Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210.

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Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210 Yong-Feng Shi, Ning Liu, Yang-Xue Li, Chun-Li Song, Xian-Jing Song, Zhuo Zhao and Bin Liu Doi: 10.3109/10715762.2015.1050588

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Abstract Background: Insulin protects cardiomyocytes from reactive oxygen species-induced apoptosis after ischemic-reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from reactive oxygen species injury. Methods: Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H2O2) or insulin at various concentrations for various times, or insulin and H2O2 for various times. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results: The percentage of viable cells were significantly and reversely associated with H2O2 concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H2O2 or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H2O2-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion: Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H2O2/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.

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Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Yong-Feng Shi1, Ning Liu1, Yang-Xue Li1, Chun-Li Song1, Xian-Jing Song1, Zhuo Zhao1 and Bin Liu1

1

Department of Cardiology, Second Hospital of Jilin University, Jilin University,


Changchun, China

Corresponding Author: Bin Liu, Second Hospital of Jilin University, 218 Ziqiang St, Nanguan, Changchun, Jilin, 130041, China. Tel: +86 431-88796739. Fax: +86 431-88796739. E-mail: [email protected] Abstract Background: Insulin protects cardiomyocytes from reactive oxygen species-induced apoptosis after ischemic-reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from reactive oxygen species injury. Methods: Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H2O2) or insulin at various concentrations for various times, or insulin and H2O2 for various times. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results: The percentage of viable cells were significantly and reversely associated with H2O2 concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H2O2 or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H2O2-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion: Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H2O2/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.

Keywords: Insulin, H2O2, microRNA-210, oxidative stress, Akt

Introduction Oxidative stress in vivo represents an imbalance in cellular free radical production. The free radical is produced by excess formation of reactive oxygen and nitrogen species, and damages lipids, proteins, and nucleic acids [1]

. Reactive oxygen and nitrogen species and hydrogen peroxide (H2O2) have

an important role in heart ischemia/reperfusion damage. H2O2 is a by-product


of ischemia/reperfusion, and a direct free radical donor in oxidative stress injury [2]. It has been reported that endogenous H2O2 has a cardioprotective effect on coronary ischemia/reperfusion injury, as a compensatory mechanism with nitric oxide, by inducing vasodilation [3]. However, the effect of H2O2 on the regulation of gene expression at the translational level in cardiomyocytes is not definitively understood, and especially the influence of microRNAs in oxidative stress injury. MicroRNAs are small non-coding RNAs of approximately 20-23 nucleotides in length, which negatively regulate gene expression in many biological and pathological processes [4]. They regulate gene functions by silencing gene expression via interaction with the 3′ untranslated region of the transcript [5], in approximately 30% of human protein-coding genes [6]. They have also been implicated in the pathophysiology of various cardiovascular diseases [7] and in improvement of hypoxia-induced cell survival [8] or heart function by upregulating angiogenesis and inhibiting apoptosis [9]. Mutharasan et al. [10] indicated a novel role for p53 and Akt in regulating the expression of

the microRNA miR-210, which is induced under hypoxia in a wide range of primary and transformed cells, as protection against oxidant stress. However, our understanding of miR-210 regulation, especially in cardiomyocytes, remains limited. Insulin has been demonstrated to be protective against ischemia-induced injury [11] and apoptosis through the phosphatidylinositol 3 kinase (PI3K)/Akt


pathway [12]. However, whether such effect is through regulation of miR-210 expression in cardiomyocytes upon oxidative stress has not been investigated. We hypothesized that insulin can inhibit oxidative stress-induced apoptosis by upregulation of miR-210 through the PI3K/Akt pathway. The purpose of the present study was to determine whether H2O2 or insulin influences the expression of miR-210 in association with PI3K/Akt activation in rat cardiomyoblast H9c2 cells. Materials and methods H9c2 cell culture Cells of the H9c2 rat myocardial cell line were obtained from the Pathophysiology Laboratory of Jilin University. Cell culture plates were purchased from Corning. The cell culture growth medium was comprised of Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Australia). H9c2 cells were grown in a humidified incubator containing 95% air and 5% CO2 at 37 °C, and subcultured before reaching confluence.

