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目的观察何首乌有效成分二苯乙烯苷(TSG)在体外实验中对α-突触核蛋白(α-synuclein,α-Syn)过表达的影响及其作用机制。方法不同浓度的TSG与α-Syn基因转染PC12细胞共孵育24 h,应用噻唑蓝(MTT)法和乳酸脱氢酶漏出法确定该药对培养细胞的无毒范围,RT-PCR法检测细胞中α-Syn mRNA含量的变化,蛋白质印迹(Western blotting)法检测α-Syn、泛素化蛋白羧基端水解酶(UCH-L1)和parkin蛋白的表达,观察TSG对它们的影响。结果 TSG对培养细胞的无毒范围为6.25～100μmol.L-1。在此剂量范围内的TSG与α-Syn基因转染PC12细胞孵育24 h,能够明显减少模型细胞α-Syn mRNA含量,抑制α-Syn蛋白表达和聚集,增强UCH-L1和parkin蛋白表达。结论 TSG在体外对基因转染细胞的α-Syn过表达和聚集有明显抑制作用,其作用机制与抑制α-Syn合成和增强α-Syn降解有关。
Objective To observe the effect of the active ingredient of Radix Polygoni Multiflori (TSG) on the overexpression of α-synuclein (α-Syn) in vitro and its mechanism. Methods Different concentrations of TSG and α-Syn gene were transfected into PC12 cells for 24 h. MTT assay and lactate dehydrogenase leakage assay were used to determine the non-toxic range of the drug to cultured cells. RT- Α-Syn mRNA expression was detected by western blotting. The expression of α-Syn, UCH-L1 and parkin protein were detected by Western blotting, and the effect of TSG on them was observed. Results The non-toxic range of TSG to cultured cells was 6.25 ~ 100μmol.L-1. Incubation of PC12 cells with TSG and α-Syn gene in this dose range for 24 h significantly reduced α-Syn mRNA expression, inhibited the expression of α-Syn protein and enhanced the expression of UCH-L1 and parkin protein. Conclusion TSG can inhibit the expression and accumulation of α-Syn in gene-transfected cells in vitro. Its mechanism is related to inhibiting the synthesis of α-Syn and enhancing the degradation of α-Syn.