Effect of NLRP3 repression on NLRP3 inflammasome activation in human corneal epithelial cells with black carbon exposure

Abstract

Purpose

To investigate the inhibitory effects of NLRP3 siRNA on NLRP3 inflammasome activation in human corneal epithelial cells (HCECs) with fresh black carbon (FBC) particles and ozone-oxidized BC (OBC) particles treatment.

Methods

HCECs were transfected with NLRP3 siRNA or control siRNA for 48 h, followed by 200 μg/ml FBC or OBC suspension for an additional 72 h. Untreated controls were cells with no siRNA transfection or BC treatment. RT-qPCR and Western blot were used to measure mRNA and protein levels of components of the NLRP3 inflammasome (NLRP3, ASC, and Caspase-1) and downstream cytokine (IL-1β), respectively.

Results

Compared with untreated control cells, mRNA levels of NLRP3, ASC, Caspase-1, and IL-1β were significantly higher (p < 0.05) in control siRNA transfected cells with BC treatments. Compared with the control siRNA transfected cells, NLRP3 siRNA transfection reduced the expression of NLRP3 and ASC, whereas it had a limited effect on the expression of Caspase-1 and IL-1β with FBC or OBC exposures. Under FBC treatment, the reductions of NLRP3 and Caspase-1 mRNA levels were 53.5% (p < 0.001) and 34.2% (p < 0. 01), respectively, and NLRP3 and ASC protein levels were lowered by 58.2% (p < 0.001) and 45.4% (p < 0.001), respectively. Under OBC treatment, the reductions of NLRP3 and Caspase-1 mRNA levels were 39.8% (p < 0.001) and 25.6% (p < 0.05), respectively, and NLRP3 and ASC protein levels were lowered by 44.8% (p < 0.001) and 41.7% (p < 0.001), respectively. Moreover, mRNA levels of ASC and IL-1β, the protein levels of Caspase-1 and IL-1β showed a tendency to decrease in NLRP3 siRNA transfected cells, it was statistically insignificant (p > 0.05).

Conclusions

NLRP3 siRNA transfection could partially reverse the increased mRNA levels of NLRP3 and Caspase-1, the protein levels of NLRP3 and ASC in HCECs with BC treatment, whereas the reductions of protein levels of Caspase-1 and IL-1β were not significant, indicating that NLRP3 siRNA has a limited inhibitory effect on the activation of NLRP3 inflammasome triggered by BC.

Keywords:

• NLRP3
• black carbon
• siRNA
• cytotoxicity
• human corneal epithelial cells

Disclosure statement

The authors report no conflict of interest.

Additional information

Funding

This research is supported by the National Natural Science Foundation of China (number 81870685), the Natural Science Foundation of Beijing Municipality (number 7172173).