Elucidation of the antipyretic and anti-inflammatory effect of 8- O -Acetyl Shanzhiside methyl ester based on intestinal flora and metabolomics analysis

Introduction Phlomoides rotata (Benth. ex Hook.f.) Mathiesen (syn. Lamiophlomis rotata (Benth. ex Hook.f.) Kudô) ( P. rotate ) is a traditional Tibetan medicine known for its hemostatic, analgesic, and anti-inflammatory effects, as well as its high content of 8- O -Acetyl Shanzhiside methyl ester (8- O aS). Clinical and experimental studies have reported gastrointestinal side effects, such as diarrhea, loose stools, even to black stools, associated with P. rotata . Given the bitter taste characteristic, laxative and antipyretic effects of iridoid glycosides, this study aims to investigate the antipyretic and anti-inflammatory effects of 8- O aS (the primary iridoid glycosides of P. rotate ) on yeast-induced pyrexia in rats. Additionally, the role 8- O aS in modulating the intestinal flora composition and metabolome profile is explored. Methods The pyretic rat model was established by injected subcutaneously with 20% dry yeast suspension. Serum, hypothalamic tissues and colon content were collected for the assessment of relevant indicators. The peripheral inflammatory factors and central thermoregulatory mediators were assessed using enzyme-linked immunosorbent assay (ELISA). The expressions of mRNA and protein in hypothalamic tissue were evaluated through polymerase chain reaction (PCR), immunohistochemistry, and western blotting. 16S rDNA sequencing and LC-MS/MS were performed to determine the alteration and correlation of the intestinal flora and neurotransmitters in the colonic contents and hypothalamus. Results and discussion: Results show that 8- O aS treatment reduced pyrogenic cytokines (such as IL-6, IL-1β), and down-regulated the level of central thermoregulatory mediators (PGE 2 ), via multiply involved in TLR4/NF-κB and HSP70/NF-κB signaling pathways. Crucially, 8- O aS treatment significantly reduced the relative abundance of Alistipes ( P < 0.01), Odoribacter ( P < 0.05) and Alistipes_finegoldii ( P < 0.05) in the intestinal flora. The correlation analysis demonstrated that 8- O aS treatment significantly correlated with the increasing on the abundance of Alistipes and levels of 5-hydroxytryptamine ( P < 0.01), and tryptamine ( P < 0.01). Our findings indicate that 8- O aS exhibits significant antipyretic and anti-inflammatory properties, potentially mediated by intestinal flora and metabolites of neurotransmitters. The results of this study may help to elucidate the antipyretic and anti-inflammatory mechanism of 8- O aS based on intestinal flora and metabolomics analysis.