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

MIAO Jiu-wang; JING Xue-ning; GAO Ying; JIANG Xu-guang; FANG Feng-li; ZHANG Qin-de

doi:10.13422/j.cnki.syfjx.20181525

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

更新时间：2024-01-04

- Effect of Autophagy on Bisdemethoxycurcumin-induced Apoptosis in Hepatocellular Carcinoma HepG2 Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 24, Issue 17, Pages: 167-171(2018)
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- DOI：10.13422/j.cnki.syfjx.20181525
  CLC： R285
- Published：2018
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MIAO Jiu-wang, JING Xue-ning, GAO Ying, et al. Effect of Autophagy on Bisdemethoxycurcumin-induced Apoptosis in Hepatocellular Carcinoma HepG2 Cells[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(17): 167-171.
DOI：

MIAO Jiu-wang, JING Xue-ning, GAO Ying, et al. Effect of Autophagy on Bisdemethoxycurcumin-induced Apoptosis in Hepatocellular Carcinoma HepG2 Cells[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(17): 167-171. DOI： 10.13422/j.cnki.syfjx.20181525.

摘要

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24 mg·L-1双去甲氧基姜黄素或联合5 mol·L-1自噬抑制剂3-甲基腺嘌呤（3-methyladenine，3-MA）对HepG2细胞作用24，48 h，以四甲基偶氮唑盐（methye thiazolye telrazlium，MTT）比色法检测细胞活力；以24 mg·L-1双去甲氧基姜黄素单用或联合3-MA对HepG2细胞作用48 h，Hoechst 33342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（Annxin V/propidium iodide，PI）双染检测细胞凋亡率，罗丹明123（Rhodamine 123，Rh123）染色检测线粒体膜电位，蛋白免疫印迹法（Western blot）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（cleaved Caspase-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cleaved Caspase-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，cleaved Caspase-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。

Abstract

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·L-1 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-1 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 cleaved Caspase-3

Bax

Beclin-1

LC3-Ⅱ(P<0.01); 3-MA could enhance the effect of BDMC-induced HepG2 cell apoptosis

increased the apoptotic rates (P<0.01)

decreased the MMP (P<0.01)

lowered the protein expression levels of pro-Caspase-3

Bcl-2 (P<0.01) 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.

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