6-Gingerol protects cardiocytes H9c2 against hypoxia-induced injury by suppressing BNIP3 expression

Abstract

Background

Cardiomyocytes loss is the predominant pathogenic characteristic in the hypoxia-induced injury. Meanwhile, it has been corroborated that Bcl-2 E1B 19-KDa interacting protein 3 (BNIP3) provokes apoptosis and autophagy. For moderating cardiomyocytes loss, we initially probed the cyto-protection effects of 6-Gingerol (6 G), meanwhile, its potential mechanisms associated with BNIP3 were elucidated in our studies.

Methods

We pretreated cardiomyocytes H9c2 cells with 6 G at different concentrations (0–100 μM) before exposure to hypoxia. Thereafter, the cell viability, lactate dehydrogenase (LDH), apoptosis and protein expression were respectively assessed using cell counting kit-8 and methyl thiazolyl tetrazolium (MTT) assay, LDH assay kit, Annexin V-fluorescein isothiocyannate/propidium iodide (Annexin V-FITC/PI) apoptosis detection kit and Western blotting analysis. In addition, we also analyzed BNIP3 level after treatment. Moreover, we enforced the exogenous overexpression of BNIP3 and then evaluated the cell viability, apoptosis, and protein level again.

Results

In our present work, we observed that the cell viability was promoted by 6 G in the hypoxia-induced H9c2 cells in a dose-dependent manner. Moreover, hypoxia-induced LDH release, apoptosis and autophagy were inhibited by 6 G pretreatment through promoting phosphorylation of PI3K, AKT and mTOR. Remarkably, accumulation of BNIP3 protein was significantly reduced by 6 G in hypoxia-induced H9c2 cells. Mechanistically, 6 G initiated the phosphorylated expression of PI3K, AKT and mTOR by down-regulating BNIP3 with reducing cardiomyocytes apoptosis and autophagy.

Conclusion

Hypoxia-induced cardiomyocytes injury was ameliorated by 6 G through suppressing BNIP3 expression with triggering PI3K/AKT/mTOR signalling pathway.

Keywords:

• 6-Gingerol
• hypoxia
• cardiomyocytes injury
• BNIP3

View addendum:

Expression of Concern

Introduction

The predominant pathogenic feature of cardiac ischemia is cardiomyocytes loss. Myocyte loss will lead to reduced pumping capacity and result in congestive heart failure and death. Ischemia controversially provokes hypoxia in cardiomyocytes. Notably, hypoxia causes cardiac dysfunction, ischemia/reperfusion injury and cardiac infarction aggravation [1,2]. In addition, hypoxia has been molecularly demonstrated to lead to dysfunction of organelles and DNA fragmentation in the pathogenesis of heart disease, ultimately causing apoptosis and autophagy which are associated with cardiomyocytes death [3–6]. Strikingly, it has been shown that dysfunction of mitochondria is associated with apoptosis with a release of cytochrome c and activation of Caspase [7].

Several natural and synthetic compounds have been introduced in previous studies for protecting cardiomyocytes against injury [8–10]. 6-Gingerol (6 G) is a natural chemical compound isolated from ginger (Zingiber officinale). Specifically, 6 G has been indicated to possess potential efficacies against tumour [11–16], oxidative stress and inflammation [17–19]. Moreover, potent protection of 6 G against hypoxia/reoxygenation-induced injury has been recently elucidated. For example, Li et al. have corroborated that 6 G exerts protective effects against hypoxia/reoxygenation-induced injury in intestinal mucosa [20]. Interestingly, an experimental study has validated that pretreatment with 6 G ameliorates myocardial injury induced by hypoxia/reoxygenation [21].

Bcl-2 E1B 19-KDa interacting protein 3 (BNIP3) has been reported as a derivable regulator of mitochondrial death and mitophagy involved in hypoxia-medicated ventricular myocytes [22]. Mitochondrial protein BNIP3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 family proteins. The ectopic expression of BNIP3 has been identified in hypoxia-medicated cardiomyocytes injury [23]. Similarly, it has been revealed that hypoxia dramatically induces the expression of pro-apoptotic gene BNIP3 in cardiomyocytes [24]. Furthermore, gradually increased BNIP3 expression has been detected in the rat model with pressure overload [25]. Previous studies demonstrated that elevated expression of BNIP3 induces a myocardial cells apoptosis in mice [26]. Importantly, results have corroborated that BNIP3 provokes a Caspase-regulated apoptosis in ventricular myocytes [22]. In addition, BNIP3 has been demonstrated to up-regulate autophagy for stimulating protective stress response [27]. However, detailed complicated genetic regulatory mechanism has scarcely been elucidated with regard to BNIP3.

