RESUMO
OBJECTIVE: To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. METHODS: Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 ß, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 ß were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. RESULTS: SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 ß, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). CONCLUSIONS: SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.
RESUMO
Immunodeficiency can disrupt normal physiological activity and function. In this study, donkey bone collagen peptide (DP) and its iron chelate (DPI) were evaluated their potential as immunomodulators in cyclophosphamide (Cytoxan®, CTX)-induced Balb/c mice. The femoral tissue, lymphocytes, and serum from groups of mice were subjected to hematoxylin and eosin (H&E) staining, methylthiazolyldiphenyl-tetrazolium bromide (MTT) cell proliferation assays, and enzyme-linked immunosorbent assay (ELISA), respectively. Furthermore, a non-targeted metabolomics analysis based on UPLC-MS/MS and a reverse transcription polymerase chain reaction (RT-qPCR) technology were used to explore the specific metabolic pathways of DPI regulating immunocompromise. The results showed that CTX was able to significantly reduce the proliferative activity of mouse splenic lymphocytes and led to abnormal cytokine expression. After DP and DPI interventions, bone marrow tissue damage was significantly improved. In particular, DPI showed the ability to regulate the levels of immune factors more effectively than Fe2+ and DP. Furthermore, metabolomic analysis in both positive and negative ion modes showed that DPI and DP jointly regulated the levels of 20 plasma differential metabolites, while DPI and Fe2+ jointly regulated 14, and all 3 jointly regulated 10. Fe2+ and DP regulated energy metabolism and pyrimidine metabolism pathways, respectively. In contrast, DPI mainly modulated the purine salvage pathway and the JAK/STAT signaling pathway, which are the key to immune function. Therefore, DPI shows more effective immune regulation than Fe2+ and DP alone, and has good application potential in improving immunosuppression.