目的 探討紫龍金片聯(lián)合埃克替尼通過表皮生長(zhǎng)因子受體（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）通路對(duì)荷瘤小鼠的抑瘤作用及細(xì)胞凋亡的機(jī)制。方法 建立Lewis肺癌荷瘤小鼠模型，隨機(jī)分為模型組、紫龍金片（375 mg/kg）組、?？颂婺幔? mg/kg）組、紫龍金片（375 mg/kg）＋?？颂婺幔? mg/kg）聯(lián)合治療組，每組10只。各給藥組ig相應(yīng)藥物（10 mL/kg），模型組ig等量生理鹽水，2次/d，間隔12 h，持續(xù)14 d。末次給藥后切除移植瘤，觀察腫瘤質(zhì)量、腫瘤體積，計(jì)算腫瘤生長(zhǎng)抑制率；蘇木素-伊紅（hematoxylin-eosin，HE）染色觀察小鼠瘤體病理形態(tài)學(xué)變化；末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的缺口末端標(biāo)記（terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling，TUNEL）法測(cè)定腫瘤細(xì)胞凋亡率；蛋白免疫印跡（Western blotting，WB）法檢測(cè)瘤組織中Bcl-2相關(guān)X蛋白（Bcl-2-associated X protein，Bax）、B細(xì)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通路相關(guān)蛋白的表達(dá)情況。結(jié)果 與模型組比較，紫龍金片組、?？颂婺峤M及聯(lián)合治療組小鼠的移植瘤質(zhì)量及腫瘤生長(zhǎng)抑制率顯著降低（P＜0.05）；與單獨(dú)給藥比較，聯(lián)合治療組的腫瘤生長(zhǎng)抑制率顯著降低（P＜0.05）。HE染色結(jié)果顯示，各給藥組腫瘤組織中均出現(xiàn)不同程度的壞死，其中聯(lián)合治療組的壞死區(qū)域最為明顯。TUNEL檢測(cè)結(jié)果顯示，與模型組比較，各給藥組的腫瘤細(xì)胞凋亡率顯著升高（P＜0.05、0.01、0.001）；與單獨(dú)給藥組比較，聯(lián)合治療組的腫瘤細(xì)胞凋亡率顯著升高（P＜0.05、0.01）。WB結(jié)果顯示，與模型組比較，紫龍金片組、?？颂婺峤M及聯(lián)合治療組的瘤組織中Bax、Caspase-9表達(dá)顯著升高（P＜0.05、0.01、0.001），Bcl-2表達(dá)顯著降低（P＜0.05、0.01、0.001）；與單獨(dú)給藥組比較，聯(lián)合治療組的變化最為明顯（P＜0.05、0.01）。與模型組比較，各給藥組的磷酸化-EGFR（phosphorylated-EGFR，p-EGFR）、p-PI3K、p-Akt和p-mTOR蛋白的表達(dá)均顯著降低（P＜0.05）；與單獨(dú)給藥比較，聯(lián)合治療組的下降幅度最大（P＜0.05）。結(jié)論 紫龍金片聯(lián)合?？颂婺崮軌蝻@著抑制荷瘤小鼠腫瘤的生長(zhǎng)，促進(jìn)腫瘤細(xì)胞凋亡，其機(jī)制可能是通過調(diào)控EGFR/PI3K/Akt信號(hào)通路的關(guān)鍵靶點(diǎn)，上調(diào)促凋亡蛋白Bax和Caspase-9的表達(dá)，下調(diào)抗凋亡蛋白Bcl-2的表達(dá)，從而協(xié)同發(fā)揮抑瘤作用。

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.

R285.5

第四屆國(guó)醫(yī)大師傳承工作室和第二屆全國(guó)名中醫(yī)傳承工作室建設(shè)項(xiàng)目[國(guó)中醫(yī)藥人教函（2022）75號(hào)];石河子大學(xué)自然科學(xué)項(xiàng)目（ZZZC201945A）