H9c2 cells (Shanghai Cell Bank, Chinese Academy of Sciences, stock no. GNR5) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin in an incubator (ESCO CelMate,CLM-170B-8-NF) at 37˚C and 5% CO₂. To establish the hypoxia-reperfusion (H/R) model, cells were first cultured under anaerobic conditions (94% N₂, 5% CO₂ and 1% O₂) for 4 h, and then transferred to a conventional oxygen incubator for 4 h for reoxygenation, with cells cultured under conventional oxygen conditions serving as controls. After successful modeling, cells were treated with 0.5, 1, 2, 4, 8 and 16 μM concentrations of atorvastatin (purchased from Tocris, UK) to screen the best concentration of atorvastatin for subsequent experiments.

The miR-26a-5p mimic, miR-26a-5p inhibitor or its negative control NC-mimic (both purchased from GeneChem, Shanghai, China) were transfected into H9c2 cells using the Lipofectamine™ 2000 transfection kit (Invitrogen, USA) according to the kit instructions. The transfection efficiency was verified after incubation at 37˚C and 5% CO₂ for 6 hours. The transfected cells were collected for subsequent experiments. The FOXO1 sequence was subcloned into pcDNA3.1 vector (Invitrogen, USA) to construct pcDNA3.1-FOXO1-GFP (pcDNA-FOXO1) and transfected with empty pcDNA3.1 (pcDNA-NC) as a control. pcDNA-FOXO1 and pcDNA-NC vectors were both synthesized in Shanghai, China. GenePharma synthesized.

Total RNA was extracted from cardiomyocytes using Trizol reagent (Invitrogen, USA). 2 μg of RNA was reverse transcribed into cDNA using cDNA synthesis kit (TaKaRa, Japan) based on cDNA sequences obtained from GenBank. The quantitative PCR (qPCR) experiments were performed using the MiniOpticon qPCR detection system (Bio-Rad Laboratories). Using GAPDH or U6 as reference control. Relative quantification results were calculated according to the 2^−ΔΔCT formula(2^−ΔΔCT = 2^{−(experimental group ΔCt − control group ΔCt)}, ΔCt = Ct target gene -Ct internal reference gene). Primer Premier 5 and miRNA Design V1.01 software were used to design primers. The primer sequence is as follows (see Table 1):

Frozen RIPA lysis buffer (Shanghai Biyuntian Biotechnology, China) was added to H9c2 cardiomyocytes. Cells were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes and lysed with FOXO1 (1:1000, ab179450, Abcam), Bax (1:1000, ab32503, Abcam), Bcl-2 (1:1000, ab32124, Abcam), caspase-3 (1:5000, ab32351, Abcam), Cytochrome C (1:5000, ab133504, Abcam), and β-actin (1:1000, ab8226, Abcam) overnight at 4˚C. After washing, membranes were treated with the corresponding secondary antibodies and incubated at 25˚C for 1 h. β-actin was used as an internal control. Protein blot imaging was viewed using the Bio-Rad VersaDocTM Imaging System (Bio-Rad Laboratories,

Freiburg, Germany).

H9c2 cells were cultured in 96-well plates at 1 × 10³ cells/well. Cell counting kit-8 (MedChem Express, USA) was used for analysis according to the kit instructions. After 48 h of cell culture, 10 μL CCK-8 reagent was added and the plates were incubated at 37˚C for another 2 h. The absorbance at 450 nm was then measured using an enzyme marker (BIO-TEK, ELX800), and cell proliferation viability was calculated.

Apoptosis was assessed using a flow cytometer (Beckman Coulter, USA) and Annexin V-FITC Apoptosis Detection Kit (Procell Life Science & Technology, China) to determine the percentage of apoptotic cells. A total of approximately 1 × 10⁶ cells were suspended in 200 µL binding buffer, 10 µL Annexin V-Fluorescein isothiocyanate and 5 µL propidium iodide (BD Biosciences, Germany) were added to the cells, followed by incubation for 30 min at room temperature and protected from light. Apoptosis was assessed using flowjo V10 software (BD Biosciences).

Cell supernatants were collected and CK and LDH activities were measured using the CK (Catalog No. A032) and (Catalog No. A020-1) LDH assay kits (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) according to the kit instructions.

The binding site of miR-26a-5p and FOXO1 gene was predicted utilizing (https://starbase.sysu.edu.cn/). After construction of the FOXO1-WT and FOXO1-MUT vectors, Lipofectamine2000 (Invitrogen) was used to transfect the vectors and miR-26a-5p mimic into cells. Cells were harvested 48 hours after transfection and lysed using Lysis Buffer (Beyotime, China), followed by measurement of luciferase activity with a dual-luciferase reporter assay system (Promega). Two parallel holes were set in each group, and the experiment was repeated three times.

