PurposeTo evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye.MethodsWe recruited 54 patients with Sjögren’s syndrome dry eye (SSDE), 50 patients with non-Sjögren’s syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting.ResultsNLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p<0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p<0.05) and NSSDE (relative 1.32-fold upregulation, p<0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold (p<0.001) in SSDE and 2.13-fold (p<0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE (p=0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p=0.001) and NSSDE (relative 2.05-fold upregulation, p=0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE (p<0.001) and NSSDE (p<0.05) groups.ConclusionsIn the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye.
Background
Particulate matter (PM) is strongly linked to human health and has detrimental effects on the eye. Studies have, however, focused on the ocular surface, with limited research on the impact of PM2.5 on intraocular pressure (IOP).
Methods
To investigate the impact of PM2.5 on IOP and the associated mechanism, C57BL/6 mouse eyes were topically exposed to a PM2.5 suspension for 3 months, and human trabecular meshwork (HTM) cells were subjected to various PM2.5 concentrations in vitro. Cell viability, NLRP3/caspase-1, IL-1β, and GSDMD expression, reactive oxygen species (ROS) production and cell contractility were measured by western blot, ELISA, cell counting kit-8, ROS assay kit or a cell contractility assay. ROS scavenger N-acetyl-L-cysteine (NAC) and caspase-1 inhibitor VX-765 were used to intervene in PM2.5-induced damages.
Results
The results revealed that the IOP increased gradually after PM2.5 exposure, and upregulations of the NLRP3 inflammasome, caspase-1, IL-1β, and GSDMD protein levels were observed in outflow tissues. PM2.5 exposure decreased HTM cell viability and affected contraction. Furthermore, elevated ROS levels were observed as well as an activation of the NLRP3 inflammasome and downstream inflammatory factors caspase-1 and IL-1β. NAC improved HTM cell viability, inhibited the activation of the NLRP3 inflammasome axis, and HTM cell contraction by scavenging ROS. VX-765 showed similar protection against the PM2.5 induced adverse effects.
Conclusion
This study provides novel evidence that PM2.5 has a direct toxic effect on intraocular tissues and may contribute to the initiation and development of ocular hypertension and glaucoma. This occurs as a result of increased oxidative stress and the subsequent induction of NLRP3 inflammasome mediated pyroptosis in trabecular meshwork cells.
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