Echinacoside ameliorates doxorubicin­induced cardiac injury by regulating GPX4 inhibition­induced ferroptosis.

Echinacoside (ECH) is a compound derived from the natural herbs Cistanche and Echinacea, which has considerable protective effects on heart failure (HF). HF is characterized by myocardial damage and abnormal ferroptosis. Glutathione peroxidase 4 (GPX4) is an important regulator of ferroptosis, which plays a role in ferroptosis-related diseases. Despite this, the therapeutic mechanisms of ECH against HF remain unknown. Therefore, the aim of the present study was to investigate the cardioprotective effect and underlying mechanisms of ECH in the treatment of doxorubicin (DOX)-induced chronic HF (CHF). Cell proliferation was assessed using a CCK-8 assay. Furthermore, cardiac cell injury and oxidative stress were determined by measuring the lactate dehydrogenase (LDH), malondialdehyde (MDA), and glutathione (GSH) levels. The levels of Fe2+ and lipid reactive oxygen species (ROS), and expression of the biomarkers of ferroptosis, including GPX4 and prostaglandin-endoperoxide synthase 2 (PTGS2), were measured to examine cardiomyocyte ferroptosis. Additionally, RNA interference was used to silence Gpx4. In vitro and in vivo, ECH considerably reduced the MDA and LDH levels and increased the GSH level, thereby attenuating DOX-induced cardiac injury and oxidative stress. Meanwhile, ECH treatment decreased the lipid ROS levels and PTGS2 expression while increasing GPX4 expression, thereby alleviating DOX-induced cardiomyocyte ferroptosis. Moreover, knockdown of Gpx4 inhibited the protective effects of ECH on DOX-induced accumulation of lipid ROS in cardiomyocytes. These findings indicate that ECH can reduce DOX-induced cardiac injury by inhibiting ferroptosis via GPX4, highlighting its value as a potentially valuable therapeutic target in the management of CHF.

Neferine protected cardiomyocytes against hypoxia/oxygenation injury through SIRT1/Nrf2/HO-1 signaling.

Acute myocardial infarction is regarded as myocardial necrosis resulting from myocardial ischemia/reperfusion (I/R) damage and retains a major cause of mortality. Neferine, which was extracted from the green embryos of mature seeds of Nelumbo nucifera Gaertn., has been reported to possess a broad range of biological activities. However, its underlying mechanism on the protective effect of I/R has not been fully clarified. A hypoxia/reoxygenation (H/R) model with H9c2 cells closely simulating myocardial I/R injury was used as a cellular model. This study intended to research the effects and mechanism underlying neferine on H9c2 cells in response to H/R stimulation. Cell Counting Kit-8 and lactate dehydrogenase (LDH) release assays were employed to measure cell viability and LDH, respectively. Apoptosis and reactive oxygen species (ROS) were determined by flow cytometry analysis. Oxidative stress was evaluated by detecting malondialdehyde, superoxide dismutase, and catalase. Mitochondrial function was assessed by mitochondrial membrane potential, ATP content, and mitochondrial ROS. Western blot analysis was performed to examine the expression of related proteins. The results showed that hypoxia/reoxygenation (H/R)-induced cell damage, all of which were distinctly reversed by neferine. Moreover, we observed that neferine inhibited oxidative stress and mitochondrial dysfunction induced by H/R in H9c2 that were concomitant with increased sirtuin-1 (SITR1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 expression. On the contrary, silencing the SIRT1 gene with its small interferingRNA eliminated the beneficial effects of neferine. It is concluded that neferine preconditioning attenuated H/R-induced cardiac damage via suppressing apoptosis, oxidative stress, and mitochondrial dysfunction, which may be partially ascribed to the activation of SIRT1/Nrf2 signaling pathway.

METTL14 promotes doxorubicin-induced cardiomyocyte ferroptosis by regulating the KCNQ1OT1-miR-7-5p-TFRC axis.

