and function. It suggests that miR-421/SIRT3 pathway plays an epigenetic regulatory role in mitochondrial damage induced by PM2.5 and that miR-421i and resveratrol exert protective effects against PM2.5-incurred cardiotoxicity.

The adverse and beneficial effects of polyphenols in green and black teas in vitro and in vivo.

Although numerous studies highlight the health benefits of tea, excessive consumption has been linked to toxic conditions. Thus, understanding the optimal consumption of tea is essential to minimize toxicity while maximizing its benefits. In this study, we investigated the effects of eight green tea samples (G1-G8) and eight black tea samples (R1-R8) from Camellia sinensis, the most popular teas in Asian culture, on RSC96 Schwann neural cells and embryonic cardiomyocyte H9c2 cells. The results showed that the IC50 (mg/ml, weight/volume) of both tea types were inversely proportional to their polyphenol content, suggesting a relationship between toxicity and polyphenol levels in both green and black tea. Interestingly, green teas generally have higher polyphenol content than black teas. We also assessed the protective effects of tea in vitro by pretreating cells with the teas at indicated doses of polyphenol and subsequently exposing them to H2O2. Both tea types significantly reduced the decline in cell viability for both cell lines, and there was no significant difference in protective polyphenol concentrations for green (G3 & G7) and black (R3 & R8) teas at effective concentrations (EC20 and EC40). To evaluate the preventative effects of tea in vivo, we examined the impact of two green (G3 & G7) and two black (R3 & R8) teas with varying polyphenol content on dextran sulfate sodium (DSS)-induced inflammatory colitis in mice. Tea-treated groups exhibited significantly lower inflammatory scores (DAI) than the control group. DSS treatment in the control group led to shortened colorectal lengths in mice, while tea co-treatment partially prevented this loss. Histological analysis revealed that G7 and R3 (with a moderate polyphenol content) treatment improved colorectal crypt structure, decreased the severity of inflammatory ulcerative colitis, and significantly reduced histological scores compared to the control group. However, G3 and R8 (with high and low doses of polyphenol content, respectively) did not show these effects, suggesting that a moderate polyphenol level in both tea types is optimal for preventative benefits.

Effects and mechanism of perinatal nonylphenol exposure on cardiac function and myocardial mitochondria in neonatal rats.

BACKGROUND: Nonylphenol (NP) is a common environmental endocrine disruptor that is associated with the development of cardiovascular disease. However, the toxic effect of NP on mitochondria in the heart of offspring to exposed individuals remains exclusive. OBJECTIVE: To investigate whether perinatal NP exposure causes mitochondrial damage in the hearts of offspring of exposed individuals and determine its mechanism of action through both animal and cell experiments. METHODS AND RESULTS: For the in vivo experiment, pregnant rats were randomly divided into four groups: the control group (corn oil, C), low dose group (2.5 mg/kg/day, L-NP group), medium dose group (50 mg/kg/day, M-NP group), and high dose group (100 mg/kg/day, H-NP group), with 12 rats in each group. The NP concentration in the hearts of offspring at PND21 and PND90 increased with the increase of the NP dose. Perinatal NP exposure induced a gradual increase in systolic blood pressure in offspring at PND90. In the H-NP group, there was a high degree of inflammatory cell infiltration, myofibril breaks, inconspicuous or absent nuclei, and pink collagen deposition. At PND90, the membrane integrity of mitochondria in the H-NP group was disrupted, the cristae disorder was aggravated, and there was internal lysis with vacuolation. Compared to the control group, the mitochondrial membrane potential of offspring at PND21 and PND90 was decreased in each of the NP exposure groups. NP exposure decreased the activity of mitochondrial respiratory enzyme complex I (CI) and increased the activity of mitochondrial respiratory enzyme complex IV (CIV) in the offspring. At PND21 and PND90, the mRNA and protein expression levels of cardiac mitochondrial PGC-1α, NRF-1, and TFAM decreased with increasing NP dose in a dose-dependent manner. In the in vitro experiment, H9C2 cells were divided into the following four groups: the blank group, RSV group (15 µg/ml), RSV + NP group (15 µg/ml RSV + 120 mmol/L NP), and NP group (120 mmol/L). With increasing NP concentration, the cell survival rate gradually decreased. Compared to the control, the membrane potential was significantly decreased in the NP group; the protein expression levels of SIRT1, PGC-1α, NRF-1, and TFAM in the NP group were significantly lower. CONCLUSION: Perinatal NP exposure caused mitochondrial damage and dysfunction in the offspring of exposed individuals in a dose-dependent manner. This toxic effect may be related to NP-induced mitochondrial pathology in the offspring and the inhibition of both gene and protein expression involved in the PGC-1α/NRF-1/TFAM mitochondrial biogenesis signaling pathway following NP exposure.

