冬凌草素诱导非小细胞肺癌细胞凋亡的作用及机制研究

Effect and mechanism of oridonin inducing apoptosis in non-small cell lung cancer cells

研究冬凌草素对非小细胞肺癌(A549、H1299)细胞增殖、凋亡及自噬效用,并明确其潜在的作用机制。使用不同浓度冬凌草素分别干预A549、H1299细胞,采用CCK8(cell counting kit-8)检测细胞活性,筛选最佳的药效浓度;以10、50μg/mL冬凌草素及顺铂5μg/mL分别干预A549、H1299细胞24 h,流式细胞计数观察冬凌草素对A549、H1299细胞凋亡的影响,蛋白质印迹(Western blot,WB)检测半胱氨酸天冬氨酸蛋白酶3(Caspase3)、半胱氨酸天冬氨酸蛋白酶3剪切体(Cleaved-Caspase3)、B淋巴细胞瘤-2蛋白(B-cell lymphoma-2,Bcl-2)、半胱氨酸天冬氨酸蛋白酶9(Caspase9)、微管相关蛋白轻链3I(microtubule-associated protein light chain 3 isoform I,LC3I)、微管相关蛋白轻链3II(microtubule-associated protein light chain 3 isoform II,LC3II)、Bax、p62、Beclin-1表达;A549细胞过表达Beclin-1并用50μg/mL冬凌草素干预,WB检测Beclin-1相关蛋白。冬凌草素浓度依赖性抑制A549及H1299细胞增殖(P<0.05),IC 50值分别为38.2、46.6μg/mL;冬凌草素可增强A549、H1299细胞中Bax、Cleaved-Caspase3、Caspase3、Caspase9、p62蛋白表达(P<0.05);抑制A549、H1299细胞中LC3-I、LC3-II、Beclin-1蛋白表达(P<0.05);冬凌草素能减少过表达Beclin-1非小细胞肺癌细胞中Beclin-1、LC3-II、p62蛋白表达(P<0.05)。冬凌草素可抑制非小细胞肺癌细胞体外活性,可能与其抑制Beclin-1蛋白表达减少自噬而诱发细胞凋亡有关。

This study aims to identify the effect of oridonin on proliferation,apoptosis and autophagy of non-small cell lung cancer(A549,H1299)cells,and to clarify its potential mechanism of action.Different concentrations of oridonin were used to intervene in A549 and H1299 cells,and the cell viability assay cell counting kit-8(CCK8)was used to detect the cell activity and screen the optimal concentration of the drug;10 and 50μg/mL oridonin and 5μg/mL cisplatin were used to intervene in A549 and H1299 cells for 24 h,and the effects of oridonin on the proliferation,apoptosis and autophagy of A549 and H1299 cells were observed by flow cytometric counting.Flow cytometric counting was performed to observe the effect of oridonin on apoptosis of A549 and H1299 cells.Detecting the expression of cysteine aspartate protease 3(Caspase3),cleaved-cysteine aspartate protease 3(Cleaved-Caspase3),B-cell lymphoma-2,Bcl-2,cysteine aspartate protease 9(Caspase9),microtubule-associated protein light chain 3 isoform I\\II(LC3I\\II),Bax,p62,Beclin-1 protein;A549 cells overexpression of Beclin-1 and addition of 50μg/mL oridonin,WB detection of Beclin-1-related proteins.oridonin concentration-dependently inhibited the proliferation of A549 and H1299 cells(P<0.05),with IC 50 values of 38.2 and 46.6μg/mL,respectively;oridonin enhanced the expression of Bax,Cleaved-Caspase3,Caspase3,Caspase9,and p62 in A549 and H1299 cells(P<0.05)and inhibited LC3-I,LC3-II,and Beclin-1 protein expression in A549 and H1299 cells(P<0.05);and oridonin reduced Beclin-1,LC3-II,and p62 protein expression in overexpressing Beclin-1 non-small cell lung cancer cells(P<0.05).The mechanism by which oridonin could inhibit the activity of NSCLC cells in vitro may be related to the inhibition of Beclin-1 protein expression to reduce autophagy and induce apoptosis.