淫羊藿苷抑制Aβ25-35诱导的N2a细胞自噬及减少细胞内外Aβ的实验研究  

Icariin Inhibited Aβ_(25-35)-induced Autophagic Systems and Decreased Aβ in N2a Cells

作　　者：姜霞 王平 JIANG Xia;WANG Ping(Department of Pathology,Hubei University of Chinese Medicine,Wuhan 430065;Institute of Gerontology,Hubei University of Chinese Medicine,Wuhan 430065)

机构地区：[1]湖北中医药大学基础医学院病理学教研室,武汉430065 [2]湖北中医药大学老年病研究所,武汉430065

出　　处：《中国中西医结合杂志》2021年第9期1080-1086,共7页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金资助项目(No.81973721,No.81303252);湖北省自然科学基金项目(No.2017CFB733)

摘　　要：目的研究淫羊藿苷(ICA)对β-淀粉样蛋白肽25-35(Aβ25-35)诱导的小鼠神经瘤母细胞(N2a)Aβ含量和自噬溶酶体途径的影响。方法 N2a细胞培养基加入不同浓度的ICA(0.05、0.1、0.2 μmol/L)孵育6 h,再将20 μmol/L老化的Aβ25-35处理24 h。实验分为5组:对照组(DMSO)、AD细胞模型组(Aβ25-35)、0.05 μmol/L ICA 干预组(Aβ25-35+ICA0.05)、0.1 μmol/L ICA 干预组(Aβ25-35+ICAO.1)、0.2 μmol/L ICA 干预组(Aβ25-35+ICA0.2)。将 0.2 μmol/L 的 ICA 预处理 N2a 细胞2、4、6 h,再加入Aβ25-35处理24 h。收集细胞和培养基,ELISA法测定细胞内外Aβ40及Aβ42含量;免疫印迹法检测LC3、P62、p70S6K及其苏氨酸389位点磷酸化(p-p70S6K,活性形式)的表达;采用细胞免疫荧光技术观察LC3和4G8(识别Aβ17-24片段)的染色强度及LAMP2和4G8共定位情况。结果与对照组比较,AD细胞模型组细胞存活率降低(P<0.05),细胞内外Aβ42及Aβ40的含量增加(P<0.01,P<0.05),LC3Ⅱ/LC3Ⅰ和P62蛋白表达均增加(P<0.05),p-p70S6K表达增加(P<0.01)。与AD细胞模型组比较,0.05、0.1和0.2 μmol/L ICA干预组细胞活力下降(P<0.05),0.2 μmol/L ICA干预组细胞内外Aβ42及Aβ40含量降低(P<0.05),0.1 和 0.2 μmol/L 干预组 ICALC3 Ⅱ/LC3 Ⅰ和 4G8 蛋白表达降低(P<0.05)。0.2 μmol/L ICA预处理4、6 h可明显降低AD细胞模型细胞内外Aβ42和Aβ40的含量,降低LC3Ⅱ/LC3 Ⅰ和P62蛋白表达及p-p70S6K/p70S6K(P<0.05)。结论ICA可抑制Aβ25-35诱导的N2a细胞自噬,降低p70S6K活性并减少细胞内外Aβ。Objective To explore the effects of icariin(ICA) on autophagy-lysosomal pathway inβ-amyloid 25-35(Aβ25-35)-induced Neuro2a(N2a) cells.Methods N2a cells were pretreated with ICA(0.05,0.1,0.2 μmol/L),then the cells were incubated with Aβ25-35 for 24 h to establish AD cell model.The cultured cells were divided into 5 groups,i.e.,the control group(DMSO),AD cell model group,0.05 μmol/L ICA group,0.1 μmol/L ICA group,and 0.2 μmol/L ICA group.Furthermore,the AD cell model were pretreated with ICA(0.2 μmol/L) for 2,4 and 6 h.Aβ40 and Aβ42 in cells extract and culture medium were determined by ELISA;Western Blot was used to detect expression of LC3,P62,p70 S6 K and its threonine 389 site phosphorylation(p-p70 S6 K,active form);the expression and distribution of LC3 and 4 G8(recognition of Aβ17-24 fragment)was detected by Immunocytochemistry.Immunofluorescence double label of LAMP2and 4 G8 were used to analyze fusion of autophagosome and lysosome.Results Compared with the control group,the cell survival rate of the AD cell model group decreased(P<0.05),the intracellular and extracellular contents of Aβ42 and Aβ40 increased(P<0.01,P<0.05),and the protein expression of LC3 Ⅱ/LC3 Ⅰ as well as P62 increased(P<0.05),the expression of p-p70 S6 K increased(P<0.01).Compared with the AD cell model group,the cell viability of 0.05,0.1 and 0.2 μmol/L ICA group decreased(P<0.05),and the intracellular and extracellular content of Aβ42 and Aβ40 of the 0.2 μmol/L ICA group decreased(P<0.05),0.1 and 0.2 μmol/L ICA group IC LC3 Ⅱ/LC3 Ⅰ and 4 G8 protein expression decreased(P<0.05).The intracellular and extracellular Aβ42 and Aβ40 contents were decreased after ICA(0.2 μmol/L)pretreatment for 4 and 6 hours,and the expression of LC3 Ⅱ/LC3 Ⅰ and P62as well as p-p70 S6 K/p70 S6 K decreased(P<0.05).Conclusion ICA could inhibit the autophagy,decrease the activity of p70 S6 Kand reduce the intracellular and extracellular Aβ induced by Aβ25-35 in vitro.

关 键 词：淫羊藿苷 小鼠神经瘤母细胞 β样淀粉蛋白肽 自噬 

分 类 号：R285[医药卫生—中药学]