Diterpenoid alkaloids from two species of Delphinium.

Four new compounds (1-4) were isolated from the whole plants of two species of Delphinium, including two C20-diterpenoid alkaloids, umbrodines A and B (1 and 2), and a dibenzoxazepinone, umbrolide A (3) from Delphinium umbrosum Hand.-Mazz. and a C20-diterpenoid alkaloid, kingiadine (4) from Delphinium kingianum Bruhl. ex Huth. Ten known diterpenoid alkaloids were also isolated. Their structures were elucidated via HR-ESIMS, IR, and NMR data. Lycoctonine (11) and delectinine (12) exhibited appreciable cardiac activity. Furthermore, 11 and 12 showed cardioprotective effects against doxorubicin-induced toxicity in H9c2 cells, with the maximum protection rates of 61.63% and 51.18%, respectively.

YB-1 Is a Novel Target for the Inhibition of α-Adrenergic-Induced Hypertrophy.

Cardiac hypertrophy resulting from sympathetic nervous system activation triggers the development of heart failure. The transcription factor Y-box binding protein 1 (YB-1) can interact with transcription factors involved in cardiac hypertrophy and may thereby interfere with the hypertrophy growth process. Therefore, the question arises as to whether YB-1 influences cardiomyocyte hypertrophy and might thereby influence the development of heart failure. YB-1 expression is downregulated in human heart biopsies of patients with ischemic cardiomyopathy (n = 8), leading to heart failure. To study the impact of reduced YB-1 in cardiac cells, we performed small interfering RNA (siRNA) experiments in H9C2 cells as well as in adult cardiomyocytes (CMs) of rats. The specificity of YB-1 siRNA was analyzed by a miRNA-like off-target prediction assay identifying potential genes. Testing three high-scoring genes by transfecting cardiac cells with YB-1 siRNA did not result in downregulation of these genes in contrast to YB-1, whose downregulation increased hypertrophic growth. Hypertrophic growth was mediated by PI3K under PE stimulation, as well by downregulation with YB-1 siRNA. On the other hand, overexpression of YB-1 in CMs, caused by infection with an adenovirus encoding YB-1 (AdYB-1), prevented hypertrophic growth under α-adrenergic stimulation with phenylephrine (PE), but not under stimulation with growth differentiation factor 15 (GDF15; n = 10-16). An adenovirus encoding the green fluorescent protein (AdGFP) served as the control. YB-1 overexpression enhanced the mRNA expression of the Gq inhibitor regulator of G-protein signaling 2 (RGS2) under PE stimulation (n = 6), potentially explaining its inhibitory effect on PE-induced hypertrophic growth. This study shows that YB-1 protects cardiomyocytes against PE-induced hypertrophic growth. Like in human end-stage heart failure, YB-1 downregulation may cause the heart to lose its protection against hypertrophic stimuli and progress to heart failure. Therefore, the transcription factor YB-1 is a pivotal signaling molecule, providing perspectives for therapeutic approaches.

Network pharmacology approach and experimental verification of Dan-Shen Decoction in the treatment of ischemic heart disease.

