四氢生物蝶呤促进肝细胞癌肿瘤血管形成的机制

Effects of tetrahydrobiopterin on the angiogenesis in hepatocellular carcinoma

目的探讨四氢生物蝶呤（BH4）对肝细胞癌（简称肝癌）肿瘤血管形成的作用及机制。方法BALB/c-nu小鼠左腋皮下接种人肝癌HepG-2细胞，并采用随机数字表法分为BH4组和对照组，其中BH4组按20 mg/kg BH4每天腹腔注射2周，对照组给予等量生理盐水。采用高效液相色谱法（HPLC）检测BH4水平，Griess试剂法检测血浆一氧化氮（NO）水平，实时荧光定量PCR检测K-ras mRNA水平，Western blot法检测鸟苷三磷酸环化水解酶Ⅰ型（GTPCH）、内皮一氧化氮合酶（eNOS）、磷酸化eNOS（p-eNOS）、蛋白激酶B （Akt）和p-Akt的表达。结果BH4组和对照组小鼠肿瘤组织中BH4水平分别为（0.24±0.02）μg/ml和（0.17±0.01）μg/ml，差异有统计学意义（P〈0.01）。BH4组和对照组的肿瘤体积分别为（191.05±8.70）mm^3和（103.10±5.03）mm^3，差异有统计学意义（P〈0.01）。BH4组和对照组小鼠肿瘤组织中NO水平分别为（51.44±2.90）mmol/L和（24.77±0.54）mmol/L，差异有统计学意义（P〈0.01）。BH4组肿瘤组织中CD34、K-ras mRNA、p-eNOS、p-Akt和GTPCH表达水平明显高于对照组，差异均有统计学意义（P〈0.01）。结论BH4作为eNOS关键辅助因子，可以激活野生型Ras-PI3K/Akt路径，促进肿瘤组织产生NO，从而诱导新生血管形成，为针对靶向BH4合成路径抑制肿瘤血管的形成提供了一些潜在的靶点。

ObjectiveTo investigate the effect and mechanism of tetrahydrobiopterin （BH4） on the angiogenesis in hepatocellular carcinoma （HCC）. MethodsBALB/c-nu mice were subcutaneously injected with HepG-2 cells and randomly divided into control and BH4 groups. The BH4 group and control group received 20 mg/kg BH4 or saline by intraperitoneal injection daily for two weeks, respectively. The level of BH4 was measured by high performance liquid chromatography （HPLC）, the level of nitric oxide （NO） was measured by Griess test array, the transcriptional level of K-ras was measured by quantitative RT-PCR, and the protein expressions of guanosine triphosphate cyclohydrolase Ⅰ（GTPCH）, endothelial nitric oxide synthase （eNOS）, phospho-Akt and Akt were determined by Western blot.ResultsBH4 level in the tumor tissues of BH4 group was （0.24±0.02） μg/ml, significantly higher than the （0.17±0.01） μg/ml in the control group （P〈0.01）. The level of NO in the tumor tissues of BH4 group was （51.44±2.90） mmol/L, significantly higher than the （24.77±0.54） mmol/L in the control group （P〈0.01）. The tumor volume of BH4 group was （191.05±8.70） mm^3, significantly higher than the （103.10±5.03） mm^3 in the control group （P〈0.01）. The expressions of CD34, K-ras, phospho-eNOS, phospho-Akt and GTPCH were significantly up-regulated in the tumor tissues of BH4 group when compared with those of the control group （P〈0.01）.ConclusionsBH4 recognized as an essential cofactor of eNOS can increase tumor-produced NO by activating the wild-type Ras-PI3K/Akt pathway, thus induces angiogenesis. This might provide a novel and promising way to control the progression of hepatocellular carcinoma through targeting BH4 synthesis pathway and inhibiting angiogenesis.