[Mechanism study on Smilax glabra flavonoids in improving cardiac aging in rats by inhibiting S100A8/A9-mediated activation of p38 MAPK/NF-κB pathway].

This study investigated the mechanism by which Smilax glabra flavonoids(SGF) ameliorate D-galactose(D-gal)-induced cardiac aging in rats through the regulation of the S100 calcium-binding protein A8/A9(S100A8/A9)-mediated p38 mitogen-activated protein kinase/nuclear factor-κB(p38 MAPK/NF-κB) inflammatory pathway, utilizing both in vivo and in vitro experiments. For the in vivo experiment, a cardiac aging model was established by subcutaneous injection of D-gal(150 mg·kg~(-1)·d~(-1)) into the neck and back of rats. The rats were randomly divided into a control group, a model(D-gal) group, a low-dose SGF(54 mg·kg~(-1)) group, a high-dose SGF(108 mg·kg~(-1)) group, and a vitamin E group. For the in vitro experiment, a cellular aging model was constructed by using D-gal-induced H9c2 cardiomyocytes, with the S100A8/A9 inhibitor paquinimod(PAQ) and S100A8/A9 overexpression plasmids employed to validate the underlying mechanisms. Cognitive function and physical strength were assessed by the Morris water maze and grip strength tests, while cardiac function was evaluated via echocardiography. The whole heart weight was measured to calculate the cardiac index. Oxidative stress markers, including superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GSH-Px), and malondialdehyde(MDA), were detected in serum and cardiac tissue. Histopathological staining [HE, Masson, dihydroethidium(DHE)] and electron microscopy were used to observe myocardial structure and mitochondrial morphology. Cellular senescence was evaluated by β-galactosidase(SA-β-gal) staining, and molecular expression levels were determined by quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot. In vivo results demonstrated that SGF effectively improved weight loss in D-gal-treated rats, shortened escape latency in the water maze, increased platform crossings, and significantly enhanced cardiac ejection fraction(EF) and fractional shortening(FS). Pathological analysis revealed that SGF markedly reduced myocardial fibrosis, reactive oxygen species(ROS)-positive areas, and SA-β-gal-positive cell counts in D-gal-treated rats. Additionally, SGF significantly upregulated antioxidant enzyme activity(SOD, CAT, and GSH-Px) while downregulating the protein expression of S100A8/A9, p-p38 MAPK, and p-NF-κB. It also suppressed the expression of senescence markers(cyclin dependent kinase inhibitor 1A [CDKN1A], CDKN2A, and tumor protein 53 [Tp53]) and inflammatory cytokines(interleukin [IL]-1β, IL-6, and tumor necrosis factor-α [TNF-α]). The in vitro experiment confirmed that SGF significantly decreased SA-β-gal-positive cell counts and the expression of senescence markers and inflammatory factors. The effects of PAQ intervention were similar to those of SGF; however, these effects were reversed by S100A8/A9 overexpression. In conclusion, SGF may inhibit the activation of the S100A8/A9-mediated p38 MAPK/NF-κB inflammatory pathway to further mitigate oxidative stress, mitochondrial dysfunction, and inflammatory responses, thereby alleviating D-gal-induced cardiac aging in rats.