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 levelsof 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.
ABSTRACT
Background Researches showed that the increase of intraocular pressure (IOP) in glaucomatous eye is associated with the increasing resistance to aqueous humor outflow effects of transforming growth factor-β (TGF-β) and CD44.Qingguangan is a traditional Chinese medicine and used to treat glaucoma.However,its mechanism of lowing-IOP effect is not elucidated.Objective This study was to investigate the lowing-IOP effect and mechanism of qingguangan granule in DBA/2J mouse,a spontaneous glaucoma model mice.Methods Ten 3 month-old female DBA/2J mice with normal IOP were chosen as control group,and 20 spontaneous ocular hypertension mice aged 9 months were randomized into high IOP group and qingguangan-treated group,with 10 mice for each group.The qingguangan (2.5 g/kg) was administered by gavaging twice per day for consecutive 15 days in the qingguangan-treated group,and normal saline solution was used in the same way in the control group and high IOP group.IOP was measured by anterior chamber injection/suction system at a perfusion rate of 2.5 and 5.0 μl/min,respectively,and the coefficient of aqueous outflow facility (C value) and outflow resistance (R value) were calculated.Another 60 3-month-old DBA/2J mice were randomized into blank control group gavaged with normal saline solution and high-,middle-and low-dose qingguangan groups gavaged with 25.00,12.50 and 6.25 g/kg drugs,respectively,and the mouse serum containing drugs was extracted 7 days after treatment.The scleral tissue with trabecular meshwork were obtained for the culture of trabecular meshwork cells and the cells were identified by immunohistochemistry of fibronectin (FN),laminin (LN) and neuronspecific enolase (NSE).TGF-β was added into the medium for 24 hours with the final concentration of 0,5,10,20,50 and 100 ng/ml,and MMT chromatometry was employed to detect the cell vitality.The cells pre-treated with 20 ng/ml TGF-β were treated with different concentration of drug serum for 24,48 and 72 hours,and the level of TGF-β2 receptor in cell supernatant and the expression of CD44 protein in the cells were detected by ELISA and Western blot assay,respectively.Results The IOPs with perfusion both 2.5 μl/min and 5.0μl/min in the qingguangan-treated group and the control group were significantly lower than those in the high IOP group (all at P＜0.01).Compared with the high IOP group,the C value was significantly reduced (2.35±1.34 vs.1.08±0.36) and the R value was evidently elevated (0.64±0.55 vs.1.05± 0.47) in the qingguangan-treated group (all at P＜0.01).Cultured cells were spindle-shaped with the positive response to FN,LN and NSE antibody.The cell vitality was lower in the 5,10 and 20 ng/ml TGF-β group than that in the 0 ng/ml TGF-β group (all at P＜0.05).Compared with the blank control group,the TGF-β2 receptor content in the supernatant and the related expression level of CD44 protein in the cells were elevated in the TGF-β-treated group (all at P＜0.01),and TGF-β2 receptor contents and CD44 expression levels in the TGF-β+high dose drug serum group was significantly lower than those in the TGF-β group and TGF-β +low dose drug serum group 24,48 and 72 hours after culture (all at P＜0.01).Conclusions Qingguangan can lower IOP of spontaneous glaucoma mice by affecting aqueous humor dynamics.Serum containing qingguangan down-regulates the expressions of TGF-β2 receptors and CD44 in trabecular meshwork cells in vitro.