自噬对双去甲氧基姜黄素诱导肝癌HepG2细胞凋亡作用的影响

Effect of Autophagy on Bisdemethoxycurcumin-induced Apoptosis in Hepatocellular Carcinoma HepG2 Cells

目的：探讨自噬对双去甲氧基姜黄素诱导肝癌HepG2细胞凋亡作用的影响。方法：以24mg·L^-1双去甲氧基姜黄素或联合5mol·L^-1自噬抑制剂3-甲基腺嘌呤（3-methyladenine，3-MA）对HepG2细胞作用24，48h，以四甲基偶氮唑盐（methye thiazolye telrazlium，MTT）比色法检测细胞活力；以24mg·L。双去甲氧基姜黄素单用或联合3-MA对HepG2细胞作用48h，Hoechst33342染色倒置荧光显微镜观察细胞凋亡形态，磷脂结合蛋白V／碘化丙啶（AnnxinV／propidiumiodide，ei）双染检测细胞凋亡率，罗丹明123（Rhodamine123，Rhl23）染色检测线粒体膜电位，蛋白免疫印迹法（Westernblot）检测自噬相关蛋白Beclin-1，LC3，半胱氨酸蛋白酶．3（Caspase-3），活化型Caspase-3（cleavedCaspase-3），B淋巴细胞瘤-2（Bcl-2），Bcl-2相关x蛋白（Bax）的活性表达。结果：与空白组比较，双去甲氧基姜黄素可抑制HepG2细胞增殖，诱导细胞凋亡，降低线粒体膜电位，使pro—Caspase-3，Bcl．2，LC3-I蛋白表达降低（P〈0．01），升高cleavedCaspase-3，Bax，Beclin-1，LC3-Ⅱ蛋白的表达（P〈0．01）；3-MA可增强双去甲氧基姜黄素所诱导的HepG2细胞凋亡，提高凋亡率（P〈0．01），增加其降低线粒体膜电位作用（P〈0．01），明显下调pro-Caspase-3，Bcl-2蛋白的表达（P〈0．01），并上调Beclin-1，LC3，cleavedCaspase-3蛋白的表达（P〈0．01）。结论：双去甲氧基姜黄素可经线粒体机制诱导HepG2细胞凋亡，并诱导细胞自噬，自噬对凋亡有抑制作用。

Objective： To observe the effect of autophagy on bisdemethoxycurcumin （BDMC） -induced apoptosis in hepatocellular carcinoma HepG2 cells. Method： Cells were treated with 24 mg. L-1 BDMC or BDMC ＋ 5 mol-L1-3-methyladenine （3-MA） for 24, 48 h, and then the cell activity was detected by methye thiazolye telrazlium （MTT） assay. Cells were treated with 24 mg. L-1 of BDMC alone or combination with 5 mol ~ L-l 3-MA for 48 h, and then the morphologies were observed by Hoechst 33342 staining by using inverted fluorescence microscope. The apoptosis rate and the mitochondrial membrane potential （MMP） were assessed by Annexin V/propidium iodide （PI） staining and Rhodamine 123 （Rh123） staining respectively; the protein expression levels of Beclin-1, LC3, cleaved Caspase-3, B-cell lymphoma-2 （Bcl-2） , and Bcl-2 associated X protein （Bax） were evaluated by Western blot. Result： As compared with the blank control group, BDMC could inhibit the proliferation and induce apoptosis in HepG2 cells, decrease the MMP, decrease the protein expression levels of pro-Caspase-3, Bcl-2, LC3- I （P 〈 0.01 ） , and increase the protein expression levels of cleavedCaspase-3, Bax, Beclin-I , LC3-11 （P 〈 0.01） ; 3-MA could enhance the effect of BDMC-induced HepG2 cell apoptosis, increased the apoptotic rates （P 〈 O. 01）, decreased the MMP （P 〈 O. O1 ）, lowered the protein expression levels of pro-Caspase-3, Bcl-2 （P 〈 O. 01 ） and increased the protein expression levels of Beclin-1, LC3, and cleaved Caspase-3 （P 〈 0.01 ）. Conclusion： BDMC could induce apoptosis via a mitochondria pathway in HepG2 cells, and could induce cells autophagy which could inhibit the cell apoptosis.