Cardiovascular ischemic disease refers to a large class of conditions that are harmful to human health. A number of previous studies have demonstrated that microRNAs (miRs) have notable roles in regulating cardiac injury. miR-144 is influential in the differentiation, growth, and metastatic processes of cells; however, the impact of miR-144 in cardiac ischemia/reperfusion (I/R) injury has not been thoroughly elucidated to date. In the present study, reverse transcription quantitative polymerase chain reaction was used to evaluate RNA expression. In addition, TTC staining was performed to detect the infarct area of the ischemic myocardia and a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay was utilized to detect the apoptosis of the myocardia. It was observed that miR-144 expression is downregulated in an I/R model in rats and that overexpression of miR-144 significantly reduced myocardial ischemic injury and apoptosis. Consistent with this result, similar findings were demonstrated in H9c2 cells subjected to hypoxia/reoxygenation. Bioinformatic analysis using MiRanda and TargetScan, and luciferase assays confirmed that forkhead box protein O1was the target of miR-144. These findings suggest that miR-144 may be exploited as a novel molecular marker or therapeutic target for myocardial I/R injury.
Context Simvastatin is the first line therapeutic drug for coronary heart disease and atherosclerosis. The protective effect mechanism of simvastatin on cardiomyocytes is unclear.Objective This study explores the effect of simvastatin on high glucose induced cardiomyocyte injury and the role of autophagy during the process.Materials and methods H9c2 cells were incubated with different doses of glucose (0, 50, 100, 200 mM) for 24 h to verify the glucose induced injury. The H9c2 cells were pre-treated with simvastatin at different dosages (0, 0.1, 0.5, 1 μM) for 30 min to rescue the injury followed by the autophagy evaluation. 3-MA was used as an autophagy inhibitor to confirm the role of autophagy in simvastatin treated process. CCK-8 assay, FACS assay, confocal microscopy, western blotting and immunofluorescence analysis were conducted to evaluate the high glucose induced injury or protective effects of simvastatin in H9c2 cell line.Results High glucose dramatically decreased H9c2 cell viability (0 mM, 0.58 ± 0.09%; vs. 50 mM, 8.67 ± 0.43%; 100 mM, 16.1 ± 3.56%; 200 mM, 32.9 ± 2.63%), induced significant cell apoptosis (0 mM, 0.96 ± 0.16%, vs. 50 mM, 7.00 ± 0.63%; 100 mM, 12.9 ± 0.78%; 200 mM, 21.8 ± 1.17%) and suppressed cell autophagy. Simvastatin decreased apoptosis and attenuate injury by decreasing cell apoptosis ratio, elevating Bcl-2 expression while decreasing Bax and caspase-3 protein expressions. Meanwhile, simvastatin restored the autophagy depicted by western blotting with increased ATG-5, Beclin1 and LC3II/LC3I protein expression and decreased p62 expression, as well as immunofluorescence with elevated LC3 fluorescence density.Discussion and conclusions The myocardial protective effect mediated by autophagy activated by simvastatin to some extent elucidated the mechanism of the protective effect of simvastatin on H9c2 cell injury, which provided a certain theoretical basis for the clinical application of simvastatin in the treatment of cardiovascular diseases. In addition, we speculate that simvastatin may be used for diabetes associated cardiovascular diseases.
To investigate the protective effect of high-density lipoprotein (HDL) on the mice cardiac myocytes induced by oxygen and glucose deprivation (OGD).Cardiac cells of primary scavenger receptor-B1 knockout mice (SR-B1-/-) and normal C57 mice (SR-B1+/+) were obtained by protease digestion and differential adhesion method. (1) The two kinds of cells were divided into normal control group (Con group), OGD group, OGD+HDL group. Propidium iodide (PI) staining were used to determine the necrosis of cardiac myocytes. (2) SR-B1+/+ cardiac cells were divided into Con group, OGD group, OGD+HDL group, and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) inhibitor LY294002 group. PI staining were used to determine the necrosis of cardiac myocytes. TUNEL staining was used to determine the cell apoptosis. The kit was used to determine the contents of MB isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) in the culture medium supernatant. The expressions of SR-B1 and Akt protein were determined by Western Blot.(1) In SR-B1+/+ cardiomyocytes, HDL could inhibit cell necrosis induced by OGD. There was no protective effect of HDL on OGD in the SR-B1-/- cardiomyocytes. (2) The study of SR-B1+/+ cells was showed that compared with Con group, necrotic cells were significantly increased and cell activity were significantly decreased, the cell viability were significantly decreased, the contents of LDH and CK-MB in supernatant were significantly increased, the expressions of phosphorylated Akt (p-Akt) and SR-B1 were significantly decreased in OGD group. Compared with OGD group, the number of necrotic cells in the OGD+HDL group was significantly decreased [PI positive cells rate: (26.71±5.94)% vs. (64.24±18.34)%], the cell activity was significantly increased [(63.84±6.95)% vs. (26.71±5.13)%], the contents of LDH and CK-MB in supernatant were significantly decreased [LDH (U/L): 896.3±161.5 vs. 1 568.3±243.5, CK-MB (U/L): 304.3±72.9 vs. 583.6±81.6], the expressions of p-Akt and SR-B1 were significantly increased (p-Akt/t-Akt: 0.84±0.13 vs. 0.18±0.06, SR-B1/β-actin: 1.23±0.19 vs. 0.09±0.02), with statistically significant differences (all P < 0.05). Compared with OGD+HDL group, necrotic cells in LY294002 group were increased, cell activity was decreased, LDH and CK-MB contents in supernatant were increased, p-Akt and SR-B1 expressions were decreased; there was no statistical difference between LY294002 group and OGD group. There was no significant difference in cell apoptosis among the 4 groups.HDL has protective effect on the mice myocardial cells. The mechanism may be related with the up regulation of the expression of SR-B1 protein by the activation of PI3K/Akt pathway.
