绿原酸通过抑制PI3K-Akt信号通路诱导肺癌A549细胞线粒体功能障碍

Chlorogenic acid induces mitochondrial dysfunction in lung cancer A549 cells by inhibiting the PI3K-Akt pathway

目的探讨绿原酸能否通过作用于PI3K-Akt信号通路造成线粒体功能障碍,从而抑制肺癌A549细胞增殖、迁移、侵袭和促进其凋亡。方法采用梯度浓度(0、25、50、100、150、200μg/ml)的绿原酸干预A549细胞48 h,CCK-8实验检测细胞增殖率并计算半抑制浓度(IC50)。将A549细胞分成空白组、绿原酸组(IC50)和绿原酸+740YP组(IC50绿原酸+50μg/ml 740YP),干预48 h后使用细胞划痕实验检测细胞迁移距离,细胞侵袭实验检测细胞侵袭能力,流式细胞术检测细胞周期、细胞凋亡水平与线粒体膜电位变化情况,酶联免疫吸附试验检测细胞上清丙二醛(MDA)含量,蛋白质印迹法检测细胞中p-PI3K、p-Akt和Caspase3蛋白表达情况。结果绿原酸对A549细胞的IC50为57.45μg/ml。细胞划痕实验结果显示,空白组、绿原酸组和绿原酸+740YP组细胞48 h迁移距离分别为(424.80±14.43)、(289.67±18.93)和(402.22±17.99)μm,细胞侵袭实验结果显示,3组细胞48 h侵袭细胞数量分别为(96.00±6.24)、(35.33±7.64)和(83.00±2.00)个,流式细胞术结果显示,3组细胞48 h凋亡率分别为(6.15±0.17)%、(54.63±0.72)%和(17.27±0.39)%,差异均具有统计学意义(F=105.98,P<0.001;F=90.62,P<0.001;F=8321.99,P<0.001);与空白组相比,绿原酸组和绿原酸+740YP组细胞迁移距离和侵袭数量均减少(均P<0.05),细胞凋亡率显著升高(均P<0.001);与绿原酸组相比,绿原酸+740YP组细胞迁移距离增加(P<0.001),细胞侵袭数量增多(P<0.001),细胞凋亡率降低(P<0.001)。流式细胞术结果显示,空白组、绿原酸组和绿原酸+740YP组细胞G0/G1期比例分别为(65.75±0.58)%、(55.84±0.78)%和(55.24±1.37)%,G2/M期比例分别为(11.21±1.03)%、(20.23±0.62)%和(9.96±0.33)%,S期比例分别为(23.04±0.49)%、(23.92±1.36)%和(34.80±1.15)%,差异均具有统计学意义(F=111.02,P<0.001;F=181.26,P<0.001;F=113.05,P<0.001);与空白组相比,绿原酸组和绿原酸+740YP组细胞G0/G1期比例减少(均P<0.001),绿原酸组G2/M期比例增加(P<0.001),绿原酸+740YP组细胞S期比例增加(P<0.001);与绿原酸组相比,绿原酸+740YP组细胞G2/M期比例减少、S期比例增加(均P<0.001)。线粒体膜电位检测结果显示,空白组、绿原酸组和绿原酸+740YP组线粒体JC-1荧光强度分别为39.51±1.32、10.05±0.19和21.85±1.45,差异具有统计学意义(F=508.82,P<0.001);与空白组相比,绿原酸组和绿原酸+740YP组荧光强度均降低(均P<0.001);与绿原酸组相比,绿原酸+740YP组荧光强度升高(P<0.001)。酶联免疫吸附试验结果显示,空白组、绿原酸组和绿原酸+740YP组细胞MDA含量分别为(0.47±0.01)、(0.61±0.01)和(0.56±0.01)nmol/ml,差异具有统计学意义(F=162.30,P<0.001);与空白组相比,绿原酸组和绿原酸+740YP组细胞MDA含量均增加(均P<0.001);与绿原酸组相比,绿原酸+740YP组细胞MDA含量减少(P=0.001)。蛋白质印迹法结果显示,空白组、绿原酸组和绿原酸+740YP组细胞p-PI3K蛋白相对表达水平分别为1.01±0.33、0.28±0.14和0.34±0.20,p-Akt蛋白相对表达水平分别为1.00±0.16、0.43±0.05和0.95±0.14,Caspase3蛋白相对表达水平分别为1.00±0.04、1.41±0.05和0.70±0.13,差异均具有统计学意义(F=8.48,P=0.018;F=19.11,P=0.002;F=57.50,P<0.001);与空白组相比,绿原酸组细胞p-PI3K、p-Akt蛋白表达均降低、Caspase3蛋白表达升高(均P<0.05),绿原酸+740YP组p-PI3K、Caspase3蛋白表达均降低(均P<0.05);与绿原酸组相比,绿原酸+740YP组细胞p-Akt蛋白表达升高、Caspase3蛋白表达降低(均P<0.05)。结论绿原酸可能通过减少PI3K与Akt蛋白磷酸化抑制PI3K-Akt信号通路,造成线粒体功能受损,引起MDA累积,最终导致肺癌A549细胞功能受损与活性降低,促进细胞凋亡。

