Objective:

2

To evaluate the protective effects of resveratrol against acute lung injury (ALI) and investigate the potential mechanisms underlying the reactive oxygen species (ROS)-triggered thioredoxin-interacting protein (TXNIP)/NOD-

LRR- and pyrin domain-containing protein 3 (NLRP3) pathway.

Methods:

2

C57BL/6 mice and J774A.1 cells were selected as the researchsubjects. Thirty Mice were randomly divided into 5 groups of 6 in each group: controlwith 0.9% saline

5 mg/kg lipopolysaccharide (LPS) 24 h

25 mg/kg resveratrol + 5 mg/kgLPS

100 mg/kg resveratrol + 5 mg/kg LPS

and 4 mg/kg NLRP3 inhibitor CY-09 + 5 mg/kgLPS. For cell stimulation

cells were pretreated with 5 and 20 μmol/L resveratrol for2 h

and stimulated with or without 1 μg/mL LPS and 3 mmol/L ATP for 2 h. The antioxidant N-acetyl-L-cysteine (NAC

2 μmol/L) was used as the positive control group. Hematoxylin and eosin staining was used to evaluate the degree of lung LPS-induced tissue damage

and enzyme-linked immunosorbent assay was used to evaluate the contents of interleukin-1β (IL-1β) and IL-18 in the serum and cell supernatant. ROS and malondialdehyde (MDA) levels in the lung tissue were detected using the corresponding kits. Western blotting was used to detect the expressions of TXNIP

high-mobility group box 1 (HMGB1)

NLRP3

as well as cysteine-aspartic acid protease 1 (caspase-1) and gasdermin D (GSDMD) along with their cleaved forms in lung tissue. Additionally

reverse transcription quantitative polymerase chain reaction was performed to analyze the expression of related inflammatory cytokines. ROS content was detected using flow cytometry and confocal laser microscopy. Mitochondrial morphological changes were observed using transmission electron microscopy

and HMGB1 expression was detected using immunofluorescence.

Results:

2

Resveratrol significantly alleviated LPS-induced lung damage with reduced inflammation

interstitial edema

and leukocyte infiltration (

P

<

0.01). It also decreased serum levels of IL-1β and IL-18 (

P

<

0.05)

while downregulating the expressions of NLRP3

IL-6

and other inflammatory markers at both the protein and mRNA levels (

P

<

0.05). Notably

the higher dose (100 mg/kg) demonstrated a better effect than the lower dose (25 mg/kg). In macrophages

resveratrol reduced IL-1β and IL-18 following LPS and ATP stimulation

suppressed HMGB1 translocation

and inhibited formation and activation of the NLRP3 inflammasome (

P

<

0.05 or

P

<

0.01). These anti-inflammatory effects were mediated through suppression ROS accumulation (

P

<

0.01) and mitochondrial dysfunction. Transmission electron microscopy revealed that resveratrol preserved mitochondrial structure

preventing the mitochondrial damage in LPS-treated groups (

P

<

0.01). The expressions of cleaved caspase-1

cleaved GSDMD

and cytoplasmic HMGB1 were all reduced following resveratrol treatment (

P

<

0.01). Moreover

resveratrol inhibited dissociation of TXNIP from thioredoxin

blocking subsequent activation of NLRP3 and downstream inflammatory cytokines (

P

<

0.01). Similarly

the higher concentration of resveratrol (20 μmol/L) exhibited superior efficacy

in vitro

.

Conclusion:

2

Resveratrol can reduce the inflammatory response following ALI and inhibit the activation of NLRP3 inflammasome and the level of HMGB1 in the cytoplasm by inhibiting ROS overproduction.