目的 探讨紫龙金片联合埃克替尼通过表皮生长因子受体（epidermal growth factor receptor，EGFR）/磷脂酰肌醇3-激酶（phosphoinositide 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）通路对荷瘤小鼠的抑瘤作用及细胞凋亡的机制。方法 建立Lewis肺癌荷瘤小鼠模型，随机分为模型组、紫龙金片（375 mg/kg）组、埃克替尼（5 mg/kg）组、紫龙金片（375 mg/kg）＋埃克替尼（5 mg/kg）联合治疗组，每组10只。各给药组ig相应药物（10 mL/kg），模型组ig等量生理盐水，2次/d，间隔12 h，持续14 d。末次给药后切除移植瘤，观察肿瘤质量、肿瘤体积，计算肿瘤生长抑制率；苏木素-伊红（hematoxylin-eosin，HE）染色观察小鼠瘤体病理形态学变化；末端脱氧核苷酸转移酶介导的缺口末端标记（terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling，TUNEL）法测定肿瘤细胞凋亡率；蛋白免疫印迹（Western blotting，WB）法检测瘤组织中Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）、半胱氨酸天冬氨酸蛋白酶-9（cystein-asparate protease-9，Caspase-9）及EGFR/PI3K/Akt通路相关蛋白的表达情况。结果 与模型组比较，紫龙金片组、埃克替尼组及联合治疗组小鼠的移植瘤质量及肿瘤生长抑制率显著降低（P＜0.05）；与单独给药比较，联合治疗组的肿瘤生长抑制率显著降低（P＜0.05）。HE染色结果显示，各给药组肿瘤组织中均出现不同程度的坏死，其中联合治疗组的坏死区域最为明显。TUNEL检测结果显示，与模型组比较，各给药组的肿瘤细胞凋亡率显著升高（P＜0.05、0.01、0.001）；与单独给药组比较，联合治疗组的肿瘤细胞凋亡率显著升高（P＜0.05、0.01）。WB结果显示，与模型组比较，紫龙金片组、埃克替尼组及联合治疗组的瘤组织中Bax、Caspase-9表达显著升高（P＜0.05、0.01、0.001），Bcl-2表达显著降低（P＜0.05、0.01、0.001）；与单独给药组比较，联合治疗组的变化最为明显（P＜0.05、0.01）。与模型组比较，各给药组的磷酸化-EGFR（phosphorylated-EGFR，p-EGFR）、p-PI3K、p-Akt和p-mTOR蛋白的表达均显著降低（P＜0.05）；与单独给药比较，联合治疗组的下降幅度最大（P＜0.05）。结论 紫龙金片联合埃克替尼能够显著抑制荷瘤小鼠肿瘤的生长，促进肿瘤细胞凋亡，其机制可能是通过调控EGFR/PI3K/Akt信号通路的关键靶点，上调促凋亡蛋白Bax和Caspase-9的表达，下调抗凋亡蛋白Bcl-2的表达，从而协同发挥抑瘤作用。

Objective To investigate the mechanism of tumour-suppressive effect and apoptosis of Zilongjin Tablets combined with icotinib through epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in tumor-bearing mice.Methods Lewis lung cancer mouse model was established, and ten mice were randomly divided into model group, Zilongjin Tablets (375 mg/kg) group, icotinib (5 mg/kg) group, and Zilongjin Tablets (375 mg/kg) + icotinib (5 mg/kg) combination treatment group, with ten mice in each group. Each administration group ig the corresponding drug (10 mL/kg), and the model group ig an equal amount of saline, twice/d, at an interval of 12 h for 14 d. The transplanted tumor was resected after the last administration of the drug, and the tumor mass and tumor volume were observed, and the tumor growth inhibition rate was calculated, the hematoxylin-eosin (HE) staining was used to observe the pathological and morphological changes of the tumors in mice, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method was used to determine the apoptosis rate of the tumor cells. Western blotting (WB) was used to detect Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), cystein-asparate protease-9 (Caspase-9), and EGFR/PI3K/Akt pathway related protein expression. Results Compared with the model group, the transplanted tumor mass and tumor growth inhibition rate of mice in the Zilongjin Tablets group, the icotinib group and the combined treatment group were significantly reduced (P < 0.05), compared with the drug alone, the tumor growth inhibition rate of the combined treatment group was significantly reduced (P < 0.05). HE results showed that necrosis of varying degrees was observed in tumor tissues of the various groups of the drug administration, with the most pronounced necrotic areas in the combined treatment group. TUNEL assay results showed that the apoptosis rate of tumor cells in each administration group was significantly higher compared with that in the model group (P < 0.05, 0.01, 0.001), and the apoptosis rate of tumor cells in the combination therapy group was significantly higher compared with that in the drug alone administration group (P < 0.05, 0.01). WB results showed that the expression of Bax and Caspase-9 was significantly higher in tumor tissues of the Zuolongjin Tablets group, the icotinib group and the combination therapy group compared with that in the model group (P < 0.05, 0.01, 0.001), and Bcl-2 expression was significantly lower (P < 0.05, 0.01, 0.001), the most obvious changes were observed in the combination therapy group compared with the drug alone administration group (P < 0.05), compared with the administration of the drug alone, the combined treatment group showed the greatest decrease (P < 0.05). Conclusion Zilongjin Tablets combined with icotinib can significantly inhibit the growth of tumors and promote apoptosis of tumor cells in tumor-bearing mice, and the mechanism may be to synergistically exert a tumor-suppressive effect by regulating the key targets of the EGFR/PI3K/Akt signalling pathway, up-regulating the expression of pro-apoptotic proteins Bax and Caspase-9, and down-regulating the expression of anti-apoptotic protein Bcl-2.

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第四届国医大师传承工作室和第二届全国名中医传承工作室建设项目[国中医药人教函（2022）75号];石河子大学自然科学项目（ZZZC201945A）