二氢杨梅素通过上调自噬活性抑制MPP^+诱导的PC12细胞损伤

Dihydromyricetin inhibits PC12 cell injury induced by 1-methyl-4-phenylpyridinium by up-regulating autophagy activity

目的观察二氢杨梅素(DMY)对1-甲基-4-苯基吡啶离子(MPP+)诱导的PC12细胞损伤的影响并探讨其可能的机制。方法含DMY 5,10,20,40和80μmol·L^(-1)的培养液预处理PC12细胞0.5 h,培养液中加入自噬抑制剂3-甲基腺嘌呤(3-MA)10 mmol·L^(-1),再加入MPP+500μmol·L^(-1)培养48 h。噻唑蓝(MTT)法检测细胞存活,丙酮酸二硝基苯腙比色法检测细胞培养液中乳酸脱氢酶(LDH)水平,透射电镜观察细胞的超微结构,Western蛋白质印迹法检测细胞中自噬相关蛋白Beclin-1和微管相关蛋白1轻链3(LC3)和P62的表达。结果与细胞对照组比较,MPP+组细胞存活率显著降低(P<0.05),细胞培养液中LDH水平显著增加(P<0.05),细胞中自噬体的数量明显减少,自噬泡占胞质总面积的百分率显著降低(P<0.05),LC3-Ⅱ和Beclin-1的蛋白表达及LC3-Ⅱ/LC3-Ⅰ的比值均显著降低(均P<0.05),P62的表达显著升高(P<0.05)。与MPP+组比较,MPP++DMY 10~80μmol·L^(-1)组细胞存活率显著升高(P<0.05),细胞培养液中LDH水平显著降低(P<0.05)。与MPP+组比较,MPP++DMY 20μmol·L^(-1)组细胞中自噬体数量明显增加,自噬泡占胞质总面积的百分率显著增加(P<0.05),LC3-Ⅱ和Beclin-1的蛋白表达和LC3-Ⅱ/LC3-Ⅰ的比值均显著增加(P<0.05),P62的表达显著降低(P<0.05)。与MPP++DMY 20μmol·L^(-1)组比较,MPP++DMY+3-MA组细胞存活率显著降低(P<0.05),细胞上清液中LDH水平显著增加(P<0.05)。结论 DMY抑制MPP+诱导的PC12细胞损伤,其机制与上调细胞自噬活性有关。

OBJECTIVE To observe the effect of dihydromyricetin （DMY） on cell injury induced by 1-methyl-4-phenylpyridinium （MPP＋） in PC12 cells and explore the possible mechanism. METHODS After being pretreated with DMY 5, 10, 20, 40 and 80 μmol· L-1 for 0.5 h, PC12 cells were incubated with DMEM culture medium including autophagy inhibitor 3-methyladenine （3-MA） 10 mmol· L-1 and MPP＋ 500 μmol·L-1 for 48 h. MTT Assay was used to test the viability of PC12 cells. The level of lactate dehydregenase （LDH） was measured colorimetrically. The ultrastructure of PC12 cells was observed under transmission electron microscope. Expressions of autophagy related protein, Beclin-1, microtubuleassociated protein light chain 3 （LC3） and p62 were measured by Western blotting. RESULTS Compared with cell control group, the cell survival rate was significantly decreased, the level of LDH was significantly increased, the number of autophagosomes was obviously decreased, the percentage of autophagic vacuoles in the total cytoplasm area was significantly decreased （P〈0.05）, expression of Beclin-1 and LC3- Ⅱ and the ratio of LC3- Ⅱ to LC3- Ⅰ were significantly down-regulated （P〈0.05） and the expression of p62 was significantly up-regulated in MPP＋ group （P〈0.05）. Compared with MPP＋ group, the cell survival rate was significantly increased, while the level of LDH was significantly decreased in MPP＋ DMY 10-80 umol· L-1 group （P〈0.05）. Compared with MPP＋ group, the number of autophagosomes obviously increased, the percentage of autophagic vacuoles in the total cytoplasm area was significantly increased, the expression of Beclin-1 and LC3- Ⅱ and the ratio of LC3- Ⅱ to LC3- Ⅰ were significantly up-regulated and the expression of p62 was significantly down-regulated in MPP ＋DMY 20 μmol· L-1 group （P〈0.05）. Compared with MPP＋＋DMY 20 μmol· L-1 group, the cell survival rate was significantly decreased, but the level of LDH was significantly increased in MPP＋＋ DMY＋3-MA group （P〈0.05）. CONCLUSION DMY may inhibit the cell injury induced by MPP＋in PC12 cells, and the mechanism may be related to the up-regulation of autophagy activity induced by DMY.