Lentiviral infection of H9c2 cells Replication-deficient lentivirus encoding rat miR-210 precursor (pre-210), miR-210 inhibitor sponge (anti-210), and miR-scramble (miR-scr) were purchased from Sangong Biotech (Shanghai, China). H9c2 cells (in 6-well plates, 3 × 104 cells/ well) were transduced with 10 µL of lentiviral vectors with a virus titer of 1 × 108 IFU/mL containing


pre-miR210 or anti-miR210, or miR-scr (without miR-210 as control). After 24 h, the medium was replaced with fresh complete medium, and culturing continued for another 48 h prior to evaluating the efficiency of transduction using an inverted fluorescent microscope. To eliminate the non-transfected cells, 0.75 µg/mL puromycin (Sigma, St. Louis, MO, USA) was added to the medium and incubated for 24 h. Effect of insulin, H2O2, or combined treatment on miR-210 expression in H9c2 cells H9c2 cells were cultured in 6-well plates at 106 cells/mL before treatment. The cultured H9c2 cells were treated with: (1) H2O2 (1000 µmol/L) for 2, 4, 6, 8, 16, 24, or 48 h; (2) H2O2 (250, 500, 1000, or 2000 µmol/L) for 8 h; (3) insulin (5, 10, 100, 500, or 1000 nmol/L) for 3 h; (4) insulin (100 nmol/L) for 1, 3, 16, or 24 h; or (5) insulin (100 nmol/L) for 3 h, washed twice with phosphate-buffered saline (PBS), and then treated with H2O2 (1000 µM) for 2, 4, 6, or 8 h. Insulin was purchased from Sigma (St Louis, MO, USA).

Total RNA was isolated from the treated cells using a TRIzol kit (Invitrogen). MiR-210 expression was determined as described below. LY294002 (50 nM), a specific inhibitor of PI3K, was used to study the role of Pkt on insulin- and H2O2-induced miR-210 expression. Measuring cardiac myocyte death and apoptosis induced by H2O2 H9c2 cells (106 cells/mL) were treated with (1) vehicle (control) or H2O2


(250, 500, 1000 and 2000 µmol/L) for 24 h; or (2) insulin (5, 10 and 100 nmol/L) for 3 h. Cell viability was then measured using the methylthiazolydiphenyl-tetrazolium bromide (MTT) method. In brief, the cells were incubated with MTT (0.2 mg/mL; Sigma) for 4 h at 37 °C in the dark. After dimethyl sulfoxide was added, the optical density was measured at 490 nm and the cell viability was calculated as a percentage of the control optical density [13]. Cells were treated with (1) the vehicle or H2O2 (250, 500 and 1000 µmol/L) for 5 h or (2) insulin for 3 h, then treated with or without H2O2 (1000 µM) for 24 h. After treatments the cells were detached from the culture dishes with 0.25% trypsin and collected by centrifugation. After washing twice with ice-cold PBS, the cells were fixed and stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodine (PI) for 15 min. The apoptotic cells were identified using EPICS XL flow cytometry (FCM; Bechman Coulter, CA, USA).

Cells determined to be both PI- and annexin V-FITC-negative (PI–/annexin V-FITC–) were judged normal and healthy. Those found to be PI-negative and annexin V-FITC-positive (PI–/annexin V-FITC+) were considered in early apoptosis. Cells that were both PI- and annexin V-FITC-positive (PI+/annexin V-FITC+) were considered late apoptotic. Quantification of cellular miRNA expression


Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. The expression of miR-210 was evaluated using an miRcute miRNA qPCR Detection Kit (Tiangen Biotech, Beijing, China). Complementary DNA (cDNA) was generated from 2 µg of total RNA using an miRcute miRNA First-strand cDNA Synthesis Kit (Tiangen Biotech, Beijing, China). The generated miR-210 cDNA was amplified via reverse transcription-PCR (RT-PCR) in accordance with the TaqMan microRNA Assay protocol (Roche LightCycler 480 II System). In brief, 20-µL reactants were incubated in a 96-well optical plate at 94 °C for 2 min, and then subjected to 40 cycles of 94 °C for 20 s, and 60 °C for 34 s. Fold changes in miR-210 expression between treatments and controls were determined using the 2–Δ Δ CT method, normalizing to U6 RNA expression as an internal reference. The PCR forward primer for mature miR-210 was 5′-TACTGTGCGTGTGACAGCGGC-3′, and the PCR reverse primer for mature miR-210 was obtained from the commercial miRcute miRNA qPCR Detection Kit (Tiangen Biotech, Beijing, China). The PCR

primers, P1 and P2, for U6 were 5′- AACGCTTCACGAATTTGCGT-3′ and 5′-CTCGCTTCGGCAGCACA-3′, respectively. Western blot analysis Cellular proteins were analyzed using western blot. Cells were lysed in buffer comprising a complete protease inhibitor cocktail. Protein contents of cell lysate were determined using Bradford’s method. Lysates equivalent to 20


µg of protein were electrophoresed on 12% SDS-PAGE gels and then transferred to polyvinylidene fluoride membranes. Each membrane was incubated for 1 h with 5% skim milk in PBS containing 0.1% Tween-20 (PBST) to block non-specific binding, and probed with primary antibodies overnight at 4 °C. After washing with PBST, membranes were incubated for 90 min at room temperature with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:3000). Blots were revealed with an electrochemiluminescence Western blotting detection kit (Beyotime Institute of Biotechnology, Haimen, China). Protein bands on radiograph film were quantified by densitometric scanning after background subtraction. Integrated densities of bands were measured using ImageJ software. Rabbit antibodies against total Akt (1:10000) and phosphorylated Akt-1(ser-473; 1:5000) were purchased from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit IgGs were from Beyotime institute of Biotechnology. Mouse antibodies against beta actin monoclonal antibody (1:2000) were from Proteintech (Chicago, USA).

Statistical analyses All data are presented as the mean and standard deviation. One-way analysis of variance and Student’s t-test were used for statistical analyses, using SPSS 13.0 software. A P-value < 0.05 was considered statistically significant. Results


Effect of H2O2 on H9c2 cell viability. The viability of H9c2 cells was tested by MTT assay after treatment with 0 (control), 250, 500, 1000, or 2000 µM H2O2 for 24 h, and recorded as a percentage of the optical density of the control (Fig. 1A). The percentage of the optical density of cells exposed to 250 µM of H2O2 (98.25% ± 0.8445%) was not significantly different from that of the blank control (P > 0.05). However, with increasing concentrations of H2O2 at 500, 1000, and 2000 µM, the difference in percentage of optical density relative to the control became progressively more significant in a dose-dependent manner (89.06 ± 2.791%, 69.96 ± 6.123%, and 33.27 ± 4.842%, respectively; all P < 0.05). These results might partially reflect the process of cell apoptosis or necrosis. Effect of H2O2 on apoptosis The rate of apoptosis in H9c2 cells treated with 0, 250, 500, or 1000 µM H2O2 for 5 h was 18.64% ± 1.402%, 21.47% ± 1.186%, 32.39% ± 2.035%, and 62.39% ± 3.323%, respectively (P < 0.05, all). Thus with increasing

concentrations of H2O2 the apoptotic rate became progressively higher (Fig. 1B and 1D). Effect of insulin on apoptosis Insulin treatment alone had no effect on cell viability as measured by MTT and FCM (Fig. 1C). Insulin treatment prior to H2O2 incubation was associated with lower apoptotic rates compared with corresponding H2O2


