背景:缺血性中风(IS)是一种有害的神经系统疾病,并且IS缺乏恢复身体功能的有价值的方法。Indobufen(IND)可以缓解IS。然而,可能的机制仍未定义。
方法:SH-SY5Y细胞在氧糖剥夺/复氧(OGD/R)环境下培养,然后用NRF2和ATG5的小干扰RNA(siRNA)处理。各种浓度的IND(50μM,100μM,200μM,和400μM)通过3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四唑溴化物测定。ELISA法检测超氧化物歧化酶(SOD)和丙二醛(MDA)水平。通过DCFH-DA染色测定活性氧(ROS)产生。LC3II/LC3I的蛋白质水平,通过蛋白质印迹检测Beclin1、p62、NRF2和ATG5。
结果:IND增加了细胞活力,同时降低OGD/R环境下SH-SY5Y细胞的凋亡率。IND通过抑制LC3II/LC3I水平抑制自噬,Beclin1蛋白,并增加了OGD/R环境下SH-SY5Y细胞中p62蛋白的表达。IND限制了ROS和MDA的含量,同时增强OGD/R暴露的SH-SY5Y细胞中SOD的活性。IND还促进OGD/R环境中的NRF2表达。
结论:IND可抑制自噬,氧化应激,OGD/R暴露对SH-SY5Y细胞凋亡的影响,通过调节转录因子NRF2和抑制ATG5的表达进一步减轻IS损伤。
BACKGROUND: Ischemic stroke (IS) is a detrimental neurological disease and IS lacks valuable methods to recover body function. Indobufen (IND) could alleviate IS. However, the possible mechanism remains undefined.
METHODS: SH-SY5Y cells were cultured under the oxygen-glucose deprivation/reoxygenation (OGD/R) environment and then were treated with small interfering RNA (siRNA) of NRF2 and ATG5. The influence of various concentrations of IND (50 μM, 100 μM, 200 μM, and 400 μM) was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide. Levels of superoxide dismutase (SOD) and malonaldehyde (MDA) were examined by ELISA. Reactive oxygen species (ROS) production was determined by DCFH-DA staining. The protein levels of LC3II/LC3I, Beclin1, p62, NRF2, and ATG5 were detected by western blot.
RESULTS: IND increased cell viability, while depressed the rate of apoptosis in SH-SY5Y cells of OGD/R environment. IND inhibited autophagy by suppressing the levels of LC3II/LC3I, Beclin1 protein, and increasing p62 protein expression in SH-SY5Y cells of OGD/R environment. IND limited the contents of ROS and MDA, while amplifying the activity of SOD in SH-SY5Y cells with OGD/R exposure. IND also promoted NRF2 expression in OGD/R environment.
CONCLUSIONS: IND could inhibit autophagy, oxidative stress, and apoptosis in SH-SY5Y cells with OGD/R exposure, further alleviating IS injury by regulating transcription factor NRF2 and inhibiting ATG5 expression.