龙胆苦苷对人胰腺癌细胞PANC-1凋亡及IL-6/JAK2/STAT3信号通路的影响

Effects of Gentiopicroside on the Apoptosis of Human Pancreatic Cancer Cells PANC-1 and IL-6/JAK2/STAT3 Signaling Pathway

目的:研究龙胆苦苷对人胰腺癌细胞PANC-1凋亡的影响,并从白细胞介素6(IL-6)/Janus激酶2(JAK2)/信号转导与转录激活因子3(STAT3)信号通路角度研究其作用机制。方法:以PANC-1细胞为研究模型,采用MTT法测定0(阴性对照)、2、4、8、16、32、64、128 mg/L龙胆苦苷作用于细胞72 h后的增殖抑制率,并计算其半数抑制浓度(IC50)。分别将细胞分为阴性对照组、吉西他滨组(阳性对照,4 mg/L)和龙胆苦苷低、中、高浓度组(15、30、60 mg/L)。分别于培养1、3、5、7 d后,采用台盼蓝拒染法进行活细胞计数,考察各组细胞的生长情况;培养72 h后,采用克隆形成试验考察细胞的克隆形成率,采用Hoechst 33258染色法检测细胞凋亡率,采用逆转录-聚合酶链式反应法和Western blotting法分别测定细胞中IL-6、JAK2、STAT3 mRNA及其蛋白表达水平。结果:4~28 mg/L龙胆苦苷均可显著抑制细胞的增殖(P<0.05或P<0.01),并具有一定的浓度依赖性趋势,IC50为9.54 mg/L。与阴性对照组比较,吉西他滨组和龙胆苦苷中、高浓度组活细胞计数(作用3、5、7 d)和细胞中IL-6、JAK2、STAT3 mRNA及其蛋白表达水平均显著降低(P<0.05或P<0.01),细胞凋亡率均显著升高(P<0.01);吉西他滨组和龙胆苦苷高浓度组细胞的克隆形成率均显著降低(P<0.01)。与吉西他滨组比较,龙胆苦苷高浓度组细胞中上述指标水平差异均无统计学意义(P>0.05)。结论:30、60 mg/L龙胆苦苷可显著抑制PANC-1细胞的增殖、诱导其凋亡,60 mg/L龙胆苦苷的作用与吉西他滨相当;其作用机制可能与抑制细胞中IL-6/JAK2/STAT3信号通路的激活有关。

OBJECTIVE:To study the effects of gentiopicroside on the apoptosis of human pancreatic cancer cells PANC-1,and to explore its mechanism from the perspective of IL-6/JAK2/STAT3 signaling pathway. METHODS:Using PANC-1 cells as model,the proliferation inhibition rate of cells was tested by MTT assay after treated with 0(negative contro),2,4,8,16,32,64,128 mg/L gentiopicroside for 72 h and IC50 were calculated. The cells were divided into negative control group,gemcitabine group(positive control,4 mg/L)and gentiopicroside low-concentration,medium-concentration and high-concentration groups(15,30,60 mg/L).After cultured for 1,3,5,7 d,Trypan blue exclusion staining was used to count the survival cell,and the growth of cells was investigated. After cultured for 72 h,colony formation assay was used to observe colony formation rate of cells;the apoptotic rate of cells was detected by Hoechst 33258 staining;the mRNA and protein expressions of IL-6,JAK2,STAT3 in cells were detected by RT-PCR and Western blotting assay. RESULTS:4-28 mg/L gentiopicroside could inhibit the proliferation of cells(P<0.05 or P<0.01),in concentration dependent trend;IC50 was 9.54 mg/L. Compared with negative control group,survival cell count(cultured from 3,5,7 d),mRNA and protein expressions of IL-6,JAK2 and STAT3 in cells were decreased significantly in gemcitabine group,gentiopicroside medium-concentration and high-concentration groups(P<0.05 or P<0.01),while the apoptotic rate was increased significantly(P<0.01). The colony formation rate of cells were decreased significantly in gemcitabine group and gentiopicroside high-concentration group(P<0.01).Compared with gemcitabine group,there was no statistical significance in above indexes of gentiopicroside highconcentration group(P>0.05). CONCLUSIONS:30,60 mg/L gentiopicroside could inhibit the proliferation and induce apoptosis of PANC-1 cells,and 60 mg/L gentiopicroside is similar to gemcitabine in the effects. Its mechanism may be related to inhibiting the activation of IL-6/JAK2/STAT3 signaling pathway.