The current study was performed to explore the effects of 6 G on hypoxia-induced cardiomyocytes injury. Given that BNIP3 has been emphasized in regulating cell death during hypoxia, raises the remarkable potential that 6 G may exert an impact on the activation of signalling pathways through modulating BNIP3 expression for protecting myocardial cells against injury. Correspondingly, we addressed whether translation products of BNIP3 are regulated by 6 G.

Materials and methods

Cell culture and hypoxia-induced injury

H9c2 cells were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured according to the supplier’s recommendation. Briefly, H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Gaithersburg, MD, USA) containing 10% (v/v) fetal bovine serum (FBS; Gibco), 100 U/mL penicillin (Invitrogen, Carlsbad, CA) and 100 μg/mL streptomycin (Invitrogen). The cells were cultured in a Sci-tive workstation (Baker, Sanford, Maine, USA). The cells in the control group were maintained in a normoxic condition (74% N₂, 5% CO₂, 21% O₂, 37 °C). As for hypoxia stimulus, H9c2 cells were incubated in a hypoxic incubator (94% N₂, 5% CO₂, 1% O₂, 37 °C) for 24 h.

Lactate dehydrogenase (LDH) release assay

To examine the release of LDH from H9c2 cells in supernatant, we exploited an LDH assay kit purchased from Beyotime (Beijing, China) with reference to the supplier’s description. The absorbance was detected using a SpectraMax M5e Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).

Cell transfection

The sequence of BNIP3 was constructed into plasmid pcDNA3.1 named pcDNA-BNIP3 and abbreviated to pcBNIP3. As for BNIP3 overexpression, pcBNIP3 and pcDNA3.1 control vector were transfected into H9c2 cells with Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). The stably transfected cells were harvested after 48 h for subsequent experiments.

Cell viability assay

Briefly, H9c2 cells were seeded in 96-well plates and grown overnight and subsequently treated with 6 G (Realin, Xi’an, China), which was dissolved in 1% tragacanth (Sigma, St Louis, MO, USA) at different concentrations (0, 5, 10, 25, 50 and 100 μM) with reference to an anterior report [28]. The culture was incubated for 24 h before hypoxia-induced injury. To confirm 6 G supported cell viability in hypoxia-stimulated H9c2 cells, cell counting kit-8 (CCK-8) (APExBIO, Houston, TX, USA) and methyl thiazolyl tetrazolium (MTT) colourimetric assay kit MMT (Sangon Biotech, Shanghai, China) assay were exploited in this part according to user manual. Each experiment was performed at least three times.

Apoptosis assay

The apoptotic cells were determined using Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) apoptosis detection kit (Biosea, Beijing, China) according to manufacturer’s instruction. The apoptotic cells were identified with a flow cytometer (Beckman Coulter, Fullerton, CA, USA).

Western blotting analysis

The protein was extracted from H9c2 cells lysed with RIPA lysis buffer (Beyotime) containing protease inhibitors (Roche Applied Science, Indianapolis, USA). The concentration of protein was determined with BCA™ protein assay kit (Pierce, Appleton, WI, USA). Proteins extracted from H9c2 cells were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Western blotting was performed with primary antibodies against p53 (ab131442, 1:1000), Cleaved-Caspase-3 (ab2302, 1 µg/mL), Cleaved-Poly(ADP-Ribose) Polymerase (PARP) (ab32064, 1:1000), Beclin-1 (ab207612, 1:2000), p62 (ab155686, 1:1000), LC3-I/II (ab48394, 1 µg/mL), BNIP3 (ab109362, 1:1000), β-actin (ab8227, 1:1000) (Abcam, Cambridge, UK), PI3K (4255, 1:1000), p-PI3K (4228, 1:1000), AKT (9272, 1:1000), p-AKT (4056, 1:1000), mTOR (2972, 1:1000), p-mTOR (5536, 1:1000) (Cell Signaling Technology, Danvers, MA), as well as with secondary antibodies (ab205718, 1:5000, Abcam; 7054, 1:1000, Cell Signaling Technology). The signals were captured by Bio-Rad ChemiDoc™ XRS system and normally quantified using Image Lab software (Bio-Rad, CA, USA).

