[摘 要] 目的：探讨积雪草苷（ASI）是否通过环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应成分结合蛋白(cAMP/PKA/CREB)信号 通路调节肝细胞癌Huh7细胞的恶性生物学行为。方法：MTT筛选合适的ASI及时间后，将Huh7细胞分为对照组、ASI低剂量组 （ASI-L组，20 μmol/L ASI）、ASI中剂量组（ASI-M组，40 μmol/L ASI）、ASI高剂量组（ASI-H组，80 μmol/L ASI）及ASI-H+Forskolin 组（80 μmol/L ASI+100 μmol/L cAMP激活剂-Forskolin），按照上述分组处理48 h后，MTT及细胞克隆实验检测Huh7细胞增殖； Transwell实验分析Huh7细胞迁移、侵袭变化；膜联蛋白V-FITC凋亡试剂盒检测细胞凋亡变化；ELISA实验、WB法检测Huh7细 胞的cAMP分泌水平及p-PKA、p-CREB蛋白的表达水平；将Huh7细胞经皮下注射于裸鼠右腹部，建立肝癌异种移植模型，以5、 15和45 mg/kg的ASI灌胃干预4周，分离肿瘤组织并称质量。结果：以接近IC50 的40 μmol/L ASI处理48 h为合适的浓度和时间。 ASI-L组、ASI-M组、ASI-H组Huh7细胞增殖及克隆数、迁移数、侵袭数、cAMP水平、p-PKA/PKA和p-CREB/CREB表达均显著低 于对照组（均P < 0.05），而凋亡率均显著高于对照组（均P < 0.05）；ASI-H+Forskolin组Huh7细胞增殖及克隆数、迁移数、侵袭 数、cAMP水平、p-PKA/PKA、p-CREB/CREB 表达均显著高于ASI-H组（均P < 0.05），而Huh7细胞凋亡率显著低于ASI-H组 （P < 0.05）；在裸鼠移植瘤实验中，5、15和45 mg/kg ASI处理组的移植瘤质量均显著低于对照组（均P < 0.05）。结论：ASI可通过 下调cAMP/PKA/CREB信号通路蛋白表达抑制Huh7细胞的恶性生物学行为、促进其凋亡，以及抑制裸鼠移植瘤的生长。

[Abstract] Objective: To investigate whether asiaticoside (ASI) regulates the malignant biological behavior of hepatocellular carcinoma Huh7 cells via the cyclic adenosine monophosphate/protein kinase A/cAMP-response element binding protein (cAMP/PKA/ CREB) signaling pathway. Methods: After determining the suitable ASI concentration and treatment duration using MTT assay, Huh7 cells were divided into the following groups: control, ASI-L (20 μmol/L), ASI-M (40 μmol/L), ASI-H (80 μmol/L), and ASI-H+ Forskolin (80 μmol/L ASI + 100 μmol/L cAMP activator Forskolin) groups. After 48 h of treatment, cell proliferation was assessed using MTT and colony formation assays; cell migration and invasion were analyzed using Transwell assays; apoptosis was detected using an Annexin V-FITC apoptosis detection kit. The secretion level of cAMP and the protein expression levels of phosphorylated PKA (p-PKA) and phosphorylated CREB (p-CREB) were evaluated using ELISA and Western blotting, respectively. A subcutaneous xenograft model was established by injecting Huh7 cells into the right abdomen of nude mice. ASI was administered by gavage at doses of 5, 15, and 45 mg/kg for 4 weeks. Tumors were then harvested and weighed. Results: Treatment with 40 μmol/L ASI for 48 hours (close to the IC??) was determined to be the appropriate concentration and duration. Compared with the control group, the ASI-L, ASI M, and ASI-H groups showed significantly reduced Huh 7 cell proliferation, colony formation, migration, invasion, cAMP levels, and expression of p-PKA/PKA and p-CREB/CREB (all P < 0.05), while apoptosis rates were significantly increased (P < 0.05). Compared with the ASI-H group, the ASI-H + Forskolin group exhibited significantly increased proliferation, colony formation, migration,invasion, cAMP level, and expression of p-PKA/PKA and p-CREB/CREB (all P < 0.05), but apoptosis was significantly reduced (all P < 0.05). In the nude mouse xenograft model, ASI at 5, 15, and 45 mg/kg markedly decreased tumor weight in nude mice (all P < 0.05). Conclusion: ASI inhibits the malignant biological behaviors and promotes apoptosis of Huh7 cells, as well as suppresses tumor growth in nude mice, by downregulating the expression of proteins in the cAMP/PKA/CREB signaling pathway.

济宁医学院教师科研扶持项目（No. JYFC2019FKJ301）