Doxorubicin (DOX) has toxic effects on the heart, causing cardiomyopathy and heart injury, but the underlying mechanisms of these effects require further investigation. This study investigated the role of DOX in promoting ferroptosis to induce myocardial injury. AC16 cardiomyocyte and neonatal rat ventricle cardiomyocytes were used as an in vitro model to study the molecules involved in myocardial injury using gene silencing, ectopic expression, and RNA immunoprecipitation. Messenger RNA and protein level analyses showed that DOX treatment resulted in the upregulation of methyltransferase-like 14 (METTL14), which catalyzes the m6A modification of the long non-coding RNA KCNQ1OT1, a miR-7-5p sponge. The RNA-binding protein IGF2BP1 is associated with KCNQ1OT1 to increase its stability and robustly inhibit miR-7-5p activity. Furthermore, a lack of miR-7-5p expression led to increased levels of transferrin receptor, promoting the uptake of iron and production of lipid reactive oxygen species and demonstrating that DOX-induced ferroptosis occurs in AC16 cells. Additionally, we found that miR-7-5p targets METTL14 in AC16 cells. Meanwhile, the role of METTL14/KCNQ1OT1/miR-7-5p axis in regulating ferroptosis in neonatal rat ventricle cardiomyocytes was also confirmed. Our results indicate that selectively inhibiting ferroptosis mediated by a METTL14/KCNQ1OT1/miR-7-5p positive feedback loop in cardiomyocytes could provide a new therapeutic approach to control DOX-induced cardiac injury.

WTAP promotes myocardial ischemia/reperfusion injury by increasing endoplasmic reticulum stress via regulating m6A modification of ATF4 mRNA.

Myocardial infarction (MI) is one of the leading causes of death. Wilms' tumor 1-associating protein (WTAP), one of the components of the m6A methyltransferase complex, has been shown to affect gene expression via regulating mRNA modification. Although WTAP has been implicated in various diseases, its role in MI is unclear. In this study, we found that hypoxia/reoxygenation (H/R) time-dependently increased WTAP expression, which in turn promoted endoplasmic reticulum (ER) stress and apoptosis, in human cardiomyocytes (AC16). H/R effects on ER stress and apoptosis were all blocked by silencing of WTAP, promoted by WTAP overexpression, and ameliorated by administration of ER stress inhibitor, 4-PBA. We then investigated the underlying molecular mechanism and found that WTAP affected m6A methylation of ATF4 mRNA to regulate its expression, and that the inhibitory effects of WTAP on ER stress and apoptosis were ATF4 dependent. Finally, WTAP's effects on myocardial I/R injury were confirmed in vivo. WTAP promoted myocardial I/R injury through promoting ER stress and cell apoptosis by regulating m6A modification of ATF4 mRNA. These findings highlight the importance of WTAP in I/R injury and provide new insights into therapeutic strategies for MI.

The long noncoding RNA THRIL knockdown protects hypoxia-induced injuries of H9C2 cells through regulating miR-99a.

BACKGROUND: Myocardial infarction (MI) is a leading cause of disease with high morbidity and mortality worldwide. Recent studies have revealed that long non-coding RNAs (lncRNAs) are involved in heart disease pathogenesis. This study aimed to investigate the effect and the molecular basis of THRIL on hypoxia-injured H9C2 cells. METHODS: THRIL, miR-99a and Brahma-related gene 1 (Brg1) expressions in H9C2 cells were altered by transient transfections. The cells were subjected to hypoxia for 4 h, and then the levels of THRIL, miR-99a and Brg1 were investigated. Cell viability, migration and invasion, and apoptotic cells were respectively measured by trypan blue exclusion assay, transwell migration assay and flow cytometry assay. Dual luciferase reporter assay was conducted to verify the interaction between miR-99a and THRIL. Furthermore, levels of apoptosis-, PI3K/AKT and mTOR pathways-related factors were measured by western blotting. RESULTS: Hypoxia induced an increase of THRIL but a reduction of miR-99a and Brg1. THRIL inhibition significantly attenuated hypoxia-induced cell injuries, as increased cell viability, migration and invasion, and decreased cell apoptosis. THRIL negatively regulated miR-99a expression through sponging with miR-99a binding site, and miR-99a inhibition abolished the protective effects of THRIL knockdown against hypoxia-induced injury in H9C2 cells. Furthermore, miR-99a positively regulated the expression of Brg1. Brg1 inhibition promoted hypoxia-induced cell injuries, while Brg1 overexpression alleviated hypoxia-induced cell injuries. Moreover, Brg1 overexpression activated PI3K/AKT and mTOR pathways. CONCLUSIONS: This study demonstrated that THRIL inhibition represented a protective effect against hypoxia-induced injuries in H9C2 cells by up-regulating miR-99a expression.

