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

Figures & data

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.

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.

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.

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.

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.

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.