CONTEXT: Dan-Shen Decoction, which is composed of Danshen, Tanxiang and Sharen, has a good therapeutic effect on ischemic heart disease (IHD). However, systematic research on the exact mechanism of action of Dan-Shen Decoction is still lacking. The anti-IHD effect of Dan-Shen Decoction was examined in this study using a systematic pharmacological method. OBJECTIVE: This study validates the efficacy and explores the potential mechanisms of Dan-Shen Decoction in treating IHD by integrating network pharmacology analyses and experimental verification. MATERIALS AND METHODS: The active components, critical targets and potential mechanisms of Dan-Shen Decoction against IHD were predicted by network pharmacology and molecule docking. H9c2 cells were pretreated with various 1 µg/mL Dan-Shen Decoction for 2 h before induction with 1000 µmol/L CoCl2 for 24 h. The cell viability was detected by CCK8, and protein expression was detected by western blots. RESULTS: The network pharmacology approach successfully identified 69 active components in Dan-Shen Decoction, and 122 potential targets involved in the treatment of IHD. The in vitro experiments indicate that the anti-IHD effect of Dan-Shen Decoction may be closely associated with targets such as AKT1 and MAPK1, as well as biological processes such as cell proliferation, inflammatory response, and metabolism. CONCLUSIONS: This study not only provides new insights into the mechanism of Dan-Shen Decoction against IHD, but also provides important information and new research ideas for the discovery of anti-IHD compounds from traditional Chinese medicine.

[Hesperetin Alleviates Doxorubicin-Induced Cytotoxicity in H9c2 Cells by Activating SIRT1/NRF2 Signaling].

Objective: To investigate whether hesperetin (Hes) alleviates doxorubicin (DOX)-induced cardiomyocytotoxicity by reducing oxidative stress via regulating silent information regulator 1 (SIRT1)/nuclear transcription factor E2-related factor 2 (NRF2) signaling in H9c2 cells. Methods: H9c2 cells were treated with DOX to establish the cardiotoxicity model and were randomly assigned to four groups, a control group (Control) and three treatment groups, receiving respectively DOX (the DOX group), Hes+DOX (the DOX+Hes group), and Hes+SIRT1 inhibitor EX527+DOX (the DOX+Hes+EX527 group). Cellular morphology was observed by the light microscope. Cell viability was evaluated by CCK-8. DOX-induced apoptosis in H9c2 cells was examined by flow cytometry. The levels of reactive oxygen species (ROS) in the H9c2 cells of the four groups were determied with 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and SIRT1 as well as the malondialdehyde (MDA) content were measured using ELISA kits. The expressions of cleaved caspase-3, cytochrome c, SIRT1, Ac-FOXO1, NRF2, and heme oxygenase 1 (HO-1) were determined by Western blot. Results: Compared with the Control group, the DOX group showed swollen cellular morphology, decreased cell density and viability, and increased LDH activity in the medium ( P<0.01); both apoptosis and the expression of cleaved caspase-3 and cytochrome c increased ( P<0.01); the activities of CAT and SOD decreased while the contents of MDA and ROS increased ( P<0.01); the expression of SIRT1, NRF2, and HO-1 decreased, the activity of SIRT1 decreased, and the expression of Ac-FOXO1 increased ( P<0.01). Compared with the DOX group, the DOX+Hes group showed improved cellular morphology, increased cell density and viability, and decreased LDH activity in the medium ( P<0.01); the apoptosis and the expression of cleaved caspase-3 and cytochrome c decreased ( P<0.01); the activities of CAT and SOD increased while the levels of MDA and ROS decreased ( P<0.01); the expression of SIRT1, NRF2, and HO-1 increased, the activity of SIRT1 increased, and the expression of Ac-FOXO1 decreased ( P<0.01). Comparison of the findings for the DOX+Hes group and the DOX+Hes+EX527 group showed that EX527 could block the protective effects of Hes against DOX-induced cell injury, oxidative stress, and SIRT1/NRF2 signaling. Conclusion: Hes inhibits oxidative stress and apoptosis via regulating SIRT1/NRF2 signaling, thereby reducing DOX-induced cardiotoxicity in H9c2 cells.

[Irisin Alleviates Inflammatory Injury in Diabetic Cardiomyopathy by Regulating NF-κB Pathway].

