Elucidation of the Antipyretic and Anti-inflammatory Effect of 8-O-Acetyl Shanzhiside methyl ester Based on Intestinal Flora and Metabolomics Analysis

Xiaolin Li¹Tianlong Liu²Keke Liang¹Renjie Wang¹Jun Yang¹Yidan Chen³Rong Wang^3*Maoxing Li^4*

• ¹College of Pharmacy, Gansu University of Traditional Chinese Medicine, Lanzhou, Gansu Province, China
• ²People's Liberation Army Joint Logistics Support Force 940th Hospital, Lanzhou, Gansu Province, China
• ³Department of Pharmacy, People's Liberation Army Joint Logistics Support Force 940th Hospital, Lanzhou, Gansu Province, China
• ⁴Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences (AMMS), Beijing, Beijing, China

Phlomoides rotata (Benth. ex Hook.f.) Mathiesen (syn. Lamiophlomis rotata (Benth. ex Hook.f.) Kudô) 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-OaS). 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-OaS (the primary iridoid glycosides of P. rotate) on yeast-induced pyrexia in rats. Additionally, the role 8-OaS in modulating the intestinal flora composition and metabolome profile is explored. 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 show that 8-OaS 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-OaS 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-OaS 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-OaS 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-OaS based on Intestinal Flora and Metabolomics Analysis.