treatments alone (26.70 ± 0.5981% cf. 31.76 ± 1.553%; P < 0.05; Fig. 1E and 1F; and 88.19 ± 0.3255% cf. 74.91% ± 1.434%; P < 0.05; Fig. 1G). The results indicate that insulin had a protective effect against H2O2-induced apoptosis of H9c2 cells. Effect of H2O2 on cellular miR-210 expression H9c2 cells were treated with 250-1000 µM H2O2 for 2-48 h, and the relative expressions of miR-210 were measured by quantitative RT-PCR (Fig. 2A). Exposure of H9c2 cells to H2O2 (1000 µM) for 4, 6, 8, or 16 h was associated with increases in miR-210 expression of 3.008 ± 0.6128-, 2.857 ± 0.9561-, 4.333 ± 0.7575-, and 2.581 ± 0.4640-fold, respectively, all P < 0.05 relative to that of the control. However, exposure of the H9c2 cells to H2O2 for 24 and 48 h resulted in lower increases in miR-210 expression (1.382 ± 0.4913- and 1.622 ± 0.1354-fold, respectively, relative to the control), and levels of miR-210 were not significantly different from that of the control. With treatments of 250, 500, or 1000 µM H2O2 for 8 h, the cellular miR-210 expressions were also significantly higher relative to the control

(2.567 ± 0.4847-, 2.293 ± 0.2027-, and 2.716 ± 0.9943-fold, respectively, all P < 0.05; Fig. 2B). Effect of insulin on cellular miR-210 expression After treatment with insulin, cellular levels of miR-210 were measured via qRT-PCR. After treatment with 5, 100, 500, or 1000 nmol/L insulin for 3 h, the levels of cellular miR-210 were 1.312 ± 0.1101-, 2.025 ± 0.1107-, 1.616 ±


0.1036-, and 1.219 ± 0.1050-fold respectively that of the control (all P < 0.05; Fig. 2C). After H9c2 cells were treated with 100 nmol/L insulin for 3, 16, or 24 h, the levels of miR-210 were 2.754 ± 0.1438-, 4.202 ± 0.1741-, and 4.084 ± 0.1091-fold that of the control, respectively (all P < 0.05; Fig. 2D). After H9c2 cells were pretreated with 100 nmol/L insulin for 3 h, washed, and then treated with 1000 µmol/L H2O2 for 2, 4, 6, or 8 h, the miR-210 levels were 1.546 ± 0.0919-fold, 1.896 ± 0.1501-fold, 2.325 ± 0.2185-fold, and 4.486 ± 0.2007-fold, respectively, that of the control (all P < 0.05; Fig. 2E). Protective effect of increased miR-210 expression on H2O2-treated H9c2 cells MiR-210 expression was manipulated by gene transfection with per-210, anti-210, or miR-scr to determine the effect of miR-210 expression on cell death. Compared with the control group, miR-210 levels in the pre-210 transfected H9c2 cells were significantly higher by 9.496 ± 1.412-fold (P < 0.05, Fig. 3A), and apoptosis and the death rate associated with H2O2 treatment was reduced from 75.65 ± 1.565% to 70.77 ± 2.362% (P < 0.05, Fig. 3B and

3C). In the anti-miR-210 transfected H9c2 cells, miR-210 levels were 0.4174 ± 0.0355-fold that of the control, a significant reduction (P < 0.05, Fig. 3A), and the apoptosis and death rate associated with H2O2 treatment was significantly increased from 63.97 ± 1.956% to 70.77 ± 2.362% (P < 0.05, Fig. 3B and 3C). MiR-210 inhibition reduces the protective effects of insulin against