Statistical analysis

Each experiment was repeated at least three times. The results of multiple experiments were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using Graphpad Prism 7.0 statistical software (GraphPad Prism Software, La Jolla, CA). Comparisons between two groups were evaluated by Student’s t-test. Comparisons among multiple groups were performed by one-way analysis of variance (ANOVA) followed by Bonferroni post-test. A p values less than .05 indicates a statistically significance.

Results

6 G promoted cell viability in the hypoxia-induced H9c2 cells

To explore the protective activity of 6 G against hypoxia-induced injury, we stimulated H9c2 cells with 6 G in different concentrations prior to hypoxia inducement. We observed that the cell viability was obviously suppressed by hypoxia inducement (p < .01) as showed in Figure 1(A,B). Further, 6 G exerted a cyto-protection against hypoxia-induced injury in a concentration-dependent manner when the concentration of 6 G was between 5 and 50 μM (p < .05 or p < .01). The cell viability was visibly weaker in H9c2 cells treated by 6 G at a higher concentration (100 μM) (p < .05) in comparison with a lower dose (50 μM) (Figure 1(A,B)). As a consequence, we selected 50 μM as a stimulant concentration for downstream experiments. Moreover, we found 6 G notably abated the release of LDH (p < .05) which was induced by hypoxia (p < .01) as shown in Figure 1(C). Given that the release of LDH negatively indicates membrane integrity of cells, our results collectively confirmed that 6 G efficiently protected H9c2 cells against hypoxia-caused damages.

Figure 1. 6 G alleviated the decline of cell viability induced by hypoxia in a concentration-dependent manner. (A and B) Cell viability was assessed using CCK-8 and MTT methods. H9c2 cells were treated with 6 G at different concentrations (0, 5, 10, 25, 50 and 100 μM) before hypoxia-induced injury. (C) The release of LDH from H9c2 cells was quantified by absorbance with a microplate reader. H9c2 cells were pre-treated with 50 μM for 24 h before stimulated in a hypoxic condition. 6 G: [6]-Gingerol. **p < .01 vs Control; #p < .05 or ##p < .01 vs Hypoxia.

Hypoxia-induced apoptosis and autophagy were inhibited by 6 G

Hypoxia-induced apoptosis of H9c2 cells was evidently suppressed by 6 G compared with that in the Hypoxia group (p < .05) (Figure 2(A)). In addition, hypoxia-induced expression of p53, Cleaved-Caspase-3 and Cleaved-PARP were obsiously (p < .05 or p < .01) suppressed by 6 G (Figure 2(B)). Likewise, 6 G possessed anti-autophagy potential indicated by the decreased Beclin-1 expression and ratio of LC3-II and LC3-I as well as increased expression of p62 in hypoxia-induced H9c2 cells (p < .05 or p < .01) (Figure 2(C)). Our subsequent results evidently showed that the cyto-protection of 6 G was associated with its anti-apoptosis and anti-autophagy activities.

Figure 2. Hypoxia-induced apoptosis and autophagy were inhibited by 6 G in the cardiomyocytes. (A) 6 G suppressed hypoxia-induced apoptosis determined using Annexin V-FITC/PI assay. The relative expression of apoptosis-related proteins (p53, Cleaved-Caspase-3 and Cleaved-PARP) (B) and autophagy-related proteins (Beclin-1, p62, LC3-I and LC3-II) (C) were quantified using Western blot assay. H9c2 cells were neither treated with 6 G nor induced by hypoxia in the Control group; H9c2 cells were induced by hypoxia for 24 h in the Hypoxia group; H9c2 cells were treated with 50 μM 6 G for 24 h before hypoxia-induced injury in the Hypoxia + 6 G group. 6 G: [6]-Gingerol. *p < .05, **p < .01 or ***p < .001 vs Control; #p < .05 or ##p < .01 vs Hypoxia.

PI3K/AKT/mTOR signalling pathway was activated by 6 G in hypoxia-induced H9c2 cells

With regard to the function of 6 G on the phosphorylation of regulatory factors in PI3K/AKT/mTOR signalling pathway, we determined the protein expression of p/t-PI3K, p/t-AKT and p/t-mTOR using Western blot assay. The phosphorylated expression of PI3K, AKT and mTOR was markedly decreased by hypoxia in comparison with that in the Control group (p < .05 or p < .01) while 6 G pretreatment significantly elevated the phosphorylation of PI3K, AKT and mTOR compared with that in the Hypoxia group (p < .05 or p < .01) (Figure 3(A,B)). Collectively, our results indicated that 6 G triggered PI3K/AKT/mTOR signalling pathway inactivated by hypoxia stimulation.

