Deciphering the molecular mechanisms underlying of Xin Xue Granule on LPS-induced pyrexia rats: A multi-omics...

Xin Xue Granule (XXGR), which have been found in the classic prescription Taiping Huimin Heji Jufang, are traditionally used for their heat-clearing and detoxifying properties and can alleviate fever caused by infection and inflammation in clinical applications. However, the molecular mechanisms underlying its antipyretic and anti-inflammatory effects remain largely unclear.

This study aimed to investigate the antipyretic and anti-inflammatory effects of XXGR in lipopolysaccharide (LPS)-induced fever in rats and to elucidate the potential mechanisms with an emphasis on vascular integrity and VEGF-related signaling pathways.

Fever was induced in adult rats by intraperitoneal injection of LPS. XXGR was administered at varying doses, followed by assessments of rectal temperature, inflammatory cytokine levels in serum and hypothalamus, and histopathological alterations. The chemical composition and blood-absorbed components of XXGR were analyzed. Multi-omics (transcriptomics and metabolomics) combined with network pharmacology were applied to identify key pathways. CD31 expression was examined by immunohistochemistry and immunofluorescence assays. The VEGFR2/PI3K/AKT/eNOS signaling pathway was validated by RT-qPCR and Western blotting.

The study indicates that XXGR treatment significantly reduces rat body temperature and alleviates inflammation-induced fever and tissue damage in a dose-dependent manner. It also markedly inhibits inflammatory factor expression in serum and hypothalamus tissues while lessening LPS - induced tissue damage. Blood component analysis suggests that Andrographis paniculata (Burm. f.) Nees, Gardenia jasminoides J.Ellis, and Bovis Calculus in XXGR may contribute to its antipyretic and anti-inflammatory effects. Subsequent analysis indicates that XXGR's potential to activate CD31 expression warrants further investigation. Activating CD31 may maintain vascular integrity, prevent excessive inflammatory cell infiltration into surrounding tissues, and reduce tissue damage. Additionally, XXGR has been shown to synergistically activate the VEGF signaling pathway, thereby activating and up-regulating the expression of genes and proteins related to VEGFR2/PI3K/AKT/eNOS. Moreover, it enhances NO production, which plays an anti-inflammatory and antipyretic role.

Overall, XXGR exhibits potent antipyretic and anti-inflammatory effects in LPS-induced febrile rats by activating the VEGFR2/PI3K/AKT/eNOS signaling pathway and enhancing vascular integrity, providing novel insights into its systemic and molecular mechanisms of action.