不同频率间歇性低氧刺激对HT22细胞活性和Hif-1α及p-NF-κB表达的影响

Effects of intermittent hypoxia stimulation with different frequencies on HT22 cell viability and expression of Hif-1αand p-NF-κB

间歇性低氧(intermittent hypoxia,IH)可通过氧化应激和炎症反应诱导认知障碍,而不同频率的IH刺激可对特定类型的细胞引起不同结果。因此本研究旨在比较3种频率IH刺激对海马神经元HT22细胞活性的影响,探讨IH刺激损伤神经元的相关分子机制。体外培养HT22细胞,分为对照组和3个不同频率IH刺激组(舱内氧浓度在21%和1%之间循环,IH1组:6循环/h;IH2组:2循环/h;IH3组:0.6循环/h)。分别在IH刺激6、12、24和48h观察细胞形态,采用CCK-8试剂盒测定细胞活性,用试剂盒测定细胞上清中乳酸脱氢酶(lactate dehydrogenase,LDH)含量,用DCFH-DA探针检测细胞内活性氧(reactive oxygen species,ROS)水平,用Western blot检测低氧诱导因子1α(hypoxia inducible factor-1α,Hif-1α)和核因子κB(nuclear factor kappaB,NF-κB)的表达水平。结果显示,与对照组相比,3个IH处理组细胞数量均减少。随IH刺激时间延长,IH处理组细胞活性均呈现不同程度的降低,细胞培养基上清中LDH含量增加,细胞内ROS水平提高,且以IH1组变化最为明显;Hif-1α蛋白表达水平和p-NF-κB/NF-κB比值也随IH刺激时间延长而升高,以IH1组变化最为显著。以上结果提示,IH刺激可导致HT22细胞氧化应激损伤,其机制与Hif-1α表达上调以及NF-κB磷酸化水平提高有关,并且高频率的IH刺激对细胞损伤更为显著。

Intermittent hypoxia(IH)could induce cognitive impairment through oxidative stress and inflammation.However,the degree of cell damage is closely related to the IH stimulus frequency.IH stimulation with different frequencies also induces opposite results on neuronal cell lines.Therefore,this study was aimed to compare the effects of IH stimulation with three different frequencies on murine hippocampal neuronal HT22 cell activity,and to explore the molecular mechanism of the IH stimulus frequency-related neuron injury.HT22 cells were cultured and divided into control group and three IH stimulation groups with different frequencies.Oxygen concentration in the chamber was circulated between 21%and 1%(IH1 group,6 cycles/h;IH2 group,2 cycles/h;IH3 group,0.6 cycle/h).Cell morphology was observed at 6,12,24 and 48 h of IH treatment.Cell viability was determined by the CCK-8 kit,lactate dehydrogenase(LDH)content in cell supernatant was determined by LDH kit,oxidative stress level was detected by the reactive oxygen species(ROS)probe,and protein expression levels of hypoxia inducible factor-1α(Hif-1α)and phosphorylated nuclear factorκB(p-NF-κB)were detected by Western blot.The results showed that,compared with control group,cell number and activity in the three IH groups were decreased,LDH content and ROS levels were increased with the prolongation of IH stimulation time,and the changes were most obvious in the IH1 group among those of the three IH groups.Hif-1αexpression and the p-NF-κB/NF-κB ratio were also up-regulated with the prolongation of IH stimulation time,and the changes of IH1 group were the most significant.These results suggest that IH stimulation induces oxidative stress injury in HT22 cells,which is related to increased Hif-1αexpression and NF-κB phosphorylation.Moreover,the higher frequency of IH stimulation induces more serious cell injury.