当归挥发油对PC12细胞缺血缺氧损伤后自噬和相关信号通路的影响
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摘要
目的研究当归挥发油对PC12细胞缺血缺氧损伤后自噬和相关信号通路的影响。方法将高分化PC12细胞分为5组:空白组、模型组和低、中、高3个剂量实验组。除空白组外,其余各组用含10 mmol·L~(-1)连二亚硫酸钠(Na2S2O4)的无糖培养基缺氧缺糖损伤1 h;同时,低、中、高3个剂量实验组加入终浓度分别为6. 25,12. 50,25. 00μg·m L~(-1)当归挥发油,复氧48 h。用噻唑蓝比色法检测当归挥发油对细胞增殖的影响,以免疫蛋白印迹法检测自噬相关蛋白[微管相关蛋白1轻链3B(LC3B-Ⅱ)、泛素结合蛋白P62(P62)]和自噬相关信号通路[磷酸化蛋白激酶B(p-Akt),磷酸化动物雷帕霉素靶蛋白(p-m TOR),磷酸化P70核糖体蛋白S6激酶(p-P70S6K)]蛋白的表达。结果空白组、模型组和低、中、高3个剂量实验组的细胞存活率分别为(100. 00±0. 00)%,(56. 37±4. 93)%,(69. 78±1. 56)%,(78. 35±2. 01)%和(89. 77±3. 03)%;模型组与空白组相比或者低、中、高剂量3个实验组与模型组相比,差异均有统计学意义(均P <0. 05)。空白组、模型组和高剂量实验组的LC3B-Ⅱ相对表达量分别为0. 30±0. 04,0. 56±0. 05和0. 42±0. 03;这3组的P62的相对表达量分别为0. 63±0. 03,0. 17±0. 01和0. 40±0. 01;模型组与空白组相比或者高剂量实验组与模型组相比,差异均有统计学意义(P <0. 01或P <0. 05,P <0. 01)。同时,p-Akt、p-m TOR与p-P70S6K的表达趋势与LC3B-Ⅱ的结果一致。结论当归挥发油可能通过激活Akt/m TOR信号转导通路抑制缺血缺氧损伤引起的PC12细胞自噬,保护受损细胞。
Objective To investigate the effect of Volatile oil from Angelica sinensis on autophagy and related signal pathway in PC12 cells after ischemia and hypoxia injury. Methods The high differentiated PC12 cells were divided into 5 groups: blank group,model group,and low-,medium-and high-doses experimental groups. Except blank group,the remaining groups were treated with sugar-free medium containing 10 mmol·L~(-1) sodium dithionite( Na_2S_2O_4) for 1 h; the three-dose experimental groups were given Volatile oils 6. 25,12. 5,25 μg·m L~(-1) reoxygenation for 48 h. The cell proliferation was measured by methyl thiazolyl tetrazolium( MTT) assay. The expression of ubiquitin-binding protein P62( P62),microtubule-associated protein 1 light chain 3 B( LC3-Ⅱ),phosphorylated protein kinase B( p-Akt),phosphorylated mammalian target protein of rapamycin( p-m TOR)and phosphorylated p70 ribosomal protein S6 kinase( p-P70 S6 K) proteins were measured by Western-blot. Results The cell viability in the blank group,model group and low-,medium-and high-doses experimental groups were( 100 ± 0. 00) %,( 56. 37 ± 4. 93) %,( 69. 78 ± 1. 56) %,( 78. 35 ± 2. 01) % and( 89. 77 ± 3. 03) %,respectively;comparison between model group and blank group or three doses experimental groups and model group,the difference were significantly( all P < 0. 05). The relative expression of LC3 B-Ⅱin the blank group,model group and high-dose experimental group were 0. 30 ± 0. 04,0. 56 ± 0. 05,0. 42 ± 0. 03; the relative expression of P62 in the 3 groups were0. 63 ± 0. 03,0. 17 ± 0. 01,0. 40 ± 0. 01; comparison between model group and blank group or high dose experimental group and model group,the difference of the factors were significantly( P < 0. 01 or P < 0. 05,P < 0. 01). Meanwhile,the expression trend of p-Akt,p-m TOR and p-P70 S6 K was consistent with that of LC3 B-II. Conclusion The Volatile oils from Angelica sinensis may inhibit autophagy induced by anoxia and ischemic in PC12 cells through activating the p-Akt/m TOR signal pathway.
