大黄素抑制人肝癌HepG2细胞血管生成作用及其机制研究

Effect of emodin on angiogenesis and its mechanism in human hepatoma cell line HepG2

目的大黄素可促进人肝癌HepG2细胞的凋亡,然而其是否可抑制肝细胞癌(hepatocellular carcinoma,HCC)血管生成及其机制罕见报道。本研究探讨大黄素对人肝癌HepG2细胞血管生成以及缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的影响,以及大黄素体内抗肝癌机制。方法采用体内鸡胚绒毛尿囊膜(chick chorioallantoic membrane,CAM)实验,实验随机分为阴性对照组(生理盐水)、大黄素低剂量组(10μmol/L)、大黄素高剂量(20μmol/L)和阳性对照组(0.15mg/mL地塞米松),每组各10只,每24h每组追加同样等量药物,共72h,观察大黄素对CAM血管生成的抑制作用。体外培养HepG2细胞,以氯化钴(CoCl2)模拟化学缺氧,设立缺氧未处理组〔阴性对照组(生理盐水)〕、缺氧大黄素低剂量组(10μmol/L)、缺氧大黄素高剂量组(20μmol/L)和缺氧阳性对照组〔10μmol/L 5-氟尿嘧啶(5-fluorouracil,5-FU)〕,每组设6个复孔,处理24h。采用小管形成实验,观察大黄素对HepG2细胞相对小管数目的影响,免疫细胞化学分析检测HIF-1α和VEGF阳性细胞的表达,Real-Time PCR分析检测HIF-1αmRNA和VEGF mRNA的表达。采用Graphpad 5.0软件进行描述性统计,组间比较采用one-way-ANOWA和Bonferroni检验。结果 CAM实验显示,实验组新生血管数目明显减少,与阴性照组比较,差异有统计学意义,F=13.374,P=0.002。阴性对照组新生血管数目为(31.47±1.81)个。给药后CAM血管生成减少,大黄素低剂量组、高剂量组和地塞米松组分别为(19.48±0.66)、(10.33±1.04)和(9.89±0.57)个,与阴性对照组比较,差异有统计学意义,P值分别为0.041、0.004和0.003;大黄素低剂量组和高剂量组与地塞米松组比较,差异无统计学意义,P值分别为0.539和1.000。小管形成实验显示,实验组相对小管数目明显减少,与阴性照组比较,差异有统计学意义,F=21.529,P<0.001。阴性对照组相对小管数目为(100.00±0.00)%。给药后,相对小管数目明显减少,大黄素低剂量、高剂量组和阳性对照组分别为(65.85±12.67)%、(46.94±8.34)%和(41.77±7.53)%,与阴性对照组比较,差异有统计学意义,P值分别为0.049、0.001和<0.001;大黄素低剂量组和高级剂量组与阳性对照组比较,差异无统计学意义,P值分别为0.104和0.069。免疫细胞化学分析显示,大黄素低剂量组、高剂量组和阳性对照组HIF-1α和VEGF阳性细胞显著减少。Real-Time PCR分析显示,实验组HIF-1αmRNA和VEGF mRNA表达降低,与阴性照组比较,差异有统计学意义(F=31.908,P<0.001;F=25.146,P<0.001)。阴性对照组HepG2细胞HIF-1αmRNA和VEGF mRNA相对表达分别为0.92±0.15和0.95±0.15;大黄素低剂量组HIF-1αmRNA和VEGF mRNA相对表达分别为0.45±0.07和0.51±0.08,与阴性对照组比较,差异有统计学意义,P值分别为0.021和0.013;大黄素高剂量组HIF-1αmRNA和VEGF mRNA相对表达分别为0.32±0.05和0.40±0.07,与阴性对照组比较,差异有统计学意义,P值分别为<0.001和0.001;阳性对照组HIF-1αmRNA和VEGF mRNA相对表达分别为0.30±0.04和0.34±0.05,与阴性对照组比较,差异有统计学意义,P值均<0.001;大黄素低剂量组、高剂量组HIF-1αmRNA和VEGF mRNA相对表达与阳性对照组比较,差异无统计学意义,P值分别为0.663、0.362、0.443和1.000。结论大黄素可能通过抑制HIF-1αmRNA的表达,从而降低VEGF mRNA的表达来抑制HepG2细胞新生血管的形成。

