目的 探讨隐丹参酮通过靶向LASP1调控Janus激酶-信号转导及转录激活因子（JAK/STAT）信号通路对胆管癌细胞恶性进展的抑制作用及其分子机制。方法 以人胆管癌HCCC-9810、RBE细胞及正常人肝内胆管上皮细胞HIBEpic为研究对象。采用细胞计数试剂盒-8法检测隐丹参酮对细胞活力的影响并计算半数抑制浓度（IC₅₀）；通过平板克隆形成实验评估细胞增殖能力；Transwell实验检测细胞迁移与侵袭；采用流式细胞术结合碘化丙啶（PI）染色分析细胞周期分布；膜联蛋白Ⅴ（Annexin Ⅴ）-异硫氰酸荧光素（FITC）/PI双染法及Hoechst 33342染色检测细胞凋亡；使用siRNA转染技术沉默LASP1表达，并通过RT-qPCR和Western blotting分别检测LASP1的mRNA及蛋白表达水平。运用Western blotting检测细胞周期蛋白B1（Cyclin B1）、细胞周期蛋白依赖性激酶1（CDK1）、剪切型半胱氨酸蛋白酶-3（cleaved Caspase-3）、cleaved Caspase-9、B细胞淋巴瘤-2蛋白（Bcl-2）、Bcl-2相关X蛋白（Bax）以及JAK/STAT信号通路关键蛋白Janus激酶2（JAK2）、磷酸化JAK2（p-JAK2）、信号转导与转录激活因子3（STAT3）、磷酸化STAT3（p-STAT3）的表达；利用JAK2激活剂broussonin E（BE）进行回复实验验证通路作用。结果 在生物学功能方面，隐丹参酮以时间和剂量相关性方式降低了细胞活力（HCCC-9810和RBE细胞的IC₅₀值分别为16.88、19.73μmol/L），并有效抑制了细胞的克隆形成、迁移和侵袭能力（P＜0.05、0.01）；细胞周期分析结果显示，隐丹参酮可将胆管癌细胞周期阻滞于G₂/M期，并下调G₂/M期检查点蛋白CDK1和Cyclin B1的表达（P＜0.01）；凋亡分析结果显示，隐丹参酮能诱导细胞凋亡，表现为凋亡形态学改变、凋亡率升高，并上调cleaved Caspase-3/9和Bax表达，下调Bcl-2表达（P＜0.05、0.01）；在机制层面，隐丹参酮能够抑制JAK/STAT信号通路的激活，表现为p-JAK2和p-STAT3蛋白水平降低而不影响其总蛋白表达，且此效应可被JAK2激活剂BE部分逆转（P＜0.05）。分子机制探索发现，LASP1在胆管癌细胞中呈低表达状态，而隐丹参酮可显著上调其表达（P＜0.01）；功能回复实验表明，沉默LASP1可部分拮抗隐丹参酮对细胞增殖的抑制和凋亡的诱导作用（P＜0.01）。结论 隐丹参酮可能通过上调LASP1表达，进而抑制JAK/STAT信号通路的激活，从而诱导胆管癌细胞发生周期阻滞和凋亡，最终发挥其其增殖、迁移和侵袭的抗肿瘤作用。

Objective To investigate the inhibition and underlying molecular mechanism of Cryptotanshinone on the malignant progression of cholangiocarcinoma cells by targeting LASP1 to regulate Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway. Methods Human cholangiocarcinoma HCCC-9810 and RBE cells and normal human intrahepatic bile duct epithelial cell line HIBEpic were used as the study subjects. The effect of cryptotanshinone on cell viability was assessed by the cell counting kit-8 (CCK-8) assay, and the half-maximal inhibitory concentration (IC₅₀) was calculated. Cell proliferation ability was evaluated by the colony formation assay. Cell migration and invasion were detected by the Transwell assay. Cell cycle distribution was analyzed by flow cytometry combined with propidium iodide (PI) staining. Cell apoptosis was detected by Annexin V- fluorescein isothiocyanate (FITC)/PI double staining and Hoechst 33342 staining. The expression of LASP1 was silenced using siRNA transfection technology, and its mRNA and protein expression levels were measured by RT-qPCR and Western blotting, respectively. The expression changes of key proteins were analyzed by Western blotting, including Cyclin B1, cyclin-dependent kinase 1 (CDK1), cleaved Caspase-3, cleaved Caspase-9, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), as well as key proteins of the JAK/STAT signaling pathway: Janus kinase 2 (JAK2), phosphorylated JAK2 (p-JAK2), signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 (p-STAT3). A rescue experiment was performed using the JAK2 activator broussonin E (BE) to validate the role of the pathway. Results In terms of biological functions, cell viability was reduced by cryptotanshinone in a time- and dose-dependent manner (the IC₅₀ values for HCCC-9810 and RBE cells were approximately 16.88 and 19.73 μmol/L, respectively), and the colony formation, migration, and invasion capabilities of the cells were effectively inhibited (P < 0.05, 0.01). Cell cycle analysis showed that cryptotanshinone induced G₂/M phase arrest in cholangiocarcinoma cells and downregulated the expression of the G₂/M checkpoint proteins CDK1 and Cyclin B1 (P < 0.01). Apoptosis analysis revealed that cryptotanshinone induced cell apoptosis, manifested as morphological changes of apoptosis, increased apoptosis rate, upregulated expression of cleaved Caspase-3/9 and Bax, and downregulated expression of Bcl-2 (P < 0.05, 0.01). At the mechanistic level, the activation of the JAK/STAT signaling pathway was inhibited by cryptotanshinone, as evidenced by decreased protein levels of p-JAK2 and p-STAT3 without affecting their total protein expression. This effect was partially reversed by the JAK2 activator BE (P < 0.05). Molecular mechanism exploration found that LASP1 was expressed at a low level in cholangiocarcinoma cells, while its expression was significantly upregulated by cryptotanshinone (P < 0.01). Functional rescue experiments indicated that the inhibitory effect of cryptotanshinone on cell proliferation and its pro-apoptotic effect were partially antagonized by silencing LASP1 (P < 0.01). Conclusion Cryptotanshinone may suppresses the activation of the JAK/STAT signaling pathway by upregulating LASP1 expression, thereby inducing cell cycle arrest and apoptosis in cholangiocarcinoma cells, ultimately exerting its anti-tumor effects by inhibiting proliferation, migration, and invasion.

R285;R286.5

国家自然科学基金资助项目（81603418，82074271）；黑龙江中医药大学“优秀创新人才支持计划”科研项目（2020YQ05）