目的 研究丹參酮Ⅱ_A對人喉癌細(xì)胞順鉑耐藥的影響作用及機(jī)制。方法 以不同質(zhì)量濃度（0、1.0、2.0、4.0、8.0、16.0、32.0 mg/L）順鉑干預(yù)對數(shù)生長期人喉癌Hep-2細(xì)胞和人喉癌順鉑耐藥細(xì)胞（Hep-2/DDP）48 h，CCK-8法檢測細(xì)胞增殖抑制率，計(jì)算半數(shù)抑制濃度（IC₅₀）和耐藥指數(shù)（RI）。以不同質(zhì)量濃度（0、0.5、1.0、2.0、4.0、8.0、16.0 mg/L）丹參酮Ⅱ_A干預(yù)對數(shù)生長期Hep-2細(xì)胞48 h，CCK-8法檢測細(xì)胞增殖抑制率，計(jì)算IC₅₀；采用丹參酮Ⅱ_A（IC₅₀5.32 mg/L）＋不同質(zhì)量濃度（0、1.0、2.0、4.0、8.0、16.0、32.0 mg/L）順鉑干預(yù)對數(shù)生長期Hep-2/DDP細(xì)胞48 h，CCK-8法檢測細(xì)胞增殖抑制率，計(jì)算2種藥物聯(lián)用的IC₅₀和逆轉(zhuǎn)倍數(shù)（RF）。取對數(shù)生長期Hep-2/DDP細(xì)胞，設(shè)置對照組、順鉑組、丹參酮Ⅱ_A＋順鉑組、丹參酮Ⅱ_A＋順鉑＋胰島素樣生長因子-1（IGF-1）組。各組分別給藥干預(yù)48 h后，采用CCK-8法、Annexin V-FITC/PI雙染法檢測細(xì)胞增殖抑制率和凋亡率，Western blotting法檢測B細(xì)胞淋巴瘤-2（Bcl-2），Bcl-2相關(guān)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）蛋白表達(dá)情況。結(jié)果 順鉑對Hep-2細(xì)胞的IC₅₀為4.58 mg/L，對Hep-2/DDP細(xì)胞的IC₅₀為14.09 mg/L，RI為3.08；丹參酮Ⅱ_A對Hep-2細(xì)胞的IC₅₀為5.32 mg/L；丹參酮Ⅱ_A聯(lián)合順鉑對Hep-2/DDP細(xì)胞的IC₅₀為3.34 mg/L，RF為4.22。與對照組比較，順鉑組Hep-2/DDP細(xì)胞增殖抑制率、凋亡率顯著升高（P<0.01）；與順鉑組比較，丹參酮Ⅱ_A＋順鉑組Hep-2/DDP細(xì)胞增殖抑制率、凋亡率顯著升高（P<0.05）；與丹參酮Ⅱ_A＋順鉑組比較，丹參酮Ⅱ_A＋順鉑＋I(xiàn)GF-1組Hep-2/DDP細(xì)胞增殖抑制率、凋亡率顯著降低（P<0.05）。與對照組相比，順鉑組Hep-2/DDP細(xì)胞Bcl-2表達(dá)量顯著降低，Bax、cleaved Caspase-3、cleaved Caspase-9表達(dá)量和Bax/Bcl-2值顯著升高（P<0.05）。與順鉑組比較，丹參酮Ⅱ_A＋順鉑組Hep-2/DDP細(xì)胞Bcl-2表達(dá)量顯著降低，Bax、cleaved Caspase-3、cleaved Caspase-9表達(dá)量和Bax/Bcl-2值顯著升高（P<0.05）。與丹參酮Ⅱ_A＋順鉑組比較，丹參酮Ⅱ_A＋順鉑＋I(xiàn)GF-1組Hep-2/DDP細(xì)胞Bcl-2表達(dá)量顯著升高，Bax、cleaved Caspase-3、cleaved Caspase-9表達(dá)量和Bax/Bcl-2值顯著降低（P<0.05）。與對照組相比，順鉑組Hep-2/DDP細(xì)胞p-PI3K、p-Akt表達(dá)量及PI3K、Akt磷酸化（p-PI3K/PI3K、p-Akt/Akt）水平顯著降低（P<0.05）。與順鉑組比較，丹參酮Ⅱ_A＋順鉑組Hep-2/DDP細(xì)胞p-PI3K、p-Akt表達(dá)量及PI3K、Akt磷酸化水平顯著降低（P<0.05）。與丹參酮Ⅱ_A＋順鉑組比較，丹參酮Ⅱ_A＋順鉑＋I(xiàn)GF-1組Hep-2/DDP細(xì)胞p-PI3K、p-Akt表達(dá)量及PI3K、Akt磷酸化水平顯著升高（P<0.05）。結(jié)論 丹參酮Ⅱ_A可逆轉(zhuǎn)人喉癌細(xì)胞順鉑耐藥，其作用機(jī)制可能與抑制PI3K/Akt通路活化而促進(jìn)喉癌細(xì)胞凋亡有關(guān)。

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

邯鄲市科學(xué)技術(shù)研究與發(fā)展計(jì)劃項(xiàng)目（21422083142）