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[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.
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Objective To investigate the process of autophagy in myocardial cells induced by methamphetamine (METH).Methods In vivo study:sixty 6-week-old male C57B1/6 J mice were randomly(random number) divided into three groups evenly,control group,three-day METH treated group and seven-day METH treated group.Mice in control group was given physiological saline through intraperitoneal injection 2 times per day and lasted 7 days.Mice in three days group and seven days group intake methamphetamine at a dose of 15 mg/kg every time through intraperitoneal injection 2 times a day,lasted 3 days and 7 days respectively.The hearts of the mice were then obtained by anatomical method 24 hours after the last intraperitoneal injection of METH,then autophagy related proteins were detected by western blotting.In vitro study:the model was established by H9C2 cells.The cells were divided into two groups,control group (cells were cultured by normal medium) and METH group (cells were cultured by medium includes 900 mmol/mL METH for 24 hours).The expressions change of autophagy related proteins in cells were tested by Western blotting.Additionally,LC3-Ⅱ was tagged by red fluorescent and then the stained cells were visualized under a Zeiss LSM710 confocal microscope.Furthermore,the numbers of autophagosomes in cells were visualized by transmission electron microscopy.Results The expression of p62,Beclin-land LC3 were significantly increased in METH group when compared with control group (P＜0.05).The level of LC3 was significantly increased in METH treated group compared with control group visualized under a Zeiss LSM710 confocal microscope.The numbers of autophagosomes in METH group are more than control group visualized by transmission electron microscopy.Conclusions Autophagy can be induced by METH in myocardial cells.
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OBJECTIVE To evaluate the cardiotoxicity of a widen-spectrum antineoplastic drug, gambogic acid, through quantitative multiple cellular phenotypic characterization. METHODS H9c2 cell line was used as a model with doxorubicin (Dox) and amiodarone (Ami) as positive controls, hypaconi?tine as negative control and 0.1% DMSO as normal control. An optimized protocol was established to identify the morphology and function of cell nuclei. The effect of drugs on cell viability, nuclear area (Hoechst33342), mitochondria mass (MitoTracker Deep Red) and cytoplasmic calcium ion mobilization (Rhod2 AM)was studied. EC50 and Z′values were calculated to evaluate the degree of toxicology and to estimate the precision and false-positive rate, respectively. RESULTS Dose-response analysis indicated that EC50 of Dox on cell viability, nuclear area, mitochondrial mass was 0.72, 0.014 and 1.21μmol · L-1, respectively. On the other hand, EC50 of Ami on the parameters of cell viability, nuclear area and mitochon?drial mass was 14.83, 6.72 and 4.54μmol·L-1, respectively with Z′value above 0.5. Hypaconitine decreased the SER ridge of mitochondria. Gambogic acid caused significant mortality of H9c2 cells and induced nuclear shrinkage as Ami did. The EC50 values of cell viability and nuclear area were 0.24 and 1.16 μmol · L- 1. Meanwhile,gambogic acid disturbed the mitochondrial function as indicated by the increased mitochondrial area (EC50=0.44 μmol · L-1), abnormal SER Ridge(EC50=0.99 μmol · L-1) and decreased mitochondrial mass(EC50=1.21 μmol · L- 1). Cellular calcium mobilization was lower than normal (EC50=0.41 μmol · L-1). CONCLUSION The EC50 values of positive controls calculated from our assessment are similar those reported in literature. A multi-parameter and simultaneous evaluation enables a comprehensive analysis of the morphology of nuclei and mitochondria of cardiomyocytes and a preliminary assessment of the mechanisms of toxicity.
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OBJECTIVE: To investigate the effect of genipin on hypoxia/reoxygenation induced oxidative stress injury in H9c2 cell line. METHODS: For mimicking myocardial ischemia/reperfusion (I/R) injury, H9c2 cells were cultured in vitro and established hypoxia/reoxygenation induced oxidative stress injury model. H9c2 cells were divided into six groups: control group, H/R group, H/R+0.5 μmol·L-1 genipin group, H/R+1.25 μmol·L-1genipin group, H/R+2.5 μmol·L-1 genipin group and H/R+10 μmol·L-1 genipin group. Cell viability was detected by cell counting kit-8 assay (CCK-8). The levels of lactate dehydrogenase (LDH), intracellular total superoxide dismutase (T-SOD) and malondialdehyde (MDA) were assessed using microplate reader. Confocal laser scanning microscope was performed to examine the production of reactive oxygen species (ROS) and the level of mitochondrial calcium (m) as well as the depolarization ratio of mitochondrial membranes potential(ΔΨm). The protein expression of cytochrome-C was detected by Western-blot. RESULTS: Comparing to control group, the cell viability of H/R group was significantly decreased in a time-dependent manner(r=-0.82,P<0.01). Low concentration(1.25-40 μmol·L-1) of genipin could improve cell viability exposed to H/R treatment (P<0.05), on the contrary, high concentration(80-320 μmol·L-1) of genipin remarkably reduced the cell viability (P<0.05). In compared with H/R group, the levels of LDH release, MDA production and ROS production, the level of m, depolarization ratio of ΔΨm and the protein expression of cytochrome-c in H/R+(1.25-10 μmol·L-1) genipin groups were notably lessened, while the level of T-SOD was increased(P<0.01 or P<0.05). CONCLUSION: Genipin could reduce H/R-induced oxidative stress injury, the mechanism might be connected with balancing the oxidative stress products and anti-oxidation enzyme system as well as improving mitochondrial dysfunction.