H2O2-induced cell apoptosis Insulin treatment prior to H2O2 incubation in the anti-210 transfected H9c2 cells induced higher apoptotic rates compared with the same treatment in the non-transfected H9c2 cells (50.17 ± 4.615% cf. 37.49 ± 5.449%, P < 0.05, Fig. 3D and 3E). The results indicate that miR-210 has a crucial protective role in insulin against H2O2-induced cell apoptosis. Insulin treatment prior to H2O2 incubation in the anti-210 transfected H9c2 cells induced lower apoptotic rates compared with H2O2 treatment in the non-transfected H9c2 cells without insulin treatment (50.17 ± 4.615% cf. 68.99 ± 4.940%, P < 0.05, Fig. 3D and 3E). These results may indicate that the protective effects of insulin against H2O2-induced cell apoptosis may not solely rely on miR-210. Regulation of miR-210 expression by Akt induced by insulin and H2O2 treatment Using western blot to determine total and phosphorylated Akt-1 content, we examined whether insulin, H2O2, or both combined can activate Akt in

H9c2 cells (Fig. 4A). Both insulin and H2O2 alone significantly stimulated Akt activity relative to the control: 4.547 ± 0.2095-fold in the insulin group, 2.793 ± 0.2316-fold for the H2O2 (500 µmol/L) group, and 5.753 ± 0.2591-fold in the H2O2 (1000 µmol/L) group (all P < 0.05). However, there was no synergistic effect after H9c2 cells were pre-exposed to 100 nmol/L insulin and then 500 µM H2O2 ((4.360 ± 0.2701-fold in the insulin+H2O2 (500 µM) group


compared with the insulin group（ 4.547 ± 0.2095-fold） or the H2O2(500 µM) group（ 2.793 ± 0.2316-fold） ). At the same concentration of insulin with 1000 µM H2O2, the phosphorylation of Akt-1 relative to the control was significantly attenuated compared with the insulin group (1.793 ± 0.2223-fold cf. 5.753 ± 0.2591-fold, P < 0.05). H9c2 cells pretreated with LY294002 (50 nM LY294002, a specific inhibitor of PI3K) could also attenuate the insulin- or H2O2- enhanced Akt activity, but there was no synergistic effect in attenuation associated with the combination of both (Fig. 4B; 1.261 ± 0.1332-fold cf. 1.920 ± 0.3376-fold, P < 0.05) and the inhibition of Akt activity could be attenuated by insulin (100 nM) and 500 µM H2O2. Insulin- and H2O2-induced cellular miR-210 expression could be significantly attenuated by LY294002 treatment. However, such attenuation was not synergistic upon a combination of both. Compared with the H2O2 treatment alone, cells with LY294002 pretreatment had a lower level of miR-210 expression (1.736 ± 0.2107-fold cf. 3.991 ± 0.3312-fold, P < 0.05; Fig. 4C). Cells given the LY294002 pretreatment had

lower levels of miR-210 compared with cells not given the pretreatment, in the insulin (1.234 ± 0.1031-fold cf. 2.840 ± 0.1198-fold, P < 0.05; Fig. 4D) and combined insulin and H2O2 treatment groups (2.539 ± 0.3088-fold cf. 4.397 ± 0.3297-fold, P < 0.05; Fig. 4E). DISCUSSION In the present study, we investigated the possible mechanisms


underlying the protective effect of insulin against H2O2-induced apoptosis of cardiomyocytes. First, we confirmed that H2O2 could induce apoptosis of H9c2 cells in a dose-dependent manner, and that insulin attenuated the apoptosis. Secondly, we found that both insulin and H2O2 treatments administered singly could promote miR-210 expression in H9c2 cells. However, there was no synergistic effect associated with the combination of insulin and H2O2. It may be that insulin and H2O2 share a common mechanism in increasing the expression of miR-210. We also assessed the potential role of miR-210 in H2O2-mediated cardiac myocyte injury. Overexpression of miR-210 via gene manipulation implied that miR-210 has a protective role against H2O2-induced cell death. MiR-210 inhibition reduced the protective effects of insulin against hydrogen peroxide-induced cell apoptosis. Our results suggest that insulin-induced miR-210 may have an anti-apoptotic effect on H2O2-mediated cardiac myocyte injury. Thirdly, we found that the ratio of p-Akt to total Akt could be increased in H9c2 cells by insulin or H2O2 stimulation. On the other hand, the inhibition of Akt brought about by the

