岩藻多糖对氧糖剥夺诱导的新生大鼠海马脑神经元的影响

Effects of fucoidan on the hippocampal neurons of neonatal rats induced by oxygen and glucose deprivation

目的:探讨岩藻多糖对氧糖剥夺(OGD)诱导的新生大鼠海马神经元的影响。方法:分离新生大鼠原代海马神经元后,分4组处理。空白组不做任何处理;Fuc组用100μg/mL岩藻多糖培养7 d;OGD组按OGD方法培养;OGD+Fuc组首先进行OGD诱导,然后用100μg/mL岩藻多糖培养7 d。分组处理后,MTT法检测细胞活力,AnnexinⅤ-FITC/PI双染法检测细胞凋亡;实时荧光定量PCR法检测白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和诱导型一氧化氮合酶(iNOS)mRNA的表达;使用试剂盒测定细胞中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;Western blot法检测微管相关蛋白1轻链3(LC3)、Beclin-1、P62,Cleaved Caspase-3和9,磷脂酰肌醇-3激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)和磷酸化Akt(p-Akt)的表达。结果:OGD可降低神经元活力,增加凋亡率和Cleaved Caspase-3、Cleaved Caspase-9蛋白表达水平(P<0.05);升高SOD活性,降低MDA含量(P<0.05);升高IL-1β、TNF-α和iNOS mRNA表达水平(P<0.05);升高LC3和Beclin-1表达水平(P<0.05);降低p-PI3K、p-Akt和P62表达(P<0.05)。岩藻多糖可拮抗上述作用(P<0.05)。结论:岩藻多糖可能通过激活PI3K/Akt信号通路,减轻OGD诱导的神经元炎症反应、自噬和氧化应激水平,发挥神经元保护作用。

Aim:To investigate the effects of fucoidan(Fuc) on the hippocampal neurons of neonatal rats induced by oxygen and glucose deprivation(OGD).Methods:The hippocampal neurons of newborn rats were isolated and allocated into Mock group(without treatment),OGD group(with OGT treatment),Fuc group(cultured with 100 μg/mL Fuc for 7 days) and OGD+Fuc group(after OGD,cultured with Fuc).MTT was used to detect cell viability,AnnexinⅤ-FITC/PI staining was used to detect cell apoptosis.RT-qPCR was used to detect the expression of inflammation markers IL-1β,TNF-α and iNOS mRNA.The kit was used to detect the levels of SOD and MDA.Western blot was used to detect the expressions of autophagy marker proteins LC3,Beclin-1 and P62,the expressions of apoptosis-related proteins Cleaved Caspase-3 and-9,and PI3K/Akt signaling pathway related proteins PI3K,p-PI3K,Akt and p-Akt.Results:In OGD group,the cell viability decreased,the apoptosis rate and the expressions of Cleaved Caspase-3,Cleaved Caspase-9 increased;SOD activity increased,and MDA content decreased(P<0.05);the expressions of IL-1β,TNF-α,iNOS mRNA increased(P<0.05);the expressions of LC3 and Beclin-1 increased(P<0.05);the expressions of p-PI3K,p-Akt and P62 decreased(P<0.05).Compared with OGD group,OGD+Fuc group showed antagonistic effect on the above indexes(P<0.05).Conclusion:Fuc can reduce inflammation,autophagy and oxidative stress by activating the PI3K/Akt signaling pathway,thus protect neurons induced by OGD.