Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy via PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway

doi:10.19852/j.cnki.jtcm.20220311.002

Abstract

Abstract:

OBJECTIVE: To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/ reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2 (PINK1/PARKIN) signaling pathway.

METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells; the hypoxia/ reoxygenation (H/R) model was established in tri-gas incubator; 2’, 7’-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species (ROS); the changes of mitochondrial membrane potential was determined by 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide staining; the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits; flow cytometry was used to detect the ratio of apoptotic cells; transmission electron microscope was used to observe the ultrastructure of H9C2 cells; Western blot was used to detect the protein changes of mitochondrial 20 kDa outer membrane protein (TOM20), translocase of inner mitochondrial membrane 23 (TIM23), presenilins associated rhomboid-like protein (PARL), PINK1, PARKIN and mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), phosphotyrosine independent ligand for the Lck SH2 domain of 62 kDa (P62), microtubule-associated protein 1 light chain 3 beta (LC3B); the mRNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction; immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin.

RESULTS: Resveratrol could inhibit the proliferation of H9C2 cells in a time- and concentration- dependent manner; however, pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels, alleviate the loss of mitochondrial membrane potential induced by H/R, inhibit H/R-induced apoptosis of H9C2 cells, and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage. Resveratrol could further increase the levels of p62, PINK1, PARKIN protein, the expression of PINK1, PARKIN mRNA and the ratio of LC3BⅡ/LC3BⅠin H/R-induced H9C2 cells, inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells, and further reduce the expression of TOM20,TIM23, PARL, Mfn1 and Mfn2 protein in H/R-induced H9C2 cells. The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells.

CONCLUSIONS: Resveratrol can protect H9C2 cells from H/R injury, which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.

Key words: resveratrol, myocytes, cardiac, hypoxia, PTEN phosphohydrolase, Parkinson disease associated proteins, mitochondrial autophagy

ZHAO Lixia, SUN Wei, BAI Decheng. Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy via PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway[J]. Journal of Traditional Chinese Medicine, 2022, 42(2): 176-186.

Figures/Tables 6

Figure 1 Resveratrol inhibited the proliferation of H9C2 cells in a time and concentration dependent manner, which was determined using an 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide cell survival assay H9c2 cells were treated with resveratrol at the concentrations of 0, 6.25, 12.5, 25, 50, 100 and 200 g/L for 24, 48 and 72 h, respectively.

Figure 2 Resveratrol can increase decrease of the mitochondrial membrane potential induced by H/R in H9C2 cells (× 200) The mitochondrial membrane potential was detected by JC-1 staining (A, F, K: control, no treatment; B, G, L: H/R, hypoxia/reoxygenation injury; C, H, M: L-dose Res, 6.25 g/L for 24 h, D, I, N: H-dose Res, 12.5 g/L for 24 h; E, J, O: BHH, 100 μM for 24 h) and the fluorescence intensity was measured with ImageProPlus software and the Red/Green ratio was analyzed by SPSS25.0 Software (P). H/R: hypoxia/reoxygenation; JC-1: 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide; L-dose Res: low-dose resveratrol; H-dose Res: high-dose resveratrol; BHH: bromhexine hydrochloride. aP < 0.01 vs control group, bP < 0.01 vs H/R group.

Figure 3 Resveratrol can activate mitochondrial autophagy (× 15 000) The ultrastructure of H9C2 cells was detected by transmission electron microscope. The red arrow indicates the mitochondrial cristae and the white arrow indicates the mitochondrial membrane. A: control, no treatment; B: H/R, hypoxia/reoxygenation injury; C: L-dose Res, 6.25 g/L for 24 h; D: H-dose Res, 12.5 g/L for 24 h; E: BHH, 100 μM for 24 h. H/R: hypoxia/reoxygenation; BHH: bromhexine hydrochloride.

Figure 4 Resveratrol promotes the expression of PINK1 and PARKIN protein and mRNA and inhibits the expression of TOM20, TIM23 and PARL protein in H9C2 cells induced by H/R, which were detected by Western blot (A, B) and qPCR (C), respectively PINK1: PTEN-induced putative kinase protein 1; PARKIN: Parkinson disease protein 2; TOM20: mitochondrial 20 kDa outer membrane protein; TIM23: translocase of inner mitochondrial membrane 23; PARL: presenilins associated rhomboid-like protein; H/R: hypoxia/reoxygenation; qPCR: quantitative polymerase chain reaction. aP < 0.01 vs control group, bP < 0.01 vs H/R group.

Figure 5 Resveratrol promotes interaction between PARKIN and ubiquitin in the H/R-induced H9C2 cells A: Co-IP assay; B: the semi-quantitative analysis of the interaction between PARKIN and ubiquitin was conducted by the ImageProPlus software. PARKIN: Parkinson disease protein 2; H/R: hypoxia/reoxygenation. aP < 0.01 vs Control group, bP < 0.01 vs H/R group.

Figure 6 Resveratrol promotes the expression of Beclin 1, p62 and LC3 in H9C2 cells induced by H/R, which were detected by Western blot (A, B) LC3: microtubule associated protein light chain 3; H/R: hypoxia/reoxygenation. aaP<0.01 vs Control group, bbP<0.01 vs H/R group.

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