Objective:

2

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:

2

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)-1α

IL-1β

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 μmol/L)

and low-dose-SIB (100 μmol/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-1β were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylin-eosin staining

and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay.

Results:

2

SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1α

IL-1β

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:

2

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.