Paeonol attenuates atherosclerosis by regulating vascular smooth muscle cells apoptosis and modulating immune cells infiltration through reducing LTβR expression.

Background: Atherosclerosis is a chronic inflammatory disease with multicellular participation, and the decrease of plaque stability induces the occurrence of clinical adverse events. In order to update the clinical treatment strategy of atherosclerosis, it is necessary to clarify the mechanism of plaque stabilization, especially to explore the targets of vascular smooth muscle cells (VSMCs) apoptosis and immune cell infiltration. Paeonol (Pae), a major phenolic compound derived from the bark of Paeonia albiflora Andr., has been proved to have anti-inflammatory properties in atherosclerosis. However, the pharmacological mechanisms of Pae in improving atherosclerosis remain unclear, particularly with regard to the role of stabilizing vulnerable plaques.

Purpose: This study is aiming to elucidate the effect of Pae against atherosclerotic unstable plaque, and to further explore the potential mechanism of Pae in inhibiting VSMCs apoptosis and immune cell infiltration.

Methods: A high-fat diet (HFD) induced atherosclerosis mice model was established in ApoE mice, Pae in two different dosages and simvastatin (SIM) were than administrated for another 4 weeks. Atherosclerotic plaque formation and lipid accumulation were assessed with hematoxylin and eosin (H&E) staining and oil red O staining. Immunofluorescence were employed to examine the general condition of mice and the protective effect of Pae on plaque progression. Cell apoptosis was assessed via TUNNEL staining and flow cytometry. The mRNA and protein expressions in aorta tissue was detected by RT-PCR and western blotting. To investigate the effect of Pae on the regulation of the LTβR/NIK/caspase-3 pathway, VSMCs were extracted from the aorta of C57BL/6 J mice and treated with LTαβ.

Results: Here, we show that Pae significantly inhibited atherosclerosis progression and stabilized vulnerable plaques in ApoE mice, in association with decreased T/B cell infiltration and VSMC apoptosis. Notably, the number of plaque-infiltrating T/B cells showed a linear positive correlation with apoptotic VSMCs, and VSMCs sensitive to apoptosis expressed LTβR, which might be activated by LTαβ-expressing T/B cells. Moreover, the protein expression of LTβR in VSMCs was decreased in plaques after treatment of Pae. Mechanistically, Pae treatment inhibited LTαβ stimulated VSMCs apoptosis via LTβR/NIK/caspase-3 signaling pathway in vitro. Importantly, LTβR overexpression increased the VSMCs apoptosis and plaque instability in ApoE mice, partially reversing the protective effect of Pae.

Conclusion: Inhibition of LTβR signaling represents a promising strategy that exerts therapeutic effects through the combined suppression of immune cell infiltration and VSMCs apoptosis, providing novel insights into the anti-atherosclerosis mechanisms of Pae.