All experimental data were processed and analyzed using GraphPad Prism 7 software, and the results were expressed as mean ± standard deviation (SD). The t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. Differences were statistically significant when P < 0.05.

To verify the expression of miR-26a-5p and FOXO1 in H/R-induced cardiomyocytes, the expression of miR-26a-5p in H/R-induced cardiomyocytes was significantly decreased (P < 0.001) as detected by RT-qPCR assay, see Figure 1(A). Western blot assay showed that FOXO1 in H/R-induced cardiomyocytes was significantly increased (P < 0.001), see Figure 1(B). As seen, the induction of H/R resulted the abnormal expression of miR-26a-5p and FOXO1 in cardiomyocytes.

To investigate the effect of atorvastatin in the treatment of myocardial ischemia-reperfusion injury, the effect of atorvastatin on cell viability was examined using CCK-8. The results showed that treatment with different concentrations of atorvastatin revealed no significant effect on the viability of H9c2 cells when the concentration of atorvastatin was less than 2 μM, and cell viability decreased significantly with increasing concentrations when the concentration of atorvastatin was higher than 2 μM (Figure 2(A)). In addition, the therapeutic effects of concentrations of 0.5 μM, 1 μM, and 2 μM atorvastatin on myocardial ischemia-reperfusion injury were examined (Figure 2(B)). CCK-8 results showed that cell viability was significantly inhibited in the H/R group compared with the NC group, and cell viability was significantly increased after treatment with 1 μM atorvastatin.The results of CK and LDH activity assays showed that after induction of H/R, the cellular CK and LDH activities were significantly higher than those in the NC group (P < 0.001), and the activities of CK and LDH were significantly decreased after the addition of 1 μM atorvastatin (P < 0.001), as shown in Figure 2(C) and Figure 2(D). Western blot experiments showed that the protein expression of Bax, caspase-3 and cyto-c were significantly increased

after the induction of H/R (P < 0.01) and the protein expression of Bcl-2 was significantly decreased (P < 0.01); while the protein expression of Bax, caspase-3 and cyto-c was least elevated and the protein expression of Bcl-2 was least decreased after treatment with 1 μM atorvastatin (Figure 2(E)). By detecting the apoptosis rate using flow cytometry, it was found that the apoptosis rate was significantly increased after H/R induction (P < 0.01), while the apoptosis rate was significantly decreased after the addition of atorvastatin treatment (P < 0.01), as shown in Figure 2(F). It is thus clear that atorvastatin can alleviate the H/R injury in cardiomyocytes, with 1 μM atorvastatin being the most effective, so subsequent experiments were performed using 1 μM atorvastatin.

To explore the molecular mechanism of atorvastatin to alleviate H/R injury in cardiomyocytes, we transfected miR-26a-5p inhibitor into H9c2 cells. As detected by RT-qPCR assay, the expression of miR-26a-5p in cells was significantly decreased after transfection of miR-26a-5p inhibitor (P < 0.01) and increased after addition of atorvastatin (P < 0.01), as shown in Figure 3(A). The results of CCK-8 assay showed that after transfection of miR 26a-5p inhibitor resulted in a significant decrease in cell viability (P < 0.05) and an increase in cell viability after treatment with atorvastatin (P < 0.001), see Figure 3(B). CK and LDH activities were detected using CK and LDH kits, and the results showed that after

transfection with miR-26a-5p inhibitor, CK and LDH activities were significantly increased (P < 0.001), and the addition of atorvastatin significantly decreased CK and LDH activities (P < 0.001), see Figure 3(C), 3 Figure 3(D). To detect the effect of atorvastatin on apoptosis of H9c2 cells, the apoptosis-related proteins and apoptosis rate were detected using western blot and flow cytometry, respectively. The results of western blot experiment showed that the protein expression of Bax, caspase-3 and cyto-c was significantly increased (P < 0.01) and that of Bcl-2 was significantly decreased (P < 0.05) after the transfection of miR-26a-5p inhibitor; The protein expression of Bax, caspase-3 and cyto-c was decreased after the addition of atorvastatin (P < 0.01) and protein expression of Bcl-2 was increased (P < 0.01), see Figure 3(E). Flow cytometry results revealed that apoptosis rate was increased after transfection with miR-26a-5p inhibitor, while apoptosis rate was significantly decreased after the addition of atorvastatin treatment (P < 0.001), see Figure 3(F). It is evident that atorvastatin can alleviate H/R injury in cardiomyocytes through upregulation of miR-26a-5p expression.