To investigate the effect of high mobility group protein B1 (HMGB1) inhibition on endoplasmic reticulum stress (ERS) after myocardial ischemia/reperfusion (I/R) in rats.Forty male Sprague-Dawley (SD) rats were randomly divided into four groups (n = 10): sham operation group, I/R model group, Gene silencing (HMGB1-siRNA) group, and empty vector (Scrambled-siRNA) group. Coronary blood flow of the rats were ligated for 30 minutes, relaxed the ligament line for 2 hours, to establish I/R injury model; not ligation with the sham operation group. Each group was injected 1 mL phosphate buffer (PBS), HMGB1-siRNA mixture or Scrambled-siRNA mixture preoperative by tail vein 0 hour, 12 hours, and 24 hours before surgery. After 2 hours reperfusion, the levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-8) in the serum were detected by enzyme linked immunosorbent assay (ELISA); the expression of HMGB1 protein in myocardium was detected by immunohistochemistry; the protein and mRNA expressions of HMGB1, GRP78, CHOP and caspase-12 in myocardium were detected by Western Blot and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR).Compared with sham operation group, the levels of serum inflammatory factor, HMGB1 positive cells, and the protein and mRNA expressions of GRP78, CHOP, caspase-12 were significantly increased in I/R model group. The levels of serum inflammatory factor in HMGB1-siRNA group were significantly lower than those in the I/R model group [TNF-α (ng/L): 783.4±203.4 vs. 963.9±214.1, IL-6 (ng/L): 358.8±94.8 vs. 452.3±103.7, IL-8 (ng/L): 180.5±73.6 vs. 347.3±90.3, all P < 0.05], HMGB1 positive cells, and the protein and mRNA expressions of GRP78, CHOP, caspase-12 in HMGB1-siRNA group were significantly lower than I/R model group (HMGB1 protein: 1.59±0.26 vs. 3.21±0.40, GRP78 protein: 2.59±0.28 vs. 4.21±0.42, CHOP protein: 2.01±0.23 vs. 3.21±0.43, caspase-12 protein: 1.48±0.22 vs. 3.01±0.48; HMGB1 mRNA: 2.35±0.26 vs. 4.67±0.45, GRP78 mRNA: 6.59±0.26 vs. 11.21±0.40, CHOP mRNA: 2.01±0.43 vs. 5.21±0.63, caspase-12 mRNA: 4.48±0.32 vs. 8.41±0.52, all P < 0.05). There was no significant difference between the Scrambled-siRNA group and the I/R model group.HMGB1 may be involved in the activation of ERS in myocardial I/R injury and increase the damage of myocardial cells.
Myocardial ischemia-reperfusion injury is accompanied by ferroptosis mediated by reactive oxygen species and iron ions, which aggravates myocardial tissue damage. The present study aims to explore the molecular mechanism underlying the mitigating effects f PCSK9 on myocardial ischemia-reperfusion injury. MI/R rat model and OGD/R induced H9c2 model were established. The interaction between PCSK9 inhibitor and LRP8 was predicted by STRING database and verified by Immunoprecipitation assay experiment. CCK-8 kit results confirmed that PCSK9 inhibitor effectively protected against cardiomyocyte damage induced by OGD/R. TTC and histological examination via H&E staining revealed a significant alleviation of myocardial infarction and pathological alterations upon treatment with the PCSK9 inhibitor. Besides, DCFH-DA staining and biochemical kit results showed that PCSK9 inhibitor could regulate the changes of ferroptosis related indicators [ROS, iron level, MDA, SOD] and inhibit ferroptosis. Rescue experiments showed that PCSK9 inhibitors targeted LRP8 expression and inhibited GPX4/ROS-mediated ferroptosis in I/R-induced rats. Our study suggested that PCSK9 inhibitors could attenuate myocardial I/R injury, with the underlying mechanism intimately tied to the targeted modulation of LRP8/GPX4-mediated ferroptosis.
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