岩藻多糖调节JAK2/STAT3信号通路保护神经元免受缺氧缺糖/再灌注损伤

Fucoidan protecting neurons from OGD/RP injury by regulating JAK2/STAT3 signal channel

目的开展岩藻多糖(Fucoidan,FU)调节JAK2/STAT3信号通路保护神经元免受缺氧缺糖/再灌注损伤(OGD/RP)研究,明确FU减轻脑缺血再灌注损伤的可能分子机制。方法以大鼠神经元PC12细胞为研究对象,通过三气培养箱和无葡萄糖的培养基建立OGD/RP细胞损伤模型。为明确JAK/STAT信号通路在FU调节PC12细胞凋亡和自噬中的作用,采用JAK/STAT信号通路抑制剂WP1066干预。实验分为对照组(Control),OGD/RP组、OGD/RP+1μM FU组、OGD/RP+μM FU+10μM WP1066(JAK/STAT抑制剂)组。用CCK-8实验检测细胞活力;用流式细胞仪分析PC12细胞凋亡、线粒体膜电位和ROS情况;用ELISA法分析细胞培养上清液中抗氧化蛋白SOD、GSH-Px水平;用Western blot法分析细胞凋亡、自噬与JAK/STAT信号通路相关的蛋白表达水平。結果OGD/RP处理能明显抑制PC12细胞增殖,经1μM FU处理能缓解OGD/RP对细胞增殖的抑制。OGD/RP组的细胞凋亡率和ROS量及线粒体膜电位明显增高,经FU处理组的情况则相反。而且通过Western blot法证实,FU处理导致Bax、Cleaved-caspase3、LC3 I/II蛋白表达量降低,Bcl-2蛋白表达量增加。此外,经FU和0GD/RP处理后,与单独0GD/RP处理相比,P-JAK2和p-STAT3表达明显下调。WP1066抑制剂能进一步下调FU和OGD/RP作用下的JAK2、STST3蛋白磷酸化水平。结怡FU通过抑制JAK2/STAT3信号通路来保护神经元免受OGD/RP引起的氧化损伤和细胞凋亡。

Objective To study the regulating effect of JAK2/STAT3 signal channel by Fucoidan(FU)to protect neurons from oxygen and glucose deprivation(OGD)/reperfusion(RP)injury so as to clarify the possible molecular mechanism of FU alleviating RP injury of cerebral ischemia.Methods Taking neuronal PC12 cells of rats as the research object,the OGD/RP cell injury model was established by three-gas incubator and glucose-free medium.The inhibitor WP1066 of JAK/STAT signal channel was used for intervention in order to clarify the role of JAK/STAT signal channel in the regulation of PC12 cells apoptosis and autophagy by FU.The cells were divided into control group,OGD/RP group,OGD/RP+1μM FU group,OGD/RP+1 FU+10μM WP 1066(JAK/STAT inhibitor)group.The viability of cell was detected by CCK-8 experiment;the apoptosis,mitochondrial membrane potential and ROS of PC12 cells were analyzed by flow cytometry;the levels of antioxidant protein Superoxide Dismutase(SOD)and Glutathione Peroxidase(GSH-Px)in cell culture supernatant were analyzed by ELISA;the protein expression levels related to apoptosis,autophagy and JAK/STAT signal channel were analyzed by Western blot.Results The proliferation of PC12 cells could be significantly inhibited by OGD/RP treatment.The inhibition of OGD/RP on cell proliferation could be alleviated after 1 μM FU treatment.The apoptotic rate,Reactive Oxygen Species(ROS)a-mount and mitochondrial membrane potential in the OGD/RP group increased significantly,while decreased greatly in the FU-treated group.It was confirmed by Western blot that FU treatment decreased the level of Bax,Cleaved-caspase 3 and LC3 I/II protein expression and increased Bcl-2 protein expression.In addition,compared with singal OGD/RP treatment,the expression of p-JAK2 and p-STAT3 was significantly decreased with FU and OGD/RP treatment.WP1066 inhibitor could further decrease the phosphorylation levels of JAK2 and STST3 proteins under the action of FU and OGD/RP.Conclusions By inhibiting JAK2/STAT3 signal channel,neurons can be protected by FU from oxidative damage and apoptosis caused by OGD/RP.