[關(guān)鍵詞]
[摘要]
目的 探究白屈菜紅堿(CHE)對(duì)腺樣囊性癌細(xì)胞(ACC2)生長(zhǎng)的抑制作用及機(jī)制。方法 利用CCK8法、EdU法、Hoechst33342/PI雙染色法、試劑盒法檢測(cè)CHE對(duì)ACC2細(xì)胞活力、細(xì)胞增殖、細(xì)胞凋亡和活性氧(ROS)水平的影響;通過(guò)Western blotting技術(shù)檢測(cè)CHE對(duì)Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表達(dá)的影響;利用斑馬魚(yú)移植瘤模型檢測(cè)CHE對(duì)斑馬魚(yú)體內(nèi)ACC2細(xì)胞生長(zhǎng)的抑制作用。結(jié)果 CCK-8結(jié)果顯示:與對(duì)照組比較,2、3、4、5、6、7、8、9、10 μmol/L的CHE顯著降低ACC2細(xì)胞的存活率(P<0.05、0.01),且呈濃度相關(guān)性; ROS檢測(cè)結(jié)果顯示:與對(duì)照組比較,5、8 μmol/L的CHE導(dǎo)致ACC2細(xì)胞內(nèi)的ROS水平顯著上升(P<0.05、0.01); EdU增殖檢測(cè)結(jié)果表明:與對(duì)照組比較,5、8 μmol/L的CHE致使ACC2細(xì)胞的增殖能力顯著下降(P<0.01);Hoechst/PI染色結(jié)果顯示:與對(duì)照組比較,CHE 5、8 μmol/L組ACC2細(xì)胞凋亡率顯著上升(P<0.01)。抗氧化劑N-乙酰半胱氨酸(NAC)顯著抑制CHE誘導(dǎo)的ROS水平升高、細(xì)胞凋亡增加(P<0.01);Western blotting結(jié)果顯示:2、5、8 μmol/L的CHE能夠顯著上調(diào)Cleaved-Caspase 3、PARP、NF-κB蛋白的表達(dá)(P<0.01),且呈現(xiàn)濃度相關(guān)性,5、8 μmol/L的CHE能夠顯著上調(diào)p-JNK的蛋白表達(dá)(P<0.01),8 μmol/L的CHE能夠顯著上調(diào)p-p38的蛋白表達(dá)(P<0.01);NAC顯著降低由CHE導(dǎo)致的Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表達(dá)增加(P<0.01),5、8 μmol/L CHE能夠有效抑制斑馬魚(yú)體內(nèi)腫瘤的生長(zhǎng)(P<0.01)。結(jié)論 體外及斑馬魚(yú)移植瘤模型證明,CHE可以有效抑制ACC2細(xì)胞生長(zhǎng),其機(jī)制與提高細(xì)胞ROS水平,上調(diào)NF-κB、p-JNK、p-p38表達(dá),從而抑制細(xì)胞增殖、誘導(dǎo)細(xì)胞凋亡相關(guān)。
[Key word]
[Abstract]
Objective To investigate the inhibitory effect of chelerythrine (CHE) on adenoid cystic carcinoma (ACC2) cells and its molecular mechanism. Methods CCK8, EdU, hoechst33342/PI double staining, and test kit method were used to detect the effects of CHE on ACC2 cell viability, cell proliferation, apoptosis and reactive oxygen species (ROS) level. The effects of CHE on the expression levels of Caspase-3, PARP, NF-κB, P-JNK and p-p38 were detected by Western blotting. The inhibitory effect of chelerythrine on the growth of ACC2 cells was detected by zebrafish xenograft tumor model. Results CCK-8 assay showed that: CHE can significantly inhibited the proliferation of ACC2 cells in a concentration dependent manner (P < 0.05 and 0.01) at the concentration of 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol/L. The results showed that ROS in ACC2 cells was significantly increased at the concentration of 5 and 8 μmol/L (P < 0.05 and 0.01). EdU assay results showed that the proliferation of ACC2 cells was significantly decreased by CHE at the concentration of 5 and 8 μmol/L (P < 0.01). Hoechst/PI staining showed that apoptosis was significantly induced by CHE at the concentration of 5 and 8 μmol/L (P < 0.01). while N-acetylcysteine (NAC) significantly inhibited the increase of ROS and apoptosis induced by CHE (P < 0.01). Western blotting results showed that the expression of Cleaved Caspase 3, PARP and NF-κB were significantly up-regulated by 2, 5, and 8 mol/L CHE (P < 0.01), the expression of p-JNK was significantly up-regulated by 5 and 8 mol/L CHE (P < 0.01), and the protein expression of p-p38 was significantly up-regulated by 8 mol/L CHE (P < 0.01). NAC significantly decreased the expression of Cleaved Caspase 3, PARP, NF-κB, p-JNK, p-p38 protein induced by CHE (P < 0.01). CHE of 5 and 8 mol/L could effectively inhibit the growth of ACC2 cells in zebrafish (P < 0.01). Conclusion CHE can effectively inhibit the proliferation of ACC2 cells in vivo and zebrafish xenograft tumor model. Elevation of the ROS level induced by CHE, upregulation of NF-κB, p-JNK and p-p38 and then lead to the inhibition of proliferation and induction of apoptosis were involved in the inhibition of ACC2 cells in vivo and in vivo.
[中圖分類(lèi)號(hào)]
[基金項(xiàng)目]
國(guó)家重點(diǎn)研發(fā)計(jì)劃(2018YFC1707300);山東省重大科技創(chuàng)新工程項(xiàng)目(2019JZZY020905);齊魯工業(yè)大學(xué)(山東省科學(xué)院)科教產(chǎn)融合創(chuàng)新試點(diǎn)工程項(xiàng)目(2020KJC-ZD08)