枸杞多糖通过抑制NF-κB/NLRP3通路减少视网膜色素变性小鼠感光细胞凋亡

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

    摘要
  • 摘要:
    目的探讨枸杞多糖可否通过抑制核因子激活的B细胞的κ-轻链增强子/NOD样受体热蛋白结构域相关蛋白3（NF-κB/NLRP3）信号通路减少视网膜色素变性（RP）小鼠视网膜光感受器细胞的凋亡。
    方法（1）体外实验将小鼠视网膜神经节细胞（661W细胞）分为正常组、模型组、枸杞多糖低剂量组（40 mg/L）、中剂量组（80 mg/L）、高剂量组（160 mg/L）和阳性药物对照组（NLRP3抑制剂，160 mg/L）；分别用50 、100 、200 和400 μmol/L四种剂量的H2O2干预661W细胞, 选出最佳 H2O2 浓度（200 μmol/L）造模，细胞计数试剂盒CCK-8 测定细胞活力，流式细胞仪检测细胞凋亡率，免疫荧光检测NLRP3标志物的表达， 酶联免疫吸附试验（ELISA）和免疫印迹法（WB）检测细胞凋亡标志物的表达。（2）选用C57/BL6小鼠和Rd10小鼠进行体内实验，分组为正常组、模型组、枸杞多糖低剂量组（0.08 g/d）、中剂量组（0.16 g/d）、高剂量组（0.32 g/d）和阳性药物对照组（NLRP3抑制剂，0.08 g/d），其中正常组为C57/BL6小鼠, 其余组为Rd10小鼠，每组10只，相对应的药物连续灌胃4周，通过视网膜电图、组织病理学检查和WB等方法观察NF-κB/NLRP3通路及细胞凋亡标志物的表达，评估枸杞多糖对视网膜光感受器细胞凋亡的影响。
    结果（1）体外实验中，与正常组相比，模型组661W细胞凋亡率明显增加（P < 0.01），且NF-κB/NLRP通路关键蛋白NLRP3、NF-κB、p-NF-κB和促凋亡蛋白caspase-3的表达水平上调（P < 0.01），Bax/Bcl-2的比值增大（P < 0.01），白细胞介素（IL）-1β 和肿瘤坏死因子（TNF）-α的浓度显著升高（P < 0.01）。与模型组相比，高剂量的枸杞多糖降低了661W细胞的凋亡率（P < 0.01），下调了NF-κB/NLRP3通路关键蛋白NF-κB、NLRP3、p-NF-κB和caspase-3的表达（P < 0.01），减小了Bax/Bcl-2的比值（P < 0.01），降低了IL-1β和TNF-α的浓度（P < 0.01）。（2）体内实验中，高剂量的枸杞多糖显著增加了Rd10小鼠外核层（ONL）厚度的形态变化以及a波和b波振幅的功能变化（P < 0.01），下调了NF-κB（P < 0.05）、NLRP3、p-NF-κB和caspase-3的表达水平（P < 0.01），减小了Bax/Bcl-2的比值（P < 0.01），降低了IL-1β （P < 0.01）和TNF-α（P < 0.05）的浓度。
    结论枸杞多糖可能通过抑制NF-κB/NLRP3通路改善视网膜形态和功能，保护光感受器免受细胞凋亡的影响。

     

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

     

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