柴胡疏肝散调节自噬 – 溶酶体途径抑制 Aβ 诱导的神经毒性

EffectsChaihu Shugan San Inhibits Aβ-induced Neurotoxicity by Regulating Autophagy-lysosome Pathway

摘要:

〔摘 要〕 目的：探讨柴胡疏肝散（CSS）通过调节自噬 - 溶酶体途径抑制 β– 淀粉样蛋白（Aβ）诱导神经毒性的作用机制。方法：选取大鼠 PC12 嗜铬细胞瘤细胞分化的类神经元细胞为研究对象，以 Aβ 诱导神经毒性为模型，采用噻唑蓝（MTT）法和乳酸脱氢酶（LDH）释放实验检测神经细胞毒性，采用单丹磺酰尸胺（MDC）染色和LysoTracker 荧光探针染色观察细胞自噬，采用 Western blot 测定自噬 – 溶酶体途径相关蛋白的表达情况。结果：Aβ 在 0.125 ~ 2.0 μmol·L-1 范围内呈浓度和时间依赖性抑制 PC12 细胞活力，而 CSS 在剂量 250 μg·mL-1 、500 μg·mL-1 均明显抑制 Aβ 诱导的细胞活力下降及乳酸脱氢酶释放（P ＜ 0.05）；荧光染色结果显示 CSS 激活 Aβ 诱导的细胞自噬和溶酶体活性；CSS 可抑制细胞 mTOR 磷酸化和增加 LC3–II/LC3–I 水平，促进自噬体形成。结论：CSS 抑制 Aβ 诱导的神经毒性，可能与其调节自噬 – 溶酶体途径有关。

Abstract:

〔Abstract〕 Objective To explore the mechanism of Chaihu Shugan San (CSS) inhibits β-amyloid (Aβ)-induced neurotoxicity via regulating autophagy-lysosomal pathway. Methods The neuron-like cells differentiated from rat PC12 pheochromocytoma cells were selected as the research object, and the neurotoxicity induced by Aβ was used as the model. The cytotoxicity of nerve cells was detected by methyl thiazol tetrazolium (MTT) assay and lactate dehydrogenase (LDH) release assay. Monodansylcadaverine (MDC) staining and LysoTracker fluorescent probe staining were used to observe autophagy, and Western blot was used to detect the expression of autophagy-lysosome pathway-related proteins. Results Aβ inhibited the viability of PC12 cells in a concentration and time-dependent manner within the range of 0.125-2.0 μmol·L-1, while CSS at 250 μg·mL-1 and 500 μg·mL-1 significantly inhibited Aβ-induced decrease in cell viability and lactate dehydrogenase release (P < 0.05). Fluorescence staining showed that CSS activated Aβ-induced autophagy and lysosomal activity. CSS can inhibit the phosphorylation of mTOR and increase the level of LC3-II/LC3-I, and promote the formation of autophagosome. Conclusion CSS inhibited Aβ-induced neurotoxicity probably by regulating autophagy-lysosomal pathway.