[Sanshentongmai Mixture Improves Oxidative Damage in Rat Cardiomyocytes H9C2 via Upregulation of microRNA-146a]. / 三参通脉合剂通过上调microRNA-146aæ”¹å–„å¤§é¼ å¿ƒè‚Œç»†èƒžH9C2的氧化损伤.

Objective: To investigate the effect of Sanshentongmai (SSTM) mixture on the regulation of oxidative damage to rat cardiomyocytes (H9C2) through microRNA-146a and its mechanism. Methods: H9C2 were cultured in vitro, H2O2 was used as an oxidant to create an oxidative damage model in H9C2 cells. SSTM intervention was administered to the H9C2 cells. Then, the changes in H2O2-induced oxidative damage in H9C2 cells and the expression of microRNA-146a were observed to explore the protective effect of SSTM on H9C2 and its mechanism. H9C2 cells cultured i n vitro were divided into 3 groups, including a control group, a model group of H2O2-induced oxidative damage (referred to hereafter as the model group), and a group given H2O2 modeling plus SSTM intervention at 500 µg/L for 72 h (referred to hereafter as the treatment group). The cell viability was measured by CCK8 assay. In addition, the levels of N-terminal pro-brain natriuretic peptide (Nt-proBNP), nitric oxide (NO), high-sensitivity C-reactive protein (Hs-CRP), and angiotensin were determined by enzyme-linked immunosorbent assay (ELISA). The expression level of microRNA-146a was determined by real-time PCR (RT-PCR). Result: H9C2 cells were pretreated with SSTM at mass concentrations ranging from 200 to 1500 µg/L. Then, CCK8 assay was performed to measure cell viability and the findings showed that the improvement in cell proliferation reached its peak when the mass concentration of SSTM was 500 µg/L, which was subsequently used as the intervention concentration. ELISA was performed to measure the indicators related to heart failure, including Nt-proBNP, NO, Hs-CRP, and angiotensin Ⅱ. Compared with those of the control group, the expressions of Nt-proBNP and angiotensin Ⅱ in the treatment group were up-regulated (P<0.05), while the expression of NO was down-regulated (P<0.05). There was no significant difference in the expression of Hs-CRP between the treatment group and the control group. These findings indicate that SSTM could effectively ameliorate oxidative damage in H9C2 rat cardiomyocytes. Finally, according to the RT-PCR findings for the expression of microRNA-146a in each group, H2O2 treatment at 15 µmol/L could significantly reduce the expression of microRNA-146a, and the expression of microRNA-146a in the treatment group was nearly doubled compared with that in the model group. There was no significant difference between the treatment group and the control group. Conclusion: SSTM can significantly resist the H2O2-induced oxidative damage of H9C2 cells and may play a myocardial protective role by upregulating microRNA-146a.

Deciphering the benefits and intensity levels of primary metabolites from Allium macrostemon Bunge and Allium chinense G. Don.

BACKGROUND: Allii Macrostemonis Bulbus is also named Xiebai in China. It is an edible vegetable, and also a famous herb for treating coronary heart disease. Allium chinense G. Don (ACGD) and Allium macrostemon Bunge (AMB) are it botanical sources. The aim of this study was to explore the cardioprotective effects, and decipher the visual spatial distribution and absolute content of primary metabolites derived from these two herbs. METHODS: H9c2 cells were used to perform the hypoxia-reoxygenation (H/R)-induced myocardial injury model. Their protective effects were evaluated by apoptosis levels. Furthermore, matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry imaging approach (MALDI-TOF MSI) was carried out to present the spatial location of primary metabolites including fatty acids, amino acids, carotenoids, and vitamins in these two Allium herbs. Multiple analytical methods were applied to perform quantitative analysis of these primary metabolites in AMB and ACGD bulbs by liquid chromatography tandem mass spectrometry (LC-MS). RESULTS: First, AMB and ACGD extracts both could increase the cell viability in H9c2 cells, and attenuate H/R-induced injury. They markedly decreased apoptosis, accompanied by activating the BCL-2/BAX pathway. Further, MALDI-TOF MSI-based relative quantification results showed several amino acids, fatty acids, carotenoids, and vitamins were largely rich in the tunics and outside scales of fresh bulbs, while some primary metabolites were abundant in their developing flower buds. Absolute quantification results displayed total contents of amino acids in ACGD bulbs were higher than those in AMB, while total contents of fatty acids and vitamins provides opposite trends in these two Allium herbs. The total contents of carotenoids and trace elements showed no significant differences between AMB and ACGD samples. CONCLUSIONS: This study would be helpful to understand the myocardial injury protection effects of these two Allium herbs, and the spatial accumulation and quantitative content levels of their main nutrients.

