Inhibitory effect and mechanism of SB225002 on NLRP3 inflammasome in retinal vascular endothelial cells under high glucose

Authors:Wang Qing, Zhuang Tongtong, Dong Lijie,Wang Linni

Corresponding author:Wang Linni, Email:wln5156@163.com

Published:2026-07-10

DOI: 10.3760/cma.j.cn115989-20250609-00190

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ABSTRACT 

Objective To observe the effect and possible mechanism of interleukin (IL)-8 antagonist SB225002 on the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in monkey retinal vascular endothelial cells (RF/6A cells) under high glucose conditions.

Methods The RF/6A cells cultured in vitro were divided into a normal control group, a mannitol group, a high glucose group, a high glucose+ dimethyl sulfoxide (DMSO) group, and high glucose+ SB225002 group, and were cultured with standard culture medium, 25 mmol/L mannitol, 25 mmol/L glucose, 25 mmol/L glucose+ 1‰ DMSO, and 25 mmol/L glucose+ 10 ng/ml SB225002 for 48 hours, respectively. The cell viability in each group was detected using Cell Counting Kit-8 and fluorescein diacetate (FDA) staining. The cell migration rate was assessed by wound healing assay. Total reactive oxygen species (ROS) and MitoSOX red mitochondrial superoxide indicator levels were measured using 2′, 7′-dichlorofluorescein diacetate fluorescent probe and MitoSOX. The mitochondrial membrane potential was evaluated with JC-10 fluorescent probe. The fluorescence intensity of IL-8, NLRP3, cysteine-aspartic acid protease-1 (Caspase-1), apoptosis-associated speck-like protein (ASC), IL-1β, and IL-18 in each group was detected by immunofluorescence. The relative protein expression levels of NLRP3, Caspase-1, ASC, IL-1β, IL-18, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) were determined by Western blot.

Results The IL-8 fluorescence intensities in the normal control group, mannitol group, high glucose group, high glucose+ DMSO group, and high glucose+ SB225002 group were 20.56±0.89, 22.20±0.91, 40.91±1.94, 40.49±2.00, and 26.15±3.57, respectively, with a statistically significant overall difference ( F=66.78, P<0.001). The IL-8 fluorescence intensity was significantly higher in the high glucose group than in the normal control group and mannitol group, while the IL-8 fluorescence intensity in the high glucose+ SB225002 group was significantly lower than that in the high glucose+ DMSO group (all P<0.05). The cell migration rate, FDA fluorescence intensity, absorbance ( A) value, total ROS fluorescence intensity, MitoSOX fluorescence intensity, monomer fluorescence intensity, as well as fluorescence intensities and relative protein expression levels of NLRP3, Caspase-1, ASC, IL-1β, and IL-18 were significantly higher and the polymer fluorescence intensity, mitochondrial membrane potential level, as well as the relative protein expression levels of Nrf2 and HO-1 were significantly lower in the high glucose group than those in the normal control group and the mannitol group (all P<0.05). The cell migration rate, FDA fluorescence intensity, A value, total ROS fluorescence intensity, MitoSOX fluorescence intensity, monomer fluorescence intensity, as well as fluorescence intensities and relative protein expression levels of NLRP3, Caspase-1, ASC, IL-1β, and IL-18 were significantly lower and the polymer fluorescence intensity, mitochondrial membrane potential level, as well as the relative protein expression levels of Nrf2 and HO-1 were significantly higher in the high glucose+ SB225002 group than those in the high glucose+ DMSO group (all P<0.05).

Conclusions SB225002 can inhibit the activation of NLRP3 inflammasome in RF/6A cells, alleviate cellular oxidative stress damage and inflammatory response under high glucose conditions, and activate the Nrf2/HO-1 signaling pathway.

KEYWORDS：

Retinal vascular endothelial cells;Nucleotide-binding oligomerization domain-like receptor protein 3;Inflammasome;Nuclear factor erythroid 2-related factor 2

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Authors Info & Affiliations 

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