Objective To investigate the effect of Volatile oil from Angelica sinensis on autophagy and related signal pathway in PC12 cells after ischemia and hypoxia injury. Methods The high differentiated PC12 cells were divided into 5 groups: blank group,model group,and low-,medium-and high-doses experimental groups. Except blank group,the remaining groups were treated with sugar-free medium containing 10 mmol·L~(-1) sodium dithionite( Na_2S_2O_4) for 1 h; the three-dose experimental groups were given Volatile oils 6. 25,12. 5,25 μg·m L~(-1) reoxygenation for 48 h. The cell proliferation was measured by methyl thiazolyl tetrazolium( MTT) assay. The expression of ubiquitin-binding protein P62( P62),microtubule-associated protein 1 light chain 3 B( LC3-Ⅱ),phosphorylated protein kinase B( p-Akt),phosphorylated mammalian target protein of rapamycin( p-m TOR)and phosphorylated p70 ribosomal protein S6 kinase( p-P70 S6 K) proteins were measured by Western-blot. Results The cell viability in the blank group,model group and low-,medium-and high-doses experimental groups were( 100 ± 0. 00) %,( 56. 37 ± 4. 93) %,( 69. 78 ± 1. 56) %,( 78. 35 ± 2. 01) % and( 89. 77 ± 3. 03) %,respectively;comparison between model group and blank group or three doses experimental groups and model group,the difference were significantly( all P < 0. 05). The relative expression of LC3 B-Ⅱin the blank group,model group and high-dose experimental group were 0. 30 ± 0. 04,0. 56 ± 0. 05,0. 42 ± 0. 03; the relative expression of P62 in the 3 groups were0. 63 ± 0. 03,0. 17 ± 0. 01,0. 40 ± 0. 01; comparison between model group and blank group or high dose experimental group and model group,the difference of the factors were significantly( P < 0. 01 or P < 0. 05,P < 0. 01). Meanwhile,the expression trend of p-Akt,p-m TOR and p-P70 S6 K was consistent with that of LC3 B-II. Conclusion The Volatile oils from Angelica sinensis may inhibit autophagy induced by anoxia and ischemic in PC12 cells through activating the p-Akt/m TOR signal pathway.
引文
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[2]MIZUSHIMA N,LEVINE B,CUERVO A M,et al.Autophagy fights disease through cellular self-digestion[J].Nature,2008,451(7182):1069-1075.
[3]BAEHRECKE E H.Autophagic programmed cell death in Drosophila[J].Cell Death Differentiat,2003,10(9):940-945.
[4]罗慧英,杨林,杨焕,等.当归挥发油对大鼠局灶性脑缺血再灌注损伤的保护作用[J].中国临床药理学与治疗学,2012,17(4):387-391.
[5]孙蓉,衣银萍,吕丽莉,等.麝香酮对连二亚硫酸钠造成PC12细胞缺氧损伤的保护作用[J].中药药理与临床,2008,24(1):15-17.
[6]CARMICHAEL J,GRAFF W G,GAZDAR A F,et al.Evaluation of a tetrazolium-based semiautomated,colorimetric assay:assessment of chemosensitivity testing[J].Cancer Res,1987,47(4):936-942.
[7]CHEN Y B,HOU J H,FENG X Y,et al.Decreased expression of Beclin 1 correlates with a metastatic phenotypic and adverse prognosis of gastric carcinomas[J].J Surg Oncol,2012,105(6):542-547.
[8]PANDURANGAN A K,ISMAIL S,ESA N M,et al.Inositol-6phosphate inhibits the m TOR pathway and induces autophagy-mediated death in HT-29 colon cancer cells[J].Archives Med Sci,2018,14(6):1281-128.
[9]BOTTI J,DJAVAHERI-MERGNY M,PILATTE Y,et al.Autophagy signaling and the cogwheels of cancer[J].Autophagy,2006,2(2):67-73.
[10]KLIONSKY D J,ABELIOVICH H,AGOSTINIS P,et al.Guidelines for the use and interpretation of assays for monitoring autophagy in highereukaryotes[J].Autophagy,2008,4(2):151-175.