Distinct membrane interaction mechanisms of gramicidin A and S in mammalian cells: A combined experimental and molecular dynamics study.

N6-methyladenosine (m6A) methylation in RNA is a dynamic and reversible modification regulated by methyltransferases and demethylases, which has been reported to participate in many pathological processes of various diseases, including cardiac disorders. This study was designed to investigate an m6A writer Mettl14 on cardiac ischemia–reperfusion (I/R) injury and uncover the underlying mechanism. The m6A and Mettl14 protein levels were increased in I/R hearts and neonatal mouse cardiomyocytes upon oxidative stress. Mettl14 knockout (Mettl14+/−) mice showed pronounced increases in cardiac infarct size and LDH release and aggravation in cardiac dysfunction post-I/R. Conversely, adenovirus-mediated overexpression of Mettl14 markedly reduced infarct size and apoptosis and improved cardiac function during I/R injury. Silencing of Mettl14 alone significantly caused a decrease in cell viability and an increase in LDH release and further exacerbated these effects in the presence of H2O2, while overexpression of Mettl14 ameliorated cardiomyocyte injury in vitro. Mettl14 resulted in enhanced levels of Wnt1 m6A modification and Wnt1 protein but not its transcript level. Furthermore, Mettl14 overexpression blocked I/R-induced downregulation of Wnt1 and β-catenin proteins, whereas Mettl14+/− hearts exhibited the opposite results. Knockdown of Wnt1 abrogated Mettl14-mediated upregulation of β-catenin and protection against injury upon H2O2. Our study demonstrates that Mettl14 attenuates cardiac I/R injury by activating Wnt/β-catenin in an m6A-dependent manner, providing a novel therapeutic target for ischemic heart disease.

<p>N6-methyladenosine (m6A) methylation in RNA is a dynamic and reversible modification regulated by methyltransferases and demethylases, which has been reported to participate in many pathological processes of various diseases, including cardiac disorders. This study was designed to investigate an m6A writer Mettl14 on cardiac ischemia–reperfusion (I/R) injury and uncover the underlying mechanism. The m6A and Mettl14 protein levels were increased in I/R hearts and neonatal mouse cardiomyocytes upon oxidative stress. Mettl14 knockout (Mettl14<sup>+/−</sup>) mice showed pronounced increases in cardiac infarct size and LDH release and aggravation in cardiac dysfunction post-I/R. Conversely, adenovirus-mediated overexpression of Mettl14 markedly reduced infarct size and apoptosis and improved cardiac function during I/R injury. Silencing of Mettl14 alone significantly caused a decrease in cell viability and an increase in LDH release and further exacerbated these effects in the presence of H<sub>2</sub>O<sub>2</sub>, while overexpression of Mettl14 ameliorated cardiomyocyte injury in vitro. Mettl14 resulted in enhanced levels of Wnt1 m6A modification and Wnt1 protein but not its transcript level. Furthermore, Mettl14 overexpression blocked I/R-induced downregulation of Wnt1 and β-catenin proteins, whereas Mettl14<sup>+/−</sup> hearts exhibited the opposite results. Knockdown of Wnt1 abrogated Mettl14-mediated upregulation of β-catenin and protection against injury upon H<sub>2</sub>O<sub>2</sub>. Our study demonstrates that Mettl14 attenuates cardiac I/R injury by activating Wnt/β-catenin in an m6A-dependent manner, providing a novel therapeutic target for ischemic heart disease.</p>

ObjectivesTo investigate the effects of EGB on advanced glycosylation end products (AGEs)-induced cardiomyocyte injury, and the role of endoplasmic reticulum stress (ERS) in the process.MethodsCultured neonatal rat cardiomyocytes were randomly...

