Inhibition of photoreceptor apoptosis in mice with retinitis pigmentosa through NF-κB/NLRP3 pathway suppression with Lycium barbarum polysaccharide

Abstract

Objective

To explore whether Lycium barbarum polysaccharide (LBP) can reduce the apoptosis of retinal photoreceptor cells in retinitis pigmentosa (RP) mice by inhibiting nuclear factor-kappa B (NF-κB)/NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) signaling pathway.

Methods

(i) In vitro experiments, mouse retinal ganglion cells (661W cells) were divided into normal, model, LBP low-dose (LBP-L, 40 mg/L), LBP middle-dose (LBP-M, 80 mg/L), LBP high-dose (LBP-H, 160 mg/L), and positive drug control (NLRP3 inhibitor, 160 mg/L) groups. And the 661W cells were exposed to varying concentrations of H₂O₂ ranging from 50 to 400 μmol/L to determine the optimal concentration for inducing apoptosis (200 μmol/L). Then the cell viability was assessed using Cell Counting Kit-8 (CCK-8), while the apoptosis rate was detected by flow cytometry; the expression of NLRP3 was detected by immunofluorescence; and the expression of apoptosis markers was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). (ii) In vivo assays were carried out with the use of C57/BL6 and Rd10 mice. The animal experimental groups were divided into normal, model, LBP-L, LBP-M, LBP-H, and NLRP3 inhibitor groups, in which the normal group was C57/BL6 mice and the other groups were Rd10 mice. Ten mice were included in each group, and the corresponding drugs were administered intragastrically for a duration of four weeks. NF-κB/NLRP3 pathway and the expression of apoptosis markers were observed by electroretinogram, histopathological examination, and WB to assess the effects of LBP on retinal photoreceptor cell apoptosis.

Results

(i) In vitro experiments, compared with the normal group, the apoptosis rate of 661W cells in model group was significantly increased (P < 0.01), and the expression levels of key proteins of NF-κB/NLRP pathway, such as NLRP3, NF-κB, p-NF-κB, and pro-apoptotic protein caspase-3, were up-regulated (P < 0.01). The rate of Bax/Bcl-2 was increased (P < 0.01), and the concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α were significantly increased (P < 0.01). Compared with the model group, high dose of LBP decreased the apoptosis rate of 661W cells (P < 0.01), and down-regulated the expression levels of the key proteins of NF-κB/NLRP3 pathway, including NF-κB, NLRP3, p-NF-κB, and caspase-3 (P < 0.01). The rate of Bax/Bcl-2 was decreased (P < 0.01), and the concentrations of IL-1β and TNF-α were decreased (P < 0.01). (ii) In vivo experiments, high dose of LBP significantly increased morphological changes in the outer nuclear layer (ONL) thickness of Rd10 mice, as well as functional changes in the amplitudes of the a-wave and b-wave (P < 0.01), which also down-regulated the expression levels of NF-κB (P < 0.05), NLRP3, p-NF-κB, and caspase-3 (P < 0.01), reduced the Bax/Bcl-2 rate (P < 0.01), and decreased the concentrations of IL-1β (P < 0.01) and TNF-α (P < 0.05).

Conclusion

LBP could improve both retinal morphology and function, providing protection to photoreceptors from apoptosis through the inhibition of the NF-κB/NLRP3 pathway.