ORJECTIVE Emodin can promote the apoptosis of the human hepatoma HepG2 cells, however,whether it inhibits hepatocellular careinoma（HCC）of angiogenesis and its mechanism has not been reported. The purpose of this study is to investigate the effect of emodin on angiogenesis and hypoxia inducible factor-1α （HIF-1a） and vascular endothelial growth factor （VEGF）in human hepatoma HepG2 cells,and discuss its possible anti-cancer mechanism. METHODS The chick chorioallantoie membrane （CAM） experiment was randomly divided into the negative control group （saline）,the low dose group （10 μmol/L）, the high dose group（ 10 μmol/L） and the positive control group（0. 15 mg/mL dexamethasone）, each group had 10 CAMs, the drug was administered respectively for 24 h,a total of 72 h,the inhibition of emodin on CAM angiogenesis was observed. HepG2 cells were cultured in vitro, cobalt chloride（CoCl2 ） was added in the medium to simulate the chemical hypoxia environment, the hypoxia untreated group was established [the negative control group（saline）J, the hypoxia low dose group （10μmol/L）, the hypoxia high close group （20μmol/L） ,and the hypoxia positive control group [10 μmol/L 5-fluorouracil（5-FU）], and each group set up 6 wells,processing 24 h. The effect of emodin on the number of relatively small tube was observed in HepG2 cells by tube formation assay,the expression of HIF-1α and VEGF positive cells was detected by immunocytochehmical analysis, the expression of HIF-1α mRNA and VEGF mRNA was detected by Real-Time PCR analysis. The Graphpad 5.0 software was used in statistics, One-way-ANOWA and Borderroni test was used to compare between the groups. RESULTS The CAM experimental results showed that the number of new blood vessels in the experimental group was significantly reduced, compared with the negative control group, the difference was statistically significant （F= 13. 374, P = 0. 002）. The number of new blood vessels of the negative control group was（31.47± 1.81）. Angiogenesis reduced significantly after administration, the number of new blood vessels of the low dose group,the high dose group and the dexamethasone group were （19.48±0.66）, （10.33±1.04） ,and （9.89±0.57）, compared with the negative control group, the difference was statistically significant, P values were 0. 041,0. 004 and 0. 003, respectively. The low dose group and the high dose group compared with the dexamethasone group, the difference was not statistically signifieant,P values were 0. 539 and 1. 000, respectively. The tubule formation assay showed that the number of relatively small pipe in the experimental group was significantly reduced, compared with the negative control group, the difference was staffstically significant（F=21. 529, P〈0. 001 ）. The number of relatively small tube of the negative control group was （100. 00± 0. 00）%. The number of relatively small tube reduced significantly after administration, the number of relatively small tube of the low dose group, the high dose group and the positive control group were （65.85 ±12.67 ）%, （46.94 ±8.34 ） % and （41.77 ± 7. 53） ^ ,compared with the negative control group, the difference was statistically significant, P values were 0. 049,0. 001 and 〈0. 001 ,respectively. The low dose group and the high dose group compared with the positive control group,the difference was not statistically significant,P values were 0. 104 and 0.069, respectively. Immunocytochemistry analysis showed that emodin can significantly reduce the expression of HIF-1a and VEGF positive cells. Real-Time PCR analysis showed that the relative expression of HIF-1a mRNA and VEGF mRNA in the experimental group was significantly reduced, compared with the negative control group,the difference was statistically significant（F= 31. 908, P〈0. 001 and F= 25. 146, P〈0. 001）. The relative expression of HIF-1a mRNA and VEGF mRNA were （0. 92±0. 15） and （0. 95±0.15） in the negative control group HepG2 cells,the relative expression of group HIF-1α mRNA and VEGF mRNA were（0. 45±0. 07） and（0. 51±0. 08） in the low dose group,compared with the negative control group, the difference was statistically significant, P values were 0. 021 and 0. 013, respectively. The relative expression of HIF-1α mRNA and VEGF mRNA were （0. 32±0. 05） and （0. 40±0. 07） in the high dose group,compared with the negative control group,the difference was statistically significant, P values were d0. 001 and 0. 001, respectively. The relative expression of HIF-1α mRNA and VEGF mRNA were （0. 304-0.04） and （0. 344-0. 05） in the positive control group, compared with the negative control group, the difference was statistically significant, all P values 〈0.001, respectively. The relative expression of HIF-1α mRNA and VEGF mRNA compared with the positive control group, the difference was not statistically signifieant,P values were 0. 663,0. 362,0. 443 and 1.000,respectively. CONCLUSION The emodin can inhibit the expression of HIF-1α mRNA mRNA, which can inhibit the expression of VEGF mRNA so as to inhibit the angiogenesis of HepG2 cells.