The protective effect of benidipine hydrochloride, a 1,4-dihydropyridine calcium antagonist, was investigated in cultured mouse fetal heart cells under the hypoxic condition (PO2, < or = 20 mmHg). The preparation of cultured fetal mouse heart muscle cells was subjected to the hypoxic condition for 24 hr. After the hypoxic treatment for 24 hr, the spontaneously beating rate of cultured heart cells was reduced subsequently and the beating almost ceased. Protection against cell injury was assessed by cell viability (NR assay), LDH release and cellular ATP levels. Under the hypoxic condition, cell viability and cellular ATP levels were markedly decreased, and LDH release was increased. Benidipine hydrochloride, at concentrations higher than 1-10 nM, strongly protected against the decreases in cell viability and cellular ATP levels as well as the increase in LDH release. The potency of the protective effect of benidipine hydrochloride was greater than those of the other tested 1,4-dihydropyridine calcium antagonists (nisoldipine, nitrendipine and nifedipine). These results suggest that benidipine hydrochloride exerts protective actions against hypoxic myocardial injury of cultured mouse fetal heart cells.

COA-Cl, a novel adenosine-like nucleic acid analog, has recently been shown to exert neuroprotective effects and to increase dopamine levels both in vivo and in vitro. Therefore, we hypothesized that COA-Cl could protect dopaminergic neurons against toxic insults. Thus, the present study aimed to investigate the protective effects of COA-Cl against hydrogen peroxide (H2O2)- and 6-hydroxydopamine (6-OHDA)-induced toxicity in PC12 cells and to elucidate the possible mechanisms. PC12 cells were incubated with COA-Cl (100μM) with or without H2O2 or 6-OHDA (200μM) for 24h. Treatment with COA-Cl attenuated the decrease in cell viability, SOD activity and the Bcl-2/Bax ratio caused by H2O2. In addition, COA-Cl attenuated the increase in LDH release, ROS production, caspase-3 activity, and apoptosis induced by H2O2. Further, COA-Cl enhanced the protection of PC12 cells against the toxicity caused by 6-OHDA, as evidenced by an increase in cell viability and the Bcl-2/Bax ratio, and a decrease in LDH release. Our results are the first to demonstrate that COA-Cl potentially protects PC12 cells against toxicity induced by H2O2 and 6-OHDA, implying that COA-Cl could be a promising neuroprotective agent for the treatment of Parkinson's disease.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial damage contributes to the initiation and pathogenesis of atherosclerosis. Salidroside can alleviate atherosclerosis and attenuate endothelial cell injury induced by ox-LDL. However, the mechanisms involved in this process are not fully understood. Therefore, the purpose of the present study was to investigate the role of the adenosine monophosphate-activated protein kinase (AMPK)/sirtuin (SIRT)1 pathway in the protection of salidroside against ox-LDL-induced human umbilical vein endothelial cells (HUVECs) injuries. The results revealed that salidroside reverses ox-LDL-induced HUVECs injury as demonstrated by the upregulation of cell viability and downregulation of LDH release. In addition, salidroside increased the expression of the SIRT1 protein in ox-LDL-treated HUVECs. Next, it was demonstrated that SIRT1 knockdown induced by transfection with small interfering (si)RNA targeting SIRT1 (siSRT1) abolished the protection of salidroside against ox-LDL-induced HUVECs injuries. This was illustrated by a decrease in cell viability and an increase in LDH release, caspase-3 activity and apoptosis rate. Furthermore, salidroside mitigated ox-LDL-induced reactive oxygen species production, upregulated malondialdehyde content and NADPH oxidase 2 expression and decreased superoxide dismutase and glutathione peroxidase activities, while these effects were also reversed by siSIRT1 transfection. In addition, it was demonstrated that salidroside suppressed ox-LDL-induced mitochondrial dysfunction as demonstrated by the increase in mitochondrial membrane potential and decreases in cytochrome c expression, and Bax/Bcl-2 reductions. However, these effects were eliminated by SIRT1 knockdown. Finally, it was demonstrated that salidroside significantly upregulated the phosphorylated-AMPK expression in ox-LDL-treated HUVECs and AMPK knockdown induced by transfection with AMPK siRNA (siAMPK) leads to elimination of the salidroside-induced increase in cell viability and the decrease in LDH release. Notably, siAMPK transfection further decreased the expression of SIRT1. In conclusion, these results suggested that salidroside protects HUVECs against ox-LDL injury through inhibiting oxidative stress and improving mitochondrial dysfunction, which were dependent on activating the AMPK/SIRT1 pathway.