inhibitor LY294002 could be attenuated by insulin treatment. Interestingly, the miR-210 levels in H9c2 cells pretreated with LY294002 was less than that of the cells treated only with insulin, H2O2, or both combined. It is known that insulin is a PI3K activator of Akt [14] and can stimulate cellular miR-210 expression. Therefore, our results may suggest that insulin and H2O2 induce miR-210 expression by activating PI3K-Akt, which then regulates miR-210


through a feedback mechanism. H2O2 has been widely used to simulate conditions of oxidative stress, to which cells show distinctive responses according to the cell type [15]. Our results showing the dose-dependent effect of H2O2 on cardiac myocytes are in accord with some previous reports [16, 17]. Insulin evokes its effects by activating a signaling cascade of protein tyrosine kinases and lipid kinases [18]. As a PI3K activator of Akt [14], insulin activates two main signaling pathways: the PI3K pathway and the Ras/mitogen-activated protein kinase pathway. The PI3K pathway has crucial roles in proliferation and survival in many kinds of cells [19, 20]. Glucose-insulin-potassium therapy has proved to be effective in cardiomyocyte protection, especially for acute myocardial infarction [21-23]. In the present study, we explored whether insulin exerts protective effects in H9c2 cardiac myocytes, and whether protection could be achieved through inhibition of H2O2-induced apoptosis via overexpression of miR-210. Our

results may further confirm that the PI3K pathway is an important factor in upregulation of miR-210, which has also been reported previously [9, 24-26]. In summary, the current study shows that insulin has a protective effect against H2O2-induced apoptosis of H9c2 cells, possibly by inducing cellular miR-210 expression, and the PI3K/Akt pathway has an important role in upregulation of miR-210 in H9c2 cells. As we only studied such protective


effects in laboratory with the concentrations of some stimulators in excess pathophysiological levels, further investigation with in vivo model(s) is warranted to confirm the important role of insulin in protecting cell death. Acknowledgments This study was supported by grants from the Finance Department of Jilin Province, China (No. 2014520), the Scientific Research Fund of Health Department of Jilin Province, China (No. 20112030) and the National Clinical Key Specialty Project. Competing interests The authors declare that no competing interests exist.

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Figure legends Fig. 1. Effects of H2O2 and insulin on H9c2 cell viability. A. The effect of H2O2 on H9c2 cardiomyocyte viability. n=3 * P < 0.05 compared with the vehicle control (0 µmol/L). B. The effect of H2O2 on the apoptosis of cultured H9c2 cells. C. The cultured H9c2 cells were treated with insulin (5, 10 and 100 nmol/L) for 3h. D. FCM analysis * P < 0.05 compared with the control. E. The


effect of insulin on the 1000 µmol/L H2O2-induced apoptosis of H9c2 cells. F. The effect of insulin on the 1000 µmol/L H2O2-induced apoptosis of H9c2 cells. * P < 0.05 compared with the H2O2 group. G. The effect of insulin on the 1000 µmol/L H2O2-induced apoptosis of H9c2 cells. n = 3; * P < 0.05 compared with the H2O2 group.

G


Fig. 2. The effects of H2O2 and insulin on miR-210 expression in H9c2 cells. H9c2 cells were treated with 1000 µM H2O2 for 2-48 h (A), with 250-2000 µM H2O2 for 8 h (B), with 5-1000 nmol/L for 3 h (C), with 100 nmol/L insulin for 1-24 h (D), and pretreated with 100 nmol/L insulin for 3 h, washed twice by PBS and then treated with 1000 µM H2O2 for 2-8 h (E). The relative expression of miR-210 was measured by qRT-PCR. Note: n = 3; * P < 0.05


compared with the vehicle control (0 µmol/L).

