Zhonghua wei zhong bing ji jiu yi xue · 2026
[Effect and mechanism of hypothermia on apoptosis and autophagy of cardiomyocytes after oxygen-glucose deprivation/restoration injury].
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Liang Zhen, Yang Song, Zhang Luying, Shao Rui, Wang Xingsheng, An Le, Hang Chenchen, Tang Ziren
Аннотация
OBJECTIVE: To establish an oxygen-glucose deprivation/restoration (OGD/R) model of cardiomyocytes to simulate myocardial ischemia reperfusion injury induced by cardiac arrest, and to explore the potential mechanism of hypothermia protective effect on cardiomyocytes.
METHODS: H9C2 cardiomyocytes in logarithmic growth phase were selected to establish the OGD/R model by selecting the most appropriate oxygen-glucose deprivation (OGD) and reoxygenation time combined with the results of the preliminary experiment. The cells were divided into four groups: blank control group without OGD treatment, 37 centigrade room temperature culture. The OGD group was subjected to OGD for 12 hours without restoration of glucose and oxygen. The OGD/R with normal temperature groups were subjected to OGD for 12 hours followed by reoxygenation for 4, 8, and 12 hours at 37 centigrade, respectively. The OGD/R hypothermia groups were subjected to OGD for 12 hours followed by reoxygenation for 4, 8, and 12 hours at 34 centigrade, respectively. Trypan blue staining was used to detect cell viability. Flow cytometry was used to analyze the change in mitochondrial membrane potential (Δ ψm). Western blotting was used to detect the protein expression of apoptosis-related protein (cleaved caspase-3/caspase-3 ratio), autophagy-related proteins [microtubule-associated protein 1 light chain 3B-II/I (LC3B-II/I), p62] and PTEN-induced kinase 1/E3 ubiquitin ligase (PINK1/Parkin) pathway proteins (PINK1, Parkin) and mitochondrial outer membrane pore protein [voltage-dependent anion channel 3 (VDAC3)] were detected.
RESULTS: Compared with the blank control group, the cell survival rate was significantly decreased in the OGD group and OGD/R groups, and the cleaved caspase-3/caspase-3 ratio, LC3B-II/I ratio and the protein expression of VDAC3 were increased, indicating that OGD and OGD/R could cause cell apoptosis and induce autophagy. In addition, OGD/R resulted in a decrease in Δψm and up-regulation of PINK1/Parkin pathway proteins. These results suggested that reoxygenation could aggravate myocardial cell injury induced by OGD, and the mechanism might be related to the PINK1/Parkin pathway. In the OGD/R groups, compared with normothermia, the survival rate of cardiomyocytes in hypothermia was increased at each time point [4 hours: (65.46±0.81)% vs. (60.26±0.71)%, 8 hours: (72.76±0.23)% vs. (62.93±0.37)%, 12 hours: (77.72±0.43)% vs. (70.75±0.61)%, all P<0.05], the decreased-Δ ψm lowed at 12 hours (green/red fluorescence intensity ratio of JC-1: 0.120±0.005 vs. 0.250±0.011, P<0.05), cleaved caspase-3/caspase-3 ratio decreased at 8 hours and 12 hours (8 hours: 0.96±0.06 vs. 1.08±0.07, 12 hours: 0.91±0.06 vs. 1.23±0.08, both P<0.05), the LC3B-II/I ratio was increased at 8 hours and 12 hours (8 hours: 1.40±0.08 vs. 1.16±0.08, 12 hours: 1.51±0.11 vs. 1.12±0.07, both P<0.05), the p62 expression was increased at 12 hours (p62/GAPDH: 0.90±0.04 vs. 0.75±0.03, P<0.05), the protein expression of VDAC3 was decreased at 12 hours (VDAC3/β-actin: 0.93±0.04 vs. 1.07±0.04, P<0.05), the protein expressions of PINK1 and Parkin were increased at 12 hours [PINK1 protein (PINK1/GAPDH): 0.98±0.06 vs. 0.80±0.05, Parkin protein (Parkin/GAPDH): 1.13±0.08 vs. 0.99±0.05, both P<0.05]. In terms of temperature influence, the expression trend of VDAC3 protein was opposite to that of autophagy proteins, while the expression trends of PINK1 and Parkin proteins were consistent with it.
