淫羊藿苷通过PERK/CHOP信号通路抑制视锥细胞内质网应激诱导的线粒体损伤的研究

Icariin protects cones by inhibiting endoplasmic reticulum-mitochondria interaction through the PERK/CHOP pathway

目的探讨在细胞内质网应激状态下,淫羊藿苷保护光感受器细胞的作用机制。方法以毒胡萝卜素(0.2μmol/L)诱导小鼠视锥细胞(661W细胞)内质网应激模型,采用阿尔玛蓝染色法检测细胞活性,蛋白免疫印迹法检测未折叠蛋白效应相关蛋白——磷酸化蛋白激酶样内质网激酶(p-PERK)、葡萄糖调节蛋白78(GRP78)、CCAAT增强子结合蛋白同源蛋白(CHOP)表达水平,荧光成像和钙比例法检测Ca2+含量,荧光染色法评价线粒体功能(线粒体通透性转换孔和膜电位)。以体外分子对接技术分析淫羊藿苷与内质网钙-ATP酶的结合及相互作用。结果毒胡萝卜素可诱导661W细胞死亡;淫羊藿苷(2.22~20.00 nmol/L)能够抑制毒胡萝卜素引起的661W细胞死亡(P<0.01)。毒胡萝卜素促进661W细胞PERK磷酸化(P<0.01)、上调转录因子CHOP的蛋白表达(P<0.01);淫羊藿苷(2.0μmol/L)能够抑制模型组细胞CHOP蛋白表达(P<0.01),有抑制PERK(Thr980)磷酸化的趋势。毒胡萝卜素作用于661W细胞的10 min内,引起胞质内钙离子含量升高;淫羊藿苷预处理(1.0μmol/L)能够抑制胞内钙离子含量的升高。毒胡萝卜素诱导661W细胞线粒体通透性转换孔的开放,进而引起线粒体膜电位降低;淫羊藿苷(2.0μmol/L)能够抑制毒胡萝卜素诱导的线粒体通透性转换孔开放和线粒体膜电位降低。体外分子对接分析显示,淫羊藿苷能够与内质网钙-ATP酶2b亚型的核苷酸结合结构域及磷酸化结构域稳定结合,结合能为-8.776 kcal/mol,且存在多个氢键结合。结论淫羊藿苷可部分拮抗毒胡萝卜素对内质网钙-ATP酶的抑制作用,维持细胞内Ca2+稳态,保护线粒体功能,进而发挥视锥细胞保护作用。

Objective To elucidate the mechanism of action of icariin to protect photoreceptor cells suffering from endoplasmic reticulum stress.Methods Thapsigargin(0.2μmol/L),was used to induce endoplasmic reticulum stress in the mouse cone cell line 661W.AlamarBlue staining was used to detect cell viability.The levels of proteins related to the unfolded protein response p-PERK,GRP78,CHOP were analyzed by Western blotting.The intracellular Ca2+level was analyzed by fluorescence imaging and the F340/F380 ratio method.Permeability transition pore protein and the membrane potential in mitochondria were analyzed by fluorescence staining.The binding and interaction between icariin and SERCA2b were analyzed in vitro by molecular docking.Results Thapsigargin induced 661W cell death.Icariin significantly inhibited 661W cell death caused by thapsigargin in the concentration of 2.22-20.00 nmol/L(P<0.01).Thapsigargin induced the unfolded protein responses in 661W cells,significantly promoting the phosphorylation of PERK(Thr980)(P<0.01)and increasing the protein levels of transcription factor CHOP(P<0.01).Icariin(2.0μmol/L)significantly downregulated the expression of CHOP protein(P<0.01)and had the trend of inhibitation of phosphorylation of PERK(Thr980).Within 10 minutes after thapsigargin acting on 661 W cells,the calcium ions in the cytosol increased.Icariin pretreatment(1.0μmol/L)inhibited an increase of intracellular calcium content.Thapsigargin induced the opening of mitochondrial permeability transition pores in 661W cells,which in turn caused a decrease in mitochondrial membrane potential.Icariin(2.0μmol/L)inhibited thapsigargin-induced mitochondrial permeability transition pore opening and mitochondrial membrane potential reduction.In vitro molecular docking analysis showed that icariin stably bound to the nucleotidebinding and phosphorylation domains of SERCA2b,with a binding energy of-8.776 kcal/mol and multiple hydrogen bonds.Conclusion Icariin partially antagonized the effect on the binding of thapsigargin and SERCA,which maintained Ca2+homeostasis in cones,contributing to mitochondrial health.This may be one mechanism of action of icariin in cone cell protection.