Fig. 3. Protective effect of miR-210 on H2O2-treated H9c2 cells. A. Modulating miR-210 expression in H9c2 cardiomyocyte cells. n = 3; * P < 0.05 compared with the control. B. miR-210 overexpression protect against hydrogen peroxide-induced cell death. n = 3; * P < 0.05 compared with the H2O2 group. ** P < 0.05 compared with the pre-210 group. C. FCM analysis. The H9c2 cells of pre-210, or anti-210 or miR-Scr transfected under the


treatment of H2O2. D. FCM analysis. The H9c2 cells of pre-210, or anti-210 or miR-Scr transfected under the treatment of insulin and then H2O2. E. miR-210 inhibition reduced the protective effects of insulin against H2O2-induced cell apoptosis. n = 3; * P < 0.05 compared with the H2O2 group. ** P < 0.05 compared with the insulin+ H2O2 group.



Fig. 4. Regulation of miR-210 expression by insulin and H2O2 treatment via Akt pathway. The ratio of P-Akt to total Akt expression in H9c2 cells treated with insulin or/and H2O2 (A) H9c2 cells were exposed to 100 nmol/L insulin with or without hydrogen peroxide. (B). H9c2 cells were pretreated with LY with or without insulin (100 nmol/L). The expression of p-Akt and total Akt were measured by Western Blot. LY pretreatment leads to decreased level of


mir-210 treated with 1000 µmol/L H2O2. H9c2 cells were pretreated with LY and then treated with 1000 µmol/L H2O2 (C) or then treated with insulin (D) or then treated with insulin and 1000 µmol/L H2O2 (E). The relative expression of miR-210 was measured by qRT-PCR. Note: n = 3 in each group; * P < 0.05 compared with the control. ** P < 0.05 compared with H2O2 or insulin or insulin+ H2O2 group.


Three pentacyclic triterpenes protect H9c2 cardiomyoblast cells against high-glucose-induced injury.

Naringenin confers protection against oxidative stress through upregulation of Nrf2 target genes in cardiomyoblast cells.

Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes.

Hydrogen sulfide protects testicular germ cells against heat-induced injury.

2 signaling pathway.

heme oxygenase 1 signaling pathway.

CIRBP protects H9C2 cells against myocardial ischemia through inhibition of NF-κB pathway.

Cardioprotective effects of fucoidan against hypoxia-induced apoptosis in H9c2 cardiomyoblast cells.

AKT Signaling Pathway.

reoxygenation‑induced H9C2 myocardioblast cells through increased autophagy.

Ghrelin protects rats against traumatic brain injury and hemorrhagic shock through upregulation of UCP2.

Mipu1 protects H9c2 myogenic cells from hydrogen peroxide-induced apoptosis through inhibition of the expression of the death receptor Fas.

NFκB pathway in H9c2 cardiac cells.

Protective effect of Boerhaavia diffusa L. against mitochondrial dysfunction in angiotensin II induced hypertrophy in H9c2 cardiomyoblast cells.

Protective effect of p-coumaric acid against doxorubicin induced toxicity in H9c2 cardiomyoblast cell lines.

reperfusion injury in diabetic mice through the insulin-PI3K-mTOR pathway.

mTOR pathway.

Cardioprotective effects of rhamnetin in H9c2 cardiomyoblast cells under H₂O₂-induced apoptosis.

MicroRNA-135a Regulates Apoptosis Induced by Hydrogen Peroxide in Rat Cardiomyoblast Cells.

reperfusion injury through its antioxidant activity.

b attenuates post-myocardial infarction remodeling and protects H9c2 cardiomyoblast against hypoxia-induced apoptosis involving Sp1 and PARP-1.

Plumbagin Protects Against Spinal Cord Injury-induced Oxidative Stress and Inflammation in Wistar Rats through Nrf-2 Upregulation.

Orientin protects myocardial cells against hypoxia-reoxygenation injury through induction of autophagy.

Rapamycin protects kidney against ischemia reperfusion injury through recruitment of NKT cells.