目的 基于Janus激酶(Janus kinase 2,JAK)/信号转导与转录激活因子(signal transducer and activator of transcription,STAT)信号通路探讨颐脑解郁复方对氧糖剥夺/再复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)大鼠海马神经干细胞(neural stem cells,NSCs)增殖和分化的影响.方法 取新生大鼠海马制成细胞悬液,原代培养海马NSCs,建立OGD/R损伤模型,实验分为正常组、OGD/R模型组、颐脑解郁复方组、抑制剂组、颐脑解郁复方+抑制剂组.增殖的细胞正常组和OGD/R模型组海马NSCs无血清培养基培养,颐脑解郁复方组用NSCs含药血清培养基培养,抑制剂组用JAK2/STAT3通路抑制剂S31-201培养,颐脑解郁复方+抑制剂组用NSCs含药血清培养基和S31-201培养.分化的细胞正常组和OGD/R模型组用NSCs诱导分化培养基培养,颐脑解郁复方组用NSCs诱导分化培养基和含药血清培养,抑制剂组用NSCs诱导分化培养基和S31-201培养,颐脑解郁复方+抑制剂组用诱导分化培养基及含药血清和S31-201培养.各组细胞培养48小时后采用CCK-8法筛选颐脑解郁复方最佳浓度及检测各组海马NSCs的增殖情况,免疫荧光法检测各组海马NSCs的分化情况,RT-PCR及Western Blot法检测JAK2、STAT3基因和p-JAK2、p-STAT3蛋白的表达.结果 (1)各组细胞培养48小时后细胞增殖情况:与正常组相比,OGD/R模型组细胞增殖数量明显降低(P＜0.01);与OGD/R模型组相比,颐脑解郁复方组、抑制剂组、颐脑解郁复方+抑制剂组海马NSCs增殖的数量明显增多(P＜0.01)o(2)各海马NSCs经诱导分化培养48小时后,与正常组相比,OGD/R模型组分化后的细胞明显减少,海马NSCs向神经元分化减少,细胞排列稀疏.与OGD/R模型组相比,颐脑解郁复方组、抑制剂组、颐脑解郁复方+抑制剂组分化后的细胞明显增多,海马NSCs向神经元分化增多,细胞密度增大,突起增多.(3)各组细胞JAK2/STAT3通路相关基因与蛋白的表达情况:与正常组相比,OGD/R模型组海马NSCs的JAK2、STAT3基因及p-JAK2、p-STAT3蛋白表达显著增多(P＜0.05,P＜0.01);与OGD/R模型组相比,颐脑解郁复方组、抑制剂组、颐脑解郁复方+抑制剂组海马NSCs的JAK2、STAT3基因及p-JAK2、p-STAT3蛋白表达均显著减少(P＜0.01).结论 颐脑解郁复方可以促进OGD/R损伤的海马NSCs的增殖与分化,作用机制可能与抑制细胞JAK2/STAT3信号通路,降低JAK2、STAT3的基因及蛋白表达水平密切相关.
Exploring the effect of Yinao Jieyu Compound on the proliferation and differentiation of hippocampal neural stem cells induced by oxygen and glucose deprivation/reoxygenation injury based on the JAK2/STAT3 pathway
Objective To investigate the effect of Yinao Jieyu Compound on the proliferation and differentiation of OGD/R rats based on JAK2/STAT3 signaling pathway.Methods The issues of newborn rat hippocampus were prepared into cell suspension,primary cultured hippocampal NSCs,to establish an OGD/R injury model.The experiments were divided into normal group,OGD/R model group,compound group,JAK2/STAT3 pathway inhibitor group,and compound+inhibitor group,hicampal NSCs in the normal group and the OGD/R model group,NSCs in the compound group with serum medium culture,JAK2/STAT3 pathway inhibitor S31-201 culture in the JAK2/STAT3 pathway inhibitor group,and NSCs with serum culture medium and S31-201 culture in the compound+inhibitor group;the differentiated cell normal group and the OGD/R model group were cultured with NSCs,NSCs and drug-containing serum,inhibitor group,NSCs and S31-201,and induced differentiation medium and drug-containing serum and S31-201.After 48 h of culture,the method of CCK-8 was used to screen the optimal concentration of compound and detect the proliferation of hippocampal NSCs of each group.The differentiation of hippocampal NSCs of each group was detected by immunofluorescence,and the expressions of JAK2,STAT3 genes,p-JAK2 and p-STAT3 protein were detected by RT-PCR and Western Blot.Results(1)After 48 h of culture in each group,the number of cell proliferation was significantly reduced in the OGD/R model(P＜0.01),and compared with the OGD/R group,the number of NSCs in the compound group,inhibitor group and compound+inhibitor group was significantly increased(P＜0.01).(2)After 48 h of induced differentiation of each hippocampal NSCs,the OGD/R model group,the hippocampal NSCs differ-entiated to neurons,and the cells were sparsely arranged.Compared with the OGD/R model group,the differentiated cells in the compound group,inhibitor group and compound+inhibitor group were increased significantly,and the hippocampal NSCs differentiated more to neurons,and the cell density and processes were increased.(3)Expression of genes and proteins involved in the JAK2/STAT3 pathway in cells of each group,compared with the normal group,OGD/R,the expression of JAK2、STAT3 genes and p-JAK2 and p-STAT3 proteins in hippocampal NSCs in the model group was increased significantly(P＜0.05,P＜0.01);Compared with the OGD/R model group,JAK2、STAT3 gene and p-JAK2 and p-STAT3 protein expression of NSCs in the hippocampus of the compound group,the inhibitor group and the compound+inhibitor group were significantly decreased(P＜0.01).Conclusion Yinao Jieyu Compound can promote the proliferation and differentiation of hippocampal NSCs with OGD/R injury,and the mechanism of action may be closely related to inhibiting the JAK2/STAT3 signaling pathway of cells and reducing the expression levels of JAK2 and STAT3 genes and proteins.
NETL
NSTL
万方数据
