雪胆素片联合阿霉素增强细胞免疫原性死亡的抗肝癌作用研究

Anti-hepatocellular carcinoma effect of combination of Xuedansu Tablets with doxorubicin on enhancing immunogenic cell death

目的研究雪胆素片联合阿霉素协同抗肝癌的作用,以及对细胞免疫原性死亡相关蛋白表达的调控。方法采用高效液相色谱法对雪胆素片主要成分进行分析;采用MTT法考察雪胆素片与阿霉素联合使用对人肝癌HepG2细胞存活率的影响;采用平板克隆观察雪胆素片与阿霉素联合使用对HepG2细胞集落形成的影响;采用流式细胞仪分析雪胆素片与阿霉素联合使用对HepG2细胞凋亡率和细胞周期的影响;采用免疫荧光检测免疫原性细胞死亡关键指标钙网蛋白(calreticulin,CRT)和高迁移率蛋白1(high mobility group protein B1,HMGB1)表达;采用ELISA法检测细胞内外三磷酸腺苷(adenosine triphosphate,ATP)浓度。建立小鼠肝癌H22细胞皮下移植瘤模型观察雪胆素片与阿霉素联合使用的体内抗肿瘤效果;采用免疫组化法检测小鼠肿瘤组织CRT和HMGB1表达;采用苏木素-伊红(HE)染色法观察雪胆素片与阿霉素联合使用对小鼠心肌毒性的影响。结果雪胆素片联合阿霉素对HepG2细胞具有浓度相关性的细胞毒性和促凋亡作用,可促进CRT膜外暴露,HMGB1向胞外分泌,ATP胞外浓度增加;雪胆素片联合阿霉素可抑制H22细胞小鼠皮下移植瘤的生长,提高免疫能力,延长其生存期,增加小鼠皮下肿瘤组织内CRT和HMGB1蛋白阳性表达的细胞数量。结论雪胆素片联合阿霉素的抗肝癌作用可能与调节免疫原性死亡相关蛋白表达有关。

Objective To study the synergistic effect of Xuedansu Tablets(雪胆素片)combined with doxorubicin against liver cancer,and the regulation of cell immunogenic death-related proteins expressions.Methods The main components of Xuedansu Tablets were analyzed by high performance liquid chromatography;Effect of Xuedansu Tablets combined with doxorubicin on survival rate of human hepatocellular carcinoma HepG2 cells was investigated by MTT method;Effect of Xuedansu Tablets combined with doxorubicin on colony formation of HepG2 cells was detected by plate cloning;Flow cytometry was used to analyze the effect of Xuedansu Tablets combined with doxorubicin on apoptosis rate and cell cycle of HepG2 cells;Immunofluorescence was used to detect expressions of calreticulin(CRT)and high mobility protein 1(HMGB1)which are key indicators of immunogenic cell death;Intracellular and extracellular adenosine triphosphate(ATP)concentrations were detected by ELISA.H22 tumor-bearing mice model was established to observe in vivo antitumor effect of Xuedansu Tablets combined with doxorubicin;CRT and HMGB1 expressions of tumor tissue in mice were detected by immunohistochemistry;Hematoxylin-eosin(HE)staining method was used to observe the effect of Xuedansu Tablets combined with doxorubicin on myocardial toxicity in mice.Results Xuedansu Tablets combined with doxorubicin had concentration-dependent cytotoxic and pro-apoptotic effects on HepG2 cells,which could promote the extracellular exposure of CRT,secretion of HMGB1 into extracellular,and increase in extracellular concentration of ATP;Xuedansu Tablets combined with doxorubicin inhibited the growth of subcutaneous transplanted tumors in H22-cell mice,improved immunity,prolonged their survival,increased the number of cells expressing CRT and HMGB1 proteins in subcutaneous tumor tissues of mice.Conclusion Anti-hepatocellular carcinoma effect of Xuedansu Tablets combined with doxorubicin may be related to the regulation of immunogenic death-related proteins expressions.