Korean J Physiol Pharmacol.  2022 Sep;26(5):325-333. 10.4196/kjpp.2022.26.5.325.

Benzoylaconine improves mitochondrial function in oxygenglucose deprivation and reperfusion-induced cardiomyocyte injury by activation of the AMPK/PGC-1 axis

• Chen L
• Yan L
• Zhang W

• KMID: 2532762
• DOI: http://doi.org/10.4196/kjpp.2022.26.5.325

Abstract

Heart failure (HF) has become one of the severe public health problems. The detailed role of mitochondrial function in HF was still unclear. Benzoylaconine (BAC) is a traditional Chinese medicine, but its role in HF still needs to be explored. In this study, oxygen-glucose deprivation and reperfusion (OGD/R) was executed to mimic the injury of H9C2 cells in HF. The viability of H9C2 cells was assessed via MTT assay. OGD/R treatment markedly decreased the viability of H9C2 cells, but BAC treatment evidently increased the viability of OGD/R-treated H9C2 cells. The apoptosis of H9C2 was enhanced by OGD/R treatment but suppressed by BAC treatment. The mitochondrial membrane potential was evaluated via JC-1 assay. BAC improved the mitochondrial function and suppressed oxidative stress in OGD/R-treated H9C2 cells. Moreover, Western blot analysis revealed that the protein expression of p-AMPK and PGC-1α were reduced in OGD/R-treated H9C2 cells, which was reversed by BAC. Rescue assays indicated that AMPK attenuation reversed the BAC-mediated protective effect on OGD/R-treated cardiomyocytes. Moreover, BAC alleviated myocardial injury in vivo. In a word, BAC modulated the mitochondrial function in OGD/R-induced cardiomyocyte injury by activation of the AMPK/PGC-1 axis. The findings might provide support for the application of BAC in the treatment of HF.

Keyword

AMP-activated protein kinases; Benzoylaconine; Cardiomyocytes; Mitochondrial function

Figure

• 

  Fig. 1 BAC alleviated OGD/R-induced H9C2 cell injury. (A) MTT assay was performed to evaluate the viability of H9C2 cells. *p < 0.05 indicates the difference compared with vehicle group; **p < 0.01 indicates the difference compared with vehicle group. (B) The LDH level was assessed via the commercial kit. *p < 0.05 indicates the difference compared with vehicle group; **p < 0.01 indicates the difference compared with vehicle group. (C) The viability of H9C2 cells was measured via MTT assay. **p < 0.01 indicates the difference compared with control group; #p < 0.05 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; ##p < 0.01 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group. (D) The LDH level was detected using the commercial kit. ***p < 0.001 indicates the difference compared with control group; #p < 0.05 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; ###p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group. (E, F) The apoptosis of H9C2 cells was tested by flow cytometry through Annexin V-FITC/PI double labeling. ***p < 0.001 indicates the difference compared with control group; #p < 0.05 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; ###p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group. BAC, benzoylaconine; OGD (2 h)/R (6 h), oxygen-glucose deprivation (2 h) and reperfusion (6 h); MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; PI, propidium iodide.

• 

  Fig. 2 BAC improved mitochondrial function in OGD/R-treated H9C2 cells. (A, B) JC-1 fluorescence assay was employed for measuring the mitochondrial membrane potential (scale bar = 50 μm). ***p < 0.001 indicates the difference compared with control group; #p < 0.05 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; ###p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group. (C, D) The ROS and ATP production were detected through using respective detection kits. ***p < 0.001 indicates the difference compared with control group; ##p < 0.01 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; ###p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group. BAC, benzoylaconine; OGD (2 h)/R (6 h), oxygen-glucose deprivation (2 h) and reperfusion (6 h); ROS, reactive oxygen species; ATP, adenosine triphosphate; LDH, lactate dehydrogenase.

• 

  Fig. 3 BAC suppressed oxidative stress in OGD/R-treated H9C2 cells. The concentration of SOD, GSH-Px, MDA and CAT was tested via ELISA. ***p < 0.001 indicates the difference compared with control group; #p < 0.05 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; ##p < 0.01 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; ###p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group. BAC, benzoylaconine; OGD (2 h)/R (6 h), oxygen-glucose deprivation (2 h) and reperfusion (6 h); SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; CAT, catalase.

• 

  Fig. 4 BAC activated AMPK/PGC-1 axis in OGD/R-treated H9C2 cells. Western blot analysis was utilized for evaluating the protein levels of AMPK, p-AMPK, and PGC-1α. ***p < 0.001 indicates the difference compared with control group; #p < 0.05 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; ###p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group. BAC, benzoylaconine; OGD (2 h)/R (6 h), oxygen-glucose deprivation (2 h) and reperfusion (6 h); SOD, superoxide dismutase.

• 

  Fig. 5 AMPK attenuation reversed BAC-mediated protective effect on OGD/R-treated cardiomyocytes. (A) The viability of H9C2 cells was assessed via MTT assay. ***p < 0.001 indicates the difference compared with control group; ##p < 0.01 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; &&p < 0.01 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (75 μM) group. (B) The mitochondrial membrane potential was detected by JC-1 fluorescence assay (scale bar = 50 μm). ***p < 0.001 indicates the difference compared with control group; ###p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; &&&p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (75 μM) group. (C, D) The ROS and ATP production were measured using respective detection kits. ***p < 0.001 indicates the difference compared with control group; ###p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (0 μM) group; &&&p < 0.001 indicates the difference compared with OGD (2 h)/R (6 h) + BAC (75 μM) group. BAC, benzoylaconine; OGD (2 h)/R (6 h), oxygen-glucose deprivation (2 h) and reperfusion (6 h); CC, compound C; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; ATP, adenosine triphosphate.

• 

  Fig. 6 BAC alleviated myocardial injury in vivo. (A) The heart infarct size was evaluated by TTC staining. ***p < 0.001 indicates the difference compared with sham group; ###p < 0.001 indicates the difference compared with ischemia reperfusion (IR) group. (B, C) The LDH and ROS levels were measured through the corresponding commercial kits. ***p < 0.001 indicates the difference compared with sham group; ###p < 0.001 indicates the difference compared with IR group. (D) The protein expression of p-AMPK, AMPK, and PGC-1α in heart tissues was assessed through Western blot. ***p < 0.001 indicates the difference compared with sham group; ###p < 0.001 indicates the difference compared with IR group. BAC, benzoylaconine; TTC, 2,3,5-triphenyltetrazolium chloride; LDH, lactate dehydrogenase; ROS, reactive oxygen species.

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