Objective: To investigate the protective effect of irisin in diabetic cardiomyopathy (DCM) and its mechanism. Methods: A mouse model of DCM was established by high-fat diet combined with the injection of streptozotocin. The mice were assigned to a control group, a DCM group, a DCM+low-dose irisin group, a DCM+high-dose irisin group, and a DCM+pyrrolidine dithiocarbamate (PDTC) (nuclear factor [NF]-κB inhibitor) group. Then, the mice received irisin intervention for 3 weeks after successful modeling. Myocardial morphologic changes were observed by hematoxylin and eosin (HE) staining and Masson staining. The levels of serum creatine kinase (CK) and creatine kinase isoenzyme CK-MB were examined by automatic biochemical analyzer. H9c2 cells were divided into the control group, high glucose and high lipid (HG/HL) group, HG/HL+low-dose irisin group, HG/HL+high-dose irisin group, and HG/HL+PDTC group. CCK-8 assay was conducted to determine cell viability. The expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 in the myocardial tissue and the cells were determined by ELISA. In addition, nuclear translocation of NF-κB p65 protein and the protein expression level of NF-κB inhibitor protein α (IκBα) in the myocardial tissue and the cells were determined by Western blot. Results: According to the results of animal experiment, low and high doses of irisin could alleviate the pathological injury and fibrosis of myocardial tissue to varying degrees. Irisin inhibited the levels of CK, CK-MB, and inflammatory factors, up-regulated IκB protein expression, and diminished NF-κB nuclear translocation. According to the results of cell experiment, low and high doses of irisin could enhance H9c2 cell viability to varying degrees, increase the level of intracellular IκB proteins, and inhibit NF-κB p65 nuclear translocation and inflammatory factor expression. The changes in these aspects in the DCM+low-dose irisin group and the DCM+high-dose irisin group were similar to those in the DCM+PDTC group. Conclusion: Through inhibiting NF-κB p65 nuclear translocation, irisin may reduce the inflammatory response in the myocardial tissue of DCM mice and H9c2 cells of myocardial injury induced by high glucose and high fat, thereby exerting a protective effect on myocardium.

LncRNA MIAT promotes hypoxia-induced H9C2 cell pyroptosis via binding to SF1 to inhibit CGRP transcription.

NEW FINDINGS: What is the central question of this study? How does long non-coding RNA myocardial infarction-associated transcript (lncRNA MIAT) function in hypoxia-induced H9C2 cells? What is the main finding and its importance? LncRNA MIAT inhibited transcription of calcitonin gene-related peptide by binding to splicing factor 1, thereby promoting hypoxia-induced H9C2 cell pyroptosis. This study provides a new theoretical basis for the treatment of acute myocardial infarction by using lncRNA MIAT as a molecular target to mediate cardiomyocyte pyrodeath. ABSTRACT: Hypoxia induces severe cardiomyocyte pyroptosis, contributing to acute myocardial infarction. The aim of this study was to analyse the molecular mechanism of long non-coding RNA myocardial infarction-associated transcript (lncRNA MIAT) in hypoxia-induced H9C2 cell pyroptosis. A hypoxic H9C2 cell model was established. Cell viability was detected via the Cell Counting Kit-8 method. Levels of lactate dehydrogenase, malondialdehyde and superoxide dismutase and expressions of pyroptotic markers, lncRNA MIAT, splicing factor 1 (SF1) and calcitonin gene-related peptide (CGRP) were detected via qRT-PCR. The subcellular localization of lncRNA MIAT was predicted and confirmed via LncATLAS and nuclear/cytosol fractionation assay. The binding relationships between lncRNA MIAT and SF1 and between SF1 and the CGRP promotor were verified via RNA immunoprecipitation. Rescue experiments were designed to confirm the role of lncRNA MIAT/SF1/CGRP in H9C2 cell pyroptosis. LncRNA MIAT was overexpressed in hypoxia-induced H9C2 cells. Hypoxia induced pyroptosis in H9C2 cells. Silencing of lncRNA MIAT enhanced cell viability and alleviated pyroptosis. LncRNA MIAT inhibited CGRP transcription via binding to SF1. Overexpression of SF1 promoted CGRP transcription and relieved H9C2 cell pyroptosis. Downregulation of CGRP reversed the role of silencing lncRNA MIAT in H9C2 cell pyroptosis. Overall, lncRNA MIAT inhibited CGRP transcription via binding to SF1, thereby promoting hypoxia-induced H9C2 cell pyroptosis.