MiR-101 has been reported to be involved in neuroinflammation, neuronal injury and neurotoxicity. However, the specific role and mechanism of miR-101 in ischemia/reperfusion (I/R)-induced neuronal injury remain largely unknown. Our study found that after oxygen-glucose deprivation/reperfusion (OGD/R) exposure, the level of miR-101 in SH-SY5Y cells was significantly decreased, which was accompanied by a decrease in cell viability, and an increase in LDH release and apoptosis. MiR-101 overexpression (miR-101 mimics) significantly promoted viability and inhibited LDH release and apoptosis in OGD/R-exposed SH-SY5Y cells. Luciferase reporter assay indicated that enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) was a direct target of miR-101, and EZH2 siRNA obviously increased the viability, inhibited LDH release and apoptosis in OGD/R-exposed SH-SY5Y cells. Besides, EZH2 siRNA could inhibit the activation of mitogen-activatedproteinkinase(MAPK14) signaling pathway and theMAPK14agonist (anisomycin) could reverse EZH2 siRNA-induced increase in cell viability, and decreases in LDH release and apoptosis. Furthermore, when the methyltransferase activity of EZH2 was inhibited by its specific inhibitorGSK126, the level of miR-101 was increased in OGD/R-exposed SH-SY5Y cells. Additionally, EZH2 siRNA upregulated miR-101 expression in OGD/R-exposed SH-SY5Y cells. Taken together, our findings reveal that miR-101/EZH2 negative feedback signaling drives OGD/R-induced injury by activating the MAPK14 signaling pathway in SH-SY5Y cells.

S262A mutation abolishes protective effects of connexin 43 against hypothermic preservation–induced injury in cardiomyocytes

Taurolidine, a derivative of the amino acid taurine, exhibits antiendotoxin, antibacterial, and antiadherence activity. We hypothesized that Taurolidine may inhibit tumor cell growth, both in an in vitro and in vivo setting. Our aim was to examine the effect of Taurolidine on the growth of a rat metastatic colorectal tumor cell line (DHD/K12/TRb) in vitro and in vivo. In the in vitro experiments, DHD/K12/TRb cells were incubated with 5, 10, 15, 25, microg/ml of Taurolidine. Cells incubated in culture medium alone were used as controls. Cell proliferation, cell viability, cell death, and cell apoptosis were measured using commercially available techniques. In the in vivo experiment, BD IX rats were randomized into two groups (n = 10/group). Group A (control) underwent laparotomy and instillation of DHD/K12/TRb tumor cells intraperitoneally followed by phosphate buffered saline (PBS). Group B received Taurolidine (100 mg/kg) instead of PBS. Animals were killed after 24 days and tumor burden assessed by counting the number of tumor nodules in the peritoneal cavity. Incubation of the tumor cells with Taurolidine resulted in a 4-fold decrease in proliferation rates (25+/-4% vs. 100+/-28% for controls) and a 4-fold increase in cell necrosis as demonstrated by the increase in LDH release (403+/-28% vs. 100+/-26% for controls), at a Taurolidine concentration of 25 microg/ml. A dose-dependent decrease in cell viability was also observed. In the in vivo study, local Taurolidine administration resulted in significant decreases in tumor burden (3+/-1 nodules in Group B animals vs. 649+/-101 nodules in Group A animals). Taurolidine inhibits the growth of a rat metastatic colorectal tumor cell line in vitro and in vivo and thus may have potential in the prevention of peritoneal metastases.

