PDF(Pubmed)
Abstract：
OBJECTIVE: To study the effect of the NLRP3/autophagy pathway on the photoreceptor inflammatory response and the protective mechanism of CY-09 and astaxanthin (AST).
METHODS: ICR mice were intraperitoneally injected NaIO3, CY-09, AST successively and divided into 5 groups, including the control, NaIO3, NaIO3+CY-09, NaIO3+AST, and NaIO3+CY-09+AST groups. Spectral domain optical coherence tomography and flash electroretinogram were examined and the retina tissues were harvested for immunohistochemistry, enzyme linked immunosorbent assay (ELISA), and Western blotting. Retinal pigment epithelium cell line (ARPE-19 cells) and mouse photoreceptor cells line (661W cells) were also treated with NaIO3, CY-09, and AST successively. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay. Apoptosis was analyzed by flow cytometry. Changes in autophagosome morphology were observed by transmission electron microscopy. Quantitative polymerase chain reaction (qPCR) was used to detect NLRP3 and caspase-1. NLRP3, caspase-1, cleaved caspase-1, p62, Beclin-1, and LC3 protein levels were measured by Western blotting. IL-1β and IL-18 were measured by ELISA.
RESULTS: Compared with the control group, the activity of NaIO3-treated 661W cells decreased within 24 and 48h, apoptosis increased, NLRP3, caspase-1, IL-1β and IL-18 levels increased, and autophagy-related protein levels increased (P<0.05). Compared with NaIO3 group, CY-09 and AST inhibited apoptosis (P<0.05), reduced NLRP3, caspase-1, IL-1β and IL-18 expression (P<0.05), and inhibited autophagy. Compared with the other groups, CY-09 combined with AST significantly decreased NLRP3 expression and inhibited the expression of the autophagy-related proteins p62, Beclin-1, and LC3 in vitro and in vivo (P<0.05).
CONCLUSIONS: CY-09 and AST inhibit NaIO3-induced inflammatory damage through the NLRP3/autophagy pathway in vitro and in vivo. CY-09 and AST may protect retina from inflammatory injury.
METHODS: ICR mice were intraperitoneally injected NaIO3, CY-09, AST successively and divided into 5 groups, including the control, NaIO3, NaIO3+CY-09, NaIO3+AST, and NaIO3+CY-09+AST groups. Spectral domain optical coherence tomography and flash electroretinogram were examined and the retina tissues were harvested for immunohistochemistry, enzyme linked immunosorbent assay (ELISA), and Western blotting. Retinal pigment epithelium cell line (ARPE-19 cells) and mouse photoreceptor cells line (661W cells) were also treated with NaIO3, CY-09, and AST successively. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay. Apoptosis was analyzed by flow cytometry. Changes in autophagosome morphology were observed by transmission electron microscopy. Quantitative polymerase chain reaction (qPCR) was used to detect NLRP3 and caspase-1. NLRP3, caspase-1, cleaved caspase-1, p62, Beclin-1, and LC3 protein levels were measured by Western blotting. IL-1β and IL-18 were measured by ELISA.
RESULTS: Compared with the control group, the activity of NaIO3-treated 661W cells decreased within 24 and 48h, apoptosis increased, NLRP3, caspase-1, IL-1β and IL-18 levels increased, and autophagy-related protein levels increased (P<0.05). Compared with NaIO3 group, CY-09 and AST inhibited apoptosis (P<0.05), reduced NLRP3, caspase-1, IL-1β and IL-18 expression (P<0.05), and inhibited autophagy. Compared with the other groups, CY-09 combined with AST significantly decreased NLRP3 expression and inhibited the expression of the autophagy-related proteins p62, Beclin-1, and LC3 in vitro and in vivo (P<0.05).
CONCLUSIONS: CY-09 and AST inhibit NaIO3-induced inflammatory damage through the NLRP3/autophagy pathway in vitro and in vivo. CY-09 and AST may protect retina from inflammatory injury.
摘要：
目的：研究NLRP3/自噬通路对光感受器炎症反应的影响及CY-09和虾青素(AST)的保护机制。
方法：ICR小鼠依次腹腔注射NaIO3、CY-09、AST，分为5组，包括控制，NaIO3,NaIO3+CY-09,NaIO3+AST,和NaIO3+CY-09+AST组。检查谱域光学相干断层扫描和闪光视网膜电图，并收集视网膜组织进行免疫组织化学，酶联免疫吸附测定(ELISA),和西方印迹。视网膜色素上皮细胞系(ARPE-19细胞)和小鼠感光细胞系(661W细胞)也相继用NaIO3、CY-09和AST处理。通过细胞计数试剂盒-8(CCK-8)测定评估细胞增殖。通过流式细胞术分析细胞凋亡。透射电镜观察自噬体形态的变化。定量聚合酶链反应（qPCR）用于检测NLRP3和caspase-1。通过Western印迹测量NLRP3、胱天蛋白酶-1、切割的胱天蛋白酶-1、p62、Beclin-1和LC3蛋白水平。通过ELISA测量IL-1β和IL-18。
结果：与对照组相比，NaIO3处理的661W细胞的活性在24和48h内降低，凋亡增加，NLRP3、caspase-1、IL-1β和IL-18水平升高,自噬相关蛋白水平升高(P<0.05)。与NaIO3组相比，CY-09和AST抑制细胞凋亡(P<0.05),NLRP3、caspase-1、IL-1β和IL-18表达降低(P<0.05),并抑制自噬。与其他组相比，CY-09联合AST可显著降低NLRP3的表达，抑制自噬相关蛋白p62、Beclin-1和LC3的表达（P<0.05）。
结论：CY-09和AST在体内外通过NLRP3/自噬通路抑制NaIO3诱导的炎症损伤。CY-09和AST可以保护视网膜免受炎症损伤。
方法：ICR小鼠依次腹腔注射NaIO3、CY-09、AST，分为5组，包括控制，NaIO3,NaIO3+CY-09,NaIO3+AST,和NaIO3+CY-09+AST组。检查谱域光学相干断层扫描和闪光视网膜电图，并收集视网膜组织进行免疫组织化学，酶联免疫吸附测定(ELISA),和西方印迹。视网膜色素上皮细胞系(ARPE-19细胞)和小鼠感光细胞系(661W细胞)也相继用NaIO3、CY-09和AST处理。通过细胞计数试剂盒-8(CCK-8)测定评估细胞增殖。通过流式细胞术分析细胞凋亡。透射电镜观察自噬体形态的变化。定量聚合酶链反应（qPCR）用于检测NLRP3和caspase-1。通过Western印迹测量NLRP3、胱天蛋白酶-1、切割的胱天蛋白酶-1、p62、Beclin-1和LC3蛋白水平。通过ELISA测量IL-1β和IL-18。
结果：与对照组相比，NaIO3处理的661W细胞的活性在24和48h内降低，凋亡增加，NLRP3、caspase-1、IL-1β和IL-18水平升高,自噬相关蛋白水平升高(P<0.05)。与NaIO3组相比，CY-09和AST抑制细胞凋亡(P<0.05),NLRP3、caspase-1、IL-1β和IL-18表达降低(P<0.05),并抑制自噬。与其他组相比，CY-09联合AST可显著降低NLRP3的表达，抑制自噬相关蛋白p62、Beclin-1和LC3的表达（P<0.05）。
结论：CY-09和AST在体内外通过NLRP3/自噬通路抑制NaIO3诱导的炎症损伤。CY-09和AST可以保护视网膜免受炎症损伤。
