| 摘要: |
| 目的：基于转录因子EB（transcription factor EB，TFEB）-溶酶体途径探讨滋阴明目地黄汤抑制视网膜神经节细胞-5（retinal ganglion cell-5，RGC-5）细胞铁死亡的分子机制。方法：诱导建立视网膜缺血再灌注损伤（retinal ischemia-reperfusion injury，RIRI）细胞模型，分别给予TFEB干扰RNA质粒（transcription factor EB siRNA Plasmid，si-TFEB）、滋阴明目地黄汤含药血清、滋阴明目地黄汤含药血清+TFEB重组腺病毒载体（recombinant adenovirus vector expressing transcription factor EB，Ad-TFEB）干预。采用3-(4，5-二甲基噻唑-2-基)-2，5-二苯基四氮唑溴盐比色［3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide，MTT］法、细胞计数试剂盒-8（Cell Counting Kit-8，CCK-8）法检测细胞存活率及增殖活性，蛋白质免疫印迹（Western Blot，WB）检测TFEB溶酶体及溶酶体相关膜蛋白1（lysosome-associated membrane protein 1，LAMP-1）、铁死亡谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）和胱氨酸/谷氨酸反向转运体（cystine/glutamate antiporter，xCT）表达;酶联免疫吸附测定（Enzyme-Linked Immunosorbent，ELISA）检测二价铁离子（Fe2+）、丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）水平。结果：与滋阴明目地黄汤含药血清+Ad-TFEB组及模型组相比，滋阴明目地黄汤药物组及TFEB沉默组RGC-5细胞存活率及细胞增殖活性增高（P＜0.05），GPX4、xCT蛋白表达增高（P＜0.05），TFEB溶酶体、LAMP-1的表达降低（P＜0.05），Fe2+、MDA含量降低（P＜0.01），GSH含量升高(P＜0.01)。结论：滋阴明目地黄汤可通过调控TFEB-溶酶体途径、抑制RGC-5细胞铁死亡对RIRI发挥保护作用。 |
| 关键词: 视网膜缺血再灌注损伤 滋阴明目地黄汤 TFEB-溶酶体途径 铁死亡 RGC-5细胞 体外研究 |
| DOI： |
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| Protective effect of Ziyin Mingmu Dihuang decoction against retinal ischemia-reperfusion injury and its mechanism: A study based on the transcription factor EB-lysosome pathway |
| LI Wei,TAN Tan,HUANG Lingfei |
| (Changde First Hospital of Traditional Chinese Medicine，Changde 415000，Hunan，China) |
| Abstract: |
| Objective：To investigate the molecular mechanism of Ziyin Mingmu Dihuang decoction in inhibiting ferroptosis of retinal ganglion cell-5 (RGC-5) based on the transcription factor EB (TFEB)-lysosome pathway.Methods: A cell model of retinal ischemia-reperfusion injury (RIRI) was induced，and transcription factor EB siRNA Plasmid (si-TFEB)，serum containing Ziyin Mingmu Dihuang decoction，and serum containing Ziyin Mingmu Dihuang decoction+recombinant adenovirus vector expressing transcription factor EB (Ad-TFEB) were used for intervention.MTT assay and CCK-8 assay were used to measure cell viability and proliferative activity;Western blot was used to measure TFEB lysosome and the expression levels of lysosome-associated membrane protein 1 (LAMP-1)，glutathione peroxidase 4 (GPX4)，and cystine/glutamate antiporter (xCT);ELISA was used to measure the levels of Fe2+，malondialdehyde (MDA)，and glutathione (GSH).Results: Compared with the serum containing Ziyin Mingmu Dihuang decoction+Ad-TFEB group and the model group，the Ziyin Mingmu Dihuang decoction group and the TFEB silencing group had significant increases in the viability and proliferative activity of RGC-5 cells (P < 0.05)，significant increases in the protein expression levels of GPX4 and xCT (P < 0.05)，significant reductions in TFEB lysosome and the expression level of LAMP-1 (P < 0.05)，significant reductions in the content of Fe2+ and MDA (P < 0.01)，and a significant increase in the content of GSH (P < 0.01).Conclusion: Ziyin Mingmu Dihuang decoction can exert a protective effect against RIRI by regulating the TFEB-lysosome pathway and inhibiting the ferroptosis of RGC-5 cells. |
| Key words: retinal ischemia-reperfusion injury Ziyin Mingmu Dihuang decoction transcription factor EB-lysosome pathway ferroptosis RGC-5 cell in vitro study |
