Synergistic effects of notoginsenoside R1 and saikosaponin B2 in atherosclerosis: a novel approach targeting PI3K/Akt/mTOR pathway and macrophage autophagy

Atherosclerosis (AS) is a significant global contributor to cardiovascular
diseases and related mortalities. The traditional treatment
primarily employs statins,  but these drugs are often associated
with side effects such as liver dysfunction and muscle
impairment. Recent studies have highlighted the potential
protective properties of saponin compounds derived from traditional herbal
sources, such as notoginsenoside R1 (NGR1) and
saikosaponin B2 (SSB2), in combating AS. However, the comprehensive
effects of these compounds against atherosclerosis and their underlying
mechanisms remain inadequately understood. Firstly, we employed network
pharmacology analysis to identify 113 common targets, including mTOR and
CASP3, for NGR1, SSB2, and atherosclerosis (AS) from databases such as
TCMSP. We constructed a protein-protein interaction (PPI) network and
performed GO and KEGG enrichment analyses, revealing key signaling
pathways involved in PI3K/Akt, inflammation, and autophagy. The
atherosclerosis model was established using ApoE-/- mice
fed with a "Western diet," followed by treatment with NGR1,
SSB2, or NS combination. Histological examinations including
Hematoxylin-Eosin (HE) staining, oil red
O (ORO) staining, and/or CD68 immunofluorescence were
conducted to evaluate the pathological conditions of the aortic root as
well as the liver and kidneys in ApoE-/- mice. Our results
indicate that the NS combination improves lipid levels, lipid transport,
and unstable plaque formation in ApoE-/- mice without adversely affecting
liver or kidney function. Finally, oxidized low-density lipoprotein
(ox-LDL) was used to culture RAW264.7 macrophages to establish an in vitro
foam cell model. The effects of NS combination on lipid
uptake, inflammatory response, apoptosis, the PI3K/Akt/mTOR signaling
pathway, and autophagy were evaluated using methods such as CCK-8 assay,
Oil Red O staining, immunofluorescence analysis, flow cytometry, RT-PCR,
and Western blot analysis. The results indicated that NS combined
treatment promoted autophagy by inhibiting the PI3K/Akt/mTOR pathway. This
significantly alleviated inflammation, reduced apoptosis and lipid
accumulation, thereby improving the pathological progression of
atherosclerosis. Collectively, this study demonstrates, for the
first time, that the NS combination synergistically activates macrophage
autophagy by suppressing the PI3K/AKT/mTOR pathway, thereby attenuating
lipid accumulation, inflammation, and apoptosis in atherosclerotic models.