目的 明确山慈菇Cremastrae Pseudobulbus Pleiones Pseudobulbus水提物的抗肝癌主要有效部位，探讨其有效部位对肝癌小鼠肿瘤血管生成的作用及机制。方法 采用正交试验优化山慈菇水提物提取条件，醇沉法将水提物分为醇沉部位与醇溶部位，苯酚硫酸法检测各部位多糖含量，在药效指导下进行有效部位筛选。将60只balb/c小鼠随机分为空白组、模型组、索拉非尼组（30 mg/kg）、山慈菇水提物组、山慈菇醇沉物组及山慈菇醇溶物组（均以生药量计1.82 g/kg），每组10只，连续ig给药16 d。通过计算抑瘤率及苏木精-伊红（hematoxylin eosin，HE）染色观察各组肿瘤组织病理变化综合评价药效；取有效部位组小鼠肿瘤组织或血清，原位末端标记（terminal deoxynucleotidyl transferase dUTP nick-end labeling，TUNEL）法测定肿瘤细胞凋亡情况；酶联免疫吸附试验（enzyme-linked immunosorben assay，ELISA）检测小鼠血清中血管内皮生长因子（vascular endothelial growth factor‌，VEGF）、基质金属蛋白酶-2（matrix metallopeptidase-2，MMP-2）和MMP-9水平；免疫组织化学法（immunohistochemistry，IHC）染色观察肿瘤组织微血管生成情况；Western blotting法检测肿瘤组织VEGF、MMP-2、MMP-9以及Wnt1、β-连环蛋白（β-catenin）的表达变化。结果 正交试验优化得山慈菇水提物最优提取条件为浸泡时间90 min、料液比1∶30、提取时间120 min、提取次数2次，山慈菇水提物、醇沉物、醇溶物得率分别为37.76%、28.00%、5.53%，含量测定得多糖含量分别为70.18%、95.19%、0.58%。动物实验结果显示，山慈菇水提物组、山慈菇醇沉物组、山慈菇醇溶物组抑瘤率分别为43.91%、43.39%、21.60%；病理切片显示，模型组肿瘤组织病变显著，各给药组均有所改善，以山慈菇水提物组与醇沉物组胞核碎裂更为明显。与模型组比较，山慈菇醇沉物组肿瘤细胞凋亡率显著增加（P＜0.01）；小鼠血清与肿瘤组织中VEGF、MMP-2、MMP-9水平显著降低（P＜0.01、0.001）；IHC结果显示，山慈菇醇沉物组较模型组微血管密度极显著降低（P＜0.001）；Western blotting结果显示，山慈菇醇沉物能抑制肿瘤组织中Wnt1、β-catenin蛋白表达（P＜0.001）。结论 山慈菇水提醇沉物是山慈菇水提物中发挥抗肝癌药效的主要有效部位，其成分主要为山慈菇多糖，作用机制可能与抑制Wnt/β-catenin通路激活，进而抑制肿瘤血管生成有关。

Objective To identify the primary anti-hepatocarcinoma active site in Shancigu (Cremastrae Pseudobulbus Pleiones Pseudobulbus) aqueous extract and investigate its effects and mechanisms on tumor angiogenesis in mice with liver cancer. Methods After optimizing the extraction conditions of Cremastrae Pseudobulbus Pleiones Pseudobulbus aqueous extract through orthogonal experiment, the aqueous extract was divided into alcohol precipitation and alcohol soluble parts using alcohol precipitation method, phenol-sulfuric acid method was used to detect the content of polysaccharides in each part, and effective parts were screened under the guidance of pharmacological effects. A total of 60 BALB/c mice were randomly assigned into a blank group, a model group, a sorafenib group (30 mg/kg), a Cremastrae Pseudobulbus Pleiones Pseudobulbus aqueous extract group, a Cremastrae Pseudobulbus Pleiones Pseudobulbus ethanol sediments group, and a Cremastrae Pseudobulbus Pleiones Pseudobulbus alcohol soluble substance group (all calculated based on the amount of raw medicine 1.82 g/kg), with 10 mice in each group. The mice were intragastrically administered continuously for 16 d. The drug efficacy was comprehensively evaluated by calculating tumor inhibition rate and observing pathological changes in tumor tissues of each group through hematoxylin-eosin (HE) staining. Tumor tissue or mouse serum after effective site administration were selected, then terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to detect apoptosis in tumor cells. Enzyme-linked immunosorben assay (ELISA) was used to determine the levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), and MMP-9 in mice serum. Immunohistochemical (IHC) was used to observe micro vessel density (MVD) in tumor tissues. Western blotting was used to detect the expression changes of VEGF, MMP-2, MMP-9, Wnt1 and β-catenin. Results The optimal extraction conditions were 90 min of soaking time, a material-to-liquid ratio of 1:30, 120 min of extraction time, and two extraction cycles. The yields of water extract, alcohol precipitate, and alcohol soluble extract of Cremastrae Pseudobulbus Pleiones Pseudobulbus were 37.76%, 28.00%, and 5.53%, respectively. The phenol sulfuric acid method determined that the polysaccharide content was 70.18%, 95.19%, and 0.58%, respectively. And the results of animal experiments showed that tumor inhibition rates were 43.91%, 43.39%, and 21.60% respectively. Pathological sections revealed significant tumor tissue lesions in the model group, while all treatment groups showed improvements, with more pronounced nuclear fragmentation in the Cremastrae Pseudobulbus Pleiones Pseudobulbus aqueous extract and ethanol sediments groups. Compared with the model group, the apoptosis rate of tumor cells in the Cremastrae Pseudobulbus Pleiones Pseudobulbus ethanol sediments group was significantly increased (P < 0.01), and VEGF, MMP-2, and MMP-9 levels were significantly decreased in mouse serum and tumor tissues (P < 0.01, 0.001). IHC results showed a significant reduction in MVD in the Cremastrae Pseudobulbus Pleiones Pseudobulbus ethanol sediments group compared to the model group (P < 0.001). Western blotting results indicated that Cremastrae Pseudobulbus Pleiones Pseudobulbus ethanol sediments inhibited the expression of Wnt1 and β-catenin proteins in tumor tissues (P < 0.001). Conclusion Cremastrae Pseudobulbus Pleiones Pseudobulbus ethanol sediments are the main active site responsible for the anti-hepatocarcinoma effect of Cremastrae Pseudobulbus Pleiones Pseudobulbus aqueous extract. Its main component is Cremastrae Pseudobulbus Pleiones Pseudobulbus polysaccharide. The mechanism of action may be related to inhibiting the activation of Wnt/β-catenin pathway and thus inhibiting tumor angiogenesis.

R285.5

国家自然科学基金资助项目（82073971）