Perturbed ER homeostasis by IGF-IIRα promotes cardiac damage under stresses.

The heart is a very dynamic pumping organ working perpetually to maintain a constant blood supply to the whole body to transport oxygen and nutrients. Unfortunately, it is also subjected to various stresses based on physiological or pathological conditions, particularly more vulnerable to damages caused by oxidative stress. In this study, we investigate the molecular mechanism and contribution of IGF-IIRα in endoplasmic reticulum stress induction in the heart under doxorubicin-induced cardiotoxicity. Using in vitro H9c2 cells, in vivo transgenic rat cardiac tissues, siRNAs against CHOP, chemical ER chaperone PBA, and western blot experiments, we found that IGF-IIRα overexpression enhanced ER stress markers ATF4, ATF6, IRE1α, and PERK which were further aggravated by DOX treatment. This was accompanied by a significant perturbation in stress-associated MAPKs such as p38 and JNK. Interestingly, PARKIN, a stress responsive cellular protective mediator was significantly downregulated by IGF-IIRα concomitant with decreased expression of ER chaperone GRP78. Furthermore, ER stress-associated pro-apoptotic factor CHOP was increased considerably in a dose-dependent manner followed by elevated c-caspase-12 and c-caspase-3 activities. Conversely, treatment of H9c2 cells with chemical ER chaperone PBA or siRNA against CHOP abolished the IGF-IIRα-induced ER stress responses. Altogether, these findings suggested that IGF-IIRα contributes to ER stress induction and inhibits cellular stress coping proteins while increasing pro-apoptotic factors feeding into a cardio myocyte damage program that eventually paves the way to heart failure.

Synthesis and in vivo evaluation of new steviol derivatives that protect against cardiomyopathy by inhibiting ferroptosis.

Cardiovascular diseases (CVDs) remain the leading cause of death globally. Inhibiting ferroptosis and thus preventing cardiac cell death is a promising and effective strategy for cardiomyopathy prevention and therapy. Steviol, an ent-kaurene diterpenoid, possesses broad-spectrum bioactivity. In the present study, with the aim to discover new agents for CVDs treatment, 30 derivatives of steviol, including 22 new ones, were synthesized, and evaluated their protective activity in vivo using the doxorubicin (DOX) induced zebrafish cardiomyopathy model. Our results firstly demonstrated that steviol has promising cardioprotective activity and further modification of steviol can greatly improve the activity. Among the new derivatives, 16d and 16e show the most potent activity. Both 16d (1 µM) and 16e (0.1 µM) effectively maintain the normal heart shape and prevent the cardiac dysfunction impaired by DOX in zebrafish. Their therapeutic efficacy is much superior to the parent natural product, steviol, and positive drug, levosimendan. Further study demonstrated that 16d and 16e inhibit DOX-induced ferroptosis and thus protect cardiomyopathy, by suppressing the glutathione depletion, iron accumulation, and lipid peroxidation, decreasing reactive oxygen species overaccumulation, and restoring the mitochondrial membrane potential. Consequently, due to their unique structure and significant cardioprotective activity with ferroptosis inhibition, new steviol derivatives 16d and 16e merit further research for the development of new cardioprotective drug candidates.

Apelin-13 Increases Functional Connexin-43 through Autophagy Inhibition via AKT/mTOR Pathway in the Non-Myocytic Cell Population of the Heart.

