徐　妍　严　旺　蔡雅卫　王继业.宁波市第二医院老年医学科，浙江宁波　500；2.宁波市第二医院神经内科，浙江宁波　500；.浙江警察学院，浙江杭州　005

异钩藤碱通过mTOR非依赖性途径诱导SH-SY5Y细胞自噬发生

徐妍1严旺2▲蔡雅卫1王继业3
1.宁波市第二医院老年医学科，浙江宁波315010；2.宁波市第二医院神经内科，浙江宁波315010；3.浙江警察学院，浙江杭州310053

目的 探讨异钩藤碱（Isorhynchophylline，IRN）人胶质瘤细胞SH-SY5Y细胞自噬的发生及其与mTOR信号通路的关系。方法 用不同浓度IRN（6、12和24 μmol/L）处理SH-SY5Y细胞24 h，免疫印迹法检测自噬标志性蛋白LC3-Ⅱ的表达，免疫荧光法观察微管相关蛋白轻链3（LC3）荧光斑点标记的自噬体形成，利用溶酶体稳定剂氯喹（CQ）以及自噬抑制剂3-MA进一步明确IRN作用的途径。Western蛋白印迹法检测mTOR相关蛋白和Beclin1的表达。结果IRN作用SH-SY5Y细胞24 h后，可显著上调LC3-Ⅱ表达，0、6、12、24 μM IRN作用后的相对表达量分别为（0.086±0.02）、（0.39±0.14）、（0.96±0.09）、（1.93±0.14）。自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ的比值增高（P＜0.01），并呈浓度依赖性，SH-SY5Y细胞内GFP-LC3荧光斑点标记的自噬体增多，在IRN不同浓度处理组（0、6、12、24 μM）中分别为2±1/细胞，4±1/细胞，8±1/细胞，和14±2/细胞。IRN联合CQ后可以促进LC3-Ⅱ的表达 （0.64±0.052）。IRN可以削弱a-Syn蛋白表达，在0、6、12、24 μM组中的相对表达量为 （0.71±0.07）、（0.69± 0.05）、（0.53±0.04）、（0.36±0.034），并呈浓度依赖性，联合3-MA后a-Syn表达上调（0.63±0.04），提示3-MA可以逆转由IRN对a-Syn的抑制效果，而CQ对a-Syn表达，与对照组比无显著影响。信号通路研究提示，对mTOR以及Beclin1表达不影响，Beclin1特异性siRNA作用后可以抑制IRN诱导的LC3-Ⅱ表达。 结论IRN可诱导SHSY5Y细胞自噬的发生，其机制为mTOR非依赖性自噬途径，Beclin1的表达可以影响IRN诱导的自噬。

异钩藤碱；SH-SY5Y细胞；自噬；Beclin1

［Abstract］Objective To explore the relationship between the occurrence of autophagy of human glioma cell SH-SY5Y induced by isorhynchophylline（IRN）and mTOR signaling pathway.Methods SH-SY5Y cells were treated by IRN with different concentrations（6，12 and 24 μmol/L）for 24h，and Western Blot method was used to test the expression of marker protein of autophagy LC3-Ⅱ.Immumofluorescence method was used to observe the formation of autophagosome marked by fluorescent spots in microtubule-associated protein LC3.Lysosome stabilizer CQ，and autophagy inhibitor 3-MA were used to further identify the action pathway of IRN.Western Blot method was used to test the expression of mTOR-associated proteins and Beclin1.Results After SH-SY5Y cells were treated by IRN，the expression of LC3-Ⅱwas significantly up-regulated.The relative expression amount after the action of 0，6，12 and 24 μM IRN was（0.086± 0.02），（0.39±0.14），（0.96±0.09），and（1.93±0.14）respectively.The ratio of autophagy-associated proteins LC3-Ⅱ/LC3-Ⅰwas increased（P＜0.01），showing concentration dependence.Autophagosome marked by GFP-LC3 fluorescent spots were increased in SH-SY5Y cells，and was 2±1/cell，4±1/cell，8±1/cell and 14±2/cell respectively in the IRN treatment groups with different concentrations（0，6，12，24 μM）.IRN combined with CQ was able to promote the expression of LC3-II（0.64±0.052）.IRN was able to reduce the expression of a-Syn protein.The relative expression amount in 0，6，12 and 24 μM groups were（0.71±0.07），（0.69±0.05），（0.53±0.04），and（0.36±0.034）respectively，showing concentration dependence.After the combination with 3-MA，a-Syn expression was up-regulated（0.63±0.04），indicating that 3-MA was able to reverse the inhibiting effects of IRN on a-Syn.The effects of CQ on the expression of a-Syn were not sig-nificant compared with the control group.The study on signaling pathway indicated that the expression of mTOR and Becline1 was not affected，and with the action of Beclin1-specific siRNA，the expression of LC3-Ⅱinduced by IRN could be inhibited.Conclusion IRN is able to induce the occurrence of autophagy of SH-WY5Y cells，and the mechanism is independent autophagy pathway of mTOR.The expression of Beclin1 is able to affect the autophagy induced by IRN.

［Key words］Isorhynchophylline；SH-SY5Y cells；Autophagy；Beclin1

细胞自噬是常见的生物学现象，是对细胞内环境稳定的高度保守的生理过程，通过降解胞浆内细胞器，长寿命蛋白和有聚集倾向的蛋白，进而满足细胞对物质的需求，一定意义上也是细胞在饥饿或者环境压力下的一种自然生理过程。……