Potential Effects of Orally Ingesting Polyethylene Terephthalate Microplastics on the Mouse Heart.

Polyethylene terephthalate microplastics (PET MPs) are widespread in natural environment, and can enter organisms and accumulate in the body, but its toxicity has not been well studied. Therefore, in order to investigate the toxic effects of PET microplastics on mammals, this study investigated the toxic effects of PET MPs on ICR mice and H9C2 cells by different treatment groups. The results indicated the cardiac tissue of mice in the PET-H (50 µg/mL) group showed significant capillary congestion, myocardial fiber breakage, and even significant fibrosis compared to the PET-C (control) group (P < 0.01). Results of the TUNEL assay demonstrated significant apoptosis in myocardial tissue in the PET-H and PET-M (5 µg/mL) groups (P < 0.01). Meanwhile, Western blotting showed increased expression of the apoptosis-related protein Bax and decreased expression of PARP, caspase-3, and Bcl-2 proteins in both myocardial tissues and H9C2 cells. In addition, flow cytometry confirmed that PET MPs decreased the mitochondrial membrane potential and apoptosis in H9C2 cells; however, this trend was reversed by N-acetylcysteamine application. Moreover, PET MP treatment induced the accumulation of reactive oxygen species (ROS) in H9C2 cells, while the MDA level in the myocardial tissue was elevated, and the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were decreased (P < 0.01), indicating a change in the redox environment. In conclusion, PET MPs promoted cardiomyocyte apoptosis by inducing oxidative stress and activating mitochondria-mediated apoptotic processes, ultimately leading to myocardial fibrosis. This study provides ideas for the prevention of PET MP toxicity and promotes thinking about enhancing plastic pollution control.

Sibjotang Protects against Cardiac Hypertrophy In Vitro and In Vivo.

Cardiac hypertrophy is developed by various diseases such as myocardial infarction, valve diseases, hypertension, and aortic stenosis. Sibjotang (, Shizaotang, SJT), a classic formula in Korean traditional medicine, has been shown to modulate the equilibrium of body fluids and blood pressure. This research study sought to explore the impact and underlying process of Sibjotang on cardiotoxicity induced by DOX in H9c2 cells. In vitro, H9c2 cells were induced by DOX (1 µM) in the presence or absence of SJT (1-5 µg/mL) and incubated for 24 h. In vivo, SJT was administrated to isoproterenol (ISO)-induced cardiac hypertrophy mice (n = 8) at 100 mg/kg/day concentrations. Immunofluorescence staining revealed that SJT mitigated the enlargement of H9c2 cells caused by DOX in a dose-dependent way. Using SJT as a pretreatment notably suppressed the rise in cardiac hypertrophic marker levels induced by DOX. SJT inhibited the DOX-induced ERK1/2 and p38 MAPK signaling pathways. In addition, SJT significantly decreased the expression of the hypertrophy-associated transcription factor GATA binding factor 4 (GATA 4) induced by DOX. SJT also decreased hypertrophy-associated calcineurin and NFAT protein levels. Pretreatment with SJT significantly attenuated DOX-induced apoptosis-associated proteins such as Bax, caspase-3, and caspase-9 without affecting cell viability. In addition, the results of the in vivo study indicated that SJT significantly reduced the left ventricle/body weight ratio level. Administration of SJT reduced the expression of hypertrophy markers, such as ANP and BNP. These results suggest that SJT attenuates cardiac hypertrophy and heart failure induced by DOX or ISO through the inhibition of the calcineurin/NFAT/GATA4 pathway. Therefore, SJT may be a potential treatment for the prevention and treatment of cardiac hypertrophy that leads to heart failure.

RBM25 binds to and regulates alternative splicing levels of Slc38a9, Csf1, and Coro6 to affect immune and inflammatory processes in H9c2 cells.

