Paeonol Attenuates Atherosclerosis by Inhibiting Vascular Smooth Muscle Cells Senescence via SIRT1/P53/TRF2 Signaling Pathway

Abstract

Atherosclerosis is a chronic inflammatory disease leading to various vascular diseases. Vascular smooth muscle cell (VSMC) senescence promotes atherosclerotic inflammation and the formation of plaque necrosis core, in part through telomere damage mediated by a high-fat diet. Our previous research found that paeonol, a potential anti-inflammatory agent extracted from Cortex Moutan, could significantly improve VSMCs dysfunction. However, the impact of paeonol on the senescence of VSMCs remains unexplored. This study presents the protective effects of paeonol on VSMCs senescence, and its potential activity in inhibiting the progression of atherosclerosis in vivo and in vitro. Sirtuin 1 (SIRT1) is a nuclear deacetylase involved in cell proliferation, senescence, telomere damage, and inflammation. Here, SIRT1 was identified as a potential target of paeonol having anti-senescence and anti-atherosclerosis activity. Mechanistic studies revealed that paeonol binds directly to SIRT1 and then activates the SIRT1/P53/TRF2 pathway to inhibit VSMCs senescence. Our results suggested that SIRT1-mediated VSMCs senescence is a promising druggable target for atherosclerosis, and that pharmacological modulation of the SIRT1/P53/TRF2 signaling pathway by paeonol is of potential benefit for patients with atherosclerosis.

Keywords: SIRT1; atherosclerosis; cell senescence; paeonol; vascular smooth muscle cells.

Figure 1

Paeonol administration mitigates the progression of atherosclerosis in ApoE^−/− mice. (A) Ultrasound images of the aortic arch and abdominal aorta in each group (the area circled by the red line indicates plaque formation). (B) Distance and (C) velocity of blood flow of mice in each group. (D) The chemical structure of paeonol. (E) Experimental design of C57BL/6J, ApoE^−/−, and ApoE^−/− + paeonol (400 and 200 mg/kg/day by intragastric administration once daily for 4 weeks). (F) Photographs of the aortic arches in each group. (G) En face Oil-Red O staining of the aortas in each group. Red: Lipid plaque deposition in intima. (H) H&E staining of the aortic sinus of mice in each group. Scale bar, 100 μm. The results are expressed as mean ± SD, ** p < 0.01 versus control; ^## p < 0.01 versus the model.

Figure 2

Paeonol reduces atherosclerosis-induced vascular senescence. (A) Paeonol significantly down-regulated the protein expression of P16 and P21 by Western blotting analysis. (B) The mRNA expression of TEL was increased after paeonol treatment by qRT-PCR. (C,D) Typical levels of SASP (TNF-α and IL-6) levels in serum and aortas of mice were decreased after paeonol treatment. The results are expressed as mean ± SD, ** p < 0.01 versus control; ^## p < 0.01 versus model.

Figure 3

Paeonol inhibits the senescence of VSMCs and activates SIRT1 signaling in ApoE^−/− mice. (A) Representative α-SMA and SA-β-gal immunofluorescence in artery intracuff lesions after treatment with paeonol in ApoE^−/− mice. DAPI was used for nuclear staining. Scale bar, 100 μm. Paeonol decreased the percentage of (B) SA-β-gal and (C) SA-β-gal colocalized with α-SMA in artery intracuff lesions of ApoE^−/− mice. (D) SIRT1, P53, and TRF2 protein levels were down-regulated after paeonol treatment by Western blotting analysis. The results are expressed as mean ± SD, ** p < 0.01 versus control; ^# p < 0.05, ^## p < 0.01 versus model.

Figure 4

Paeonol delays t-BHP stimulated senescence of VSMCs. Effects of t-BHP (A) and paeonol (B) on the activity of VSMCs by the CCK-8 assay. (C) After treatment with paeonol, cellular senescence was significantly reduced by SA-β-gal staining (senescent cells were stained blue). Scale bar, 10 μm. (D) The expression level of senescence-associated proteins (P21, P53, and P16) in t-BHP stimulated VSMCs was significantly down-regulated by paeonol. The results are expressed as mean ± SD, * p < 0.05, ** p < 0.01 versus control; ^# p < 0.05, ^## p < 0.01 versus t-BHP.

Figure 5

Paeonol increases the expression of SIRT1 and attenuates telomere damage in t-BHP-induced senescent VSMCs. VSMCs were exposed to t-BHP and then treated with paeonol, protein expression levels of SIRT1 (A) and TRF2 (B) were analyzed by Western blotting. (C) The level of TEL mRNA was increased after paeonol treatment by real-time PCR. (D,E) Paeonol decreased the levels of the SASP markers (IL-6 and TNF-α) in the cell supernatant using ELISA assay. (F) The cell proliferation capacity of VSMCs was determined by the EdU staining assay. The results are expressed as mean ± SD, ** p < 0.01 versus control; ^# p < 0.05, ^## p < 0.01 versus t-BHP.

Figure 6

SIRT1 is a potential cellular target of paeonol in VSMCs. (A) Paeonol promoted the resistance of SIRT1 to different temperature gradients, which was detected by CETSA in VSMCs. (B) Paeonol enhanced the resistance of SIRT1 to proteases, which was investigated by DARTS. (C) SIRT1 was successfully silenced in VSMCs by immunofluorescence staining and Western blot. Protein expression levels of senescence-associated proteins (P53, P16, P21) (D) and TRF2 (E) were analyzed by Western blotting in the control, t-BHP, siSIRT1, paeonol, and siSIRT1 + paeonol groups. (F) The level of TEL mRNA was assessed by real-time PCR. The results are expressed as mean ± SD, ** p < 0.01 versus control; ^# p < 0.05, ^## p < 0.01 versus t-BHP; ^Δ p < 0.05, ^ΔΔ p < 0.01 versus paeonol.

Figure 7

Paeonol inhibits VSMC senescence by activating the SIRT1/P53/TRF2 signaling pathway. The results are expressed as mean ± SD, ** p < 0.01 versus control; ^# p < 0.05, ^## p < 0.01 versus t-BHP; ^ΔΔ p < 0.01 versus t-BHP + paeonol.