复方四黄液通过调控TLR2/4-IL-6/TNF信号轴及铁死亡通路抑制骨折术后感染的机制研究OA

Objective To investigate the mechanism of Compound Sihuang Liquid in inhibiting postoperative fracture infection via modulating the TLR2/4-IL-6/TNF signaling axis and ferroptosis pathway.Methods Network pharmacology was used to identify active components of Compound Sihuang Liquid and their potential targets related to bacterial infection.Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were conducted.Molecular docking was integrated to verify binding affinities of core targets and active components.In animal experiments,40 SPF-grade white rabbits were divided into a control group,a Compound Sihuang Liquid group,a vancomycin group,a Compound Sihuang Liquid+Fer-1 group,and a Compound Sihuang Liquid+Erastin group using the random number table method,with 8 animals in each group.After modelling postoperative fracture infection,the control group was administered normal saline via gavage;the Compound Sihuang Liquid group was administered the corresponding liquid via gavage;the vancomycin group was administered vancomycin via intraperitoneal injection.The Compound Sihuang Liquid+Fer-1 group was administered the corresponding liquid via gavage and the ferroptosis inhibitor Ferrostatin-1 via intraperitoneal injection,while the Compound Sihuang Liquid+Erastin group was administered the corresponding liquid via gavage and the ferroptosis activator Erastin via intraperitoneal injection.Intervention was given once daily for 15 consecutive days.Then,Western blot was employed to measure Acyl-CoA synthetase long-chain family member 4(ACSL4),cyclooxygenase-2(COX-2),glutathione peroxidase 4(GPX4),interleukin-6(IL-6),Toll-like receptor 2(TLR2),Toll-like receptor 4(TLR4),tumor necrosis factor-alpha(TNF-α),and glyceraldehyde-3-phosphate dehydrogenase(GAPDH)expression levels.Enzyme-linked immunosorbent assay(ELISA)was performed to examine serum malondialdehyde(MDA),IL-6,and TNF-α expression levels.Results According to network pharmacology results,117 active components were screened in Compound Sihuang Liquid,and 81 intersected with targets related to bacterial infection,with IL-6 and TNF being the core targets.Molecular docking revealed favorable binding affinities between the key active components and core targets.GO and KEGG enrichment analyses indicated that Compound Sihuang Liquid was primarily involved in responses to xeno-stimuli,reactive oxygen species,and oxidative stress,while modulating IL-17,TNF,and Toll-like receptor signaling pathways.Animal experiment results revealed that,compared with the control group,the Compound Sihuang Liquid group exhibited significantly decreased ACSL4,COX-2,IL-6,TLR2,TLR4,and TNF-α expression,and significantly increased GPX4 expression in synovial tissues(P<0.001),along with significantly decreased serum MDA,IL-6,and TNF-α expression(P<0.001).Compared with the vancomycin group,the Compound Sihuang Liquid group showed significantly decreased ACSL4,TLR2,and TLR4 expression and significantly increased GPX4 expression in synovial tissues(P<0.001),while no significant intergroup difference was observed in serum MDA,IL-6,or TNF-α expression(P>0.05).Compared with the Compound Sihuang Liquid group,the Compound Sihuang Liquid+Fer-1 group demonstrated significantly decreased ACSL4,COX-2,IL-6,TLR2,TLR4,and TNF-α expression and significantly increased GPX4 expression in synovial tissues(P<0.001),along with significantly decreased serum MDA,IL-6,and TNF-α expression(P<0.05).Similarly,the Compound Sihuang Liquid+Erastin group displayed significantly increased ACSL4,COX-2,IL-6,TLR2,TLR4,and TNF-α expression and significantly decreased GPX4 expression in synovial tissues(P<0.001),along with significantly increased serum MDA,IL-6,and TNF-α expression(P<0.05).Conclusion Compound Sihuang Liquid alleviates postoperative fracture infection by regulating the TLR2/4‒IL-6/TNF signaling axis and ferroptosis pathway,providing a novel strategy for clinical treatment.