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目的:观察梓醇对纤维状β-淀粉样蛋白(amyliod-beta protein,Aβ_(1-42))诱导的小鼠脑微血管内皮细胞(b End.3)损伤的保护作用,并探讨其保护作用机制。方法:将b End.3细胞分为溶剂组,模型组(Aβ_(1-42)20μmol·L~(-1)),梓醇低、中、高浓度组(Aβ_(1-42)20μmol·L~(-1)+梓醇50,200,500μmol·L~(-1));噻唑蓝(MTT)比色法检测梓醇及其对Aβ_(1-42)诱导的b End.3细胞活性的影响;Hoechst33258染色法检测梓醇对Aβ_(1-42)诱导的b End.3细胞凋亡情况的影响;蛋白免疫印迹法(Western blot)检测自噬及凋亡相关蛋白(Beclin~(-1),LC3),B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax),细胞色素-C(cytochrome-C),半胱氨酸蛋白酶-3(Caspase-3)及切割活化型Caspase-3(cleaved Caspase-3)的表达水平。结果:MTT显示,梓醇对b End.3细胞无毒副作用;MTT与Hoechst33258染色结果显示,与模型组比较,梓醇低、中、高剂量组能明显提高b End.3细胞活性,抑制Aβ_(1-42)介导的b End.3细胞凋亡(P<0.05);Western blot显示,与模型组比较,梓醇预处理后,可明显降低Bax/Bcl-2,cytochrome-C/β-actin,cleaved Caspase-3/Caspase-3,Beclin~(-1)/β-actin,LC3-Ⅱ/LC3-Ⅰ(P<0.05)。结论:梓醇可能通过抑制Aβ_(1-42)诱导的b End.3细胞凋亡及过度自噬发挥其神经保护作用。
AIM: To observe the protective effect of catalpol on the injury of brain endothelium cells (b.3) induced by fibrillar β-amyloid protein (Aβ 1-42) in mice and its protective effect mechanism. Methods: The b.3 cells were divided into solvent group, model group (Aβ_ (1-42) 20μmol·L -1), catalpol low, medium and high concentration groups (Aβ 1-42) 20μmol · L -1 and catalpol 50,200 and 500 μmol·L -1), and the effect of catalpol on the activity of Aβ 1-42 -induced b End.3 cells by MTT assay ; Hoechst33258 staining was used to detect the effect of catalpol on Aβ 1-42-induced apoptosis in b. End3 cells; Western blot was used to detect the expression of autophagy and apoptosis-related protein Beclin ~ (-1) , LC3, Bcl-2, Bax, cytochrome-C, Caspase-3 and cleavage Expression level of activated caspase-3 (cleaved Caspase-3). Results: The results of MTT showed that catalpol had no toxic side effects on bEnd.3 cells. The results of MTT and Hoechst33258 staining showed that compared with the model group, catalpol low, medium and high dose groups could significantly increase the activity of b.3, (1-42) (P <0.05). Western blot showed that compared with the model group, pretreatment with catalpol significantly decreased the expression of Bax / Bcl-2 and cytochrome-C / β -actin, cleaved Caspase-3 / Caspase-3, Beclin -1 / β-actin and LC3-Ⅱ / LC3-Ⅰ (P <0.05). Conclusion Catalpol can exert its neuroprotective effect by inhibiting Aβ 1-42-induced apoptosis of b. End-3 cells and excessive autophagy.