RETRACTED: TUG1 alleviates hypoxia injury by targeting miR-124 in H9c2 cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article "This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Corin protects H2O2-induced apoptosis through PI3K/AKT and NF-κB pathway in cardiomyocytes.

BACKGROUND: The functional role of corin in H2O2-induced apoptosis is largely unexplored. The present study investigated the protective role of corin against cell injury by possible involvement of PI3K/AKT and NF-kB signaling pathways in cardiomyocytes. METHOD: Cardiomyocytes H9c2 and HL-1 cells were used in the study. Cell viability was measured using CCK-8 assay; cell apoptosis was analyzed by flow cytometry, TUNEL assay, and western blot; and cell migration was measured using wound healing assay. The fluorescent intensities of reactive oxygen species (ROS) were measured using a flow cytometer. Quantitative RT-PCR was used to measure the mRNA expression of corin. Western blot was used to measure the protein expression of corin, apoptosis-related proteins (Bax, cleaved-Caspase-3 and -9), and PI3K/AKT and NF-κB signaling pathway proteins. RESULTS: Treatment with H2O2 (150µM, 6h) significantly decreased cell viability and relative migration, increased apoptosis, and decreased the expression of corin in H9c2 and HL-1 cells. Overexpression of corin alleviated the H2O2-induced cell injury by increasing cell viability and migration and decreasing apoptosis in the cardiomyocytes. Overexpression of corin also decreased the ROS level in the cardiomyocytes likely through upregulating HIF-1α. These effects of corin on the cell injury might be mediated via the corin-induced activations of PI3K/AKT and NF-κB signaling pathways. CONCLUSION: Overexpression of corin protected cardiomyocytes from H2O2-induced injury by decreasing apoptosis and ROS level via activations of the PI3K/AKT and NF-κB signaling pathways and upregulating HIF-1α.

Impact of GGCX polymorphisms on warfarin dose requirements in atrial fibrillation patients

Background/aim: Warfarin is a common anticoagulant with large interindividual differences and a narrow therapeutic range. The polymorphisms of gamma-glutamyl carboxylase (GGCX) are important genetic factors for warfarin dose requirements. Materials and methods: Polymerase chain reaction and direct sequencing methods were used to detect the GGCX rs699664 genotype in 215 atrial fibrillation (AF) patients with warfarin administration. The effects on warfarin dose by different genotypes were analyzed. A warfarin dosing algorithm was developed based on age, height, CYP2C9, VKORC1, and GGCX genotype. Results: In 215 AF patients, there were 104 cases of wild-type GG genotype (48.4%), 92 cases of GA genotype (42.8%), and 19 cases of AA genotype (8.8%). Patients with the GGCX rs699664 A allele (GA or AA genotypes) needed higher warfarin doses than those with the GG genotype (P < 0.05). A warfarin dosing algorithm showed that age, height, CYP2C9, VKORC1, and GGCX genotype were the best variables for estimating warfarin dose (R2 = 41.2%). Another independent cohort of 60 AF patients showed a significant linear correlation between predicted warfarin maintenance dose and actual dose (R = 0.660, P < 0.01). Conclusion: AF patients with the GA and AA genotypes in GGCX rs699664 required significantly higher warfarin doses. GGCX rs699664 is a potential predictor for the warfarin dose of AF patients.