Studies have shown a link between the downregulation of connexin 43 (Cx43), the predominant isoform in cardiac gap junctions, and high susceptibility to cardiac arrhythmias and cardiomyocyte death. Non-myocytic cells (NMCs), the most abundant component of the heart, exert multiple cardiac functions and represent an important therapeutic target for diseased cardiac tissue. A few studies have investigated the effect of Apelin-13, an endogenous peptide with a key role in various cardiovascular functions, on Cx43 expression in cardiomyocytes. However, it remained unknown whether Apelin-13 influences Cx43 expression in NMCs. Here, we found that in NMCs, Cx43 protein expression increased after Apelin-13 treatment (100 nM for 48 h). Furthermore, dye transfer assays proved that Apelin-13-treated NMCs had a greater ability to communicate with surrounding cardiomyocytes, and this effect was abrogated by carbenoxolone, a gap junction inhibitor. Interestingly, we showed that Apelin-13 increased Cx43 through autophagy inhibition, as proved by the upregulation of p62 and LC3I, acting as 3-MA, a well-known autophagy inhibitor. In addition, Apelin-13-induced AKT and mTOR phosphorylation was abolished by LY294002 and rapamycin inhibitors resulting in Cx43 increased suppression. These results open the possibility of targeting gap junctions in NMCs with Apelin-13 as an exciting therapeutic approach with great potential.

Aidi injection reduces doxorubicin-induced cardiotoxicity by inhibiting carbonyl reductase 1 expression.

CONTEXT: Aidi injection (ADI), a traditional Chinese medicine antitumor injection, is usually combined with doxorubicin (DOX) for the treatment of malignant tumours. The cardiotoxicity of DOX is ameliorated by ADI in the clinic. However, the relevant mechanism is unknown. OBJECTIVE: To investigate the effects of ADI on DOX-induced cardiotoxicity and its mechanism. MATERIALS AND METHODS: ICR mice were randomly divided into six groups: control, ADI-L, ADI-H, DOX, DOX + ADI-L and DOX + ADI-H. DOX (i.p., 0.03 mg/10 g) was administered in the presence or absence of ADI (i.p., 0.1 or 0.2 mL/10 g) for two weeks. Heart pathology and levels of AST, LDH, CK, CK-MB and BNP were assessed. H9c2 cells were treated with DOX in the presence or absence of ADI (1, 4, 10%). Cell viability, caspase-3 activity, nuclear morphology, and CBR1 expression were then evaluated. DOX and doxorubicinol (DOXol) concentrations in heart, liver, kidneys, serum, and cells were analysed by UPLC-MS/MS. RESULTS: High-dose ADI significantly reduced DOX-induced pathological changes and the levels of AST, LDH, CK, CK-MB and BNP to normal. Combined treatment with ADI (1, 4, 10%) improved the cell viability, and IC50 increased from 68.51 µM (DOX alone) to 83.47, 176.9, and 310.8 µM, reduced caspase-3 activity by 39.17, 43.96, and 61.82%, respectively. High-dose ADI inhibited the expression of CBR1 protein by 32.3%, reduced DOXol levels in heart, serum and H9c2 cells by 59.8, 72.5 and 48.99%, respectively. DISCUSSION AND CONCLUSIONS: ADI reduces DOX-induced cardiotoxicity by inhibiting CBR1 expression, which provides a scientific basis for the rational use of ADI.

Knockdown of SGLT1 prevents the apoptosis of cardiomyocytes induced by glucose fluctuation via relieving oxidative stress and mitochondrial dysfunction.

Fluctuations in the concentration of glucose in the blood is more detrimental than a constantly high level of glucose with respect to the development of cardiovascular complications associated with diabetes mellitus (DM). Sodium glucose cotransporter 2 (SGLT2) inhibitors have been developed as antidiabetic drugs with cardiovascular benefits; however, whether inhibition of SGLT1 protects the diabetic heart remains to be determined. This study investigated the role of SGLT1 in rat H9c2 cardiomyocytes subjected to fluctuating levels of glucose and the underlying mechanisms. The results indicated that knockdown of SGLT1 restored cell proliferation and suppressed the cytotoxicity associated with fluctuating glucose levels. Oxidative stress was induced in H9c2 cells subjected to fluctuating glucose levels, but these changes were effectively reversed by knockdown of SGLT1, as manifested by reductions in the level of intracellular reactive oxygen species and increased antioxidant activity. Further study demonstrated that knockdown of SGLT1 attenuated the mitochondrial dysfunction in H9c2 cells exposed to fluctuating glucose levels, by restoring mitochondrial membrane potential and promoting mitochondrial fusion. In addition, knockdown of SGLT1 downregulated the expression of Bax, upregulated the expression of Bcl-2, and reduced the activation of caspase-3 in H9c2 cells subjected to fluctuating levels of glucose. Collectively, our results show that knockdown of SGLT1 ameliorates the apoptosis of cardiomyocyte caused by fluctuating glucose levels via regulating oxidative stress and combatting mitochondrial dysfunction.