Figure 3. The PI3K/AKT/mTOR signalling pathway was activated by 6 G in hypoxia-induced cardiomyocytes. (A) The relative phosphorylated expression of regulatory factors (PI3K, AKT and mTOR) were normalized after Western blot assay. (B) Regulatory factors were separated using SDS-PAGE according to their molecular masses. The size of p-PI3K is about 60 kDa. H9c2 cells were neither treated with 6 G nor induced by hypoxia in the Control group; H9c2 cells were induced by hypoxia for 24 h in the Hypoxia group; H9c2 cells were treated with 50 μM 6 G for 24 h before hypoxia-induced injury in the Hypoxia + 6 G group. 6 G: [6]-Gingerol; p-: phospho-; t-: total-; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis. *p < .05 or **p < .01 vs Control; #p < .05 or ##p < .01 vs Hypoxia.

Accumulation of BNIP3 protein was reduced by 6 G in hypoxia-induced H9c2 cells

For investigating the regulatory effects of 6 G on BNIP3 expression, we treated cardiomyocytes with 6 G before the cells were subjected to hypoxia for 24 h. The Western blot analyzes showed that hypoxia strongly stimulated the expression of BNIP3 (p < .001) while 6 G pretreatment ameliorated the expression of intracellular BNIP3 at protein level (p < .05, p < .01 or p < .001). Meanwhile, 6 G remarkably attenuated the expression of BNIP3 in a concentration-dependent manner (Figure 4(A,B)). Summarily, 6 G repressed the expression of BNIP3 induced by hypoxia.

Figure 4. Hypoxia-induced expression of BNIP3 at protein level was inhibited by 6 G in a concentration-dependent manner. (A) The relative expression of BNIP3 was normalized after Western blot assay. (B) BNIP3 protein was separated by SDS-PAGE according to its molecular masses. H9c2 cells were neither treated with 6 G nor induced by hypoxia in the Control group; H9c2 cells were treated with 0, 5, 10, 25 and 50 μM 6 G for 24 h before hypoxia-induced injury. 6 G: [6]-Gingerol; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ***p < .001 vs Control; #p < .05, ##p < .01 or ###p < .001 vs Hypoxia.

The cyto-protection of 6 G was realized by regulating BNIP3

To characterize the cyto-protection of 6 G by regulating BNIP3, we initially stimulated the expression of BNIP3 by transfecting pcBNIP3 into H9c2 cells. Relative BNIP3 expression at protein level was evidently elevated in comparison to that in the cardiomyocytes transfected with pcDNA3.1 (p < .001) (Figure 5(A)). Besides, we confirmed that up-regulated BNIP3 promoted apoptosis of H9c2 cells compared with that in the negative control (Hypoxia + 6 G + pcDNA3.1) (p < .05), illustrating that 6 G exerted an anti-apoptotic function on hypoxia-induced H9c2 cells by suppressing BNIP3 expression (Figure 5(B)). Compared with that in the Hypoxia + 6 G + pcDNA3.1 group, the expression of p53, Cleaved-Caspase-3 and Cleaved-PARP was distinctly promoted by BNIP3 in H9c2 cells transfected with pcBNIP3 and pretreated with 6 G (p < .05 or p < .01) (Figure 5(C)). Furthermore, overexpression of BNIP3 increased Beclin-1, reduced p62, and elevated the ratio of LC3-II and LC3-I (p < .05, p < .01 or p < .001) (Figure 5(D)), indicating that 6 G possessed anti-autophagy activity by down-regulating BNIP3. Consequently, we concluded that 6 G exerted a cyto-protection by regulating BNIP3.

