Fan Ying, Zhang Yi-na, Zhang Yan-qiao, Teng Zong-yan, Li Zhi-wei, Wu Xiao-wei, Li Hu-lun
Geriatric Department, the Second Affiliated Hospital, Harbin Medical University, Harbin 150086, China.
Zhonghua Yi Xue Za Zhi. 2006 Dec 5;86(45):3173-6.
To investigate the role of phosphorylation of protein kinase C (PKC) delta in the toxicity of 6-hydroxydopamine (6-OHDA) to the death of dopaminergic neurons.
Human neuroblastoma cells of the line SH-SY5Y were cultured 6-OHDA of the concentrations of 0, 50, 100, 200, and 400 micromol/L was added to observe its toxicity. Rottlerin (PKCdelta inhibitor, 2 micromol/L), bisindolylmaleimide (Bis, general PKC inhibitor, 10 nmol/L), Gö6976 (calcium-dependent PKC inhibitor, 5 nmol/L), and phobol-12-myristate-13-acetate (PMA, PKC activator, 100 nmol/L) were added into the culture fluid of another SH-SY5Y cells respectively, and then (1) culture fluid of equal volume was added for 18 h so as to observe there effects on the survival of the SH-SY5Y cells, or (2) 100 micromol/L 6-OHDA was added to observe the effects of intervention on PKC on the survival of the SH-SY5Y cells by using MTT assay. Cell lysis solution with phosphatase inhibitor was used to lyse the culture cells to extract plasma protein. Western blotting was used to detect the expression of phosphorylated PKCdelta.
MTT assay showed that all different concentrations (50 - 400 micromol/L) of 6-OHDA significantly and dose-dependently caused cell death with an EC50 of 92 micromol/L. Pretreatment with rottlerin and Bis alone did not influence the survival of the cells significantly,. However, the survival rate of the cells pretreated by Gö6976 alone was 92.3% +/- 3.2% that of the control group (P < 0.01), and the survival rate of the cells pretreated by PMA was 49.5% +/- 1.0% that of the control group (P < 0.01) Pretreatment of rottlerin decreased the death rate of the cells treated with 6-OHDA to 30.4% +/- 1.6% and conferred significant protection against 6-OHDA neurotoxicity by 57% +/- 6% compared to that of the cells treated by 6-OHDA alone (P < 0.01). However, Bis and Gö6976 did not affect the 6-OHDA-induced cell damage. Pretreatment of PMA increased the death rate of the cells treated with 6-OHDA to 67.1% +/- 2.2% and significantly aggravated 6-OHDA-induced cell toxicity by 66% +/- 9% (P < 0.01). Western blotting showed that 6-OHDA administration increased the expression of phosphorylated PKCdelta, pretreatment with Rottlerin inhibited such increase, PMA promoted such increase, and Bis and Gö6976 did not influence such increase.
Inhibition of PKCdelta phosphorylation with rottlerin ameliorates the neurotoxicity evoked by 6-OHDA, and activation of PKCdelta phosphorylation by PMA aggravates neurotoxicity, which implicating that this kinase participates in the 6-OHDA-induced neurotoxicity and Parkinsonian neurodegeneration.
研究蛋白激酶C（PKC）δ磷酸化在6-羟基多巴胺（6-OHDA）对多巴胺能神经元毒性作用中的作用。
培养人神经母细胞瘤SH-SY5Y细胞系，分别加入浓度为0、50、100、200和400 μmol/L的6-OHDA，观察其毒性作用。在另一批SH-SY5Y细胞培养液中分别加入罗特列素（PKCδ抑制剂，2 μmol/L）、双吲哚马来酰胺（Bis，PKC通用抑制剂，10 nmol/L）、Gö6976（钙依赖性PKC抑制剂，5 nmol/L）和佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA，PKC激活剂，100 nmol/L），然后（1）加入等体积培养液培养18 h，观察其对SH-SY5Y细胞存活的影响，或（2）加入100 μmol/L 6-OHDA，采用MTT法观察PKC干预对SH-SY5Y细胞存活的影响。用含磷酸酶抑制剂的细胞裂解液裂解培养细胞，提取胞浆蛋白。采用蛋白质免疫印迹法检测磷酸化PKCδ的表达。
MTT法显示，不同浓度（50～400 μmol/L）的6-OHDA均能显著且呈剂量依赖性地导致细胞死亡，半数有效浓度（EC50）为92 μmol/L。单独用罗特列素和Bis预处理对细胞存活无显著影响。然而，单独用Gö6976预处理的细胞存活率为对照组的92.3%±3.2%（P<0.01），用PMA预处理的细胞存活率为对照组的49.5%±1.0%（P<0.01）。用罗特列素预处理可将6-OHDA处理的细胞死亡率降至30.4%±1.6%，与单独用6-OHDA处理的细胞相比，对6-OHDA神经毒性的保护作用显著提高57%±6%（P<0.01）。然而，Bis和Gö6976不影响6-OHDA诱导的细胞损伤。用PMA预处理可使6-OHDA处理的细胞死亡率增至67.1%±2.2%，并使6-OHDA诱导的细胞毒性显著增加66%±9%（P<0.01）。蛋白质免疫印迹法显示，给予6-OHDA可增加磷酸化PKCδ的表达，用罗特列素预处理可抑制这种增加，PMA可促进这种增加，而Bis和Gö6976不影响这种增加。
用罗特列素抑制PKCδ磷酸化可改善6-OHDA诱发的神经毒性，用PMA激活PKCδ磷酸化可加重神经毒性，这表明该激酶参与了6-OHDA诱导的神经毒性和帕金森病神经变性。