Majonoside-R2 extracted from Vietnamese ginseng protects H9C2 cells against hypoxia/reoxygenation injury via modulating mitochondrial function and biogenesis.

Vietnamese ginseng has a therapeutic effect on various diseases; however its bioactivity against cardiac hypoxia/reoxygenation (HR) injury remains unclear. In this study, we evaluated the protective roles of total saponin extract (TSE) and majonoside-R2 (MR2) targeting mitochondria in HR-induced rat cardiomyocyte H9C2 cells. The results showed that both TSE and MR2 effectively protected the cells from HR damage. Particularly, 9 µM of MR2 significantly increased the viability of HR-induced cells (p < 0.05). Interestingly, MR2 treatment markedly prevented the loss of mitochondrial membrane potential and cardiolipin content, and an increase in reactive oxygen species production in HR-treated H9C2 cells. Moreover, MR2 treatment altered the mRNA expression of genes involved in mitochondrial biogenesis under HR conditions. The present study documented for the first time the cardioprotective effects of MR2 against HR injury by maintaining mitochondrial function and modulating mitochondrial biogenesis.

An in vivo and in vitro model on the protective effect of corilagin on doxorubicin-induced cardiotoxicity via regulation of apoptosis and PI3-K/AKT signaling pathways.

Globally, doxorubicin (DOX)-induced cardio dysfunction is a serious cause of morbidity and mortality in cancerous patients. An adverse event of cardiotoxicity is the main deem to restrict in the clinical application by oncologists. Corilagin (CN) is well known for its antioxidative, anti-fibrosis, and anticancer effects. Herein, we aimed to evaluate the action of CN on DOX-induced experimental animals and H9c2 cells. The myocardium-specific marker, CK-MB, and the influx of mitochondrial calcium levels were measured by using commercial kits. Biochemical indices reflecting oxidative stress and antioxidant attributes such as malondialdehyde, glutathione peroxidase, reduced glutathione, superoxide dismutase, and catalase were also analyzed in DOX-induced cardiotoxic animals. In addition, mitochondrial ROS were measured by DCFH-DA in H9c2 cells under fluorescence microscopy. DOX induction significantly increased oxidative stress levels and also modulated apoptosis/survival protein expressions in myocardial tissues. Western blots were used to measure the expressional levels of Bax/Bcl-2, caspase-3, PI3-K/AKT, and PPARγ signaling pathways. Histological studies were executed to observe morphological changes in myocardial tissues. All of these DOX-induced effects were attenuated by CN (100 mg/kg bw). These in vitro and in vivo results point towards the fact that CN might be a novel cardioprotective agent against DOX-induced cardiotoxicity through modulating cardio apoptosis and oxidative stress.

Zerumin A attenuates the inflammatory responses in LPS-stimulated H9c2 cardiomyoblasts.

