目的 优化鄂尔多斯钝顶螺旋藻多糖（Spirulina platensis polysaccharides，SPP）提取工艺并探讨SPP通过调控细胞乙酰化水平抑制肝细胞癌（hepatocellular carcinoma，HCC）的作用。方法 采用热水提取法、反复冻融法、超声辅助提取法提取SPP，单因素实验考察3种方法对SPP提取率的影响；利用响应面法优化SPP的提取工艺。利用DEAE-纤维素分离SPP得到中性糖级分（neutral polysaccharides form S. platensis，SPPN）和酸性糖级分（acidic polysaccharides form S. platensis，SPPA），通过CCK-8实验检测SPP及其各多糖级分对HCC细胞增殖的影响；利用1 mg/mL SPP或SPPN处理HepG2细胞24 h，检测细胞内多种酰化修饰水平；利用1 µmol/L去乙酰化酶抑制剂（histone deacetylase inhibitor，HDACI）及1 mg/mL SPPN联合处理HepG2细胞，检测细胞活力及半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）蛋白表达；构建Hepa1-6肝癌荷瘤小鼠模型，连续7 d ig SPPN，考察SPPN的体内抗肿瘤作用。结果 超声辅助提取法SPP的提取率最高；通过响应面试验得到最优工艺条件为超声时间10 min、超声功率300 W、液料比25∶1，此条件下SPP提取率为（10.42±0.30）%。SPPA显著促进HepG2细胞增殖（P＜0.01），而SPPN对HCC细胞均有显著杀伤作用（P＜0.01），对正常小鼠肝细胞无明显影响；荷瘤小鼠模型进一步证实SPPN具有抗肿瘤作用（P＜0.05）。SPP和SPPN能够改变细胞内多种酰化修饰水平，其中对乙酰化水平的影响较大。HDACI与SPPN联合处理显著增强SPPN对HCC细胞的杀伤作用（P＜0.01），并显著降低细胞内Caspase-3、Bcl-2蛋白表达水平（P＜0.01）。结论 响应面优化后SPP的提取率为（10.42±0.30）%，且分级后的SPPN可通过调控细胞乙酰化水平抑制HCC细胞增殖。

Objective To optimize the extraction process of Spirulina platensis polysaccharides (SPP) and explore the role of SPP in inhibiting hepatocellular carcinoma (HCC) through the regulation of cellular acetylation levels. Methods Hot water extraction method, repeated freeze-thaw method and ultrasound assisted extraction method were used to extract SPP. Single factor experiments were conducted to investigate the effects of these three methods on the extraction rate of SPP. The extraction process was optimized using response surface methodology. The crude polysaccharides were fractionated by DEAE cellulose chromatography, yielding neutral (SPPN) and acidic (SPPA) polysaccharides. The effects of various fractions on the proliferation of HCC cells were detected by CCK-8 experiment. HepG2 cells were treated with 1 mg/mL SPP or SPPN for 24 h, the levels of various acylation modifications in cells was detected. HepG2 cells were treated with 1 µmol/L histone deacetylase inhibitor (HDACI) and 1 mg/mL SPPN, the cell viability and protein expressions of cysteine aspartate protease-3 (Caspase-3) and B-cell lymphoma-2 (Bcl-2) were detected. Hepa1-6 liver cancer mouse model was constructed and continuously ig SPPN for 7 d to investigate the in vivo anti-tumor effect of SPPN. Results The ultrasound assisted extraction method had the highest extraction rate for SPP. The optimal process conditions obtained through response surface testing were ultrasonic time of 10 min, ultrasonic power of 300 W, and liquid to material ratio of 25∶1. Under these conditions, the SPP extraction rate was (10.42 ±0.30)%. SPPA significantly promoted proliferation of HepG2 cells (P < 0.01), while SPPN had a significant killing effect on HCC cells (P < 0.01), with no significant effect on normal mouse liver cells. The tumor bearing mouse model further confirmed that SPPN had anti-tumor effects (P < 0.05). SPP and SPPN could alter various levels of intracellular acylation modifications, with a significant impact on acetylation levels. The combined treatment of HDACI and SPPN significantly enhanced the killing effect of SPPN on HCC cells (P < 0.01), and significantly reduced the expression levels of Caspase-3 and Bcl-2 proteins in cells (P < 0.01). Conclusion The extraction rate of SPP after response surface optimization is (10.42 ±0.30)%, and the graded SPPN could inhibit HCC cell proliferation by regulating cell acetylation levels.

R285.5

国家自然科学基金资助项目（32060217）；内蒙古自治区青年拔尖人才项目；内蒙古医科大学致远人才项目（ZY20242103）；内蒙古医科大学面上项目（2025MS08075）；内蒙古医科大学“创客培育”项目（101322025082）