目的 研究石蒜碱对肝癌H₂₂细胞荷瘤小鼠红细胞膜结构功能的影响。方法 通过抑瘤率实验、生命延长率实验、流式细胞仪检测肿瘤细胞凋亡率实验，验证石蒜碱的体内抗肿瘤活性；采用试剂盒测定红细胞膜总蛋白、胆固醇和唾液酸含量，考察石蒜碱对H₂₂荷瘤小鼠红细胞膜主要组分的影响；采用荧光偏振法检测红细胞膜流动性，荧光染色法检测红细胞膜封闭度，验证石蒜碱对H₂₂荷瘤小鼠红细胞膜结构功能的影响；采用三磷酸腺苷（adenosine triphosphate，ATP）酶活性检测试剂盒、荧光分光光度计、激光共聚焦扫描显微镜、Western blotting法，检测石蒜碱对H₂₂荷瘤小鼠红细胞膜离子通道相关因子活性的影响；采用Western blotting法检测石蒜碱对H₂₂荷瘤小鼠肿瘤细胞内活化的半胱氨酸蛋白酶-3（cleaved Caspase-3）、cleaved Caspase-9、B淋巴细胞瘤2（B-cell lymphoma 2，Bcl-2）和Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）表达的影响。结果 石蒜碱能够显著抑制H₂₂荷瘤小鼠肿瘤生长、有效延长H₂₂荷瘤小鼠生存时间，并且能够诱导H₂₂荷瘤小鼠肿瘤细胞凋亡（P<0.05、0.01）；石蒜碱能够增加H₂₂荷瘤小鼠红细胞膜总蛋白、唾液酸和胆固醇含量，同时提高红细胞膜流动性和封闭度，增强H₂₂荷瘤小鼠红细胞膜Na⁺， K⁺-ATP、Ca²⁺， Mg²⁺-ATP酶活性，升高H₂₂荷瘤小鼠红细胞内pH值，降低红细胞内H⁺、Ca²⁺、Cl⁻浓度，增强H₂₂荷瘤小鼠红细胞对HCO³⁻/Cl⁻转运活性，升高Band 3蛋白表达水平（P<0.01）；石蒜碱能够升高H₂₂荷瘤小鼠肿瘤细胞内cleaved Caspase-3、cleaved Caspase-9蛋白表达水平，升高促凋亡蛋白Bax表达、降低抑凋亡蛋白Bcl-2表达，使Bcl-2/Bax降低（P<0.01）。结论 石蒜碱对H₂₂荷瘤小鼠具有抗肿瘤作用，能够通过调控H₂₂荷瘤小鼠红细胞膜组成结构，提高细胞膜离子通道的活性，在离子通道之间建立联系相互促进，通过这种相互促进作用来调节胞内酸碱度、离子平衡，使红细胞膜结构功能得到改善，进而增强红细胞免疫功能，并调控肿瘤细胞内凋亡相关蛋白表达，激活肿瘤细胞凋亡相关因子，启动肿瘤细胞凋亡程序，从而发挥抗肿瘤作用。

ObjectiveTo study the effect of lycorine on structure and function of red blood cell membranes in H₂₂ tumor-bearing mice.Methods Anti-tumor activity of lycorine in vivo was verified by tumor inhibition rate experiment, life extension rate experiment, and tumor cells apoptosis rate test by flow cytometry. Contents of total protein, cholesterol and sialic acid of erythrocyte membrane were determined by assay kit to detect the effect of lycorine on content of main components of erythrocyte membrane in H₂₂ tumor-bearing mice. The erythrocyte membrane fluidity was detected by fluorescence polarization method, and erythrocyte membrane sealing degree was determined by Zamudio method to verify the effect of lycorine on erythrocyte membrane structure function of H₂₂ tumor-bearing mice. Adenosine triphosphate (ATP) activity assay kit, fluorescence spectrophotometer, laser confocal scanning microscopy and Western blotting method were used to detect the effect of lycorine on erythrocyte membrane ion channel activity of H₂₂ tumor-bearing mice. Western blotting was used to detect the effects of lycorine on protein expressions of cleaved Caspase-3, cleaved Caspase-9, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) in tumor cells of H₂₂ tumor-bearing mice. ResultsLycorine inhibited tumor growth in H₂₂ tumor-bearing mice, prolonged the survival time of H₂₂ tumor-bearing mice, and induced tumor cell apoptosis in H₂₂ tumor-bearing mice (P < 0.05, 0.01); Lycorine increased erythrocyte membrane total protein, sialic acid and cholesterol contents in H₂₂ tumor-bearing mice, improved the fluidity and tightness of erythrocyte membrane, enhanced the erythrocyte membrane Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activity of H₂₂ tumor-bearing mice, increased the intracellular pH of H₂₂ tumor-bearing mice, decreased H⁺, Ca²⁺, Cl⁻ concentrations in red blood cells, enhanced the HCO³⁻/Cl⁻ transport activity of H₂₂ tumor-bearing mice, and increased Band 3 protein expression (P < 0.01). Lycorine increased the protein expressions of cleaved Caspase-3 and cleaved Caspase-9, increased the expression of pro-apoptotic protein Bax, decreased the expression of anti-apoptotic protein Bcl-2, and reduced the ratio of Bcl-2/Bax (P < 0.01). ConclusionLycorine has anti-tumor effect on H₂₂tumor-bearing mice, and can improve the activity of cell membrane ion channel by changing the erythrocyte membrane structure of H₂₂ tumor-bearing mice, and establish a relationship between ion channels to promote each other. Through this mutual promotion, the intracellular pH and ion balance are adjusted, and the structural function of erythrocyte membrane is improved, thereby enhancing the immune function of red blood cells. In addition, lycorine regulates the activity of apoptosis-related proteins in tumor cells, activates the apoptosis-related factors, initiates the apoptosis-related process of tumor cells, and exerts the anti-tumor effect.

R285.5

中国博士后面上项目（2019M651296）；黑龙江省自然科学基金联合引导项目（LH2019H066）；哈尔滨商业大学青年创新人才项目（18XN024，2019CX37）；中央支持地方高校改革发展基金优秀青年人才项目（2020YQ12）；黑龙江省博士后科研启动资助项目（LBH-Q20026）；2021年哈尔滨商业大学教师“创新”项目支持计划项目