In vivo and in vitro study on the regulatory mechanism of XiaoChaiHu decoction on PANoptosis in sepsis-induced cardiomyopathy

Ethnopharmacological relevance: In accordance with the tenets of traditional Chinese medicine, sepsis is categorized into three distinct syndromes: heat syndrome, blood stasis syndrome, and deficiency syndrome. Xiaochaihu decoction (XCHD) has many functions, including the capacity to protect the liver, cholagogue, antipyretic, anti-inflammatory, and anti-pathogenic Microorganisms. XCHD exerts the effect of clearing heat and reconciling Shaoyang. The XCHD contains many efficacious active ingredients, yet the mechanism of sepsis-induced cardiomyopathy (SIC) remains elusive.

Aim of the study: To investigate the molecular mechanisms underlying the protective effects of XCHD against SIC using an integrated approach combining network pharmacology and Molecular Biology techniques.

Materials and methods: Network pharmacology methods identified the active ingredients, target proteins, and pathways affected by XCHD in the context of SIC. We conducted in vivo experiments using mice with lipopolysaccharide-induced SIC, evaluating cardiac function through echocardiography and histology. XCHD-containing serum was analyzed to determine its principal active components using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The effects of XCHD-containing serum on SIC were further tested in vitro in LPS-treated H9c2 cardiac cells. Protein expression levels were quantified via Western blotting and enzyme-linked immunosorbent assay (ELISA). Additionally, molecular docking was performed between the active components and ZBP1, a potential target protein. Overexpression of ZBP1 in H9c2 cells allowed for a deeper exploration of its role in modulating SIC-associated gene expression.

Results: UPLC-MS/MS identified 31 shared XCHD and XCHD-containing serum components. These included organic acids, Terpenoids, and Flavonoids, which have been identified as the active components of XCHD. Our findings revealed that XCHD alleviated LPS-induced myocardial injury, improved cardiac function, and preserved cardiomyocyte morphology in mice. In vitro studies, we demonstrated that XCHD-containing serum significantly suppressed the expression of inflammatory cytokines (IL-6, IL-1β, and TNF-α) in LPS-induced H9c2 cells. Mechanistic investigations showed that XCHD downregulated genes associated with PANoptosis, a novel cell death pathway, suggesting its protective role in sepsis-damaged hearts. Conversely, overexpression of ZBP1 abolished the protective effects of XCHD and amplified PANoptosis-related gene expression.

Conclusions: Our study provides the first evidence supporting the protective effects of XCHD against SIC, both in vitro and in vivo. The underlying mechanism involves the inhibition of ZBP1-initiated PANoptosis, offering new insights into treating SIC using XCHD.

Drug-containing serum; Network pharmacology; PANoptosis; Sepsis-induced cardiomyopathy; XiaoChaiHu decoction.