Background: Alternative splicing (AS) is a biological process that allows genes to be translated into diverse proteins. However, aberrant AS can predispose cells to aberrations in biological mechanisms. RNA binding proteins (RBPs), closely affiliated with AS, have gained increased attention in recent years. Among these RBPs, RBM25 has been reported to participate in the cardiac pathological mechanism through regulating AS; however, the involvement of RBM25 as a splicing factor in heart failure remains unclarified. Methods: RBM25 was overexpressed in H9c2 cells to explore the target genes bound and regulated by RBM25 during heart failure. RNA sequencing (RNA-seq) was used to scrutinize the comprehensive transcriptional level before identifying AS events influenced by RBM25. Further, improved RNA immunoprecipitation sequencing (iRIP-seq) was employed to pinpoint RBM25-binding sites, and RT-qPCR was used to validate specific genes modulated by RBM25. Results: RBM25 was found to upregulate the expression of genes pertinent to the inflammatory response and viral processes, as well as to mediate the AS of genes associated with cellular apoptosis and inflammation. Overlap analysis between RNA-seq and iRIP-seq suggested that RBM25 bound to and manipulated the AS of genes associated with inflammation in H9c2 cells. Moreover, qRT-PCR confirmed Slc38a9, Csf1, and Coro6 as the binding and AS regulatory targets of RBM25. Conclusion: Our research implies that RBM25 plays a contributory role in cardiac inflammatory responses via its ability to bind to and regulate the AS of related genes. This study offers preliminary evidence of the influence of RBM25 on inflammation in H9c2 cells.

Puerarin Inhibits NLRP3-Caspase-1-GSDMD-Mediated Pyroptosis via P2X7 Receptor in Cardiomyocytes and Macrophages.

Diabetic cardiomyopathy (DCM) is a critical complication of long-term chronic diabetes mellitus, and it is characterized by myocardial fibrosis and myocardial hypertrophy. Previous studies have shown that the pyroptosis pathway was significantly activated in DCM and may be related to the P2X7 receptor. However, the role of the P2X7 receptor in the development of DCM with pyroptosis is still unclear. In this study, we aimed to explore the mechanism of puerarin and whether the P2X7 receptor can be used as a new target for puerarin in the treatment of DCM. We adopted systematic pharmacology and bioinformatic approaches to identify the potential targets of puerarin for treating DCM. Additionally, we employed D-glucose-induced H9C2 rat cardiomyocytes and lipopolysaccharide-treated RAW264.7 mouse mononuclear macrophages as the in vitro model on DCM research, which is close to the pathological conditions. The mRNA expression of cytokines in H9C2 cells and RAW264.7 macrophages was detected. The protein expressions of NLRP3, N-GSDMD, cleaved-caspase-1, and the P2X7 receptor were investigated with Western blot analysis. Furthermore, molecular docking of puerarin and the P2X7 receptor was conducted based on CDOCKER. A total of 348 puerarin targets and 4556 diabetic cardiomyopathy targets were detected, of which 218 were cross targets. We demonstrated that puerarin is effective in enhancing cardiomyocyte viability and improving mitochondrial function. In addition, puerarin is efficacious in blocking NLRP3-Caspase-1-GSDMD-mediated pyroptosis in H9C2 cells and RAW264.7 cells, alleviating cellular inflammation. On the other hand, similar experimental results were obtained by intervention with the P2X7 receptor antagonist A740003, suggesting that the protective effects of puerarin are related to the P2X7 receptor. The molecular docking results indicated key binding activity between the P2X7 receptor and puerarin. These findings indicate that puerarin effectively regulated the pyroptosis signaling pathway during DCM, and this regulation was associated with the P2X7 receptor.

The retinoic acid response is a minor component of the cardiac phenotype in H9c2 myoblast differentiation.

