Korean J Physiol Pharmacol.  2020 Jan;24(1):69-79. 10.4196/kjpp.2020.24.1.69.

Quercetin-induced apoptosis ameliorates vascular smooth muscle cell senescence through AMP-activated protein kinase signaling pathway

• Kim SG
• Sung JY
• Kim JR
• Choi HC

• KMID: 2466571
• DOI: http://doi.org/10.4196/kjpp.2020.24.1.69

Abstract

Aging is one of the risk factors for the development of cardiovascular diseases. During the progression of cellular senescence, cells enter a state of irreversible growth arrest and display resistance to apoptosis. As a flavonoid, quercetin induces apoptosis in various cells. Accordingly, we investigated the relationship between quercetin-induced apoptosis and the inhibition of cellular senescence, and determined the mechanism of oxidative stress-induced vascular smooth muscle cell (VSMC) senescence. In cultured VSMCs, hydrogen peroxide (H₂O₂) dose-dependently induced senescence, which was associated with increased numbers of senescence-associated β-galactosidase-positive cells, decreased expression of SMP30, and activation of p53-p21 and p16 pathways. Along with senescence, expression of the anti-apoptotic protein Bcl-2 was observed to increase and the levels of proteins related to the apoptosis pathway were observed to decrease. Quercetin induced apoptosis through the activation of AMP-activated protein kinase. This action led to the alleviation of oxidative stress-induced VSMC senescence. Furthermore, the inhibition of AMPK activation with compound C and siRNA inhibited apoptosis and aggravated VSMC senescence by reversing p53-p21 and p16 pathways. These results suggest that senescent VSMCs are resistant to apoptosis and quercetin-induced apoptosis attenuated the oxidative stress-induced senescence through activation of AMPK. Therefore, induction of apoptosis by polyphenols such as quercetin may be worthy of attention for its anti-aging effects.

Keyword

Aging; AMP-activated protein kinase; Apoptosis; Quercetin; Vascular smooth muscle cell

MeSH Terms

Aging
AMP-Activated Protein Kinases*
Apoptosis*
Cardiovascular Diseases
Cell Aging*
Hydrogen Peroxide
Muscle, Smooth, Vascular*
Polyphenols
Quercetin
Risk Factors
RNA, Small Interfering
AMP-Activated Protein Kinases
Hydrogen Peroxide
Polyphenols
Quercetin
RNA, Small Interfering

Figure

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  Fig. 1 Induction of senescence in vascular smooth muscle cells by hydrogen peroxide (H2O2). (A) The chemical structure of quercetin. (B) Cells were incubated with different concentrations of H2O2 for 1 h. The cell viability was examined by MTT assay. (C) The cells stained with senescence-associated β-galactosidase and the expression of SMP30 was determined by western blot analysis to evaluate the senescent status (scale bar = 100 µM). (D) Results of western blot analysis indicate that the H2O2 treatment induced p53-p21 and p16 expressions. (E) H2O2-induced senescent cells displayed resistance to apoptosis by increasing Bcl-2 expression and inhibiting the apoptosis pathway. Representative results from three independent experiments are shown (n = 3); *p < 0.05 vs. control.

• 

  Fig. 2 The effects of quercetin on vascular smooth muscle cell (VSMC) senescence. (A) The protein level of AMPK was not changed by hydrogen peroxide (H2O2) (10, 20, and 50 µM). After treatment with H2O2 (50 µM, 1 h), cells were incubated with quercetin (50 µM, 6 h). (B) Western blot analysis indicated that quercetin induced the AMPK signaling pathway in VSMCs. (C and D) The senescence of VSMCs was observed to be delayed by quercetin (scale bar = 100 µM). (E) The protein levels of p53, p21, and p16 were determined by western blot analysis. Representative results from three independent experiments are shown (n = 3).

• 

  Fig. 3 The effects of quercetin on apoptosis in vascular smooth muscle cells. After treatment with hydrogen peroxide (H2O2) (50 µM, 1 h), cells were incubated with quercetin (50 µM, 6 h). (A) Quercetin inhibited H2O2-increased protein level of Bcl-2 and induced the apoptosis pathway. (B) Apoptosis was assessed with Annexin V-FITC staining by flow cytometric analysis followed by determination of the percentage of apoptotic cells. (C) The apoptotic cells were stained with acridine orange solution. Representative results from three independent experiments are shown (n = 3); *p < 0.01 vs. control, #p < 0.01 vs. H2O2 alone.

• 

  Fig. 4 The inhibitory effect of quercetin-induced AMPK activation on vascular smooth muscle cell (VSMC) senescence. (A–C) Cells were incubated with quercetin (50 µM, 6 h) alone or quercetin + compound C (C.C; 10 µM, 1 h) after pretreatment with hydrogen peroxide (H2O2) (50 µM, 1 h). (A) The activation of p-AMPK is observed to be inhibited by C.C. (B) Senescence-associated β-galactosidase (SA-β-gal) staining and expression of SMP30 were restored by inhibiting AMPK activation. (C) The expressions of p53, p21, and p16 were determined by western blot analysis. (D–F) VSMCs were transfected with the control or AMPK siRNA for 48 h, and incubated with H2O2 and quercetin. (D) VSMCs were subjected to western blot analysis to determine the level of p-AMPK. (E) After transfection, cells were stained with SA-β-gal and expression of SMP30 was confirmed. (F) Results of western blot analysis indicated that p53-p21 and p16 pathways were restored in the AMPK siRNA-transfected cells. Scale bar = 100 µM. Representative results from three independent experiments are shown (n = 3).

• 

  Fig. 5 The inhibitory effect of compound C on quercetin-induced apoptosis in vascular smooth muscle cell (VSMC). Cells were incubated with quercetin (50 µM, 6 h) alone or quercetin + compound C (C.C; 10 µM, 1 h) after pretreatment with hydrogen peroxide (H2O2) (50 µM, 1 h). (A) Bcl-2 and pro-apoptotic proteins were determined by western blot analysis. (B) Results of flow cytometric analysis indicated that quercetin induced apoptosis by activating AMPK. (C) VSMCs were stained with acridine orange solution to visualize apoptotic cells. Representative results from three independent experiments are shown (n = 3); *p < 0.05 vs. control, #p < 0.05 vs. H2O2 alone, &p < 0.05 vs. C.C.

• 

  Fig. 6 The inhibitory effect of AMPK siRNA transfection on quercetin-induced apoptosis in vascular smooth muscle cell (VSMC). VSMCs were transfected with control or AMPK siRNA for 48 h and incubated with hydrogen peroxide (H2O2) and quercetin. (A) The protein levels of Bcl-2, Bax, cytochrome C, Caspase-3 and cleaved caspase-3 were determined by western blot analysis. (B) Apoptosis was confirmed by flow cytometric analysis followed by indication of the percentage of apoptotic cells. (C) Transfected cells stained with anti-Bax (green) and anti-Bcl-2 (red). VSMCs transfected with AMPK siRNA showed lower Bax expression and higher Bcl-2 expression. (D) The graphic abstract of quercetin-induced anti-senescence mechanism. Representative results from three independent experiments are shown (n = 3); *p < 0.05 vs. control, #p < 0.05 vs. H2O2 alone, &p < 0.05 vs. CON siRNA.

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