Zerumin A (ZA) is one of the potential components of Curcuma amada rhizomes, and it has been shown to possess a variety of pharmacological activities. This study deals with the beneficial activity of ZA in lipopolysaccharide (LPS)-stimulated inflammation in H9c2 cardiomyoblasts. Herein, H9c2 cells were preincubated with ZA for 1 h and stimulated with LPS for 24 h. The cells were analyzed for the expression of various pro-inflammatory mediators and signaling molecules. Results showed that the cell viability was significantly improved and reactive oxygen species production was alleviated remarkably with ZA pretreatment. We also found that ZA pretreatment significantly suppressed the upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein levels, and nitric oxide (NO) release in LPS-stimulated cells. In addition, ZA significantly ameliorated LPS-elicited overexpression of pro-inflammatory chemokines and cytokines such as monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor α (TNF- α), interferon-γ (IFN-γ), and interleukin-1 (IL-1) in H9c2 cells, and it upregulated the synthesis of the anti-inflammatory cytokine interleukin-10 (IL-10). Moreover, pretreatment with ZA and the mitogen-activated protein kinases (MAPK) pathway inhibitors also reduced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK), and p38. ZA significantly inhibited IkB-a phosphorylation and nuclear factor (NF)-kB p65 subunit translocation into nuclei. Overall data demonstrated that ZA protects cardiomyocytes against LPS injury by inhibiting NF-kB p65 activation via the MAPK signaling pathway in vitro. These findings suggest that ZA may be a promising agent for a detailed study for the prevention or treatment of myocardial dysfunction in sepsis.

LncRNA SNHG12 downregulates RAGE to attenuate hypoxia-reoxygenation-induced apoptosis in H9c2 cells.

Ischemia-reperfusion (I/R) injury causes cardiac dysfunction through several mechanisms including the irregular expression of some long noncoding RNA. However, the role of SNHG12 in myocardial I/R injury remains unclear. Here, we found the increase of the SNHG12 level in hypoxia-reoxygenation (H/R)-injured-H9c2 cells. SNHG12 silencing enhanced the apoptosis of H/R-injured H9c2 cells, while SNHG12 overexpression relieved the cardiomyocyte apoptosis induced by H/R stimulation. Additionally, the suppression of SNHG12 significantly boosted the H/R-induced expression and the production of TNF-α, IL-6, and IL-1ß, as well as the activation of NF-κB, which were fully reversed after overexpression of SNHG12. Mechanistically, SNHG12 adversely regulated the production of receptor for advanced glycation end products (RAGE) in H/R-stimulated H9c2 cells. Antibody blocking of RAGE alleviated the apoptosis of H/R-injured H9c2 cells. Collectively, we have determined a valuable mechanism by which the high level of SNHG12 contributes to H9c2 cells against H/R injury through the reduction of RAGE expression.

miR-375-3p contributes to hypoxia-induced apoptosis by targeting forkhead box P1 (FOXP1) and Bcl2 like protein 2 (Bcl2l2) in rat cardiomyocyte h9c2 cells.

miRNAs have been pointed to play critical role in the development of congenital heart disease (CHD). miRNA-375-3p (miR-375-3p) was involved in cardiac dysfunction and cardiogenesis. However, no prior study had established a therapeutic role of miR-375-3p in CHD. We intended to investigate the effect and mechanism of miR-375-3p on apoptosis in hypoxic cardiomyocytes in vitro. Expression of miR-375-3p, forkhead box P1 (FOXP1) and Bcl2 like protein 2 (Bcl2l2) was detected using real-time quantitative PCR and western blot. Apoptosis was measured with MTT assay, flow cytometry and caspase-3 activity assay. The potential target binding between miR-375-3p and FOXP1/Bcl2l2 was predicted on DianaTools, and was validated by luciferase reporter assay and RNA pull-down assay. As a result, miR-375-3p was upregulated and FOXP1/Bcl2l2 was downregulated in maternal serum of women with fetal CHD and hypoxia-induced rat cardiomyocyte h9c2 cells. Hypoxia induced apoptosis rate elevation, caspase-3 activity promotion and viability inhibition in h9c2 cells; overexpression of miR-375-3p promoted, whereas knockdown of miR-375-3p antagonized hypoxia-induced effects in h9c2 cells. In addition, miR-375-3p was validated to negatively regulate FOXP1 and Bcl2l2 expression through target binding, and silencing of FOXP1 and Bcl2l2 could independently abate the anti-apoptosis role of miR-375-3p knockdown in hypoxic h9c2 cells. Collectively, blocking miR-375-3p suppressed hypoxia-evoked apoptosis of cardiomyocytes by targeting and upregulating FOXP1 and Bcl2l2. Our results might suggest maternal serum miR-375-3p as a potential biomarker for prenatal detection of fetal CHD.