石菖蒲远志有效成分组合抑制β淀粉样蛋白25-35神经毒性的钙相关机制

Calcium-related mechanism of inhibiting neurotoxicity of Aβ_(25-35) with compositions of effective components in grass leaf sweetflag rhizome and milkwort root

目的:探讨石菖蒲、远志的有效成分β-细辛醚、丁香酚和远志皂苷组合抑制β淀粉样蛋白(Aβ)神经细胞毒性与钙离子相关的保护机制。方法:利用培养的胎生大鼠海马神经细胞,以Aβ25-35 1μmol/L处理,建立神经毒性模型;以β-细辛醚40μmol/L、丁香酚2μmol/L、远志皂苷20μmol/L与Aβ25-35 1μmol/L同时处理神经细胞,检测药物的抗Aβ神经毒性作用。利用Hoechst-33258及Caspase-3染色检测神经细胞损害及凋亡。利用Fluo-3 AM荧光探针,测定神经细胞内钙离子浓度,考察β-细辛醚、丁香酚和远志皂苷对Aβ引起的钙浓度变化的影响。结果:Aβ25-35 1μmol/L使培养海马神经细胞发生核固缩及碎片化、Capase-3染色阳性细胞增多、细胞内钙离子浓度显著升高(P<0.01),而同时给予β-细辛醚40μmol/L、丁香酚2μmol/L、远志皂苷20μmol/L组合抑制了Aβ25-35 1μmol/L介导的神经细胞损害,翻转细胞内钙离子浓度至正常水平,而对正常细胞钙离子浓度没有影响。结论:石菖蒲、远志有效成分β-细辛醚40μmol/L、丁香酚2μmol/L、远志皂苷20μmol/L组合能抑制Aβ25-35神经细胞毒性,稳定细胞内钙离子浓度,对受损神经细胞有保护作用。

Objective：To explore the calcium ion related protection mechanism of compositions of β-asarone,eugenol and tenuigenin in grass leaf sweetflag rhizome and milkwort root on Aβ-induced neurotoxicity.Methods：The cell model of neurotoxicity was established by cultivating hippocampus neurons from fetal rat with 1μ mol/L Aβ25-35.The neuronal cells were treated with β-asarone（40μ mol/L）,eugenol（2μ mol/L） and tenuigenin（20μ mol/L） and Aβ 25-35（1μ mol/L）simultaneously,in order to test the antagonism of drugs on Aβ-induced neurotoxicity.The damage and apoptosis of cells were tested by using Hoechst-33258 and Caspase-3 staining methods.The Fluo-3 AM fluorescent probe was used to determine intracellular calcium ion concentration,in order to demonstrate the influence of β-asarone,eugenol and tenuigenin on the changes of calcium concentration.Results：There were obvious manifestations as karyopyknosis,debris,increasing of positive cells of Caspase-3 staining and raising intracellular calcium ion concentration in hippocampus neurons cultivated with 1μ mol/L Aβ25-35（P〈0.05）.The compositions of β-asarone（40μmol/L）,eugenol（2μmol/L） and tenuigenin（20μmol/L） could inhibit the neuronal damage mediated by Aβ_（25-35）（1μ mol/L） and recover the intracellular calcium ion concentration,and with no influence on calcium ion concentration of normal cells.Conclusion：The effective components as β-asarone（40μ mol/L）,eugenol（2μ mol/L） and tenuigenin（20μ mol/L） from grass leaf sweetflag rhizomeand milkwort root could inhibit the Aβ_（25-35）-induced neurotoxicity,stabilize intracellular calcium ion concentration,and protect the damaged nerve cells.