摘要
目的探讨花椒籽油对喉癌细胞自噬和凋亡的影响及其作用机制。方法分别以不同体积分数(v/v)花椒籽油(0,0.02%,0.04%,0.06%,0.08%和0.10%)干预人支气管上皮细胞BEAS-2B和人喉癌细胞Hep-2,细胞计数试剂盒-8法(CCK-8)检测细胞活力。取对数生长期Hep-2随机分为对照组、花椒籽油低、中、高剂量组和花椒籽油高剂量+胰岛素样生长因子1(IGF-1)(PI3K激动剂)组,流式细胞术(FCM)检测细胞凋亡和细胞周期进程;丹酰尸胺(MDC)法细胞自噬染色检测自噬小泡形成;蛋白质印迹(WB)法检测凋亡、自噬和磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/雷帕霉素机械靶蛋白(mTOR)通路相关蛋白表达。结果与对照组Hep-2(99.03%±0.82%)相比,花椒籽油(0.02%,0.04%,0.06%,0.08%和0.10%)(v/v)处理可降低细胞存活率(84.63%±0.73%,57.34%±0.84%,19.76%±0.62%,17.22%±0.72%,12.19%±0.81%),差异具有统计学意义(t=22.718~133.559,均P<0.001),而对BEAS-2B活力无抑制作用(t=0.283~1.980,均P>0.05)。与对照组相比,花椒籽油低、中和高剂量组Hep-2凋亡率、G1/G0期细胞占比、自噬小泡积分光密度值(IOD)、活化半胱胺酸蛋白酶-3(Cleaved-caspase-3)、Bcl-2相关X蛋白(Bax)、微管相关蛋白1A/1B-轻链3I/II(LC 3I/II)和苄氯素1(Beclin-1)表达均升高(t=4.270~58.425);G2/M期细胞占比、泛素结合蛋白p62和Bcl-2表达及p-PI3K/PI3K,p-AKT/AKT和p-mTOR/mTOR均降低(t=3.041~58.765),差异具有统计学意义(均P<0.05)。与花椒籽油高剂量组比较,花椒籽油高剂量+IGF-1组Hep-2凋亡率、G1/G0期细胞占比、Cleaved-caspase-3,Bax,LC3II/I和Beclin-1表达、自噬小泡IOD值均降低(t=4.931~39.507);Bcl-2,p62表达、G2/M期细胞占比、p-PI3K/PI3K,p-AKT/AKT和p-mTOR/mTOR比值均升高(t=3.402~14.207),差异具有统计学意义(均P<0.05)。结论花椒籽油可促进Hep-2细胞自噬和凋亡,其作用机制可能与抑制PI3K/AKT/mTOR通路激活有关。
Objective To investigate the effect and mechanism of zanthoxylum bungeanum seed oil on autophagy and apoptosis of laryngeal cancer cells.Methods Human bronchial epithelial cells BEAS-2B and human laryngeal cancer cells Hep-2 were treated with different volume fractions(v/v)of Zanthoxylum bungeanum seed oil(0,0.02%,0.04%,0.06%,0.08%,0.10%),respectively,cell viability was detected by cell counting kit-8 method(CCK-8).Hep-2 cells in logarithmic growth phase were randomly divided into control group,Zanthoxylum bungeanum seed oil low,medium and high dose groups and Zanthoxylum bungeanum seed oil high dose+insulin-like growth factor 1(IGF-1)(PI3K agonist)group,cell apoptosis and cell cycle progression were detected by flow cytometry(FCM)assay,the formation of autophagic vesicles was detected by monodansyl cadaverine(MDC)staining,the expression of apoptosis,autophagy and phosphatidylinositide 3-kinases(PI3K)/protein kinase B(AKT)/mechanistic target of rapamycin(mTOR)pathway-related proteins were detected by Western blotting(WB).Results Compared with the control group Hep-2(99.03%±0.82%),treatment with zanthoxylum bungeanum seed oil(0.02%,0.04%,0.06%,0.08%,0.10%)(v/v)could reduce cell survival rate(84.63%±0.73%,57.34%±0.84%,19.76%±0.62%,17.22%±0.72%,12.19%±0.81%),and the differences were statistically significant(t=22.718~133.559,all P<0.001),while it has no inhibitory effect on BEAS-2B activity(t=0.283~1.980,all P>0.05).Compared with the control group,the Hep2 apoptosis rate,G1/G0 phase cell proportion,autophagic vesicle integrated optical density(IOD)value,Cleaved-caspase-3,Bcl-2 associated X protein(Bax),microtubule-associated protein 1A/1B 1ight chain 3I/II(LC 3I/II)and Beclin-1 expression were all increased in the low,medium,and high-close groups of zantheoxylum bungeanum seed oil(t=4.270~58.425);the proportion of G2/M phase cells,ubiquitin-binding protein P62,Bcl-2 expression and p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR expression were all decreased(t=3.041~58.765),and the differences were statistically significant(all P<0.05).Compared with the high-dose zanthoxylum bungeanum seed oil group,the apoptosis rate of Hep-2,the proportion of G1/G0 phase cells,the expression of Cleaved-caspase-3,Bax,LC3II/I and Beclin-1,the IOD value of autophagic vesicles(t=4.931~39.507),the expression of Bcl-2 and ubiquitin-binding protein P62,the proportion of G2/M phase cells,the ratio of p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR in the high-dose zanthoxylum bungeanum seed oil+IGF-1 group were decreased(t=3.402~14.207),and the differences were statistically significant(all P<0.05).Conclusion Zanthoxylum bungeanum seed oil can promote autophagy and apoptosis of Hep-2 cells,and its mechanism may be related to the inhibition of PI3K/AKT/mTOR pathway activation.
作者
李琳
王文红
侯凯
刘兆芳
孙强
LI Lin;WANG Wenhong;HOU Kai;LIU Zhaofang;SUN Qiang(Department of Pharmacology,Heze Medical College,Shandong Heze 274000,China;Department of Otolaryngological,Heze Municipal Hospital,Shandong Heze 274000,China)
出处
《现代检验医学杂志》
2025年第4期91-96,共6页
Journal of Modern Laboratory Medicine
基金
山东省医药卫生科技发展计划项目(编号:20204010257)。
关键词
喉癌
花椒籽油
自噬
凋亡
磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素机械靶蛋白通路
laryngeal cancer
zanthoxylum bungeanum seed oil
autophagy
apoptosis
phosphatidylinositide 3-kinases(PI3K)/protein kinase B(AKT)/mechanistic target of rapamycin(mTOR)pathway
