姜黄素对小鼠肝癌细胞体外增殖和凋亡的影响

Effects of curcumin on in vitro proliferation and apoptosis of mouse hepatocellular carcinoma cells

目的探讨姜黄素对小鼠肝癌Hepa1-6细胞体外增殖和凋亡的影响。方法取处于对数生长期的Hepa1-6细胞,随机分为对照组、10μmol/L组、20μmol/L组和40μmol/L组,分别采用0、10、20、40μmol/L的姜黄素进行干预,通过MTT法检测细胞增殖,流式细胞术检测细胞凋亡。RT-qPCR法检测Hepa1-6细胞中凋亡相关分子Bcl-2和Caspase-3及Toll样受体2(TLR2)、IFN-α、IFN-β基因表达。ELISA法检测Hepa1-6细胞培养上清IFN-Ⅰ。对照组、40μmol/L组中Hepa1-6细胞分别与CD4+T细胞、CD8+T细胞和NK细胞共孵育,流式细胞术检测CD4+T细胞、CD8+T细胞和NK细胞CD69表达。结果10、20、40μmol/L组Hepa1-6细胞增殖抑制率呈剂量依赖性增加,抑制率依次为(36.45±2.11)%、(46.40±2.08)%、(82.16±3.32)%,与对照组比较差异均有统计学意义(P均<0.05);10、20和40μmol/L组Hepa1-6细胞凋亡比例分别为10.47%、26.66%和49.81%,与对照组比较差异均有统计学意义(P均<0.01)。RT-qPCR结果显示,10、20和40μmol/L组中抗凋亡基因Bcl-2转录水平分别是对照组的(0.77±0.06)倍、(0.44±0.06)倍和(0.25±0.08)倍,与对照组比较差异均有统计学意义(P均<0.01);同时,促凋亡基因Caspase-3表达增高,10、20和40μmol/L组中促凋亡基因Caspase-3转录水平分别是对照组的(1.73±0.31)倍、(2.52±0.45)倍和(3.25±0.20)倍,与对照组比较差异均有统计学意义(P均<0.01)。随姜黄素浓度的增加,TLR2转录水平逐渐升高,10、20和40μmol/L组TLR2转录水平分别是对照组的(3.56±0.59)倍、(4.93±0.04)倍和(6.60±0.47)倍,与对照组比较差异均有统计学意义(P均<0.05);10、20和40μmol/L组IFN-α转录水平升高,分别为对照组的(4.21±0.22)倍、(5.54±0.39)倍和(8.05±0.93)倍,与对照组比较差异均有统计学意义(P均<0.05);IFN-β转录水平分别为对照组的(3.08±0.51)倍、(5.31±0.55)倍和(8.35±0.59)倍,与对照组比较差异均有统计学意义(P均<0.01)。10、20和40μmol/L组细胞分泌IFN-α水平分别为(57.00±8.8)、(82.47±4.42)、(100.37±8.99)pg/mL,对照组为(36.26±6.12)pg/mL,10、20和40μmol/L组细胞分泌IFN-α水平高于对照组(P均<0.01);10、20和40μmol/L组细胞分泌IFN-β水平分别为(60.67±6.81)、(91.33±5.51)、(144.00±10.15)pg/mL,对照组为(44.67±1.15)pg/mL,10、20和40μmol/L组细胞分泌IFN-β水平高于对照组(P均<0.01)。对照组CD4+T细胞、CD8+T细胞和NK细胞表达CD69水平分别为(24.53±1.39)%、(23.40±2.32)%、(32.33±3.08)%,40μmol/L组CD4+T细胞、CD8+T细胞和NK细胞表达CD69水平分别为(39.90±3.51)%、(31.47±3.88)%、(50.83±4.74)%,对照组与40μmol/L组CD4+T细胞、CD8+T细胞和NK细胞CD69水平比较差异均有统计学意义(P均<0.05)。结论姜黄素通过调控TLR2分子表达,促进IFN-Ⅰ分泌,从而抑制肝癌细胞增殖,促进肝癌细胞凋亡,同时促进免疫细胞活化。

