硫化氢对 6-OHDA 诱导的 SH-SY5Y 细胞损伤的保护作用涉及 PKC/PI3K/Akt 通路。
Protective effect of hydrogen sulphide against 6-OHDA-induced cell injury in SH-SY5Y cells involves PKC/PI3K/Akt pathway.
机构信息
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
出版信息
Br J Pharmacol. 2010 Sep;161(2):467-80. doi: 10.1111/j.1476-5381.2010.00887.x.
BACKGROUND AND PURPOSE
Hydrogen sulphide (H(2)S) is a novel neuromodulator. The present study aimed to investigate the protective effect of H(2)S against cell injury induced by 6-hydroxydopamine (6-OHDA), a selective dopaminergic neurotoxin often used to establish a model of Parkinson's disease for studying the underlying mechanisms of this condition.
EXPERIMENTAL APPROACH
Cell viability in SH-SY5Y cells was measured using MTT assay. Western blot analysis and pharmacological manipulation were employed to study the signalling mechanisms.
KEY RESULTS
Treatment of SH-SY5Y cells with 6-OHDA (50-200 microM) for 12 h decreased cell viability. Exogenous application of NaHS (an H(2)S donor, 100-1000 microM) or overexpression of cystathionine beta-synthase (a predominant enzyme to produce endogenous H(2)S in SH-SY5Y cells) protected cells against 6-OHDA-induced cell apoptosis and death. Furthermore, NaHS reversed 6-OHDA-induced loss of tyrosine hydroxylase. Western blot analysis showed that NaHS reversed the down-regulation of PKCalpha, epsilon and Akt and the up-regulation of PKCdelta in 6-OHDA-treated cells. Blockade of PKCalpha with Gö6976 (2 microM), PKCepsilon with EAVSLKPT (200 microM) or PI3K with LY294002 (20 microM) reduced the protective effects of H(2)S. However, inhibition of PKCdelta with rottlerin (5 microM) failed to affect 6-OHDA-induced cell injury. These data suggest that the protective effects of NaHS mainly resulted from activation of PKCalpha, epsilon and PI3K/Akt pathway. In addition, NaHS-induced Akt phosphorylation was significantly attenuated by Gö6976 and EAVSLKPT, suggesting that the activation of Akt by NaHS is PKCalpha, epsilon-dependent.
CONCLUSIONS AND IMPLICATIONS
H(2)S protects SH-SY5Y cells against 6-OHDA-induced cell injury by activating the PKCalpha, epsilon/PI3K/Akt pathway.
背景与目的
硫化氢（H₂S）是一种新型的神经调节剂。本研究旨在探讨 H₂S 对 6-羟多巴胺（6-OHDA）诱导的细胞损伤的保护作用，6-OHDA 是一种选择性多巴胺能神经毒素，常用于建立帕金森病模型以研究该疾病的潜在机制。
实验方法
通过 MTT 测定法测量 SH-SY5Y 细胞的细胞活力。采用 Western blot 分析和药理学操作研究信号转导机制。
主要结果
6-OHDA（50-200μM）处理 SH-SY5Y 细胞 12 小时会降低细胞活力。外源性应用 NaHS（H₂S 供体，100-1000μM）或过表达半胱氨酸-β-合酶（SH-SY5Y 细胞中产生内源性 H₂S 的主要酶）可防止 6-OHDA 诱导的细胞凋亡和死亡。此外，NaHS 逆转了 6-OHDA 诱导的酪氨酸羟化酶丢失。Western blot 分析显示，NaHS 逆转了 6-OHDA 处理细胞中 PKCalpha、epsilon 和 Akt 的下调以及 PKCdelta 的上调。用 Gö6976（2μM）阻断 PKCalpha、用 EAVSLKPT（200μM）阻断 PKCepsilon 或用 LY294002（20μM）阻断 PI3K 会降低 H₂S 的保护作用。然而，用罗特林（5μM）抑制 PKCdelta 并未影响 6-OHDA 诱导的细胞损伤。这些数据表明，NaHS 的保护作用主要源于激活 PKCalpha、epsilon 和 PI3K/Akt 途径。此外，Gö6976 和 EAVSLKPT 显著减弱了 NaHS 诱导的 Akt 磷酸化，表明 NaHS 通过 PKCalpha、epsilon 依赖性激活 Akt。
结论和意义
H₂S 通过激活 PKCalpha、epsilon/PI3K/Akt 途径保护 SH-SY5Y 细胞免受 6-OHDA 诱导的细胞损伤。
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