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Published on: July 6, 2019
Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell
Jin-Ying Ou1,2, Shan-Hong Liu1,2, Dong-Kai Tang1,2
1Third Level Research Laboratory of State Administration of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510515, China.
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 β, 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 β 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 β, 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.
Insights
Silibinin (SIB) effectively reduces pyroptosis in lipopolysaccharide (LPS)-induced endotoxemia by inhibiting caspase-11 and GSDMD. This compound protects against LPS-mediated lethality by preventing inflammatory cytokine release and improving survival rates.
Area of Science:
- Biochemistry
- Immunology
- Pharmacology
Background:
- Endotoxemia is a life-threatening condition caused by bacterial lipopolysaccharide (LPS).
- Pyroptosis, a pro-inflammatory form of programmed cell death, plays a critical role in endotoxemia pathogenesis.
- Targeting pyroptosis pathways presents a potential therapeutic strategy for endotoxemia.
Purpose of the Study:
- To investigate the protective effects of silibinin (SIB) against LPS-induced endotoxemia.
- To elucidate the underlying molecular mechanisms of SIB's action, focusing on pyroptosis inhibition.
Main Methods:
- In vitro studies using mouse peritoneal macrophages to assess cell viability, cytotoxicity, and inflammatory cytokine (IL-1α, IL-1β, IL-18) levels.
- Analysis of intracellular LPS levels, LPS-caspase-11 interaction, and expression of pyroptosis-related proteins (caspase-11, GSDMD) and genes.
- In vivo studies using mouse and zebrafish models of endotoxemia to evaluate survival rates, histopathology, and inflammatory markers.
Main Results:
- SIB significantly reduced caspase-11 and GSDMD-mediated pyroptosis in LPS-treated cells.
- SIB inhibited the translocation of intracellular LPS and its binding with caspase-11.
- SIB treatment attenuated inflammatory cytokine secretion, prolonged survival, and improved survival rates in endotoxemia models.
Conclusions:
- Silibinin exhibits protective effects in LPS-mediated endotoxemia by inhibiting pyroptosis.
- SIB acts by suppressing caspase-11-mediated GSDMD cleavage and preventing the interaction between LPS and caspase-11.
- These mechanisms contribute to the overall attenuation of LPS-induced lethality by silibinin.
