目的 研究丹参酮Ⅱ_A对人喉癌细胞顺铂耐药的影响作用及机制。方法 以不同质量浓度（0、1.0、2.0、4.0、8.0、16.0、32.0 mg/L）顺铂干预对数生长期人喉癌Hep-2细胞和人喉癌顺铂耐药细胞（Hep-2/DDP）48 h，CCK-8法检测细胞增殖抑制率，计算半数抑制浓度（IC₅₀）和耐药指数（RI）。以不同质量浓度（0、0.5、1.0、2.0、4.0、8.0、16.0 mg/L）丹参酮Ⅱ_A干预对数生长期Hep-2细胞48 h，CCK-8法检测细胞增殖抑制率，计算IC₅₀；采用丹参酮Ⅱ_A（IC₅₀5.32 mg/L）＋不同质量浓度（0、1.0、2.0、4.0、8.0、16.0、32.0 mg/L）顺铂干预对数生长期Hep-2/DDP细胞48 h，CCK-8法检测细胞增殖抑制率，计算2种药物联用的IC₅₀和逆转倍数（RF）。取对数生长期Hep-2/DDP细胞，设置对照组、顺铂组、丹参酮Ⅱ_A＋顺铂组、丹参酮Ⅱ_A＋顺铂＋胰岛素样生长因子-1（IGF-1）组。各组分别给药干预48 h后，采用CCK-8法、Annexin V-FITC/PI双染法检测细胞增殖抑制率和凋亡率，Western blotting法检测B细胞淋巴瘤-2（Bcl-2），Bcl-2相关X蛋白（Bax）、激活型半胱氨酸蛋白酶-3（cleaved Caspase-3）、cleaved Caspase-9、磷脂酰肌醇3激酶（PI3K）、p-PI3K、蛋白激酶B（Akt）蛋白表达情况。结果 顺铂对Hep-2细胞的IC₅₀为4.58 mg/L，对Hep-2/DDP细胞的IC₅₀为14.09 mg/L，RI为3.08；丹参酮Ⅱ_A对Hep-2细胞的IC₅₀为5.32 mg/L；丹参酮Ⅱ_A联合顺铂对Hep-2/DDP细胞的IC₅₀为3.34 mg/L，RF为4.22。与对照组比较，顺铂组Hep-2/DDP细胞增殖抑制率、凋亡率显著升高（P<0.01）；与顺铂组比较，丹参酮Ⅱ_A＋顺铂组Hep-2/DDP细胞增殖抑制率、凋亡率显著升高（P<0.05）；与丹参酮Ⅱ_A＋顺铂组比较，丹参酮Ⅱ_A＋顺铂＋IGF-1组Hep-2/DDP细胞增殖抑制率、凋亡率显著降低（P<0.05）。与对照组相比，顺铂组Hep-2/DDP细胞Bcl-2表达量显著降低，Bax、cleaved Caspase-3、cleaved Caspase-9表达量和Bax/Bcl-2值显著升高（P<0.05）。与顺铂组比较，丹参酮Ⅱ_A＋顺铂组Hep-2/DDP细胞Bcl-2表达量显著降低，Bax、cleaved Caspase-3、cleaved Caspase-9表达量和Bax/Bcl-2值显著升高（P<0.05）。与丹参酮Ⅱ_A＋顺铂组比较，丹参酮Ⅱ_A＋顺铂＋IGF-1组Hep-2/DDP细胞Bcl-2表达量显著升高，Bax、cleaved Caspase-3、cleaved Caspase-9表达量和Bax/Bcl-2值显著降低（P<0.05）。与对照组相比，顺铂组Hep-2/DDP细胞p-PI3K、p-Akt表达量及PI3K、Akt磷酸化（p-PI3K/PI3K、p-Akt/Akt）水平显著降低（P<0.05）。与顺铂组比较，丹参酮Ⅱ_A＋顺铂组Hep-2/DDP细胞p-PI3K、p-Akt表达量及PI3K、Akt磷酸化水平显著降低（P<0.05）。与丹参酮Ⅱ_A＋顺铂组比较，丹参酮Ⅱ_A＋顺铂＋IGF-1组Hep-2/DDP细胞p-PI3K、p-Akt表达量及PI3K、Akt磷酸化水平显著升高（P<0.05）。结论 丹参酮Ⅱ_A可逆转人喉癌细胞顺铂耐药，其作用机制可能与抑制PI3K/Akt通路活化而促进喉癌细胞凋亡有关。

