Objective:

2

To investigate the anti-inflammatory effect of rutaecarpine (RUT) on monosodium urate crystal (MSU)-induced murine peritonitis in mice and further explored the underlying mechanism of RUT in lipopolysaccharide (LPS)/MSU-induced gout model

in vitro

.

Methods:

2

In MSU-induced mice

36 male C57BL/6 mice were randomly divided into 6 groups of 8 mice each group

including the control group

model group

RUT low-

medium-

and high-doses groups

and prednisone acetate group. The mice in each group were orally administered the corresponding drugs or vehicle once a day for 7 consecutive days. The gout inflammation model was established by intraperitoneal injection of MSU to evaluate the anti-gout inflammatory effects of RUT. Then the proinflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and the proportions of infiltrating neutrophils cytokines were detected by flow cytometry. In LPS/MSU-treated or untreated THP-1 macrophages

cell viability was observed by cell counting kit 8 and proinflammatory cytokines were measured by ELISA. The percentage of pyroptotic cells were detected by flow cytometry. Respectively

the mRNA and protein levels were measured by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot

the nuclear translocation of nuclear factor κB (NF-κB) p65 was observed by laser confocal imaging. Additionally

surface plasmon resonance (SPR) and molecular docking were applied to validate the binding ability of RUT components to tumor necrosis factor α (TNF-α) targets.

Results:

2

RUT reduced the levels of infiltrating neutrophils and monocytes and decreased the levels of the proinflammatory cytokines interleukin

1β (IL-1β) and interleukin 6 (IL-6

all

P

<

0.01).

In vitro

RUT reduced the production of IL-1β

IL-6 and TNF-α. In addition

RT-PCR revealed the inhibitory effects of RUT on the mRNA levels of IL-1β

IL-6

cyclooxygenase-2 and TNF-α (

P

<

0.05 or

P

<

0.01). Mechanistically

RUT markedly reduced protein expressions of tumor necrosis factor receptor (TNFR)

phospho-mitogen-activated protein kinase (p-MAPK)

phospho-extracellular signal-regulated kinase

phospho-c-Jun N-terminal kinase

phospho-NF-κB

phospho-kinase α /β

NOD-like receptor thermal protein domain associated protein 3 (NLRPS)

cleaved-cysteinyl aspartate specific proteinase-1 and cleaved-gasdermin D in macrophages (

P

<

0.05 or

P

<

0.01). Molecularly

SPR revealed that RUT bound to TNF-α with a calculated equilibrium dissociation constant of 31.7 μmol/L. Molecular docking further confirmed that RUT could interact directly with the TNF-α protein via hydrogen bonding

van der Waals interactions

and carbon-hydrogen bonding.

Conclusion:

2

RUT alleviated MSU-induced peritonitis and inhibited the TNFR1-MAPK/NF-κB and NLRP3 inflammasome signaling pathway to attenuate gouty inflammation induced by LPS/MSU in THP-1 macrophages

suggesting that RUT could be a potential therapeutic candidate for gout.