神经药理学报

神经药理学报››2022,Vol. 12››Issue (2): 1-.DOI:10.3969/j.issn.2095-1396.2022.02.001

• 研究论文 •下一篇

S14G-humanin 对氧糖剥夺/ 复氧神经细胞损伤的保护作用

孙丽，张晓莹，高广生

  1. 1 山东第一医科大学附属中心医院重症医学科，济南，250013，中国 2 山东省泰安市中心医院住院准备中心，泰安，271000，中国 3 山东省泰安市中心医院重症医学科，泰安，271000，中国
  • 出版日期:2022-04-26发布日期:2022-04-26
  • 通讯作者:高广生，E-mail：shenggao163@163.com
  • 作者简介:孙丽，硕士；E-mail：mail0987123@126.com
  • 基金资助:
    山东省医药卫生科技发展计划项目（No.2018WS150）；山东省保健科技协会科学技术课题立项项目（No.SDBJKT20180024）； 泰安市科技发展计划（引导计划）项目（No.2018NS1068）

Neuroprotective Effect of S14G-Humanin on SH-SY5Y Cells induced by Oxygen Glucose Deprivation/Reoxygenation

SUN Li，ZHANG Xiao-ying，GAO Guang-sheng

  1. 1. Intensive Care Unit，Central Hospital Affiliated to Shandong First Medical University，Jinan，250013，China 2. Admission Preparation Center，Taian City Central Hospital，Taian，271000，China 3. Intensive Care Unit，Taian City Central Hospital，Taian，271000，China
  • Online:2022-04-26Published:2022-04-26

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摘要：

目的：评估S14G-humanin（HNG）在SH-SY5Y 神经母细胞瘤细胞体外氧糖剥夺/ 复氧（oxygen glucose deprivation/reoxygenation，OGD/R）模型中潜在的神经保护作用的相关分子机制。 方法：将SH-SY5Y 细胞分为 对照组、OGD/R 模型组、HNG 干预组、HNG 联合FLLL32 抑制剂组。三气培养箱建立SH-SY5Y 细胞OGD/R 损 伤模型；抑制剂组给予不同剂量HNG、FLLL32（JAK2/STAT3 通路抑制剂）。CCK8 细胞检测试剂盒检测细胞活性， 流式细胞仪检测细胞凋亡率，Western Blot 分析JAK2、p-STAT3（Y705）、p-STAT3（S727）的表达。 结果：OGD/R 导致SH-SY5Y 细胞存活率显著降低（ P<0.01），凋亡率显著增加（ P<0.01）。给予0.1、1、5、10 μg·L -1HNG 干预的 SH-SY5Y 细胞与未行HNG 干预的OGD/R 细胞相比，存活率更高（ P<0.01），凋亡明显减少（ P<0.01），1 μg·L -1HNG 干预组效果最好。与对照组比较，OGD/R 降低JAK2 和p-STAT3（Y705）蛋白水平（ P<0.01）。给予HNG 干预的细胞表达JAK2（ P<0.01）和p-STAT3（Y705）（ P<0.01）蛋白水平增加。给予1、5 μg·L -1HNG 干预的细 胞表达JAK2（ P<0.01）、p-STAT3（Y705）（ P<0.01）和p-STAT3（S727）（ P<0.05）蛋白水平有更显著的增加。给予 FLLL32 和HNG 干预的细胞无法增加细胞存活率。 结论：HNG 通过激活JAK2/STAT3 信号通路抑制OGD/R 诱 导的细胞凋亡，有希望用于治疗脑梗死的药物。

关键词:S14G-humanin,Janus 激酶2,信号转导与转录活化因子3,细胞凋亡,SH-SY5Y

Abstract:

Objective：To establish an in vitro oxygen glucose deprivation/reoxygenation （OGD/R） model using SH-SY5Y neuroblastoma cells to mimic the in vitro ischemia/reperfusion injury in stroke. To evaluate the potential neuroprotective effect of S14G-humanin （HNG） from ischemia/reperfusion in vitro，and further to identify the molecular mechanism underlying the protection，so as to provide a theoretical basis for the treatment of cerebral ischemia/reperfusion injury with HNG. Methods：SH-SY5Y cells were divided into control group，oxygen-glucose deprivation reperfusion OGD/R model group and inhibitor group. The present study established an in vitro OGD/R model using SH-SY5Y neuroblastoma cells by three gas incubator. HNG and FLLL32，an inhibitor of JAK2/STAT3 signal，were added to the cells in indicated experiments. CCK8 kit was used to detect the cell viability，and flow cytometry was used to detect the cell apoptosis rate. The expression of JAK2，p-STAT3（Y705） and p-STAT3（S727） were analyzed by Western Blot. Results：The results showed that OGD/R resulted in a significant decrease in the survival rate of SH-SY5Y cells （P<0.01） and a significant increase in the apoptosis rate of SH-SY5Y cells （ P<0.01）. SH-SY5Y cells incubated 0.1，1，5 or 10 μg·L -1HNG showed higher cell survival （ P<0.01） and significantly decreased apoptosis （ P<0.01） compared with the cells without HNG treatment in OGD/R processes，with the most evident effects at 1 μg·L -1HNG. OGD/R lowered protein levels of JAK2 （ P<0.01） and p-STAT3 at Y705 site （ P<0.01），compared to control. Adding HNG to the cells increased protein levels of JAK2 （ P<0.01） and p-STAT3 （Y705）（ P<0.01） in OGD/R conditions. Moreover，protein levels of JAK2（ P<0.01），p-STAT3 （Y705）（ P<0.01） and p-STAT3（ S727）（ P<0.05） had notable increase at values of 1 and 5 μg·L -1HNG in OGD/R conditions. Co-treatment with HNG and FLLL32 was unable to restore the cell viability and abolishing the inhibition of apoptosis by HNG（ P>0.05）. HNG inhibited OGD/ R-induced reduction of JAK2，p-STAT3（Y705） and p-STAT3（S727） protein expression， whereas the inhibitory effect was not observed with co-treatment with HNG and FLLL32. Conclusion：These data collectively indicate that HNG has neuroprotective effects against OGD/ R by activating JAK2/STAT3 signaling and suggest that HNG may be a promising drug for the treatment of stroke.

Key words:S14G-humanin,JAK2,STAT3,apoptosis,SH-SY5Y cells

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