目的 分析山慈菇多糖（Cremastrae Pseudobulbus Pleiones Pseudobulbus polysaccharide，CPP）的化学组成并进行多糖结构表征，并探究其抗肝细胞癌（hepatocellular carcinoma，HCC）的药效及作用机制。方法 采用苯酚-硫酸法、考马斯亮蓝法检测CPP样品的总糖及蛋白含量，结合紫外光谱法、红外光谱法、扫描电子显微镜分析、PMP柱前衍生化-HPLC法、凝胶渗透色谱对CPP进行结构表征。雄性Balb/c小鼠于右前肢腋下接种H22原代细胞，随机分为模型组、索拉非尼（30 mg/kg）组及CPP低、中、高剂量（200、400、800 mg/kg）组，每组10只，另取10只正常小鼠作为对照组。连续给药16 d，计算抑瘤率；采用苏木素-伊红（hematoxylin-eosin，HE）染色法观察小鼠肿瘤组织病理变化；取模型组和CPP中剂量组小鼠肿瘤组织，利用转录组学测序技术分析CPP发挥抗HCC活性的作用机制，结合京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）、基因本体（gene ontology，GO）富集分析等探究CPP抗HCC的关键信号靶点及作用通路；通过Western blotting、qRT-PCR、ELISA法进行体内机制验证。结果 CPP中的总糖质量分数为95.19%，蛋白质量分数为0.33%。结构表征结果显示，CPP是以α构型糖苷键连接的吡喃型糖环，呈不规则卷曲状，由甘露糖和葡萄糖（物质的量比为2.388∶1）组成，重均相对分子质量为1.27×10⁵。动物实验结果显示，CPP低、中、高剂量组对H22荷瘤小鼠模型的抑瘤率分别为45.13%、48.68%、35.94%，抗HCC最佳剂量为400 mg/kg；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CPP中剂量组胞核碎裂更为明显。转录组学结果显示CPP可能通过调节NOD样受体热蛋白结构域3（NOD like receptor family pyrin domain containing 3，NLRP3）信号通路发挥抗HCC作用。Western blotting结果显示，与模型组比较，CPP中剂量组小鼠肿瘤组织中NLRP3、凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing a CARD，ASC）、半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）和消皮素D（gasdermin D，GSDMD）表达水平显著降低（P＜0.05、0.01、0.001）；ELISA结果显示，与模型组比较，CPP中剂量组肿瘤组织中白细胞介素-1β（interleukin 1β，IL-1β）和IL-18水平显著降低（P＜0.001）。结论 CPP的抗HCC作用可能与抑制NLRP3炎症小体的激活，阻断Caspase-1/GSDMD介导的细胞焦亡通路有关。

Objective To analyze the chemical composition and characterize the polysaccharide structure of Shancigu (Cremastrae Pseudobulbus Pleiones Pseudobulbus) polysaccharide (CPP), and investigate its anti-hepatocellular carcinoma (HCC) efficacy and mechanism. Methods Total sugar and protein contents of CPP samples were determined using phenol-sulfuric acid method and Coomassie brilliant blue method, respectively. Structural characterization was performed by UV spectroscopy, infrared spectroscopy, scanning electron microscopy, PMP pre-column derivatization-HPLC and gel permeation chromatography. Male Balb/c mice were inoculated with H22 primary cells under the right forelimb axilla, mice were randomly divided intomodel, sorafenib (30 mg/kg) group and CPP low-, medium-. high-dose (200, 400, 800 mg/kg) groups, with 10 mice per group, another 10 normal mice were selected as the control group. Administration was performed for 16 consecutive days, tumor inhibition rates were calculated. Hematoxylin-eosin (HE) staining was used to observe histopathological changes in tumor tissues. Tumor tissues from model group and CPP medium-dose group were analyzed using transcriptomic sequencing to elucidate the mechanism of CPP’s anti-HCC activity. Key signaling targets and pathways of CPP against HCC were investigated through Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analysis. In vivo mechanism validation was performed using Western blotting, qRT-PCR and ELISA. Results CPP contained 95.19% total sugar and 0.33% protein. The structural characterization results showed that CPP was a pyran sugar ring connected by α-glycosidic bonds, irregularly coiled, composed of mannose and glucose (with a mass ratio of 2.388∶1), and had a weight average relative molecular weight of 1.27 × 10⁵. The animal experiment results showed that CPP low-, medium- and high-dose groups had tumor inhibition rates of 45.13%, 48.68% and 35.94% on H22 tumor bearing mouse models, respectively. The optimal dose for anti-HCC was 400 mg/kg. Histopathological sections revealed significant tumor tissue lesions in model group, with improvements observed across all treatment groups, particularly marked nuclear fragmentation in CPP medium-dose group. Transcriptomic analysis suggested CPP may exert its anti-HCC effects by modulating NOD like receptor family pyrin domain containing 3 (NLRP3) signaling pathway. Western blotting results showed that compared with model group, the expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cystein-asparate protease-1 (Caspase-1) and gasdermin D (GSDMD) in tumor tissues of mice in CPP medium-dose group were significantly reduced (P < 0.05, 0.01, 0.001). ELISA results showed that compared with model group, the levels of interleukin-1β (IL-1β) and IL-18 in tumor tissues of CPP medium-dose group were significantly reduced (P < 0.001). Conclusion The anti-HCC effect of CPP may be related to the inhibition of NLRP3 inflammasome activation and blockade of Caspase-1/GSDMD mediated cell apoptosis pathway.

R285.5

国家自然科学基金资助项目（82073971）