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目的本实验以冈田酸(OA)损伤的HT22细胞作为氧化应激损伤的体外模型,探讨小檗碱对OA氧化应激损伤作用的影响及其机制。方法应用CCK-8法检测不同浓度冈田酸(0、20、40、60、80、160 nmol/L)损伤HT22细胞12 h后的细胞活力;不同浓度小檗碱(2.5、5.0、10.0、20.0μmol/L)预处理HT22细胞12 h后加入OA损伤12 h,应用CCK-8比色法检测细胞的增殖状况;倒置显微镜下观察细胞形态学变化;应用试剂盒对细胞丙二醛(MDA)和抗氧化酶(SOD-1和GSH-Px)活性的进行测定;2′,7′-dichlorfluorescein-diacetate(DCFH-DA)染色检测HT22细胞内ROS水平;Western blot法检测Cleaved caspase-3蛋白的表达。结果 OA(0~160 nmol/L)处理HT22细胞12 h后,细胞活力分别为(100.00±0.42)%、(91.96±1.18)%、(72.79±1.51)%、(57.89±3.18)%、(41.50±1.58)%、(33.83±1.59)%,细胞活力呈浓度依赖性下降;小檗碱(2.5~20.0μmol/L)预处理细胞12 h后,再给予OA作用12 h,细胞活力由OA单独损伤组(76.48±3.94)%,分别上升至(89.18±3.38)%、(97.06±5.56)%、(94.85±3.16)%、(87.52±4.81)%;小檗碱(5.0、10.0μmol/L)预处理组,减少了MDA的产生,抑制了细胞内ROS的积聚,提高了抗氧化酶(SOD-1和GSH-Px)活性,下调了Cleaved caspase-3蛋白表达。结论小檗碱可以通过对抗OA诱导的HT22细胞氧化应激损伤和凋亡发挥其神经保护作用。
OBJECTIVE: To investigate the effect and mechanism of berberine on the oxidative stress-induced injury induced by oxidative stress in HT22 cells injured by okadaic acid (OA) as an in vitro model. Methods The cell viability of HT22 cells injured by different concentrations of okadaic acid (0, 20, 40, 60, 80 and 160 nmol / L) for 12 h was determined by CCK-8 assay. Different concentrations of berberine (2.5, 20.0μmol / L) for 12 h, cells were injured by adding OA for 12 h. Cell proliferation was detected by CCK-8 colorimetric assay. Cell morphological changes were observed under inverted microscope. Malondialdehyde (MDA) ) And antioxidant enzymes (SOD-1 and GSH-Px). The level of ROS in HT22 cells was detected by 2 ’, 7’-dichlorfluorescein-diacetate (DCFH-DA) staining. Cleaved caspase-3 protein expression. Results The viability of HT22 cells treated with OA (0 ~ 160 nmol / L) for 12 h was (100.00 ± 0.42)%, (91.96 ± 1.18)%, (72.79 ± 1.51)%, (57.89 ± 3.18)%, 41.50 ± 1.58%, and (33.83 ± 1.59)% respectively. Cell viability was decreased in a concentration-dependent manner. After pretreated with berberine (2.5-20.0μmol / L) for 12 h, (89.18 ± 3.38)%, (97.06 ± 5.56)%, (94.85 ± 3.16)% and (87.52 ± 4.81)% respectively in the group of single injury (76.48 ± 3.94)%; berberine L) decreased the production of MDA, inhibited the accumulation of intracellular ROS, increased the activities of antioxidant enzymes (SOD-1 and GSH-Px), and down-regulated the expression of Cleaved caspase-3. Conclusion Berberine can exert its neuroprotective effect against OA-induced oxidative stress injury and apoptosis in HT22 cells.