Objective To study the effect and mechanism of tanshinone Ⅱ_A on cisplatin resistance in human laryngeal carcinoma cells. Methods Human laryngeal carcinoma Hep-2 cells and human laryngeal carcinoma cisplatin-resistant cells (Hep-2/DDP) were treated with different concentrations of cisplatin (0, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 mg/L) for 48 h. The inhibition rate of cell proliferation

was detected by CCK-8 method, and the semi-inhibitory concentration (IC₅₀) and drug resistance index (RI) were calculated. Hep-2 cells were treated with different concentrations of tanshinone Ⅱ_A (0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L) in logarithmic growth phase for 48 h. The cell proliferation inhibition rate was detected by CCK-8 method, and the IC₅₀ was calculated. Hep-2/DDP cells were treated with tanshinone Ⅱ_A IC₅₀ (5.32 mg/L) + cisplatin at different concentrations (0, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 mg/L) for 48 h, and the cell proliferation inhibition rate was detected by CCK-8 method. The IC₅₀ and reversion fold (RF) of two drug combinations were calculated. Hep-2/DDP cells in logarithmic growth phase were divided into control group, cisplatin group, tanshinone Ⅱ_A + cisplatin group, and tanshinone Ⅱ_A + cisplatin + IGF-1 group. The cell proliferation inhibition rate and apoptosis rate were detected by CCK-8 method and Annexin V-FITC/PI double staining method, and the expression of Bcl-2, Bax, Cleaved caspase-3, Cleaved caspase-9, PI3K, p-PI3K, Akt,and p-Akt was detected by Western blotting. Results The IC₅₀ of cisplatin on Hep-2 cells was 4.58 mg/L, the IC₅₀ of cisplatin on Hep-2/DDP cells was 14.09 mg/L, and the RI was 3.08. The IC₅₀ of tanshinone Ⅱ_A on Hep-2 cells was 5.32 mg/L. The IC₅₀ and RF of tanshinone Ⅱ_A combined with cisplatin were 3.34 mg/L and 4.22, respectively. Compared with the control group, the proliferation inhibition rate and apoptosis rate of Hep-2/DDP cells in cisplatin group were significantly increased (P < 0.01). Compared with cisplatin group, the proliferation inhibition rate and apoptosis rate of Hep-2/DDP cells in tanshinone Ⅱ_A + cisplatin group were significantly increased (P < 0.05). Compared with tanshinone Ⅱ_A + cisplatin group, the proliferation inhibition rate and apoptosis rate of Hep-2/DDP cells in tanshinone Ⅱ_A + cisplatin + IGF-1 group were significantly decreased (P < 0.05). Compared with the control group, the expression level of Bcl-2 in Hep-2/DDP cells in cisplatin group was significantly decreased, while the expression levels of Bax, Cleaved caspase-3, Cleaved caspase-9, and Bax/Bcl-2 ratio were significantly increased (P < 0.05). Compared with cisplatin group, the expression level of Bcl-2 in Hep-2/DDP cells in tanshinone Ⅱ_A + cisplatin group was significantly decreased, and the expression levels of Bax, Cleaved caspase-3, Cleaved caspase-9, and Bax/Bcl-2 ratio were significantly increased (P < 0.05). Compared with tanshinone Ⅱ_A + cisplatin group, the expression level of Bcl-2 in Hep-2/DDP cells in tanshinone Ⅱ_A + cisplatin + IGF-1 group was significantly increased, and the expression levels of Bax, Cleaved caspase-3, Cleaved caspase-9, and Bax/Bcl-2 ratio were significantly decreased (P < 0.05). Compared with the control group, the expressions of p-PI3K and p-Akt and the phosphorylation of PI3K and Akt (p-PI3K/PI3K, p-Akt/Akt) in Hep-2/DDP cells in cisplatin group were significantly decreased (P < 0.05). Compared with cisplatin group, the expressions of p-PI3K and p-Akt and the phosphorylation levels of PI3K and Akt in Hep-2/DDP cells in tanshinone Ⅱ_A + cisplatin group were significantly decreased (P < 0.05). Compared with tanshinone Ⅱ_A + cisplatin group, the expressions of p-PI3K and p-Akt and the phosphorylation levels of PI3K and Akt in Hep-2/DDP cells in tanshinone Ⅱ_A + cisplatin + IGF-1 group were significantly increased (P < 0.05). Conclusions Tanshinone Ⅱ_A can reverse the cisplatin resistance of human laryngeal cancer cells, which mechanism may be related to inhibiting the activation of PI3K/Akt pathway and promoting the apoptosis of laryngeal cancer cells.

R285.5

邯郸市科学技术研究与发展计划项目（21422083142）