This study explored the relationship among lncRNA NR_027324, miR-103-3p, and autophagy-related protein 5 (ATG5) in cardiomyocytes under hypoglycemic/hypoxic conditions.Bioinformatics and luciferase assays were used to investigate the interaction between NR_027324 and miR-103-3p. H9C2 cells cultured under hypoglycemic/hypoxic conditions were transfected with NR_027324 siRNA, overexpression vectors, and miR-103-3p mimics/inhibitors. Cell viability and damage were measured using MTT and LDH assays, respectively. qRT-PCR was performed to assess the mRNA levels of NR_027324, miR-103a-3p, and ATG5. Protein expressions were analyzed by Western blotting, and immunofluorescence was used to observe LC3-I/II expression.In hypoglycemic/hypoxic conditions, H9C2 cell viability decreased significantly, and LDH levels increased, indicating cell damage. Simultaneously, NR_027324 and ATG5 expressions were upregulated, while miR-103-3p was downregulated. Overexpression of NR_027324 enhanced cell viability and reduced LDH release. Conversely, knockdown of NR_027324 resulted in decreased cell viability and increased LDH release. In addition, knockdown of NR_027324 led to upregulation of miR-103-3p and downregulation of ATG5. Furthermore, the luciferase activity was significantly lower when miR-103-3p interacted with wildtype NR_027324, validating the binding between NR_027324 to miR-103-3p. The overexpression of NR_027324 led to downregulation of miR-103-3p, while its knockdown resulted in the upregulation of miR-103-3p. Lastly, NR_027324 overexpression alone significantly upregulated ATG5 expression, which was counteracted when NR_027324 and miR-103-3p were co-overexpressed.The findings of this study highlight the significance of NR_027324, miR-103-3p, and ATG5 in mediating the autophagy and apoptosis response of H9C2 cells under hypoglycemic/hypoxic stress, providing valuable insights into potential targets for therapeutic interventions in related cardiovascular conditions.

Dimethyl fumarate (DMF) is a newly approved drug for the treatment of relapsing forms of multiple sclerosis and relapsing-remitting multiple sclerosis. Here, we investigated the effects of DMF and its metabolites mono-methylfumarate (MMF and methanol) on different gastrointestinal cancer cell lines and the underlying molecular mechanisms involved. Cell viability was measured by the MTT or CCK8 assay. Protein expressions were measured by Western blot analysis. LDH release, live- and dead-cell staining, intracellular GSH levels, and mitochondrial membrane potential were examined by using commercial kits. DMF but not MMF induced cell necroptosis, as demonstrated by the pharmacological tool necrostatin-1, transmission electron microscopy, LDH and HMGB1 release in CT26 cells. The DMF-induced decrease in cellular GSH levels as well as cell viability and increase in reactive oxygen species (ROS) were inhibited by co-treatment with GSH and N-acetylcysteine (NAC) in CT26 cells. DMF activated JNK, p38 and ERK MAPKs in CT26 cells and JNK, p38 and ERK inhibitors partially reversed the DMF-induced decrease in cell viability. GSH or NAC treatment inhibited DMF-induced JNK, p38, and ERK activation in CT26 cells. DMF but not MMF increased autophagy responses in SGC-7901, HCT116, HT29 and CT26 cancer cells, but autophagy inhibition did not prevent the DMF-induced decrease in cell viability. DMF but not its metabolite MMF induced necroptosis in colon cancer cells through a mechanism involving the depletion of GSH, an increase in ROS and activation of MAPKs.

