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ABSTRACT OBJECTIVE：To investigate the reversal effects and potential mechanism of levoshikonin（L-SHK）on cisplatin
（DDP） resistance of human cervical carcinoma HeLa cells. METHODS：Human cervical carcinoma HeLa cells were used as
research objects，and drug-resistant HeLa/DDP cells were induced by DDP. CCK-8 assay was used to determine drug resistance
index of HeLa/DDP cells，the inhibition rate of different doses of L-SHK（0.125，0.25，0.5，1，2，4，8，16 μmol/L）on cell
proliferation，IC50 and the reversion index of L-SHK on HeLa/DDP cells. Effects of low，medium and high doses of L-SHK（0.3，
0.6，1.2 μmol/L）combined with DDP on cell cycle and apoptosis rate were determined by flow cytometry. Western blotting assay
was used to detect the effects of low，medium and high doses of L-SHK（0.3，0.6，1.2 μmol/L）combined with DDP on the
expression of apoptosis-related protein（Cleaved caspase-3，Bcl-2 and Bax）. RESULTS：The drug resistance index of HeLa/DDP
cells was 11.8. The inhibition rate of L-SHK on HeLa/DDP cells increased with the increase of dose. Compared with DDP alone，
IC50 of DDP+low-dose，medium-dose and high-dose L-SHK groups were decreased significantly，with a dose-dependent manner
（P＜0.05）. The reversion indexes were 1.38，2.80，6.71 in DDP+low-dose，medium-dose and high-dose L-SHK groups. Compared
with blank control group，the proportion of cells at phase G0/G1 and phase S in administration groups，as well as early and late
apoptosis rate and total apoptosis rate of cells，the protein expression of Bax and Cleaved caspase-3 in L-SHK combination groups
were increased significantly；the proportion of cells at phase G2/M in administration group as well as the protein expression of
Bcl-2 in L-SHK combination groups were decreased significantly（P＜0.05）. Compared with DDP group，the proportion of cells at
phase S and G2/M and the protein expression of Bcl-2 in L-SHK combination groups were significantly decreased；the proportion of
cells at phase G0/G1，early and late apoptosis rate and total apoptosis rate，the protein expression of Bax and Cleaved caspase-3 in
L-SHK combination groups were significantly increased（P＜0.05）. CONCLUSIONS：HeLa/DDP cells are resistant to DDP，and
L-SHK can reverse the drug resistance. L-SHK combined with DDP can promote the apoptosis of HeLa/DDP cells，which is better
than DDP alone. Its mechanism may be related to the influence of cell cycle and the regulation of apoptosis-related protein
expression.
KEYWORDS Human cervical carcinoma HeLa cells；Levoshikonin；Cisplatin；Resistance；Apoptosis；Cell cycle；Apoptosis-
related protein；Reversal effect；Mechanism

宫颈癌是妇科常见的恶性肿瘤，发病率在全球女性 能的分子机制，以期为耐药逆转剂的筛选以及SHK类化
常见恶性肿瘤中位居第3位，最新的统计显示，全球每年 合物新药的开发奠定理论基础。
新发宫颈癌56.9万例，死亡约31.1万例 [1-2] 。化疗是当前 1 材料
治 疗 宫 颈 癌 的 主 要 手 段 ，顺 铂（Cisplatin，以 下 简 称 1.1 仪器
“DDP”）为宫颈癌化疗的一线用药。然而，由于 DDP 长 31 型 CO2 培养箱（美国 Thermo Fisher Scientific 公
期应用易导致耐药的发生，严重影响了宫颈癌的治疗效 司）；Eclipse TS100型显微镜、GPJ9-TS100-F型倒置荧光
[3]
果 。由此可见，DDP 耐药性问题是宫颈癌临床治疗中 显微镜（日本Nikon公司）；Epoch 2型微孔板分光光度计
亟待解决的难题之一。紫草（Arnebiae Radix）为紫草科 （美国 BioTek 公司）；FACSCanto Ⅱ型流式细胞仪（美国
多年生草本植物新疆紫草[Arnebia ecuchroma（Royle） BD公司）；PowerPac型细胞电泳仪（美国Bio-Rad公司）；
Johnst.]或内蒙紫草（Arnebia guttata Bunge）的干燥根，具 C-DIGIT 凝胶成像仪（美国 LI-COR 公司）；MH1 型微量
有凉血、活血、解毒、透疹的功效，常用于治疗血热毒盛、 振荡仪（海门市其林贝尔仪器制造有限公司）；DK-8D型
[4]
斑疹紫黑、麻疹不透、疮疡、湿疹、水火烫伤等症 。紫草 水浴锅（上海精宏实验设备有限公司）；PMC-060型迷你
素（Shikonin，以下简称“SHK”）是从紫草中分离得到的 掌上离心机（日本 Tomy 公司）；AC2-6S1AirstreamⅡ级
一种天然萘醌类化合物。有研究证明，该化合物可抑制 A2 型生物安全柜（美国 SCCO 公司）；AB 135-S 型十万
多种肿瘤细胞的增殖，并可通过促进肿瘤细胞凋亡和坏 分之一分析天平（瑞士Mettler Toledo公司）。
死等途径来发挥对肿瘤的治疗作用 [5-6] 。目前有新的研 1.2 药品与试剂
究表明，SHK 对部分耐药肿瘤细胞具有一定的逆转作 L-SHK 对照品（天津一方科技有限公司，批号：
用，在耐药性卵巢癌、胶质母细胞瘤、肺癌等的治疗方面 AB072S，纯度：＞99％）；DDP 注射液（江苏豪森药业集
表现突出 [7-14] 。天然提取的SHK包含左旋体和右旋体两 团有限公司，批号：601191902，规格：6 mL∶ 30 mg）；RP-
种旋光异构体，由于其右旋体不溶于水，故多以左旋紫 MI 1640培养液、胎牛血清（美国Gibco公司，批号分别为
草素（L-SHK）进行实验研究。本课题组前期研究显示， 1181753、C2027005）；含乙二胺四乙酸（EDTA）的0.25％
L-SHK 联合 DDP 能够增强 DDP 对宫颈癌 HeLa 细胞的 胰酶（天津百诺克医药生物技术科技有限公司，批号：
[15]
抑制作用 。基于此，本研究拟进一步研究 L-SHK 对 280514）；不含 ETDA 的胰酶（美国 Hyclone 公司，批号：
DDP耐药宫颈癌细胞（HeLa/DDP）耐药性的影响及其可 1715826）；膜联蛋白Ⅴ-硫氰酸荧光素/碘化丙啶（Annex-

·1868 · China Pharmacy 2020 Vol. 31 No. 15 中国药房 2020年第31卷第15期