The H9c2 myoblast cell line, isolated from the left ventricular tissue of rat, is currently used in vitro as a mimetic for skeletal and cardiac muscle due to its biochemical, morphological, and electrical/hormonal signaling properties. During culture, H9c2 cells acquire a myotube phenotype, where a critical component is the inclusion of retinoic acid (RA). The results from some authors on H9c2 suggested that thousands of genes respond to RA stimuli, while others report hundreds of genes responding to RA over different cell types. In this article, using a more appropriate experimental design, we first confirm the H9c2 cardiac phenotype with and without RA and report transcriptomic and physiological changes regarding calcium handling, bioenergetics, and other biological concepts. Interestingly, of the 2360 genes showing a transcriptional change, 622 genes were statistically associated with the RA response. Of these genes, only 305 were RA-specific, and the rest also showed a culture-time component. Thus, the major expression changes (from 74 to 87%) were indeed due to culture conditions over time. Unexpectedly, only a few components of the retinol pathway in KEGG responded to RA. Our results show the role of RA in the H9c2 cultures impacting the interpretation using H9c2 as an in vitro model.

Bioactive Aspergteroids G-J from Soft-Coral-Associated Symbiotic and Epiphytic Fungus Aspergillus terreus EGF7-0-1.

Two new disubstituted maleimides, aspergteroids G-H (1-2), and two trisubstituted butenolides aspergteroids I-J (3-4), along with four known analogs, were isolated and structurally identified from the fermentation extract of soft-coral-associated symbiotic and epiphytic fungus Aspergillus terreus EGF7-0-1. The structures of the new compounds were established mainly via spectroscopic data analyses, and their absolute configurations were determined via X-ray diffraction analysis and comparison of the calculated and experimental electronic circular dichroism. Myocardial protection assays showed that compounds 1, 2, 5, and 6 possess protective effects against tert-butyl hydroperoxide (TBHP)-induced H9c2 (rat myocardial cells) apoptosis at low concentrations. Based on the analyses of the protein-protein interaction (PPI) network and Western blotting, compound 1 may inhibit the apoptosis and inflammatory response of cardiomyocytes after TBHP induction and improve the antioxidant capacity of cardiomyocytes. We speculate that the anti-inflammatory response of compound 1 is suppressed by the glycogen synthase kinase-3 beta (GSK-3ß), downregulated by the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome activation, and suppressed by the expression of cysteinyl aspartate specific proteinase-3 (caspase-3) and B-cell lymphoma-2 associated X protein (Bax).

Uncovering the molecular mechanisms of Fructus Choerospondiatis against coronary heart disease using network pharmacology analysis and experimental pharmacology.

Fructus Choerospondiatis (FC), a Mongolian medicine, was mainly used in Mongolian medical theory for the treatment of coronary heart disease (CHD). Nonetheless, the main components and mechanisms of action of FC in the treatment of coronary artery disease have not been studied clearly. AIM OF THE STUDY: The aim of this study is to identify the components of FC and analyze the pathways affected by the targets of these components to probe into the potential mechanisms of action of FC on coronary heart disease. MATERIALS AND METHODS: Identification of compounds in FC employing high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS) method, then further investigate the network pharmacology and molecular docking to obtain potential targets and elucidate the potential mechanism of action of FC in the therapy of CHD. Experimental validation was established to verify the mechanism of FC in vitro. RESULTS: 21 FC components were identified and 65 overlapping targets were gained. In addition, these ingredients regulated AMPK and PPAR signaling pathway by 65 target genes including IL6, AKT1 and PPARg, etc. Molecular docking displayed that the binding ability of the key target PPARg to FC components turned out to be better. Experimental validation proved that FC treatment decreased the expression of PPARg (p < 0.05) compare with model group, which may be involved in the PPAR signaling pathway. CONCLUSIONS: This study was the first to elucidate the mechanism of action of components of FC for the treatment of CHD using network pharmacology. It alleviated CHD by inhibiting the expression of PPARg to attenuate hypoxia/reoxygenation injury, and the results give a basis for elucidating the molecular mechanism of action of FC for the treatment of coronary heart disease.

Highly Efficient Cardiac Differentiation and Maintenance by Thrombin-Coagulated Fibrin Hydrogels Enriched with Decellularized Porcine Heart Extracellular Matrix.

Biochemical and biophysical properties instruct cardiac tissue morphogenesis. Here, we are reporting on a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen that is suitable for investigations employing an in vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin facilitates the gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. Our investigations revealed enhanced cardiogenic differentiation in the H9c2 myoblast cells when using the system in a co-culture with Nor-10 fibroblasts. Further enhancement of differentiation efficiency was achieved by 3D embedding of rat neonatal cardiomyocytes in the 3D system. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony, and the maintenance of spontaneous beating, as compared to various controls including Matrigel, pure fibrin and collagen I as well as a fibrin-collagen I blend.