To verify the targeting relationship between miR-26a-5p and FOXO1, we used the StarBase website to predict the binding site of miR-26a-5p to FOXO1 (Figure 4(A)). As detected by dual luciferase reporter gene assay, miR-26a-5p reduced the luciferase activity of wild-type FOXO1 (P < 0.01) and had almost no effect on the luciferase activity of mutant FOXO1 (Figure 4(B)). The transfection efficiency of pc-DNA3.1-FOXO1 was examined by RT-qPCR assay, and the results showed that transfection of pc-DNA3.1-FOXO1 significantly increased the expression of FOXO1 (P < 0.001) (Figure 4(C)). The mRNA and protein expression levels of FOXO1 were detected by RT-qPCR and Western blot assays, respectively, and both results showed that the mRNA and protein expression of FOXO1 were significantly decreased (P < 0.001) after transfection with miR-26a-5p mimic, and after transfection with miR-26a-5p inhibitor, FOXO1 mRNA and protein expression were elevated (Figure 4(D), Figure 4(F)). CCK-8 results showed that cell viability was significantly higher after transfection with miR-26a-5p mimic compared to NC group (P < 0.01). Cell viability was reduced after transfection with miR-26a-5p mimic + pc-DNA3.1-FOXO1 compared with the miR-26a-5p mimic group (Figure 4(E)). Western blot detection of apoptosis-related proteins showed that compared with the NC group, the expression of Bax, caspase-3 and cyto-c proteins were significantly reduced (P < 0.01) and the expression of Bcl-2 protein was significantly increased (P < 0.01) after miR-26a-5p mimic transfection, while after transfection with miR-26a-5p mimic + pc-DNA3.1-FOXO1, Bax, caspase-3 and cyto-c protein expression was increased and Bcl-2 protein expression was decreased, indicating that transfection with miR-26a-5p inhibited apoptosis, while FOXO1 decreased this inhibition (Figure 4(G)). In addition, the same results were obtained for apoptosis detection by flow cytometry (Figure 4(H)). CK and LDH activity assays showed that CK

and LDH activities were significantly reduced after transfection with miR-26a-5p mimic compared with the NC group (P < 0.001). In contrast, CK and LDH activities were significantly increased after transfection with miR-26a-5p mimic+pc-DNA3.1-FOXO1 compared with the miR-26a-5p mimic group (P < 0.001), as shown in Figure 4(I) and Figure 4(J). It is evident that miR-26a-5p alleviates H/R injury in cardiomyocytes by targeting negative regulation of FOXO1.

To verify whether atorvastatin could alleviate myocardial ischemia-reperfusion injury through miR-26a-5p/FOXO1, we transfected miR-26a-5p inhibitor into cells and detected the expression of FOXO1 by RT-qPCR and Western blot assay, and the results showed that the expression of FOXO1 in the cells decreased after adding atorvastatin (P < 0.01) (Figure 5(A), Figure 5(B)). And the expression of FOXO1 was significantly increased (P < 0.01) after the addition of atorvastatin in cells transfected with miR-26a-5p inhibitor compared with AT+NC inhibitor group, see Figure 5(A). CCK-8 results showed that the cell viability was significantly increased (P < 0.001) after the addition of atorvastatin. In contrast, cell viability was decreased in cells transfected with miR-26a-5p inhibitor by addition of atorvastatin compared with the AT + NC inhibitor group (Figure 5(E)). CK and LDH activity assay results showed that the addition of atorvastatin significantly decreased CK and LDH activity (P < 0.001), while compared with the AT + NC inhibitor group, CK and LDH activities were increased after the addition of atorvastatin treatment in cells transfected with miR-26a-5p inhibitor (P < 0.001), as shown in Figure 5(C) and Figure 5(D). Western blot assay results showed that the protein expression of Bax, caspase-3, cyto-c and FOXO1 were significantly decreased (P < 0.01) and the protein expression of Bcl-2 increased significantly (P < 0.001); after transfection with miR-26a-5p inhibitor and addition of atorvastatin, the protein expression of Bax, caspase-3, cyto-c and FOXO1 increased and the protein expression of Bcl-2 decreased (Figure 5(F)). The results of flow cytometry apoptosis assay showed that the apoptosis rate was decreased after the addition of atorvastatin (P < 0.001) and increased after the addition of atorvastatin after transfection with miR-26a-5p inhibitor (P < 0.001), as shown in Figure 5(G). Thus, it is clear that atorvastatin can reply to the effect of transfection with miR-26a-5p inhibitor induced of H/R injury in cardiomyocytes, and atorvastatin alleviated H/R injury in cardiomyocytes through miR-26a-5p/FOXO1.