Figure 5. Increased BNIP3 expression reversed inhibitory effects of 6 G in hypoxia-induced apoptosis and autophagy of cardiomyocytes. (A) The relative BNIP3 protein expression was increased by transfecting pcBNIP3 into H9c2 cells. (B) Increased BNIP3 expression inhibited the anti-apoptotic activity of 6 G in the hypoxia-induced H9c2 cells. (C) The expression of apoptosis-related proteins (p53, Cleaved-Caspase-3 and Cleaved-PARP) was increased by BNIP3 in the hypoxia-induced H9c2 cells after 6 G treatment. (D) Increased BNIP3 expression reversed the effects of 6 G in the autophagy-related protein (Beclin-1, p62, LC3-II and LC3-I) expression in the hypoxia-induced H9c2 cells. H9c2 cells were neither treated with 6 G nor induced by hypoxia in the Control group; H9c2 cells were induced by hypoxia for 24 h in the Hypoxia group; Hypoxia-induced H9c2 cells were transfected with pcDNA3.1 or pcBNIP3 before 6 G treatment in the Hypoxia + 6 G + pcDNA3.1 or Hypoxia + 6 G + pcBNIP3 groups. 6 G: [6]-Gingerol. **p < .01 or ***p < .001 vs Control; #p < .05 or ##p < .01vs Hypoxia; +p < .05, ++p < .01 or +++p < .001 vs cells transfected with pcDNA3.1.

6 G activated PI3K/AKT/mTOR signalling pathway by suppressing BNIP3 expression

We have demonstrated that hypoxia blocked PI3K/AKT/mTOR signalling pathway and 6 G pretreatment could activate PI3K/AKT/mTOR signalling pathway. To further validate whether 6 G participates in activating PI3K/AKT/mTOR signalling pathway by regulating BNIP3, the phosphorylation of regulatory factors in cardiomyocytes pretreated with 6 G under hypoxic condition were examined using Western blot analysis. Our results showed that 6 G evidently promoted the phosphorylated expression of PI3K, AKT and mTOR (p < .05 or p < .01) (Figure 6(A,B)) while overexpression of BNIP3 reversed the activation effect of 6 G on PI3K/AKT/mTOR signalling pathway by inhibiting the phosphorylation (p < .01) (Figure 6(A,B)). From the above, we expected that 6 G triggered PI3K/AKT/mTOR signalling pathway through inhibiting BNIP3 expression.

Figure 6. Increased BNIP3 expression blocked the PI3K/AKT/mTOR signalling pathway activated by 6 G in hypoxia-induced cardiomyocytes. (A) The relative phosphorylated expression of regulatory factors (PI3K, AKT and mTOR) were normalized after Western blot assay. (B) Regulatory factors were separated by SDS-PAGE according to their molecular masses. The size of p-PI3K is about 60 kDa. H9c2 cells were neither treated with 6 G nor induced by hypoxia in the Control group; H9c2 cells were induced by hypoxia for 24 h in the Hypoxia group; Hypoxia-induced H9c2 cells were transfected with pcDNA3.1 or pcBNIP3 before 6 G treatment in the Hypoxia + 6 G + pcDNA3.1 or Hypoxia + 6 G + pcBNIP3 groups. 6 G: [6]-Gingerol; p-: phospho-; t-: total-; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis. *p < .05 or **p < .01 vs Control; #p < .05 or ##p < .01 vs Hypoxia; ++p < .01 vs cells transfected with pcDNA3.1.

Discussion

In the present work, we collectively demonstrated that 6 G exhibited a pre-protective effect on hypoxia-induced cardiomyocytes at a lower concentration. Mechanistically, the results indicated that 6 G might repress apoptosis and autophagy by triggering PI3K/AKT/mTOR signalling pathway via negatively regulating BNIP3 expression.

Hypoxia is an elementary physiological and pathological stimulus in the establishment of cardiomyocytes injury model. Meanwhile, cell death is the primary response in reaction to hypoxia, suggesting decreased cell viability. As a consequence, inhibition of cell death is regarded as an attractive approach to control cardiac disease. It has been reported that 6 G exerts cellular protective effects against radiation-induced cell damage [29], 4-hydroxynonenal-induced cells mortality [19], amyloid-β-induced pheochromocytoma cells injury [30], and chlorpyrifos-induced oxidative damage in the brain, ovary and uterus [18]. The cyto-protection of 6 G has also been elucidated in hypoxia-induced injury [20]. Our observation, that 6 G pretreatment enhanced the cell viability which was reduced by hypoxia in a concentration-dependent manner, suggesting that 6 G moderated hypoxia-induced cardiomyocytes injury at a lower concentration. Consistently, a recent study showed that administrating 6 G notably reduces ST section of cardiogram in cardiac ischemia/reperfusion model, meanwhile improves cardiac function and reduces myocardial infarction area [31].