Preparation of Novel Nanoformulation to Enhance Efficacy in the Treatment of Cardiovascular Disease.

Despite many efforts over the last few decades, cardiac-based drug delivery systems are experiencing major problems, such as the effective delivery of the precise amount of a drug. In the current study, an effort has been made to prepare a nano-herbformulation (NHF) to overcome the major problem of conventional intervention. Copper oxide-based NHF was prepared using plant extract of Alternanthera sessilis and characterized using physicochemical techniques such as Transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Dynamic light scattering (DLS), UV-Vis spectroscopy, and Fourier-transform infrared spectroscopy (FTIR). TEM analysis revealed that spherical NHF obtained of size 20-50 nm. In addition, XRD and FTIR confirmed the presence of phytochemicals with biological properties over the surface of copper oxide-based NHF. It was demonstrated that dose-dependent antiapoptotic activity was shown against DOX-induced cardiomyocytes, where ROS levels were significantly reduced to 0.29% from 37.99%. The results of the flow cytometry analysis using PI and Annexin staining further confirmed the antiapoptotic activity of NHF against DOX-induced cardiomyocytes by ROS scavenging. Thus, NHF might be used for cardiovascular disease treatment.

Gene Electrotransfer into Mammalian Cells Using Commercial Cell Culture Inserts with Porous Substrate.

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we expand the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. We first use multiscale numerical modeling to determine the pulse parameters, substrate pore size, and other factors that are expected to result in successful gene electrotransfer. Based on the numerical results, we design a simple device combining an insert with substrate containing pores with 0.4 µm or 1.0 µm diameter, a multiwell plate, and a pair of wire electrodes. We test the device in three mammalian cell lines and obtain transfection efficiencies similar to those achieved with conventional bulk electroporation, but at better cell viability and with low-voltage pulses that do not require the use of expensive electroporators. Our combined theoretical and experimental analysis calls for further systematic studies that will investigate the influence of substrate pore size and porosity on gene electrotransfer efficiency and cell viability.

Quality by design-based optimization of formulation and process parameters for berberine nanosuspension for enhancing its dissolution rate, bioavailability, and cardioprotective activity.

Berberine (BER) possesses dissolution rate limited oral bioavailability. The present study deciphers the formulation of nanosuspension loaded with BER for enhancing its cardioprotective potential. The nanosuspension was prepared by a liquid antisolvent precipitation technique using sodium lauryl sulfate as a surfactant and polyvinyl pyrrolidone K30 (PVP K30) as a polymer. The optimized formulation showed a particle size of 251.32 ± 4.18 nm, zeta potential of -24.10 ± 1.16 mV, and drug loading capacity of 98.22 ± 2.24%. The results showed about 6.01-fold and 3.54-fold enhancement in the dissolution rate and permeability, respectively, upon loading berberine into nanosuspension. About 8.44-fold increase in Cmax , 27.22-fold increase in AUC0-t , and 27.38-fold increase in AUC0-∞ were observed in the case of BER nanosuspension, compared to its naïve form. The results of particle size, zeta potential, and drug loading showed a nonsignificant change in the response of fresh and aged nanosuspension, which indicated that the formulation was stable. In vitro results on H9C2 cell line indicated a lower cellular proliferation rate after treatment with BER nanosuspension with decreased cytoplasmic expression of angiotensin converting enzyme (ACE) protein. Overall, the results indicated the successful development of BER nanosuspension with an enhanced dissolution rate, permeability, bioavailability, and cardioprotective activity. Practical applications The present study provides the evidence that the formulation of nanosuspension loaded with berberine enhance the cardioprotective activity of berberine. The results of the study supports the improved bioavailability of nanosuspension of berberine showed enhanced cardioprotective activity.

Azadirachta indica-wrapped copper oxide nanoparticles as a novel functional material in cardiomyocyte cells: An ecotoxicity assessment on the embryonic development of Danio rerio.

