目的 探讨鹿药皂苷japonicoside B（JaB）抑制肝癌细胞生长和转移的作用及机制。方法 以人肝癌SMMC-7721和HepG2细胞为研究对象，设置对照组和JaB（2.5、5.0μmol/L）组，采用Transwell小室实验检测细胞迁移、侵袭能力；扫描电镜观察细胞形态变化；Western blotting检测上皮细胞间充质转化（epithelial-mesenchymal transition，EMT）过程和磷脂酰肌醇3激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）通路相关蛋白表达。以裸鼠移植瘤模型和裸鼠肝癌肺转移模型为研究对象，设置模型组及JaB组（5、10 mg/kg）和5-氟尿嘧啶（10 mg/kg）组，观察JaB对体内肿瘤生长和肺转移的影响；免疫组化法检测肿瘤组织中Ki67表达；Western blotting检测凋亡、EMT过程和PI3K/Akt通路相关蛋白表达。结果 与对照组比较，JaB显著抑制肝癌细胞的迁移和侵袭（P＜0.01），上调E-钙黏蛋白（E-cadherin）表达（P＜0.01），下调N-钙黏蛋白（N-cadherin）和Snail、Slug、Twist表达（P＜0.01）；JaB干预后，肝癌细胞形态发生变化，由梭型或伸展状态逐渐变圆，伪足变短消失；PI3K抑制剂LY294002处理肝癌细胞后，效果与JaB相似，均可抑制PI3K磷酸化（P＜0.01），同时上调E-cadherin表达（P＜0.01），下调N-cadherin表达（P＜0.01），从而抑制EMT过程。裸鼠移植瘤模型中，与模型组比较，JaB能够明显抑制肿瘤生长（P＜0.01），其中JaB高剂量（10 mg/kg）组抑制肿瘤生长作用最为显著，效果优于5-氟尿嘧啶（P＜0.01）；JaB组肿瘤组织中Ki67、B细胞淋巴瘤2蛋白（B-cell lymphoma 2，Bcl-2）表达显著降低（P＜0.01），剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved cystein-asparate protease-3，cleaved Caspase-3）、cleaved Caspase-9、Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）表达显著升高（P＜0.01），Akt的磷酸化水平显著降低（P＜0.05、0.01），与细胞实验结果一致，JaB可能通过调节Akt的磷酸化影响PI3K/Akt通路活性，进一步抑制EMT过程。裸鼠肝癌肺转移模型中，JaB显著抑制肝癌细胞肺转移的发生，与模型组比较，JaB组肿瘤组织中Ki67、N-cadherin、Snail、Slug、Twist表达显著降低（P＜0.05、0.01），E-cadherin表达显著升高（P＜0.01），Akt的磷酸化水平显著降低（P＜0.05、0.01），肿瘤细胞EMT过程受到了抑制。结论 JaB通过调控PI3K/Akt通路，上调E-cadherin，抑制N-cadherin以及Snail、Slug、Twist等转录因子表达来提高细胞间的黏附力，并抑制其向间质细胞转化，逆转了EMT过程，从而抑制肝癌细胞的迁移和侵袭，以及其在体内生长和转移，进而发挥抗肿瘤的作用。

Objective To explore the inhibitory effect and mechanism of japonicoside B (JaB) in Smilacina japonica on the growth and metastasis of hepatocellular carcinoma cells (HCC). Methods SMMC-7721 and HepG2 cells were used as the research objects, and control group and JaB (2.5, 5.0 μmol/L) groups were set up. Transwell chamber experiments were used to detect migration and invasion ability of cells; Cellular morphological changes were observed using scanning electron microscopy; Western blotting was used to detect the epithelial-mesenchymal transition (EMT) process and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway related protein expressions. Nude mouse transplant tumor models and nude mouse liver cancer lung metastasis models were used as research objects, model group, JaB (5, 10 mg/kg) groups and 5-fluorouracil (10 mg/kg) group were set up to observe the effects of JaB on tumor growth and lung metastasis in vivo; Immunohistochemistry was used to detect Ki67 expression in tumor tissues; Western blotting was used to detect apoptosis, EMT process and PI3K/Akt pathway related protein expressions. Results Compared with control group, JaB significantly inhibited the migration and invasion of HCC (P < 0.01), up-regulated the expression of E-cadherin (P < 0.01), and down-regulated the expressions of N-cadherin and Snail, Slug, Twist (P < 0.01); After JaB intervention, the morphology of HCC was changed, gradually becoming round from spindle shaped or stretched state, and pseudopodia became shorter and disappeared; After treating with PI3K inhibitor LY294002, the effect was similar to that of JaB, both of which could inhibit PI3K phosphorylation (P < 0.01), while up-regulating E-cadherin expression (P < 0.01) and down-regulating N-cadherin expression (P < 0.01), thereby inhibiting the EMT process. In the nude mouse transplant tumor model, compared with model group, JaB could significantly inhibit tumor growth (P < 0.01), among which the high-dose JaB (10 mg/kg) group had the most significant inhibitory effect on tumor growth, with a better effect than 5-fluorouracil (P < 0.01); The expressions of Ki67 and B-cell lymphoma 2 protein (Bcl-2) in tumor tissues of JaB group were significantly reduced (P < 0.01), while the expressions of cleaved cysteine aspartate protease-3 (cleaved Caspase-3), cleaved Caspase-9 and Bcl-2 associated X protein (Bax) were significantly increased (P < 0.01). The phosphorylation level of Akt was significantly reduced (P < 0.05, 0.01), which was consistent with the results of cell experiments, JaB may affect the PI3K/Akt pathway activity by regulating Akt phosphorylation, further inhibiting the EMT process. In the nude mouse liver cancer lung metastasis model, JaB significantly inhibited the occurrence of liver cancer cell lung metastasis. Compared with model group, the expressions of Ki67, N-cadherin, Snail, Slug and Twist in tumor tissue of JaB group were significantly reduced (P < 0.05, 0.01), the expression of E-cadherin was significantly increased (P < 0.01), and the phosphorylation level of Akt was significantly reduced (P < 0.05, 0.01), the EMT process of tumor cells was inhibited. Conclusion JaB enhances intercellular adhesion and inhibits their transformation into interstitial cells by regulating PI3K/Akt pathway, up-regulating E-cadherin, inhibiting N-cadherin as well as transcription factors such as Snail, Slug, and Twist, and reversing the EMT process, and then inhibits the migration and invasion of liver cancer cells, as well as their growth and metastasis in vivo, thereby exerting anti-tumor effects.

R285.5

国家自然科学基金资助项目(81973192);西安医学院校级科研项目(2020DOC31);西安医学院科技能力提升计划(2022NLTS061);陕西省教育厅青年创新团队项目(22JP076);国家中管局中药化学高水平重点学科建设项目(zyyzdxk-2023202)