Catalpol Alleviates HFpEF via Inhibition of the S100A8-RAGE-NOX4 Inflammatory Axis in Murine Hearts

Yu Cao,1,* Sudan Li,2,* Yi Zhang,2 Menghua He,3 Yan Shen2 1Department of Geriatrics, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China; 2Department of Cardiology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China; 3Department of Teaching Affairs, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yan Shen, Department of Cardiology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China, Email [email protected]: This study aimed to elucidate the therapeutic effects of catalpol (CAT) on heart failure with preserved ejection fraction (HFpEF) and explore its underlying mechanisms.Methods: An HFpEF mouse model was established by combining a high-fat diet with N^ω^-nitro-L-arginine methyl ester administration, followed by CAT treatment for four weeks. Cardiac function was assessed by echocardiography, while histopathological changes were evaluated using hematoxylin–eosin and Masson’s trichrome staining. Network pharmacology and transcriptomic analyses were integrated to identify the core targets and signaling pathways of CAT in HFpEF. In vitro, H9C2 were stimulated with angiotensin II (Ang II), followed by CAT treatment, with recombinant rat S100A8 used to specifically activate the TLR4/RAGE pathway. Western blotting (WB) and quantitative real-time PCR were performed to validate the predicted signaling mechanisms both in vivo and in vitro.Results: In vivo, CAT markedly improved multiple pathological and functional abnormalities in HFpEF mice, including obesity, glucose intolerance, hypertension, diastolic dysfunction, myocardial inflammation, and fibrosis. Integrated network pharmacology and transcriptomic analyses consistently identified the RAGE signaling pathway as the key pathway of CAT. Molecular docking revealed strong binding affinities between CAT and S100A8, RAGE, NOX4, ERK1, and MMP2. WB results further demonstrated that CAT significantly downregulated the S100A8/RAGE/NOX4 axis and its downstream effector MMP2 in cardiac tissue. In vitro, CAT alleviated Ang II–induced cardiomyocyte hypertrophy, reduced the expression of inflammatory cytokines (TNF-α, IL-1β, IL-6) and the heart failure marker ANP, and inhibited S100A8, RAGE, and NOX4 protein levels. Notably, recombinant S100A8 activation weakened the protective effects of CAT.Conclusion: CAT exerts cardioprotective effects against HFpEF by suppressing myocardial inflammation and fibrosis through modulation of the S100A8/RAGE/NOX4 signaling axis and its downstream effectors. Keywords: catalpol, heart failure with preserved ejection fraction, network pharmacology, S100A8-RAGE-NOX4 axis, transcriptomics