This research reports on the production of copper oxide nanoparticles (CuO NPs) through the green synthesis method using Azadirachta indica (Ai) flower extract. Synthesized Ai-CuO NPs are characterized by Zeta Potential, TGA, SEM and TEM analysis. The Ai-CuO NPs gave a maximum peak at 270 nm. As per XRD studies, the Ai-CuO NPs obtained were crystalline. FTIR spectrum Ai-CuO NPs showed the presence of functional groups like the O-H group, aromatic group, etc. TEM and SEM assist in investigating the size and morphology of the Ai-CuO NPs, which were spherical and varied in size between 10.11 nm and 17.54 nm. EDAX showed that Ai-CuO NPs were pure with no impurities. The synthesized Ai-CuO NPs were then analyzed for their cytotoxicity at various concentrations (5, 10, 20, 30, 40 and 50 µg/mL) against H9c2 cardiomyocyte cells using MTT assay. DOX-induced H9c2 cell damage of apoptosis and ROS. The nanoparticle formed by Ai-CuO was cured with different concentrations (5, 10 and 20 µg/mL). In zebrafish, 48 hpf and 72 hpf were measured at 75 µM to reduce dysfunction and mortality during organ development. These results can have a beneficial impact on eco-toxicological effects.

Direct cardio-protection of Dapagliflozin against obesity-related cardiomyopathy via NHE1/MAPK signaling.

Obesity is an important independent risk factor for cardiovascular diseases, remaining an important health concern worldwide. Evidence shows that saturated fatty acid-induced inflammation in cardiomyocytes contributes to obesity-related cardiomyopathy. Dapagliflozin (Dapa), a selective SGLT2 inhibitor, exerts a favorable preventive activity in heart failure. In this study, we investigated the protective effect of Dapa against cardiomyopathy caused by high fat diet-induced obesity in vitro and in vivo. Cultured rat cardiomyocyte H9c2 cells were pretreated with Dapa (1, 2.5 µM) for 1.5 h, followed by treatment with palmitic acid (PA, 200 µM) for 24 h. We showed that Dapa pretreatment concentration-dependently attenuated PA-induced cell hypertrophy, fibrosis and apoptosis. Transcriptome analysis revealed that inhibition of PA-activated MAPK/AP-1 pathway contributed to the protective effect of Dapa in H9c2 cells, and this was confirmed by anti-p-cJUN fluorescence staining assay. Using surface plasmon resonance analysis we found the direct binding of Dapa with NHE1. Gain and loss of function experiments further demonstrated the role of NHE1 in the protection of Dapa. In vivo experiments were conducted in mice fed a high fat diet for 5 months. The mice were administered Dapa (1 mg·kg-1·d-1, i.g.) in the last 2 months. Dapa administration significantly reduced the body weight and improved the serum lipid profiles. Dapa administration also alleviated HFD-induced cardiac dysfunction and cardiac aberrant remodeling via inhibiting MAPK/AP-1 pathway and ameliorating cardiac inflammation. In conclusion, Dapa exerts a direct protective effect against saturated fatty acid-induced cardiomyocyte injury in addition to the lowering effect on serum lipids. The protective effect results from negative regulating MAPK/AP-1 pathway in a NHE1-dependent way. The current study highlights the potential of clinical use of Dapa in the prevention of obesity-related cardiac dysfunction.

Effects of levosimendan on the hypoxic / reoxygenated cardiomyocyte fibrosis by regulating long chain noncoding RNA / microRNA-641 / 解剖学报

