蛇床子素通过PI3K-AKT信号通路诱导HaCaT细胞凋亡的研究
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摘要
目的观察蛇床子素对人永生化角质形成细胞株(HaCaT细胞)增殖和凋亡的影响并探讨其相关机制。方法采用CCK-8法检测HaCaT细胞增殖情况;Hoechst33258荧光染色法观察细胞凋亡形态;流式细胞仪检测HaCaT细胞周期变化及细胞凋亡水平;采用Western blotting检测蛇床子素对Ha CaT细胞磷酸肌醇3激酶(PI3K),人磷酸化磷酸肌醇3激酶(p-PI3K),蛋白激酶B(AKT),磷酸化蛋白激酶B(p-AKT)蛋白以及通路下游凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2),Bcl相关X蛋白(Bax),Cleaved-caspase3表达的影响。结果在所选质量浓度范围内(20、40、60μg/mL),蛇床子素对HaCaT细胞的增殖有明显抑制作用且能显著促进HaCaT细胞的凋亡;其中,20、40、60μg/mL质量浓度组蛇床子素可明显抑制PI3K和AKT的磷酸化,但对PI3K和AKT的表达无明显影响;蛇床子素能促进促凋亡蛋白Bax,Cleaved-caspase3的表达,抑制抗凋亡蛋白Bcl-2的表达。结论蛇床子素能抑制HaCaT细胞增殖,促进细胞凋亡,可能与其能抑制PI3K和AKT的磷酸化,阻断PI3K-AKT通路,激活下游相关促凋亡蛋白有关。
Objective: To investigate the effect of osthole on the proliferation and apoptosis of HaCaT cells and its related mechanisms. Methods: CCK-8 method was used to detect the proliferation of HaCaT cells and Hoechst 33258 fluorescence staining was used to observe the apoptotic morphology. The cell cycle and apoptotic level of HaCaT cells were detected by flow cytometry. Western blotting was used to detect the expression of PI3 K,p-PI3 K,AKT,p-AKT,the expression of Bcl-2,Bax and Cleaved-caspase3. Results: Within the selected concentration(60 μg/mL,40 μg/mL,20 μg/mL),osthole inhibited the proliferation of HaCaT cells and promoted cell apoptosis. Among them,osthole at 20,40,60 μg/mL concentration significantly inhibited the phosphorylation of PI3 K and AKT,but had no significant effect on the expression of PI3 K and AKT. Osthole could promote the expression of Bax and Cleaved-caspase3,and inhibited the expression of Bcl-2. Conclusion: Osthole can inhibit the proliferation of HaCaT cells and promote cell apoptosis,which may be related to its inhibition of phosphorylation of PI3 K and AKT,blockade of PI3 K-AKT pathway and activation of downstream apoptotic proteins.
Objective: To investigate the effect of osthole on the proliferation and apoptosis of HaCaT cells and its related mechanisms. Methods: CCK-8 method was used to detect the proliferation of HaCaT cells and Hoechst 33258 fluorescence staining was used to observe the apoptotic morphology. The cell cycle and apoptotic level of HaCaT cells were detected by flow cytometry. Western blotting was used to detect the expression of PI3 K,p-PI3 K,AKT,p-AKT,the expression of Bcl-2,Bax and Cleaved-caspase3. Results: Within the selected concentration(60 μg/mL,40 μg/mL,20 μg/mL),osthole inhibited the proliferation of HaCaT cells and promoted cell apoptosis. Among them,osthole at 20,40,60 μg/mL concentration significantly inhibited the phosphorylation of PI3 K and AKT,but had no significant effect on the expression of PI3 K and AKT. Osthole could promote the expression of Bax and Cleaved-caspase3,and inhibited the expression of Bcl-2. Conclusion: Osthole can inhibit the proliferation of HaCaT cells and promote cell apoptosis,which may be related to its inhibition of phosphorylation of PI3 K and AKT,blockade of PI3 K-AKT pathway and activation of downstream apoptotic proteins.
引文
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[15]黎萍,郑百红,陈鹏,等.硒对病毒性心肌炎小鼠心肌细胞凋亡的抑制作用及其作用机制[J].吉林大学学报:医学版,2016,42(1):54-58.
[16]张岩,宋惠珠,温浩,等.蛇床子素通过过氧化物酶体增殖物活化受体γ抑制乳腺癌MCF-7细胞增殖的研究[J].肿瘤研究与临床,2015,27(6):375-380.
[17]王可,苑振亭,王金海,等.蛇床子素抑制HaCaT细胞增殖及诱导凋亡的影响[J].中国现代医药杂志,2009,11(2):53-55.
[2]郝勇,石继海,韩慧,等.他克莫司软膏对银屑病皮损角质形成细胞增殖分化的影响[J].中国皮肤性病学杂志,2012, 26(9):773-775.
[3]韦柳婷,冯洁,莫书荣. PI3K-Akt信号通路与肿瘤相关性的研究进展[J].肿瘤学杂志,2014,20(4):331-336.
[4]黄秀兰,崔国辉,周克元. PI3K-Akt信号通路与肿瘤细胞凋亡关系的研究进展[J].癌症, 2008,27(3):331-336.
[5]张淼,苗朝阳,张晓艳. PI3K/AKT信号通路与寻常型银屑病表皮增殖失调的相关性研究[J].医学研究杂志,2017,46(7):171-173.
[6]张晓平,陈新梅,崔清华,等.蛇床子素药理作用及其相关机制研究进展[J].中国合理用药探索,2013,10(11):28-32.
[7]夏曙,于世英. PI3K/Akt信号转导通路在恶性肿瘤中的研究进展[J].肿瘤,2006,26(6):576-578.
[8]范文斌,包倪荣,赵建宁. PI3K/Akt信号通路在骨代谢中的作用[J].中国矫形外科杂志,2013,21(19):1958-1962.
[9]王笑,王甄真,陈雁. PI3K/AKT信号通路在维持血糖平衡中的作用[J].生命科学,2013,25(2):133-139.
[10]周思远,陈大帅,刘婷,等. PI3K-AKT信号转导通路在针灸效应中的作用[J].中华中医药杂志,2014,29(2):524-527.
[11]陈银涛,于秉治,武迪迪. PI3K/Akt/mTOR信号通路及临床相关肿瘤抑制剂[J].中国生物化学与分子生物学报,2014,30(10):949-956.
[12]王昊,冉立伟,惠珂,等. Survivin和PI3K、AKT在寻常型银屑病皮损角质形成细胞中的表达及其相关性[J].南方医科大学学报,2017,37(11):1512-1516.
[13]黄莉宁,薛汝增,罗光浦,等. p-Akt、p-FoxO1在寻常型银屑病皮损中的表达[J].中国麻风皮肤病杂志,2014,30(12):737-739.
[14]张晓艳,周平,尤立平,等.银屑病皮损表皮内Akt的激酶活性增强[J].中华皮肤科杂志,2009,42(6):413-416.
[15]黎萍,郑百红,陈鹏,等.硒对病毒性心肌炎小鼠心肌细胞凋亡的抑制作用及其作用机制[J].吉林大学学报:医学版,2016,42(1):54-58.
[16]张岩,宋惠珠,温浩,等.蛇床子素通过过氧化物酶体增殖物活化受体γ抑制乳腺癌MCF-7细胞增殖的研究[J].肿瘤研究与临床,2015,27(6):375-380.
[17]王可,苑振亭,王金海,等.蛇床子素抑制HaCaT细胞增殖及诱导凋亡的影响[J].中国现代医药杂志,2009,11(2):53-55.