Objective To explore the effects of curcumin on the in vitro proliferation and apoptosis of mouse liver can-cer Hepa1-6 cells.Methods Hepa1-6 cells in the logarithmic growth phase were divided into the control group,10μmol/L group,20μmol/L group and 40μmol/L group,respectively,which were treated with 0,10,20,and 40μmol/L of cur-cumin.Cell proliferation was assessed by the MTT assay,and apoptosis level was detected by FACS.Real-time quantitative PCR(RT-qPCR)was employed to detect the levels of apoptosis-related molecules Bcl-2,Caspase-3,Toll-like receptor 2(TLR2),IFN-α,and IFN-βin Hepa1-6 cells.The secretion levels of IFN-αand IFN-βin the cell culture supernatant were determined using ELISA.Hepa1-6 cells were incubated with CD4+T cells,CD8+T cells and NK cells in the control group and 40μmol/L curcumin group,respectively.FACS was used to detect the expression levels of CD69 in CD4+T cells,CD8+T cells and NK cells.Results The proliferation inhibition rates of Hepa1-6 cells in the 10,20 and 40μmol/L groups increased in a dose-dependent manner,and the inhibition rates were(36.45±2.11)%,(46.40±2.08)%and(82.16±3.32)%,respectively.There was statistically significant difference in comparison with that of the control group(P<0.05).The apoptotic proportions of Hepa1-6 cells in the 10μmol/L,20μmol/L and 40μmol/L groups were 10.47%,26.66%and 49.81%,respectively.And the difference was statistically significant in comparison with that of the control group(P<0.01).RT-qPCR results showed that the transcription levels of anti-apoptotic gene Bcl-2 in the 10μmol/L,20μmol/L and 40μmol/L groups were(0.77±0.06)times,(0.44±0.06)times and(0.25±0.08)times that of the control group,respectively,with statistically significant difference(all P<0.01).At the same time,the expression of pro-apoptotic gene Caspase-3 significantly increased,and the transcription levels of pro-apoptotic gene Caspase-3 in the 10μmol/L,20μmol/L and 40μmol/L groups was(1.73±0.31)times,(2.52±0.45)times and(3.25±0.20)times that of the control group,respectively,with statistically significant difference(all P<0.01).With the increase of curcumin concentrations,TLR2 transcription levels increased gradually.TLR2 transcription levels in the 10μmol/L,20μmol/L and 40μmol/L groups were(3.56±0.59)times,(4.93±0.04)times and(6.60±0.47)times that of the control group,respectively,with statistically significant difference(all P<0.05).In addition,the transcription levels of IFN-αin the 10μmol/L,20μmol/L and 40μmol/L groups significantly increased,which were(4.21±0.22),(5.54±0.39)and(8.05±0.93)times that of the control group,respectively,and there were statistically significant differences among these groups(all P<0.05).The transcription levels of IFN-βwere(3.08±0.51),(5.31±0.55)and(8.35±0.59)times that of the control group,and the difference was statistically significant(all P<0.01).For IFN-α,compared with the control group[(36.26±6.12)pg/mL],the levels of IFN-αsecreted by cells in the 10μmol/L,20μmol/L and 40μmol/L groups were(57.00±8.8),(82.47±4.42),and(100.37±8.99)pg/mL,respectively;the levels of IFN-αsecreted by cells in the 10,20,and 40μmol/L groups were higher that that of the control group(all P<0.01).The levels of IFN-βsecreted by the cells in the 10,20,and 40μmol/L groups were(60.67±6.81),(91.33±5.51),and(144.00±10.15)pg/mL,respec-tively,while that in the control group was(44.67±1.15)pg/mL.The levels of IFN-βsecreted by the cells in the 10,20,and 40μmol/L groups were higher than that in the control group(all P<0.01).In the control group,CD69 expression lev-els of CD4+T cells,CD8+T cells and NK cells were(24.53±1.39)%,(23.40±2.32)%,(32.33±3.08)%,respectively.The CD69 expression levels of CD4+T cells,CD8+T cells and NK cells in the 40μmol/L group were(39.90±3.51)%,(31.47±3.88)%,(50.83±4.74)%,respectively.There were significant differences in CD69 levels of CD4+T cells,CD8+T cells and NK cells between control group and 40μmol/L group(all P<0.05).Conclusion Curcumin promotes the secretion of typeⅠinterferon by regulating the expression of TLR2,thus inhibits the proliferation of hepatocellular car-cinoma cells,induces the apoptosis of hepatocellular carcinoma cells,as well as promotes the activation of immune cells.