[Abstract] Objective To investigate whether levosimendan (Lev) affects hypoxia / reoxygenation (H / R) - induced cardiomyocyte proliferation, apoptosis and fibrosis by regulating the molecular axis of long chain noncoding RNA (LncRNA) eosinophil granule ontogeny transcript (EGOT) / microRNA (miR) -641. Methods Rat cardiomyocytes H9C2 were cultured in vitro, and H / R-treated cells were used to establish cell damage models, which were randomly divided into control group, H / R group, H / R + Lev 1 μmol / L (H / R + Lev-L) group, H / R + Lev 5 μmol / L (H / R + Lev-M) group, and H / R + Lev 10 μmol / L (H / R + Lev-H) group, 9 samples per group. MTT method was used to detect cell proliferation. Flow cytometry was used to detect the apoptosis rate. Real-time P CR was used to detect the expression levels of EGOT and miR-641 mRNA. P cDNA-EGOT and EGOT small interfering RNA (si-EGOT) were transfected into H9 C2 cells respectively, and the cell proliferation and apoptosis rates were detected by the above method. The dual luciferase report experiment verified the targeting relationship between EGOT and miR-641. Western blotting was used to detect the expression levels of Bax, Bcl-2, collagen I (colI), collagen Ⅲ (col Ⅲ), tissue inhibitor of matrix metalloproteinase 2 (TIMP 2), matrix metalloproteinase-2 (MMP -2) . Results Compared with the control group, the cell survival rate of the H / R group reduced significantly (P < 0. 05), the apoptosis rate increased significantly (P < 0. 05), and the protein levels of Bax, c I, col Ⅲ, TIMP 2, and MMP -2 increased significantly (P < 0. 05), the level of Bcl-2 protein reduced significantly (P < 0. 05), the expression level of EGOT reduced significantly (P < 0. 05), the expression level of miR-641 increased significantly (P < 0. 05) . Compared with the H / R group, the cell survival rate of the H / R + Lev-L group, H / R + Lev-M group, and H / R + Lev-H group increased significantly (P < 0. 05), and the apoptosis rate decreased significant (P < 0. 05), the protein levels of Bax, colI, colⅢ, TIMP 2, MMP -2 reduced significantly (P < 0. 05), the level of Bcl-2 protein increased significantly (P < 0. 05), the expression level of EGOT increased significantly (P < 0. 05), the expression level of miR-641 reduced significantly (P < 0. 05), and each index of H / R + Lev-L group, H / R + Lev-M group, H / R + Lev-H group, the difference was statistically significant (P < 0. 05) . The dual luciferase report experiment confirmed that EGOT ccould target and bind to miR-641. The effect of transfecting pcDNA-EGOT and Lev was similar. Transfection of si-EGOT could reduce the effect of Lev on H / R-induced proliferation, apoptosis and fibrosis of H9 C2 cells. Conclusion Levosimendan may promote H / R-induced H9 C2 cell proliferation and inhibit apoptosis and fibrosis by up-regulating EGOT expression and down-regulating miR-641 expression.

[Pharmacokinetic study of Polygonum orientale extract in H9c2 cells by UPLC-MS/MS].

A detection method of ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was established to detect concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-ß-D-glucoside in H9 c2 cells and applied to the pharmacokinetic study of Polygonum orientale extract in the cells. H9 c2 cells were treated with 100 µg·mL~(-1) P. orientale extract and then they and the corresponding nuclei, mitochondria and Golgi bodies were collected at the set time. After protein precipitation, UPLC-MS/MS was used to determine concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-ß-D-glucoside in the whole cells and subcellular structures. Also, related pharmacokinetic parameters were calculated. The results showed that the peak time was 8 h for all these components. Orientin, vitexin, quercetin and isoorientin have high affinities to nuclei and mitochondria, while the affinity of kaempferol-3-O-ß-D-glucoside is higher with mitochondria compared to nuclei. It is suggested that these chemical components of P. orientale may mainly act on nuclei or mitochondria to exert pharmacological effects of protecting cardiomyocytes.

Long Non-Coding RNA SOX2 Overlapping Transcript Aggravates H9c2 Cell Injury via the miR-215-5p/ZEB2 Axis and Promotes Ischemic Heart Failure in a Rat Model.

Heart failure is a common cardiovascular disease, which has been regarded as one of the highest health care costs with high morbidity and mortality in the western countries. Long noncoding RNAs have been widely reported to regulate the initiation or progression of cardiovascular diseases. However, the specific role of SOX2 overlapping transcript (SOX2-OT) in ischemic heart failure remains uncharacterized. The present study aimed to explore the function and mechanism of SOX2-OT in ischemic heart failure. The starBase website was used to predict potential miRNAs or target mRNAs. Western blot assay was implemented to test collagen protein levels. Functional assays were conducted to evaluate the effects of SOX2-OT on H9c2 cell viability and apoptosis. RNA pull down and luciferase reporter assays were used to confirm the combination between miR-215-5p and SOX2-OT. We found out that SOX2-OT level was increased by oxygen glucose deprivation/reoxygenation treatment in H9c2 cells. Silencing of SOX2-OT ameliorated cell injury by promoting cell viability, inhibiting cell apoptosis and reducing productions of collagens. Mechanistically, miR-215-5p was confirmed to bind with SOX2-OT after prediction and screening. In addition, we discovered that miR-215-5p negatively regulated zinc finger E-box binding homeobox 2 (ZEB2) protein level by directly binding with ZEB2 3' untranslated region. Finally, we verified that SOX2-OT aggravated cell injury by targeting ZEB2 in H9c2 cells. In conclusion, SOX2-OT aggravated heart failure in vivo and promoted H9c2 cell injury via the miR-215-5p/ZEB2 axis in vitro, implying a novel insight into heart failure treatment.

