健脾消癌方通过PI3K/Akt信号通路抑制结肠癌血管生成的研究＊

目的 观察结肠癌HCT116细胞健脾消癌方的条件培养液对HUVEC细胞管腔形成的影响,从PI3K/Akt生物轴调控角度探讨其作用机制。方法 培养HCT116细胞,细胞设3组：对照组,健脾消癌方组（加入15%健脾消癌方含药血清）及人参皂苷Rg3组;制备HCT116细胞健脾消癌方条件培养液（分组及制备方法见实验方法）,用条件培养液干预HUVEC（脐静脉内皮细胞,Human Umbilical Vein Endothelial Cells）,Matrigel基质胶法检测HCT116细胞健脾消癌方条件培养液对HUVEC小管形成的影响。随后采用蛋白免疫印迹法（Western blot）检测各组HCT116细胞磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）、p-Akt、VEGF（血管内皮生长因子,Vascular endothelial growth factor）蛋白表达。最后在结肠癌HCT116荷瘤小鼠中验证健脾消癌方对肿瘤生长速度的影响,并经瘤组织VEGF蛋白表达、CD31免疫组化染色检测肿瘤内血管生成情况。结果 模型组HUVEC细胞管腔形成较空白血清组显著增加（P<0.05）;健脾消癌方组及人参皂苷Rg3组较模型组HUVEC细胞管腔形成显著减少（P<0.01）。p-Akt和VEGF蛋白表达水平模型组高于空白血清组（P<0.05）,健脾消癌方组及人参皂苷Rg3组显著低于模型组（P<0.01）;PI3K、Akt蛋白表达量组间差异无统计学意义。与对照组比较,模型组荷瘤小鼠肿瘤体积显著性增大,瘤组织内VEGF表达、CD31阳性面积显著性增加,差异有统计学意义（P<0.05）;与模型组比较,健脾消癌方组及人参皂苷Rg3组荷瘤小鼠肿瘤体积显著减小,瘤组织内VEGF表达、CD31阳性面积降低,差异有统计学意义（P<0.05）。结论 健脾消癌方可抑制肿瘤的血管生成和生长,其作用机制可能与PI3K/Akt生物轴调控VEGF表达有关。

Study on Jianpi Xiaoai Decoction Inhibiting Angiogenesis of Colon Cancer through PI3K/Akt Signaling Pathway

Objective To observe the effects of Jianpi Xiaoai Decoction conditioned medium of HCT116 cells on tube formation of HUVEC cells, and to explore the mechanism of action from the perspective of PI3K/Akt biological axis regulation.Methods HCT116 cells were cultured and divided into three groups: control group, Jianpi Xiaoai Decoction group (adding 15% Jianpi Xiaoai Decoction containing serum) and ginsenoside Rg3 group. The conditioned culture medium of HCT116 cell Jianpi Xiaoai Decoction was prepared (grouping and preparation method were presented in experimental method), HUVEC cells were treated with conditioned medium. Matrigel lumen formation experiment was used to observe the effect of HCT116 cell Jianpi Xiaoai Decoction conditioned-medium on HUVEC cell lumen formation. The expression of PI3K, Akt, p-Akt and VEGF in each group were detected by western blotting. Effects of Jianpi Xiaoai Decoction on growth rate of tumor in HCT116 cell line tumor-bearing mice was verified, and angiogenesis condition in the tumor was detected by VEGF expression in tumor tissue and CD31 immunohistochemical staining.Results Compared with the blank serum group, the lumen formation of HUVEC in model groups was significantly reduced (P<0.01); compared with the model group, the lumen formation of HUVEC in the Ginsenoside Rg3 and Jianpi Xiaoai Formula group was significantly increased (P<0.01). There was no significant difference in PI3K and Akt protein expression among all groups. The protein expression of p-Akt and VEGF in model group was much higher than that in blank serum group (P<0.01), and the protein expression of p-Akt and VEGF in Ginsenoside Rg3 group and Jianpi Xiaoai Decoction group was much lower than that in model group (P<0.01). Compared with the control group, the tumor volume, VEGF expression and CD31 positive area of the model group were significantly increased (P<0.05); compared with the model group, the tumor volume, VEGF expression and CD31 positive area in tumor tissue were significantly decreased in the Jianpi Xiaoai Decoction group and Ginsenoside Rg3 group (P<0.05).Conclusion Jianpi Xiaoai Decoction can regulate the expression of VEGF by regulating PI3K/Akt signaling pathway, and then inhibit tumor angiogenesis and growth.