黄芪-莪术抑制肺癌血管生成及其对EGFR/PI3K/AKT和HIF-1α/VEGF信号通路的影响

Inhibition of angiogenesis of lung cancer by Astragali radix-Curcumae rhizoma and its effects on EGFR/PI3K/AKT and HIF-1α/VEGF signaling pathways

目的:探讨黄芪-莪术抑制肺癌血管生成作用及其是否与抑制EGFR/PI3K/AKT及HIF-1α/VEGF信号通路有关。方法:制备含中药生药0.3 g/mL生黄芪煎液、0.1 g/mL莪术煎液及0.4 g/mL黄芪-莪术煎液;腋下接种Lewis肺癌细胞构建Lewis肺癌移植瘤小鼠模型,然后随机分成对照组、贝伐单抗组、黄芪组、莪术组、黄芪-莪术组。接种次日,各中药组分别按照0.2mL/10g剂量灌胃,对照组及贝伐单抗组予等量0.9%氯化钠溶液灌胃,连续14d;贝伐单抗组于第5、8、11天腹腔注射贝伐单抗15mg/kg,对照组和各中药组同时腹腔注射等量0.9%氯化钠溶液。隔日观察各组小鼠一般体征及体质量,于第15天处死,称瘤重并计算抑瘤率,免疫组化法检测肿瘤组织中微血管密度(MVD),ELISA及Real-time PCR法检测EGFR/PI3K/AKT及HIF-1α/VEGF信号通路相关蛋白及mRNA表达。结果:黄芪-莪术组抑瘤率为53.77%,其次分别为贝伐单抗组41.88%、黄芪组30.48%及莪术组26.67%;贝伐单抗组、黄芪组、黄芪-莪术组MVD值均低于对照组(P<0.01,P<0.05),其中贝伐单抗组最低(P<0.01),黄芪-莪术组低于黄芪组、莪术组(P<0.05);与对照组比较,黄芪-莪术组VEGF、HIF-1α、EGFR、P-PI3K、AKT蛋白表达显著降低(P<0.01,P<0.05),VEGF、EGFR、AKT mRNA表达显著降低(P<0.01,P<0.05),黄芪-莪术组HIF-1α蛋白及EGFR mRNA低于黄芪组及莪术组(P<0.05),黄芪-莪术组对于EGFR、AKT mRNA的下调作用优于贝伐单抗组(P<0.01,P<0.05)。结论:黄芪-莪术能通过抑制肺癌血管生成抑制肿瘤生长,配伍应用优于单药,其机制与抑制EGFR/PI3K/AKT及HIF-1α/VEGF信号通路中EGFR、AKT、VEGF蛋白表达及EGFR、AKT、VEGF mRNA转录有关。

Objective: To investigate the inhibitory effects of Astragali radix-Curcumae rhizoma on lung cancer angiogenesis and whether it is related to the inhibition of EGFR/PI3K/AKT and HIF-1α/VEGF signaling pathways.Methods: Firstly, 0.3 g/mL raw Astragali radix solution, 0.1 g/mL Curcumae rhizoma solution and 0.4 g/mL Astragali radixCurcumae rhizoma solution containing traditional Chinese medicine crude drugs were prepared;Then Lewis lung cancer cells were inoculated into the armpits to construct a mouse model of Lewis lung cancer transplantation tumor, and then they were randomly divided into control group, Bevacizumab group, Astragali radix group, Curcumae rhizoma group, Astragali radixCurcumae rhizoma group. From the second day of inoculation, each traditional Chinese medicine group was intragastrically administered at a dose of 0.2 mL/10 g. The control group and Bevacizumab group were given the same amount of normal saline for 14 consecutive days;the Bevacizumab group was intraperitoneally injected on d5, d8, and d11 Bevacizumab 15mg/kg, the control group and each traditional Chinese medicine group were injected intraperitoneally with the same amount of normal saline at the same time. The general signs and body weights of the mice in each group were observed every other day. They were sacrificed on d15. The tumors were weighed to calculate the tumor inhibition rate and counted lung metastases. The microvessel density in the tumor tissues was detected by immunohistochemistry, and EGFR/PI3K/AKT and HIF-1α/VEGF signaling pathway related protein and mRNA expression were detected by ELISA and Real-time PCR. Results:The tumor inhibition rate was 53.77% in Curcumae rhizoma group, followed by 41.88% in Bevacizumab group, 30.48% in Astragali radix group and 26.67% in Curcumae rhizoma group. The MVD value in Bevacizumab group, Astragali radix group and Astragali radix-Curcumae rhizoma group was lower than that in control group(P<0.01, P<0.05), and the lowest value was in Bevacizumab group(P<0.01), and the MVD value in Astragali radix-Curcumae rhizoma group was lower than that in Astragali radix and Curcumae rhizoma group(P<0.05). Compared with the control group, the protein expressions of VEGF, HIF-1α, EGFR, P-PI3K and AKT in Astragali radix-Curcumae rhizoma group were significantly decreased(P<0.01, P<0.05), the mRNA expressions of VEGF, EGFR and AKT in Astragali radix-Curcumae rhizoma group were significantly decreased(P<0.01, P<0.05). HIF-1α protein and EGFR mRNA in Astragali radix-Curcumae rhizoma group was lower than that in single Astragali radix group and Curcumae rhizoma group(P<0.05), and the down-regulation effect of Astragali radix-Curcumae rhizoma group on EGFR and AKT mRNA was better than that in Bevacizumab group(P<0.01, P<0.05). Conclusion: Astragali radix-Curcumae rhizoma can inhibit tumor growth by inhibiting lung cancer angiogenesis. The combination of Astragali radix-Curcumae rhizoma is better than single-agent Astragali radix and Curcumae rhizoma. Its mechanism is related to the inhibition the protein expression of EGFR, AKT and EGFR, and the mRNA transcription of EGFR, AKT, and VEGF in the EGFR/PI3K/AKT and HIF-1α/VEGF signaling pathways.