Cai Meiyun, Zhuang Wenxin, Lyu E, Fu Wenyu
Department of Histology and Embryology, School of Basic Medical Sciences, Shangdong Provincial Key Laboratory for Neurologic Disorders and Regenerative Repair, Weifang Medical University, Weifang 261053, China.
Center for Experimental Medical Research, Weifang Medical University, Weifang 261053, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2020 Jul;36(7):583-589.
Objective To investigate the protective effects of kaempferol (KAE) on PC12 cells against neurotoxin 6-hydroxydopamine (6-OHDA)-induced inflammatory damage and explore whether it is related to the inhibition of the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Methods After being cultured for 24 hours, PC12 cells were divided into 4 groups: control, 6-OHDA (100 μmol/L), 6-OHDA combined with (20, 40, 60, 80, 100) μmol/L KAE and KAE groups. The morphological features of PC12 cells were observed under an inverted microscope, and the cell viability was detected by CCK-8 assay. In the case of the optimal effective dose of KAE, the expression of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), nuclear factors (NF)-κB were evaluated by immunocytochemical staining and Western blot analysis. To explore the mechanism underlying the protective role of KAE, KAE and SB203580 (p38 MAPK pathway inhibitor) (10 μmol/L) were separately added into 6-OHDA culture medium of PC12 cells. The protein levels of iNOS, COX2, NF-κB, p38 MAPK and p-p38 MAPK were tested by Western blotting. Results Compared with the control group, the number of cells in the 6-OHDA group was significantly reduced, with most cell bodies shrunk and aggregated into clumps. Compared with the 6-OHDA group, the number of cells increased and the morphology was effectively improved in the 6-OHDA combined with KAE (20, 40, 60, 80, and 100 μmol/L) group. KAE at 80 μmol/L was demonstrated the best protective effects in the present work. In the 6-OHDA group, the expression of COX2, iNOS, NF-κB significantly increased. Compared with the 6-OHDA group, the expression of the above molecules decreased markedly in the 6-OHDA combined with 80 μmol/L KAE group. The protein expression levels of COX2, iNOS, NF-κB, p38 MAPK and p-p38 MAPK went up significantly in the 6-OHDA group as compared with the control group, while they were down-regulated obviously by both KAE and SB203580. Conclusion KAE exerts neuroprotective effects on PC12 cells against the damage induced by 6-OHDA probably through the p38 MAPK signaling pathway.
目的 探讨山柰酚（KAE）对PC12细胞抗神经毒素6-羟基多巴胺（6-OHDA）诱导的炎性损伤的保护作用，并探究其是否与抑制p38丝裂原活化蛋白激酶（p38 MAPK）信号通路有关。方法 PC12细胞培养24小时后，分为4组：对照组、6-OHDA（100 μmol/L）组、6-OHDA联合（20、40、60、80、100）μmol/L KAE组和KAE组。在倒置显微镜下观察PC12细胞的形态特征，采用CCK-8法检测细胞活力。在KAE最佳有效剂量情况下，通过免疫细胞化学染色和蛋白质印迹分析评估环氧合酶-2（COX2）、诱导型一氧化氮合酶（iNOS）、核因子（NF）-κB的表达。为探究KAE保护作用的潜在机制，将KAE和SB203580（p38 MAPK通路抑制剂）（10 μmol/L）分别加入PC12细胞的6-OHDA培养基中。通过蛋白质印迹法检测iNOS、COX2、NF-κB、p38 MAPK和p-p38 MAPK的蛋白水平。结果 与对照组相比，6-OHDA组细胞数量显著减少，多数细胞体萎缩并聚集成团。与6-OHDA组相比，6-OHDA联合KAE（20、40、60、80和100 μmol/L）组细胞数量增加，形态有效改善。本研究中80 μmol/L的KAE显示出最佳保护作用。在6-OHDA组中，COX2、iNOS、NF-κB的表达显著增加。与6-OHDA组相比，6-OHDA联合80 μmol/L KAE组上述分子的表达明显降低。与对照组相比，6-OHDA组中COX2、iNOS、NF-κB、p38 MAPK和p-p38 MAPK的蛋白表达水平显著升高，而KAE和SB203580均使其明显下调。结论 KAE可能通过p38 MAPK信号通路对PC12细胞抗6-OHDA诱导的损伤发挥神经保护作用。