Objective To investigate whether chlorogenic acid can inhibit the proliferation,migration,invasion and promote apoptosis of lung cancer A549 cells by causing mitochondrial dysfunction through PI3K-Akt pathway.Methods A549 cells were treated with chlorogenic acid at concentrations of 0,25,50,100,150,and 200μg/ml for 48 h.CCK-8 assay was used to detect the cell proliferation rate and calculate the half maximal inhibitory concentration(IC50).A549 cells were divided into three groups:control group,chlorogenic acid group(IC50)and chlorogenic acid+740-YP group(IC50 chlorogenic acid+50μg/ml 740YP).After 48 h of intervention,the cell migration distance was detected by cell scratch assay.Cell invasion assay was used to detect cell invasion ability.Cell cycle,apoptosis and mitochondrial membrane potential were detected by flow cytometry.The content of malondialdehyde(MDA)in cell supernatant was detected by enzyme-linked immunosorbent assay(ELISA).Western blotting was used to detect the protein expression of p-PI3K,p-Akt and Caspase3.Results The IC50 of chlorogenic acid to A549 cells was 57.45μg/ml.The results of cell scratch assay showed that the 48 h migration distances of the control group,chlorogenic acid group and chlorogenic acid+740YP group were(424.80±14.43),(289.67±18.93)and(402.22±17.99)μm,respectively.The results of cell invasion assay showed that the numbers of invasive cells after 48 h were 96.00±6.24,35.33±7.64 and 83.00±2.00,and the results of flow cytometry showed that the 48 h apoptosis rates were(6.15±0.17)%,(54.63±0.72)%and(17.27±0.39)%,respectively,among the three groups with statistically significant differences(F=105.98,P<0.001;F=90.62,P<0.001;F=8321.99,P<0.001).Compared with the control group,the cell migration distances and invasive numbers of chlorogenic acid group and chlorogenic acid+740YP group were decreased(all P<0.05),while the apoptosis rates were significantly increased(both P<0.001).Compared with chlorogenic acid group,the cell migration distance of chlorogenic acid+740YP group increased(P<0.001),the number of cell invasion increased(P<0.001),and the apoptosis rate decreased(P<0.001).The results of flow cytometry showed that the proportions of cells in G0/G1 phase in the control group,chlorogenic acid group and chlorogenic acid+740YP group were(65.75±0.58)%,(55.84±0.78)%and(55.24±1.37)%,respectively.The proportions of G2/M phase were(11.21±1.03)%,(20.23±0.62)%and(9.96±0.33)%,and the proportions of S phase were(23.04±0.49)%,(23.92±1.36)%and(34.80±1.15)%,respectively,with statistically significant differences(F=111.02,P<0.001;F=181.26,P<0.001;F=113.05,P<0.001).Compared with the control group,the proportions of G0/G1 phase cells in chlorogenic acid group and chlorogenic acid+740YP group decreased(both P<0.001),and the proportion of G2/M phase in chlorogenic acid group increased(P<0.001),and the proportion of S phase cells in chlorogenic acid+740YP group increased(P<0.001).Compared with chlorogenic acid group,the proportion of G2/M phase cells decreased and the proportion of S phase cells increased in chlorogenic acid+740YP group(both P<0.001).The results of mitochondrial membrane potential detection showed that the JC-1 fluorescence intensity of mitochondria in the control group,chlorogenic acid group and chlorogenic acid+740YP group were 39.51±1.32,10.05±0.19 and 21.85±1.45,respectively,with a statistically significant difference(F=508.82,P<0.001).Compared with the control group,the fluorescence intensity of chlorogenic acid group and chlorogenic acid+740YP group decreased(both P<0.001).Compared with chlorogenic acid group,the fluorescence intensity of chlorogenic acid+740YP group increased(P<0.001).ELISA results showed that the MDA contents of the control group,chlorogenic acid group and chlorogenic acid+740YP group were(0.47±0.01),(0.61±0.01)and(0.56±0.01)nmol/ml,respectively,with a statistically significant difference(F=162.30,P<0.001).Compared with the control group,MDA contents in chlorogenic acid group and chlorogenic acid+740YP group increased(both P<0.001).Compared with chlorogenic acid group,MDA content in chlorogenic acid+740YP group decreased(P=0.001).Western blotting results showed that the relative protein expression levels of p-PI3K in the control group,chlorogenic acid group and chlorogenic acid+740YP group were 1.01±0.33,0.28±0.14 and 0.34±0.20,respectively.The relative protein expression levels of p-Akt were 1.00±0.16,0.43±0.05 and 0.95±0.14,and the relative protein expression levels of Caspase3 were 1.00±0.04,1.41±0.05 and 0.70±0.13,respectively,and there were statistically significant differences(F=8.48,P=0.018;F=19.11,P=0.002;F=57.50,P<0.001).Compared with the control group,the expressions of p-PI3K and p-Akt protein in chlorogenic acid group decreased,and the expression of Caspase3 protein increased(all P<0.05).The expressions of p-PI3K and Caspase3 protein in chlorogenic acid+740YP group decreased(both P<0.05).Compared with chlorogenic acid group,the expression of p-Akt protein in chlorogenic acid+740YP group increased,and the expression of Caspase3 protein decreased(both P<0.05).Conclusion Chlorogenic acid may inhibit the PI3K-Akt pathway by reducing the phosphorylation of PI3K and Akt proteins,resulting in the damage of mitochondrial function and the accumulation of MDA,which eventually leads to the damage of lung cancer A549 cells function and the reduction of cells activity,and then promotes cells apoptosis.