目的考察地骨皮甲素(kukoamine A,KuA)对鱼藤酮诱导的PC12细胞损伤的保护作用并对其作用机制进行初步研究,为发现具有抗帕金森病的活性先导化合物提供依据。方法通过体外建立鱼藤酮诱导的帕金森模型,运用MTT、LDH、Hoechst33342染色等方法对Ku A体外抗鱼藤酮诱导的PC12细胞损伤进行初步确认。利用比色法和荧光染色分别考察KuA对超氧化物歧化酶(SOD)活性、丙二醛(MDA)及活性氧(ROS)含量、线粒体膜电位(MMP)的影响。采用Western blotting探讨KuA体外抗鱼藤酮诱导PC12细胞损伤的作用机制。结果 0.5μmol/L鱼藤酮可降低PC12细胞存活率,不同浓度Ku A可减轻鱼藤酮诱导的PC12细胞损伤。与模型组相比,KuA可降低细胞内ROS及MDA含量,并提高SOD活性。此外,KuA可提高基质金属蛋白酶(MMP)水平,下调Bax/Bcl-2值、抑制细胞色素C释放以及下调Caspase-3、Caspase-9、α-synuclein的蛋白表达。结论 KuA对鱼藤酮诱导的PC12细胞帕金森病模型具有保护作用,其可能的机制与抑制ROS生成、保护MMP、调控线粒体凋亡途径相关蛋白表达和降低α-synuclein蛋白表达有关。
Objective To investigate the neuroprotective effects of kukoamine A(KuA) on rotenone-induced PC12 cells damage and to preliminary verify its potential action mechanisms. The present study may lay the foundation for finding leading compounds with anti-Parkinson’s disease(PD) effects. Methods A PD model induced by rotenone was established in vitro, and MTT, LDH, and Hoechst33342 staining were used for preliminary confirmation of KuA resistance to rotenone-induced PC12 cell injury in vitro. The effects of KuA on superoxide dismutase(SOD) activity, malondialdehyde(MDA) and reactive oxygen species(ROS) content, and mitochondrial membrane potential(MMP) were investigated by colorimetric method and fluorescence staining, respectively. Western blotting was applied to explore the underlying mechanisms of protective effects of KuA against rotenone-induced PC12 cells damage. Results The PC12 cell viability was significantly decreased after exposure to 0.5 μmol/L rotenone, whereas pretreatment with different concentrations of KuA could attenuate the cell injury induced by rotenone. Compared with the rotenone-treated group, KuA could decrease the ROS production and MDA level, while increase the SOD activity. In addition, KuA could effectively increase the MMP, decrease the cytochrome c release and the Bax/Bcl-2 ratio as well as inhibit caspase-3, caspase-9, and α-synuclein protein expressions. Conclusion KuA showed neuroprotective ability on rotenone-induced PC12 cells PD model and the potential protective mechanisms of KuA can be related with inhibition of ROS generation, protection of MMP, regulation of protein expressions involved in the mitochondrial apoptosis pathway and reduction of α-synuclein expression.
Chinese Traditional and Herbal Drugs