Clinical application of a new warfarin-dosing regimen based on the CYP2C9 and VKORC1 genotypes in atrial fibrillation patients.

The polymorphisms of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) are important genetic factors for warfarin dose determinations. The present study aimed to investigate the contribution of the CYP2C9 and VKORC1 genotypes to warfarin dose requirement in atrial fibrillation (AF) patients, and to evaluate the clinical application of a warfarin-dosing algorithm. A total of 122 AF patients with a target international normalized ratio of 2.0 to 3.0 were included to determine the genotypes of CYP2C9 (rs1057910) and VKORC1 (rs9923231). A warfarin-dosing algorithm was developed based on age, height, and the CYP2C9 and VKORC1 genotypes of AF patients. The results indicated that the mean warfarin daily dose requirement was lower in the CYP2C9*1/*3 genotype compared with those in the homozygous wild-type CYP2C9*1/*1 patients (P<0.05), and was higher in patients with the VKORC1 AG and GG genotypes compared with those with the AA genotype (P<0.05). The multivariate regression model showed that age, height, and the CYP2C9 and VKORC1 genotypes were the best variables for estimating warfarin dose (R2=56.4%). A new warfarin-dosing algorithm was developed and its validity was confirmed in a second cohort of AF patients. During the 50-day follow-up, 63.3% (19/30) of control group patients and 86.7% (26/30) of patients in the experimental group acquired the warfarin maintenance dose. Among all the patients who acquired the warfarin maintenance dose, the mean time elapse from initiation until warfarin maintenance dose was significantly less in the experimental group (25.8±1.7 day) compared to the control group (33.1±1.9 day) (P<0.05). There was significant linear correlation between predicted warfarin maintenance dose and actual dose (r=0.822, P<0.01). In conclusion, a new warfarin-dosing algorithm was developed based on the CYP2C9 and VKORC1 genotypes, and it can shorten the time elapse from initiation until warfarin maintenance dose in AF patients with warfarin therapy.

Differential N-glycosylation of kallikrein 6 derived from ovarian cancer cells or the central nervous system.

Ovarian cancer causes more deaths than any other gynecological disorder. Perturbed glycosylation is one of the hallmarks of this malignancy. Kallikrein 6 (KLK6) elevation in serum is a diagnostic and prognostic indicator in ovarian cancer. The majority of ovarian carcinomas express high levels of KLK6, which diffuses into the circulation. Under physiological conditions, KLK6 is expressed highly in the central nervous system and found at high levels in cerebrospinal fluid from where it enters the circulation. Our aim was to characterize and compare the N-glycosylation status of this protein in ovarian cancer ascites fluid and cerebrospinal fluid. Anion-exchange chromatography was used to reveal different post-translational modifications on the two isoforms. Mobility gel shift Western blot analysis coupled with glycosidase digestion showed that the molecular weight difference between the two isoforms was because of differential glycosylation patterns. The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with alpha2-6-linked sialic acid. The structure and composition of glycans of both KLK6 isoforms was elucidated by glycopeptide monitoring with electrospray ionization-Orbitrap tandem mass spectrometry. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma.

[Vitamin K epoxide reductase complex 1 gene polymorphism and warfarin dose requirement in Chinese patients].

OBJECTIVE: To investigate the possible relationship between vitamin K epoxide reductase complex 1 (VKORC1) gene polymorphism and warfarin dose requirements in Chinese patients. METHOD: Genotyping for the C1173T polymorphism in the VKORC1 gene was performed using a restriction enzyme digestion of PCR-amplified DNA in 102 Chinese patients treated with warfarin. RESULTS: TT genotype was found in 83 patients (81.4%), CT genotype in 16 patients (15.7%) and CC genotype in 3 patients (2.9%). To achieve similar target INR range (1.5 - 2.5), the warfarin dose requirement was significantly lower in patients with TT genotype than that in patients with CT/CC genotypes (P < 0.01). CONCLUSION: C1173T polymorphism in the VKORC1 gene might be one important determinant for warfarin dose requirement in Chinese patients.