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浙江大学学报（医学版）
浙江大学学报（医学版）  2021, Vol. 50 Issue (5): 591-600    DOI: 10.3724/zdxbyxb-2021-0162
专题报道     
α-细辛醚、β-细辛醚改善Aβ25-35诱导的PC12细胞损伤及机制
石坚宏1,李瑞芝2,杨元宵3,姬丽婷1,李昌煜1,*()
1.浙江中医药大学药学院，浙江 杭州 310053
2.海宁市中医院科教科，浙江 嘉兴 314400
3.杭州医学院药学院，浙江 杭州 310053
Protective effect of α-asarone and β-asarone on Aβ 25-35-induced inflammatory response in PC12 cells and its mechanism
SHI Jianhong1,LI Ruizhi2,YANG Yuanxiao3,JI Liting1,LI Changyu1,*()
1. College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 310053, China;
2. Department of Science and Education, Haining Traditional Chinese Medicine Hospital, Jiaxing 314400, Zhejiang Province, China;
3. School of Pharmacy, Hangzhou Medical College, Hangzhou 310053, China
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摘要：

目的：探究α-细辛醚、β-细辛醚对β淀粉样蛋白活性片段Aβ25-35诱导的PC12细胞损伤模型的保护作用及相关作用机制。方法：采用Aβ25-35诱导PC12细胞建立Aβ毒性损伤细胞模型。将PC12细胞分为空白对照组、模型对照组、α-细辛醚组（0.5、1.0、1.5?μg/mL）、β-细辛醚组（6.3、12.5、25.0?μg/mL）、血管活性肠肽（VIP）组，并设VIP拮抗剂对照。采用细胞计数试剂盒（CCK-8）法检测细胞存活率；流式细胞术检测细胞凋亡率；酶联免疫吸附试验检测炎症因子白介素（IL）-1、IL-10、肿瘤坏死因子（TNF）-α，氧化因子诱生型一氧化氮合酶（iNOS）、一氧化氮（NO）和凋亡因子caspase-3、p53水平；蛋白质印迹法检测细胞c-Jun氨基端激酶（JNK）、p38丝裂原活化蛋白激酶（p38MAPK）蛋白表达。结果：与模型对照组比较，α-细辛醚组、β-细辛醚组和VIP组细胞存活率增加，细胞凋亡率下降，凋亡因子caspase-3、p53和炎症因子IL-1、TNF-α水平下降，IL-10水平升高，氧化因子iNOS和NO水平下降，c-Jun氨基端激酶（JNK）、p38MAPK蛋白表达减少（均P<0.05）。VIP拮抗剂干预后，β-细辛醚组细胞存活率下降，细胞凋亡率增加，凋亡因子caspase-3、p53和炎症因子IL-1、TNF-α水平升高，IL-10水平下降，氧化因子iNOS和NO水平升高，JNK、p38MAPK蛋白表达增加（均P<0.05）；α-细辛醚组无显著变化（均P>0.05）。结论：α-细辛醚、β-细辛醚对Aβ25-35诱导的PC12细胞损伤模型具有保护作用，β-细辛醚可通过促进VIP的分泌，调控JNK/MAPK通路从而抑制炎症因子、氧化因子水平，改善PC12细胞凋亡；α-细辛醚作用机制与VIP分泌水平无明显联系。
关键词： 阿尔茨海默病;  α-细辛醚;  β-细辛醚;  PC12细胞;  炎症;  氧化因子;  JNK/MAPK通路;  血管活性肠肽    
Abstract:

Objective: To investigate effects of α-asarone and β-asarone on Aβ25-35-induced PC12 cell injury and related mechanisms. Methods:Aβ toxic injury cell model was induced by Aβ25-35 in PC12 cells. PC12 cells were divided into blank control group, model control group, α-asarone group (0.5, 1.0, 1.5?μg/mL), β-asarone group (6.3, 12.5, 25.0?μg/mL), vasoactive intestinal peptide (VIP) group, and VIP antagonist control group. Cell survival rate was detected by CCK-8 kit; cell apoptosis rate was detected by flow cytometry. The levels of inflammatory cytokines interleukin (IL)-1, IL-10, tumor necrosis factor (TNF)-α, oxidation-related inducible nitric oxide synthase (iNOS), nitric oxide (NO), apoptosis factors caspase-3 and p53 were detected by ELISA method. The expressions of C-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) were detected by Western blotting. Results:Compared with model control group, cell survival rates of α-asarone group, β-asarone group and VIP group increased; the cell apoptosis rate decreased; levels of apoptosis-related factors caspase-3, p53, inflammatory factors IL-1, TNF-α decreased; IL-10 level increased; levels of oxidization-related factors iNOS and NO decreased; the expression of JNK and p38MAPK protein decreased (all P<0.05). After VIP antagonist intervention, the survival rate of β-asarone group decreased; apoptosis rate increased; apoptosis related factors caspase-3, p53, inflammatory factors IL-1, TNF-α increased; IL-10 decreased; oxidation related factors iNOS and NO increased; the expression of JNK and p38MAPK protein increased (allP<0.05); while there were no significant changes in these indicators of α-asarone group (allP>0.05).Conclusion: α-asarone and β-asarone have protective effects on PC12 cell injury induced by Aβ25-35. β-asarone may inhibit inflammatory factors and oxidation-related factors through promoting VIP secretion, regulating JNK/MAPK pathway, and reducing PC12 cell apoptosis; however, the effect of α-asarone may be not related to VIP secretion.
Key words: Alzheimer’s disease    α-Asarone    β-Asarone    PC12 cell    Inflammation    Oxidation factor    JNK/MAPK pathway    Vasoactive intestinal peptide
收稿日期: 2021-06-11 出版日期: 2021-12-29
:  R285.5  
基金资助: 浙江省自然科学基金（Q21H280028）
通讯作者: 李昌煜     E-mail: lcyzcmu@sina.com
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石坚宏
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引用本文:

石坚宏,李瑞芝,杨元宵,姬丽婷,李昌煜. α-细辛醚、β-细辛醚改善Aβ25-35诱导的PC12细胞损伤及机制[J]. 浙江大学学报（医学版）, 2021, 50(5): 591-600.

SHI Jianhong,LI Ruizhi,YANG Yuanxiao,JI Liting,LI Changyu. Protective effect of α-asarone and β-asarone on Aβ 25-35-induced inflammatory response in PC12 cells and its mechanism. J Zhejiang Univ (Med Sci), 2021, 50(5): 591-600.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0162    或    https://www.zjujournals.com/med/CN/Y2021/V50/I5/591

图 1  各组细胞存活率比较（=3）n与空白对照组比较，<0.01；与模型对照组比较，<0.05，<0.01；与无VIP拮抗剂比较，<0.05，<0.01. VIP：血管活性肠肽.
图 2  流式细胞术检测各组细胞凋亡结果（=3）nVIP：血管活性肠肽.
图 3  各组细胞凋亡率和凋亡因子表达水平比较（=3）nA：各组细胞凋亡率比较；B：各组caspase-3水平比较；C：各组p53水平比较. 与空白对照组比较，<0.05，<0.01；与模型对照组比较，<0.05，<0.01；与无VIP拮抗剂比较，<0.05，<0.01. VIP：血管活性肠肽；caspase：胱天蛋白酶.
图 4  各组细胞炎症因子比较（=3）nA：各组IL-1水平比较；B：各组IL-10水平比较；C：各组TNF-α水平比较. 与空白对照组比较，<0.05，<0.01；与模型对照组比较，<0.05，<0.01；与无VIP拮抗剂比较，<0.05，<0.01.VIP：血管活性肠肽；IL：白介素；TNF：肿瘤坏死因子.
图 5  各组细胞氧化因子比较（=3）nA：各组iNOS水平比较；B：各组NO水平比较. 与空白对照组比较，<0.01；与模型对照组比较，<0.05，<0.01；与无VIP拮抗剂比较，<0.05，<0.01.VIP：血管活性肠肽；iNOS：诱生型一氧化氮合酶；NO：一氧化氮.
图 6  各组JNK、p38MAPK蛋白表达电泳图A：α-细辛醚组JNK、p38蛋白条带图；B：β-细辛醚组JNK、p38蛋白条带图.VIP：血管活性肠肽；JNK：c-Jun氨基端激酶；p38MAPK：p38丝裂原活化蛋白激酶.
图 7  各组JNK、p38MAPK蛋白表达水平比较（=3）nA：α-细辛醚组与其他各组JNK蛋白表达水平比较；B：α-细辛醚组与其他各组p38蛋白表达水平比较；C：β-细辛醚组与其他各组JNK蛋白表达水平比较；D：β-细辛醚组与其他各组p38蛋白表达水平比较. 与空白对照组比较，<0.01；与模型对照组比较，<0.01；与无VIP拮抗剂比较，<0.01.VIP：血管活性肠肽；JNK：c-Jun氨基端激酶；p38MAPK：p38丝裂原活化蛋白激酶.
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