Flavonoids of S. suberectus Dunn Regulate Cyclophosphamide-Induced Immunosuppression Through NF-κB Pathways

Simple SummaryImmunosuppressive diseases are frequently encountered in animal husbandry, including porcine reproductive and respiratory syndrome, swine fever, and infectious bursal disease in chickens. Due to their high mortality rates, these diseases pose a significant threat to the healthy development of animal husbandry. The total flavonoids ofSpatholobus suberectus Dunn(TFSD) exhibit significant immunomodulatory properties, demonstrating their therapeutic potential against immunosuppressive conditions. The study results indicate that TFSD administration significantly mitigates cyclophosphamide-induced immunotoxicity by preserving splenic and thymic architecture while restoring immune competence in treated subjects. Additionally, network pharmacology, molecular docking, and in vivo experiments demonstrated that TFSD can reduce immunosuppression-induced damage by regulating the NF-κB signaling pathway. These findings suggest that TFSD is a promising immunotherapeutic candidate for managing immunosuppression-related immune dysfunction in veterinary medicine.This study investigated the immunomodulatory effects and underlying mechanisms of total flavonoid ofSpatholobus suberectus Dunn(TFSD) and its primary constituent formononetin (FMN) in immunosuppressed mouse models.Spatholobus suberectus Dunn(S. suberectus Dunn) was first analyzed qualitatively and quantitatively by broadly targeted metabolomics using Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Integrated network pharmacology and molecular docking approaches were used to investigate the potential mechanisms underlyingS. suberectus Dunn’s immunomodulatory effects. Subsequently, mice were pretreated with TFSD and FMN for seven days before constructing an immunosuppression model through intraperitoneal injection of 200 mg/kg.bw cyclophosphamide (CTX). In vivo experiments validated the findings and investigated the mechanisms underlying the effects of TFSD and FMN on immunosuppression. Metabolomic analysis identified 501 distinct flavonoids inS. suberectus Dunn, with FMN exhibiting the highest relative abundance among all detected compounds. The primary active components ofS. suberectus Dunnagainst immunosuppression are flavonoids, including FMN and vestitol. The core targets of these components were identified as NF-κB and IKKβ, with the NF-κB signaling pathway being suggested as the most probable mechanism of action. FMN exhibited strong binding affinity to the core targets, NF-κB p65 and IKKβ. In vivo experiments indicated that pretreatment with TFSD and FMN prevented CTX-induced pathological damage in the spleen and thymus and increased immune cell counts and immunoglobulin levels. Additionally, it significantly upregulated the secretion and expression of key cytokines and the mRNA expression of NF-κB p65, IKKα, and IKKβ (p< 0.05 orp< 0.01). In conclusion, TFSD and FMN can protect mice from CTX-induced immunosuppression by regulating the NF-κB signaling pathway, making them promising drug candidates for preventing and treating immunosuppression-related diseases.