CONCLUSIONS: Hypothermia can alleviate OGD/R-induced cardiomyocyte apoptosis and regulate the level of autophagy, which may be related to the regulation of VDAC3 and PINK1/Parkin pathways. VDAC3 may be involved in the hypothermic protection of cardiomyocytes after OGD/R injury and reduce the injury of cardiomyocytes.
METHODS: H9C2 cardiomyocytes in logarithmic growth phase were selected to establish the OGD/R model by selecting the most appropriate oxygen-glucose deprivation (OGD) and reoxygenation time combined with the results of the preliminary experiment. The cells were divided into four groups: blank control group without OGD treatment, 37 centigrade room temperature culture. The OGD group was subjected to OGD for 12 hours without restoration of glucose and oxygen. The OGD/R with normal temperature groups were subjected to OGD for 12 hours followed by reoxygenation for 4, 8, and 12 hours at 37 centigrade, respectively. The OGD/R hypothermia groups were subjected to OGD for 12 hours followed by reoxygenation for 4, 8, and 12 hours at 34 centigrade, respectively. Trypan blue staining was used to detect cell viability. Flow cytometry was used to analyze the change in mitochondrial membrane potential (Δ ψm). Western blotting was used to detect the protein expression of apoptosis-related protein (cleaved caspase-3/caspase-3 ratio), autophagy-related proteins [microtubule-associated protein 1 light chain 3B-II/I (LC3B-II/I), p62] and PTEN-induced kinase 1/E3 ubiquitin ligase (PINK1/Parkin) pathway proteins (PINK1, Parkin) and mitochondrial outer membrane pore protein [voltage-dependent anion channel 3 (VDAC3)] were detected.
RESULTS: Compared with the blank control group, the cell survival rate was significantly decreased in the OGD group and OGD/R groups, and the cleaved caspase-3/caspase-3 ratio, LC3B-II/I ratio and the protein expression of VDAC3 were increased, indicating that OGD and OGD/R could cause cell apoptosis and induce autophagy. In addition, OGD/R resulted in a decrease in Δψm and up-regulation of PINK1/Parkin pathway proteins. These results suggested that reoxygenation could aggravate myocardial cell injury induced by OGD, and the mechanism might be related to the PINK1/Parkin pathway. In the OGD/R groups, compared with normothermia, the survival rate of cardiomyocytes in hypothermia was increased at each time point [4 hours: (65.46±0.81)% vs. (60.26±0.71)%, 8 hours: (72.76±0.23)% vs. (62.93±0.37)%, 12 hours: (77.72±0.43)% vs. (70.75±0.61)%, all P<0.05], the decreased-Δ ψm lowed at 12 hours (green/red fluorescence intensity ratio of JC-1: 0.120±0.005 vs. 0.250±0.011, P<0.05), cleaved caspase-3/caspase-3 ratio decreased at 8 hours and 12 hours (8 hours: 0.96±0.06 vs. 1.08±0.07, 12 hours: 0.91±0.06 vs. 1.23±0.08, both P<0.05), the LC3B-II/I ratio was increased at 8 hours and 12 hours (8 hours: 1.40±0.08 vs. 1.16±0.08, 12 hours: 1.51±0.11 vs. 1.12±0.07, both P<0.05), the p62 expression was increased at 12 hours (p62/GAPDH: 0.90±0.04 vs. 0.75±0.03, P<0.05), the protein expression of VDAC3 was decreased at 12 hours (VDAC3/β-actin: 0.93±0.04 vs. 1.07±0.04, P<0.05), the protein expressions of PINK1 and Parkin were increased at 12 hours [PINK1 protein (PINK1/GAPDH): 0.98±0.06 vs. 0.80±0.05, Parkin protein (Parkin/GAPDH): 1.13±0.08 vs. 0.99±0.05, both P<0.05]. In terms of temperature influence, the expression trend of VDAC3 protein was opposite to that of autophagy proteins, while the expression trends of PINK1 and Parkin proteins were consistent with it.
CONCLUSIONS: Hypothermia can alleviate OGD/R-induced cardiomyocyte apoptosis and regulate the level of autophagy, which may be related to the regulation of VDAC3 and PINK1/Parkin pathways. VDAC3 may be involved in the hypothermic protection of cardiomyocytes after OGD/R injury and reduce the injury of cardiomyocytes.