目的 明確山慈菇Cremastrae Pseudobulbus Pleiones Pseudobulbus水提物的抗肝癌主要有效部位，探討其有效部位對(duì)肝癌小鼠腫瘤血管生成的作用及機(jī)制。方法 采用正交試驗(yàn)優(yōu)化山慈菇水提物提取條件，醇沉法將水提物分為醇沉部位與醇溶部位，苯酚硫酸法檢測(cè)各部位多糖含量，在藥效指導(dǎo)下進(jìn)行有效部位篩選。將60只balb/c小鼠隨機(jī)分為空白組、模型組、索拉非尼組（30 mg/kg）、山慈菇水提物組、山慈菇醇沉物組及山慈菇醇溶物組（均以生藥量計(jì)1.82 g/kg），每組10只，連續(xù)ig給藥16 d。通過計(jì)算抑瘤率及蘇木精-伊紅（hematoxylin eosin，HE）染色觀察各組腫瘤組織病理變化綜合評(píng)價(jià)藥效；取有效部位組小鼠腫瘤組織或血清，原位末端標(biāo)記（terminal deoxynucleotidyl transferase dUTP nick-end labeling，TUNEL）法測(cè)定腫瘤細(xì)胞凋亡情況；酶聯(lián)免疫吸附試驗(yàn)（enzyme-linked immunosorben assay，ELISA）檢測(cè)小鼠血清中血管內(nèi)皮生長(zhǎng)因子（vascular endothelial growth factor‌，VEGF）、基質(zhì)金屬蛋白酶-2（matrix metallopeptidase-2，MMP-2）和MMP-9水平；免疫組織化學(xué)法（immunohistochemistry，IHC）染色觀察腫瘤組織微血管生成情況；Western blotting法檢測(cè)腫瘤組織VEGF、MMP-2、MMP-9以及Wnt1、β-連環(huán)蛋白（β-catenin）的表達(dá)變化。結(jié)果 正交試驗(yàn)優(yōu)化得山慈菇水提物最優(yōu)提取條件為浸泡時(shí)間90 min、料液比1∶30、提取時(shí)間120 min、提取次數(shù)2次，山慈菇水提物、醇沉物、醇溶物得率分別為37.76%、28.00%、5.53%，含量測(cè)定得多糖含量分別為70.18%、95.19%、0.58%。動(dòng)物實(shí)驗(yàn)結(jié)果顯示，山慈菇水提物組、山慈菇醇沉物組、山慈菇醇溶物組抑瘤率分別為43.91%、43.39%、21.60%；病理切片顯示，模型組腫瘤組織病變顯著，各給藥組均有所改善，以山慈菇水提物組與醇沉物組胞核碎裂更為明顯。與模型組比較，山慈菇醇沉物組腫瘤細(xì)胞凋亡率顯著增加（P＜0.01）；小鼠血清與腫瘤組織中VEGF、MMP-2、MMP-9水平顯著降低（P＜0.01、0.001）；IHC結(jié)果顯示，山慈菇醇沉物組較模型組微血管密度極顯著降低（P＜0.001）；Western blotting結(jié)果顯示，山慈菇醇沉物能抑制腫瘤組織中Wnt1、β-catenin蛋白表達(dá)（P＜0.001）。結(jié)論 山慈菇水提醇沉物是山慈菇水提物中發(fā)揮抗肝癌藥效的主要有效部位，其成分主要為山慈菇多糖，作用機(jī)制可能與抑制Wnt/β-catenin通路激活，進(jìn)而抑制腫瘤血管生成有關(guān)。

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

國(guó)家自然科學(xué)基金資助項(xiàng)目（82073971）