It has been well demonstrated that 6 G stimulates apoptosis in several cancer cells [11–14]. Meanwhile, 6 G induced autophagy in human cervical adenocarcinoma cells [15] and induced autophagy to protect human umbilical vein endothelial cells against apoptosis [32] while inhibited autophagy in lung cancer cells [33]. The effect of 6 G on autophagy in hypoxia-induced myocardial cells remains indistinct. Accordingly, we examined the apoptosis and autophagy of hypoxia-induced myocardial cells pretreated with 6 G. In consistent with previous studies that 6 G represses apoptosis to alleviate hypoxia/reoxygenation-induced myocardial injury [21], we also proved that 6 G lessened the production of apoptosis-related proteins, suggesting that 6 G restrained hypoxia-induced myocardial apoptosis. Unexpectedly, our results showed that 6 G reversed the regulatory effects of hypoxia on autophagy-related proteins, indicating 6 G exerted an anti-autophagy function in hypoxia-induced myocardial injury. With regard to regulatory effects on signalling pathways, we paid more attention to PI3K/AKT/mTOR signalling pathway since studies have demonstrated that PI3K/AKT/mTOR signalling pathway was suppressed by 6 G in human umbilical vein endothelial cells [32]. Although phosphorylated mTOR expression was repressed by 6 G in osteosarcoma cells [12], Lv et al. strikingly indicated that 6 G activates PI3K/AKT signalling pathway and inhibits apoptosis to alleviate hypoxia/reoxygenation-induced myocardial injury mechanistically [21]. In consistent with the study of Lv et al., we also proved that 6 G triggered PI3K/AKT/mTOR signalling pathway which was blocked by hypoxia.

According to a recent study, Guo et al. have shown that hypoxia dramatically induces the expression of pro-apoptotic gene BNIP3 in cardiomyocytes [24]. Moreover, results suggested that BNIP3 results in ischemia/reperfusion injury, initiating a protective response by promoting autophagy [27,34]. Whereas, quite a few evidence have emerged about the modulatory function of 6 G on BNIP3, particularly in cardiomyocytes exposed to hypoxia circumstance. Consequently, we quantified the expression of BNIP3. Our results verified that 6 G repressed BNIP3 expression in a dose-dependent relationship. It has revealed that overexpression of BNIP3 leads to an unusual type of apoptosis as Caspase inhibitors are unable to prevent cell and cytochrome c does not release from mitochondrial [24]. In an attempt to investigate cellular function of BNIP3 response to 6 G, we forced BNIP3 overexpression. The results indicated that 6 G conferred protection against hypoxia-medicated myocardial injury by lessening apoptosis and autophagy through down-regulating BNIP3.

Previous studies have demonstrated that hypoxia induces the dephosphorylation of PI3K/AKT/mTOR in cardiomyocytes and activation of PI3K/AKT/mTOR signalling pathway sufficiently protect cardiomyocytes against injury [35–39], resembling what we have proved with 6 G in hypoxia-medicated myocardial injury. Growing evidence have demonstrated that BNIP3 participates in regulating PI3K/AKT/mTOR signalling pathway [40,41]. Absorbing effects of BNIP3 on the autophagy and apoptosis through signalling pathways has been conducted by sequential explorations that demonstrated in certain cases that small interfering RNA-mediated ablation of BNIP3 motivates mTOR phosphorylation [41] and JNK dominates over AKT for promoting BNIP3 expression in order to subsequently induce apoptosis and autophagy [25]. Centered on PI3K/AKT/mTOR signalling pathway, we estimated whether BNIP3 was involved in modulation of 6 G on PI3K/AKT/mTOR signalling pathway. By regulating BNIP3 overexpression through transfection with pcBNIP3, we validated that increased BNIP3 scarcely ameliorated the phosphorylated expression of PI3K, AKT and mTOR in hypoxia-induced myocardial cells pretreated with 6 G.

We initially presumed that 6 G might activate PI3K/AKT/mTOR signalling pathway by down-regulating BNIP3 expression. Accordingly, the assumption was verified after a series of experiments. We also elucidated a possible mechanism, that 6 G activated PI3K/AKT/mTOR signalling pathway by repressing BNIP3 expression, by which hypoxia-induced cardiomyocytes injury was ameliorated.

Author contributions

Conceived and designed the experiments: Qi Ren and Shaojun Zhao; Performed the experiments and analyzed the data: Qi Ren; Contributed reagents/materials/analysis tools: Changjie Ren; Wrote the manuscript: Qi Ren; Revised the manuscript: Shaojun Zhao.

Disclosure statement

No potential conflict of interest was reported by the authors.