二苯乙烯苷对基因转染PC12细胞α-突触核蛋白过表达和泛素-蛋白酶体系统的影响

Effects of Tetrahydroxy-stilbene Glycoside on α-Synuclein Over-expression and Ubiquitin-Proteasome System in Gene-transfected PC12 Cells

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 explore the effects and mechanisms of 2,3,5,4＇-tetrahydroxy stilbene-2-13-D- glycoside （TSG）, an ef- fective component of Polygonum multiflorum, on a-synuelein （a-Syn） over-expression in vitro. METHODS Different concentrations of TSG were incubated with a-Syn gene-transfeeted PC12 cells for 24 h. The nontoxicity range of TSG to cultured cells was determined by MTT method and lactate dehydrogenase leakage method. The change in oL-Syn mRNA content in the cells was measured by RT-PCR. The protein expression of a-Syn, UCH-L1 and parkin was detected by Western blotting. RESULTS The nontoxieity range of TSG to cultured cells was 6. 25 - 100 μmol·L^-1. When incubated with a-Syn gene-transfected PC12 ceils within this dose range for 24 h, TSG significantly decreased oL-Syn mRNA content, inhibited a-Syn protein expression and aggregation, and enhanced the protein expression of UCH-L1 and parkin in the model cells. CONCLUSION TSG inhibited a-Syn over-expression and aggregation in gene-tranfected PC12 cells in vitro, and the mechanisms may be related to the inhibition of synthesis and the enhancement of degradation of a-synuclein.