梓醇对氧糖剥夺_复氧复糖损...胞瘤细胞的修复作用及其机制_刘燕.pdf

山东医药2023 年第 63 卷第 3 期梓醇对氧糖剥夺/复氧复糖损伤成熟人神经母细胞瘤细胞的修复作用及其机制刘燕1，高春辰1，2，厉励2，冯清华1，吴明华3，李文磊31 南京中医药大学附属医院南京中医药大学第一临床医学院，南京210023；2 东部战区空军医院；3 南京中医药大学附属医院/江苏省中医院摘要：目的观察梓醇对氧糖剥夺/复氧复糖损伤的人神经母细胞瘤细胞SH-SY5Y（SH-SY5Y细胞）的修复作用，并探讨其作用机制。方法将SH-SY5Y细胞分为对照组、诱导组、模型组、梓醇组。对照组：SH-SY5Y细胞不做任何处理；诱导组：用ATRA诱导SH-SY5Y细胞72 h，将其诱导为成熟神经元；模型组：ATRA诱导SH-SY5Y细胞72 h后，将完全培养基换成无糖的EBSS溶液置于三气培养箱（氧气1%，二氧化碳5%，氮气94%）4 h，氧糖剥夺结束后换回完全培养基（含糖），复氧 12 h；梓醇组：细胞经模型组同样处理后，加入含不同浓度梓醇（50、100、200 mol/L）的完全培养基常规培养72 h。采用CCK-8法检测各组细胞的活力；细胞划痕实验观察细胞的迁移、穿越能力；-tubulin 免疫荧光染色标记轴突，并测量其长度；Western blotting 法检测细胞中神经生长相关蛋白（GAP43）、骨桥蛋白（OPN）、磷酸化的胰岛素生长因子 1 受体（p-IGFR）、抑癌基因（PTEN）、雷帕霉素分子靶点（mTOR）、磷酸化核糖体S6蛋白（p-S6）。结果与对照组相比，诱导组存活率升高（P0.0001）；与诱导组相比，模型组存活率降低（P0.0001）；不同浓度梓醇组存活率均有升高（P均0.05。对照组细胞胞体圆润，轴突较短，胞体与胞体之间几乎没有轴突连接；诱导组细胞轴突较长并且相互交织，形成神经网络；与诱导组相比，模型组数量减少，胞体扁平，轴突回缩；与模型组比较，梓醇组神经元数量增多，轴突长度获得一定的恢复；与空白对照组相比，诱导组轴突长度延长；与诱导组相比，模型组轴突长度缩短（P0.001）；不同浓度梓醇组神经元轴突长度均延长（P均0.05。与模型组相比，不同浓度梓醇组48 h后的划痕面积均小（P均0.05。与诱导组比较，模型组PTEN相对表达量升高（P0.05），OPN降低（P0.05）；与模型组比较，不同浓度梓醇组GAP43、OPN、p-IGFR、mTOR及p-S6蛋白相对表达量均升高，PTEN蛋白相对表达量降低（P均0.05）。结论梓醇对氧糖剥夺/复氧复糖损伤的SH-SY5Y细胞具有修复作用，其作用机制可能是通过上调OPN表达，诱导细胞表面IGFR受体磷酸化成为p-IGFR，进而激活mTOR通路，同时降低PTEN的表达以减轻其对mTOR通路的抑制作用，使细胞内在生长能力被充分启动，从而达到促进神经细胞迁移、轴突发芽和生长的作用。50、100、200 mol/L梓醇对氧糖剥夺/复氧复糖损伤的SH-SY5Y细胞修复作用相当。关键词：梓醇；SH-SY5Y细胞；氧糖剥夺/复氧复糖；细胞增殖；细胞迁移；轴突生长；骨桥蛋白；PTEN；雷帕霉素分子靶点；类胰岛素生长因子受体doi：10.3969/j.issn.1002-266X.2023.03.002 中图分类号：R730.2 文献标志码：A 文章编号：1002-266X（2023）03-0006-06Repair effect and mechanism of catalpol on mature human neuroblastoma cells after oxygen-glucose deprivation/reoxygenation injuryLIU Yan1，GAO Chunchen，LI Li，FENG Qinghua，WU Minghua，LI Wenlei1 The Affiliated Hospital of Nanjing University of Chinese Medicine，Nanjing 210029，ChinaAbstract：Objective To observe the repair effect of catalpol on human neuroblastoma cells（SH-SY5Y）after oxygen-glucose deprivation/reoxygenation injury，and to investigate its mechanism.Methods SH-SY5Y cells were divided into the control group，all-trans retinoic acid（ATRA）group，model group，and catapol group.Control group：SH-SY5Y cells did not receive any treatment；ATRA group：the naive SH-SY5Y cells were induced by ATRA for 72 h to differentiate 基金项目：国家自然科学基金面上项目（81973794）；循证能力建设项目（2019XZZX NB007）；江苏省中医药局管理局资助项目（JD201803；ZT202102）。第一作者简介：刘燕（1994-），女，硕士研究生，主要研究方向为神经损伤后的再生与修复。E-mail：通信作者简介：李文磊（1980-），女，副主任医师，主要研究方向为脑血管疾病。E-mail： 吴明华（1966-），男，主任医师，主要研究方向为脑血管疾病。E-mail：6山东医药2023 年第 63 卷第 3 期into mature neurons.Model group：after ATRA induction，the complete medium was replaced by sugar-free EBSS solution，and these cells were then placed in a three-gas incubator（oxygen 1%，carbon dioxide 5%，nitrogen 94%）for 4 h；after the deprivation of oxygen and sugar was over，SH-SY5Y cells were cultured in original condition with oxygen and sugar for 12 h.Catalpol group：after receiving the same treatment as the model group，cells were placed in the complete medium containing different concentrations of catalpol（50，100，and 200 mol/L）for 72 h，respectively.The cell viability in each group was detected by CCK-8.-III-tubulin immunofluorescence staining was used to label the axons，and we measured their length.Western blotting was used to detect the protein levels of growth associated protein 43（GAP 43），osteopontin（OPN），phosphorylation-insulin-like growth factor 1 receptor（p-IGFR），phosphatase and tensin homolog deleted on chromosome ten（PTEN），mammalian target of rapamycin（mTOR），and phosphorylated ribosomal protein S6（p-S6）.Results The survival rate was higher in the ATRA group than in the control group（P0.0001）.The survival rate of the model group was lower than that of the ATRA group（P0.0001）.The survival rates in different concentrations of catalpol groups were higher than that of the model group（all P0.05）.The cell bodies of the control group were round and the axons were short.The cell axons in the ATRA group were long and connected to form neural networks.Compared with the ATRA group，the number of cells in model group decreased，the cell body was flat，and the axon was retracted.Compared with model group，the number of cells in the catalpol group increased and the length of axons was longer.Axon length was longer in the induction group than in the blank control group.Compared with the ATRA group，the axon length in the model group was shorter（P0.001），and the length of axon in different concentrations of catalpol groups was extended（P0.01）.The scratch area of different concentrations of catalpol groups was smaller than that of the model group after 48 h（P0.05）.Compared with ATRA group，the relative expression level of PTEN increased，while the OPN decreased in the model group（both P0.05）.In catalpol groups with different concentrations，the relative expression levels of GAP43，OPN，p-IGFR，mTOR，and p-S6 protein increased，while the relative expression level of PTEN protein decreased in comparison to those of the model group（all P0.05）.Conclusions SH-SY5Y cells after oxygen-glucose deprivation/reoxygenation injury can be repaired by catalpol.The underlying mechanism may be that catalpol induces the p