Purified antioxidant from the medicinal mushroom Phellinus pini protects rat H9c2 cell against H2 O2 -induced oxidative stress.

In this study, through the combination of AB-8 macroporous resin, Sephadex LH-20 column chromatography and semi-preparative HPLC, an antioxidant component was purified from the crude extract of Phellinus pini, thereby evaluating the cardioprotective effect of the fraction. As a result, total phenolic content of the 60% ethanol elution was increased by 4.8-fold after one run treatment on Sephadex LH-20 chromatography with gradient elution. After semi-preparative HPLC separation, the first peak (PP-S4-1) showed that inhibition ratio of erythrocyte hemolysis was 91.9%, and inhibition ratio of lipid peroxidation was also increased by 87.6%, at 50 µg/ml (p < .01). Based on the results of ESI-MS, 1 HNMR, 13 CNMR, and RP-HPLC compared to many published results, PP-S4-1was identified as catechin (MW 290.015, C15 H14 O6 ). The results showed that PP-S4-1 pretreatment made cell viability increased, and the generation of reactive oxygen species (ROS) inhibited. Meanwhile, PP-S4-1 remarkably decreased the fluorescence intensity of Ca2+ , and increased mitochondrial membrane potential (MMP; ΔΨm). In addition, PP-S4-1 could significantly inhibit the decrease of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity as well as the increase of MDA content in H9c2 cells induced by H2 O2 . Moreover, pretreatment with PP-S4-1 significantly improved the morphological changes and prevented H2 O2 -induced DNA damage. Therefore, this study clarifies the ability of PP-S4-1 to treat H9c2 cell oxidative stress damage induced by H2 O2 through its antioxidant effect. PRACTICAL APPLICATIONS: This research is not only helpful to elaborate the cardioprotective effect of Phellinus pini but also can contribute to the development of health foods or drug supplements for heart disease in the future. This is the first report dealing with phenolic component and cardioprotective activity of a medicinal mushroom P. pini belonging to the genus Phellinus.

Metformin protects cardiomyocytes against oxygen-glucose deprivation injury by promoting autophagic flux through AMPK pathway.

Metformin has been shown to protect myocardial ischaemia/reperfusion or hypoxia/reoxygenation injury. In our current study, we investigated the effects of metformin on autophagy and its possible underlying mechanisms in in vivo myocardial infarction (MI) model and in vitro oxygen-glucose deprivation (OGD) model. A rat model of MI was made by ligating coronary artery in vivo study. Metformin (200 mg/kg/day) could improve cardiac function, prevent rats from MI-induced injury by reducing myocardial infarct size and apoptosis. Moreover, metformin furtherly promoted autophagy by increasing the protein expression of LC3-II, ATG5, ATG7 and Beclin1, and by involving AMPK pathway during MI. H9c2 cells were treated with metformin (4 mM) in vitro study to assess its effects after exposure to OGD. Metformin increased cell viability and inhibited OGD-induced LDH synthesis and cell apoptosis. Furthermore, metformin increased autophagosome formations as well as expression of autophagy-related proteins, promoted autophagic flux. In addition, metformin augmented the protein level of Bcl-2 and diminished the protein levels of Bax and cleaved caspase-3. Metformin also upregulated p-AMPK expression. Nevertheless, the above-mentioned effects of metformin on H9c2 cells were remarkably eliminated by compound C (an AMPK inhibitor). In summary, we displayed that metformin protected cardiomyocytes against OGD-induced injury and apoptosis by promoting autophagic flux through the AMPK pathway.