过表达JAK1对岩藻多糖保护神经元免受糖氧剥夺/复氧损伤的影响

Effect of overexpression of JAK1 on protection of fucoidan-exposed neurons from glucose-oxygen deprivation/reoxygenation injury

关键词:  岩藻多糖  线粒体自噬  Janus激酶1/信号转导和转录激活因子3信号通路  肾上腺嗜铬瘤细胞  氧化应激

Key words:  fucoidan  mitophagy  JAK1/STAT3 pathway  PC12 cell  oxidative stress

摘要:

[目的] 研究探讨岩藻多糖是否通过抑制Janus激酶1/信号转导和转录激活因子3（JAK1/STAT3）信号通路来保护神经元免受糖氧剥夺/复氧损伤（OGD/RP）引起的氧化损伤和细胞凋亡。[方法] 建立大鼠肾上腺嗜铬瘤细胞（PC12）OGD/RP模型，采用CCK8法筛选出岩藻多糖对OGD/RP模型的最大浓度；流式细胞术检测细胞凋亡；活性氧（ROS）荧光探针（DCFH-DA）法测定细胞ROS水平；酶联免疫吸附实验（ELISA）检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）含量；JC-1法测定线粒体膜电位（MMP）变化；透射电镜观察线粒体自噬情况；蛋白免疫印记法（Western Blot）检测线粒体自噬、细胞凋亡及JAK1/STAT3通路等相关蛋白的表达。[结果] 通过CCK8筛选出OGD/RP损伤模型下400 mg/kg岩藻多糖组的细胞活性及增殖能力最强，用于后续实验研究；与OGD/RP组相比，OGD/RP+岩藻多糖组细胞中SOD、GSH-Px的含量、线粒体自噬体数量、MMP值及PTEN诱导假定激酶1（PINK1）、帕金蛋白（Parkin）及微管相关蛋白轻链3（Lc3B）蛋白的相对表达增加，细胞凋亡率、细胞凋亡相关蛋白、ROS含量及p-JAK1/JAK1、p-STAT/STAT3蛋白的相对表达量降低；与OGD/RP+岩藻多糖- pcDNA-NC组相比，OGD/RP+岩藻多糖+pcDNA3.1-JAK1组的细胞中SOD、GSH-Px的含量、线粒体自噬体数量、MMP值及PINK1、Parkin及Lc3B蛋白的相对表达下降，而ROS含量增加。[结论] 岩藻多糖能够通过抑制JAK/STAT信号通路，降低细胞凋亡及氧化损伤，促进线粒体自噬，达到保护神经元免受OGD/RP损伤的目的。

Abstract:

[Objective] In this study，we investigated whether fucoidan protects neurons from oxidative damage and apoptosis caused by glucose-oxygen deprivation/reoxygenation injury（OGD/RP） by inhibiting JAK1/STAT3 signal pathway. [Methods] A rat model of glucose-oxygen deprivation/reoxygenation injury（OGD/RP） in PC12 cells was established，and the maximum concentration of fucoidan on OGD/RP model was selected by CCK8 method；apoptosis was detected by flow cytometry；ROS levels were determined by reactive oxygen species（ROS） fluorescent probe（DCFH-DA）；superoxide dismutase（SOD） and glutathione peroxidase（GSH-Px） contents were detected by enzyme-linked immunosorbent assay（ELISA）；mitochondrial membrane（MMP） potential changes were determined by JC-1 assay；mitophagy was observed by transmission electron microscopy；mitophagy，apoptosis and JAK1/STAT3 pathway and other related proteins were detected by Western blot. [Results] The 400 mg/kg fucoidan group under OGD/RP injury model was screened by CCK8 to have the strongest cell activity and proliferation ability，which was used for subsequent experimental research. Compared with the OGD/RP group，the contents of SOD and GSH-Px，the quantity of mitophagosomes，the MMP value and the relative expressions of PINK1，Parkin and Lc3II/Lc3I proteins in the cells of the OGD/RP+fucoidan group were increased. Apoptosis rate，apoptosis-related proteins，ROS content and the relative expression of p-JAK1/JAK1，p-STAT/STAT3 proteins were decreased. Compared with the OGD/RP+fucoidan group，the SOD，GSH-Px contents，the quantity of mitophagosomes，MMP value and the relative expressions of PINK1，Parkin and Lc3II/Lc3I proteins of the cells in the OGD/RP+fucoidan+pcDNA3.1-JAK1 group were decreased，while ROS content was increased. [Conclusion] Fucoidan can protect neurons from OGD/RP injury by inhibiting JAK/STAT signal pathway，reducing apoptosis and oxidative damage，and promoting mitophagy.