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

[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。

[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.