Cross talk between oxidized LDL (ox-LDL) and angiotensin II (Ang II) may be relevant in atherosclerosis. In this study, we examined the presence of a specific endothelial receptor for ox-LDL (LOX-1) and Ang II receptors in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of Ang II on LOX-1 gene and protein expression. LOX-1 was consistently identified in HCAECs by reverse transcriptase-polymerase chain reaction (RT-PCR), cDNA sequence, Western blot, and 125I-labeled ox-LDL binding assay (Bmax, 29.7 ng/mg protein). The HCAECs also exhibited Ang II receptors (AT1>AT2), as determined by RT-PCR and 125I-labeled Ang II binding assay (Bmax, 2.21 and 1.19 fmol/mg protein, respectively). Incubation of HCAECs with Ang II markedly increased LOX-1 mRNA (RT-PCR) and protein (Western blot) expression. The increase in LOX-1 expression was dependent on Ang II concentration (10(-12) to 10(-6) mol/L). Ang II caused a concentration-dependent increase in 125I-labeled ox-LDL uptake by HCAECs and enhanced ox-LDL-mediated cell injury, as evident from an increase in LDH release and a decrease in cell viability. These effects of Ang II were completely blocked by pretreatment of HCAECs with losartan, a specific AT1 blocker, but not by PD123319, a specific AT2 blocker. These observations indicate the following: (1) HCAECs possess abundant LOX-1 as well as Ang II (AT1>AT2) receptors, (2) Ang II upregulates LOX-1 receptor and ox-LDL uptake, (3) the effects of Ang II are mediated by AT1 activation, and (4) Ang II enhances ox-LDL-mediated injury to HCAECs.

Cerebral ischemia-reperfusion (CI/R) is a complex process that frequently results in neuronal oxidative stress and apoptosis. Dipsacoside B (DB) possesses antimicrobial and detoxifying properties and can improve mitochondrial function. Nevertheless, the potential neuroprotective effects of DB in stroke remain uncertain. This investigation aims to elucidate the impact of DB on CI/R, as well as its underlying regulatory mechanism. In our study, results showed that DB lessened the decrease in cell viability and increase in LDH release in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated HT22 cells. DB attenuated OGD/R-tempted oxidative stress, apoptosis, and mitochondrial dysfunction. DB enhanced mitophagy in HT22 cells following OGD/R treatment by enhancing the levels of LC3-II/LC3-I, PINK1, and Parkin. Blocking of mitophagy by mdivi-1 or silencing PINK1 abolished the protective effect of DB against OGD/R-induced oxidative stress damage and mitochondrial dysfunction in HT22 cells was found to be dependent on the activation of PINK1/Parkin-mediated mitophagy, as evidenced by the loss of protection when mitophagy was blocked by mdivi-1 or PINK1 silencing. Additionally, the neuroprotective effects of DB were confirmed in the middle cerebral artery occlusion mouse model, indicated by alleviation of oxidative stress, apoptosis, and mitochondrial dysfunction. In conclusion, DB attenuated OGD/R-tempted oxidative stress, apoptosis, and mitochondrial dysfunction in HT22 hippocampal neurons by activating PINK1/Parkin-mediated mitophagy.

Reactive oxygen species mediated ginsenoside Rg3- and Rh2-induced apoptosis in hepatoma cells through mitochondrial signaling pathways

BackgroundExposure to airborne fine particulate matter (PM2.5) has been reported to be harmful to the human kidney. However, whether the activation of oxidative stress and cell apoptosis plays key roles in the nephrotoxicity caused by PM2.5 exposure is still poorly understood. The aim of this study was to explore the mechanism of cytotoxicity after PM2.5 exposure in human proximal tubule epithelial cells (HK-2 cells).ResultsPM2.5 exposure resulted in a significant decrease in cell viability, with an increase in LDH release and the early kidney damage marker kidney injury molecule-1 (KIM-1) expression in a dose-dependent manner and time-dependent manner. PM2.5 exposure induced reactive oxygen species (ROS) generation and markedly elevated apoptosis in HK-2 cells. In addition, PM2.5 exposure resulted in the activation of antioxidant pathway, as evidenced by the increased expressions of Nrf2, HO-1 and NQO1 and decreased expression of Keap1. Moreover, PM2.5 exposure also induced the activation of apoptotic pathway, as evidenced by the increased expressions of pro-apoptotic proteins Bax, caspase-3 and caspase-8 and decreased expression of antiapoptotic protein Bcl-2.ConclusionsOur results demonstrated that both antioxidant pathway and apoptotic pathway played critical roles in the damage mediated by PM2.5 in HK-2 cells. This study would give us a strategy to prevent the impairment of renal function by PM2.5 induced through repression of oxidative stress and apoptosis.