Xinmaikang-mediated mitophagy attenuates atherosclerosis via the PINK1/Parkin signaling pathway

The Chinese herbal compound Xinmaikang (XMK) is effective in treating atherosclerosis (AS), although the associated mechanisms of action remain unclear. We hypothesize that XMK increases mitophagy via the PINK1/Parkin signaling pathway and decreases reactive oxygen species (ROS), thus treating AS.

To explore the above-mentioned mechanisms of action of XMK in AS.

Ultra-performance liquid chromatography assay was performed to clarify the composition of XMK. A 16-week high-fat diet was fed to APOE^-/- mice to form an AS model. Next, mice were given XMK(0.95 g/kg/d, 1.99 g/kg/d, 3.98 g/kg/d, i.g.) or Atorvastatin(3 mg/kg/d, i.g.) or Rapamycin(4 mg/kg/d, i.p.) or XMK with Mdivi-1(40 mg/kg/d, i.p.) or an equivalent amount of normal saline for 4 weeks. Then mice were examined for AS plaque area, lesion area, collagen fiber, pro-inflammatory cytokines, lipid level, ROS level and mitophagy level. We assessed AS using Oil Red O, hematoxylin and eosin, and Sirius red staining, as well as ROS measurements. Mitophagy was evaluated by transmission electron microscopy, real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, single-cell Western blot, and immunofluorescence staining. In vitro, by oxidizing low-density lipoprotein, formation of RAW264.7 macrophage-derived foam cells induced. we induced foam cell formation in RAW264.7 macrophages. Then cells were incubated with XMK-medicated serum with or without Mdivi-1. We examined foam cell formation, ROS level, mitophagy level in cells. Finally, we knocked down the PINK1, and examined foam cell formation and PINK1/Parkin level in RAW264.7 macrophages.

UPLC analysis revealed 102 main ingredients in XMK. In vivo, XMK at medium-dose or high-dose significantly reduced AS plaques, lipids, pro-inflammatory cytokines, and ROS and increased mitophagy. In further study, Single-cell western blot showed that mitophagy level in macrophages sorted from AS mice was lower than the control mice. While XMK improved mitophagy level. In vitro, XMK reduced foam cell formation and ROS and increased mitophagy. When PINK1 was knocked down, XMK's effects on foam cell formation and PINK1/Parkin pathway activation were reduced.

The study shows that XMK is effective against AS by mediating macrophage mitophagy via the PINK1/Parkin signaling pathway. For the treatment of AS and drug discovery, it provides an experimental basis and target.