南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (3): 375-382.doi: 10.12122/j.issn.1673-4254.2023.03.06

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酒川芎增强阿美替尼对EGFR突变型非小细胞肺癌脑移植瘤的抑制作用

牛雯雯，荣翔宇，赵 倩，刘雪柔，徐廉松，李姗姗，李 娴   

  1. 蚌埠医学院药学院，安徽 蚌埠 233030；中药饮片制造新技术安徽省重点实验室，安徽 亳州 236800；安徽协和成药业饮片有限公司博士后工作站，安徽 亳州 236800
  • 出版日期:2023-03-20 发布日期:2023-03-20

Wine-processed Chuanxiong Rhizoma enhances efficacy of aumolertinib against EGFR-mutant non-small cell lung cancer xenografts in nude mouse brain

NIU Wenwen, RONG Xiangyu, ZHAO Qian, LIU Xuerou, XU Liansong, LI Shanshan, LI Xian   

  1. School of Pharmacy, Bengbu Medical College, Bengbu 233030, China; Anhui Provincial Key Laboratory of New Technology of Chinese Herbal Pieces Manufacturing, Bozhou 236800, China; Postdoctoral Workstation of Anhui Xiehecheng Pharmaceutical Yinpian Co Ltd, Bozhou 236800, China
  • Online:2023-03-20 Published:2023-03-20

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摘要： 目的 探讨酒川芎（WCR）增强阿美替尼抑制EGFR突变型非小细胞肺癌脑移植瘤的作用。方法 建立人脑内皮细胞（hCMEC/D3）和PC9非小细胞肺癌细胞共培养体系，采用流式细胞术考察酒川芎水提液（2 mg/mL）联合阿美替尼（10、20 μmol/L）对PC9细胞凋亡的影响；通过ABCB1-MDCK单层细胞模型评估酒川芎水提液（0.5、1、2 mg/mL）对阿美替尼（8 μmol/L）跨膜转运的影响。以Western blot法检测血脑屏障完整性相关的紧密连接蛋白表达；建立裸鼠非小细胞肺癌脑移植瘤模型，考察酒川芎（1 mg/g）联合阿美替尼（10 mg/kg）抑制EGFR突变型非小细胞肺癌脑移植瘤的作用。结果 与单用阿美替尼组（20 μmol/L）对比，酒川芎（2 mg/mL）联合阿美替尼（20 μmol/L）组给药可致细胞凋亡百分率增加21%（P<0.01）。酒川芎（0.5、1、2 mg/mL）联合阿美替尼的双侧转运Papp（A→B）较单用阿美替尼均显著增加（P<0.05）。与酒川芎联合后，紧密连接蛋白zonula occludens（ZO）-1，claudin-5和外排蛋白P-glycoprotein的蛋白表达明显减少（P<0.05）。在PC9-Luc脑移植瘤裸鼠模型中，酒川芎联合阿美替尼可明显提升单用阿美替尼的抑瘤效果，缓解脑移植瘤裸鼠体质量的下降，并延长脑移植瘤裸鼠的生存时间（P<0.05）；病理组织学染色结果显示，酒川芎联合阿美替尼可明显增加脑移植瘤裸鼠脑组织中肿瘤细胞的凋亡百分率（P<0.05），并改善瘤周脑组织神经损伤。免疫组化染色结果显示，酒川芎能明显下调脑组织中紧密连接蛋白ZO-1和claudin-5的蛋白表达。结论 酒川芎能增加阿美替尼抑制EGFR突变型非小细胞肺癌脑移植瘤的作用，其机制可能与下调ZO-1，claudin-5和P-glycoprotein蛋白表达，从而促进阿美替尼透过血脑屏障有关。

关键词: 非小细胞肺癌；酒川芎；阿美替尼；血脑屏障

Abstract: Objective To investigate the effect of wine-processed Chuanxiong Rhizoma (WCR) for enhancing the efficacy of aumolertinib against xenografts of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) in the brain of nude mice. Methods In a co-culture system of hCMEC/D3 and PC9 NSCLC cells, the effect of aqueous extract of WCR (2 mg/mL) combined with aumolertinib (10 and 20 μmol/L) on apoptosis of PC9 cells was investigated using flow cytometry. The effects of WCR extract (0.5, 1, and 2 mg/mL) on transmembrane transport of 8 μmol/L aumolertinib was examined in ABCB1-MDCK monolayer cells. Western blotting was used to detect the expressions of the tight junction proteins related with blood- brain barrier integrity. A nude mouse model bearing NSCLC xenograft in the brain was established to observe the inhibitory effect of WCR (1 mg/g) combined with aumolertinib (10 mg/kg) on tumor growth. Results Compared with aumolertinib (20 μmol/L) alone, WCR extract (2 mg/mL) combined with aumolertinib significantly increased the apoptosis rate of PC9 cells by 21% (P<0.01). The combined treatment with WCR (0.5, 1, 2 mg/mL) obviously increased apical-basolateral transport of aumolertinib in ABCB1-MDCK monolayer cells (P<0.05) and significantly lowered the expression levels of zonula occludens-1, claudin-5 and P-glycoprotein (P<0.05). In the tumor-bearing mice, compared with aumolertinib alone, the combined treatment with WCR and aumolertinib produced stronger inhibitory effect on tumor growth, improved weight loss, and prolonged the survival time of the nude mice (P<0.05). Pathological examination showed that the combined treatment obviously increased the apoptosis rate of the tumor cells and alleviated neural injuries in the brain. Immunohistochemistry revealed that WCR treatment significantly reduced the expressions of ZO-1 and claudin-5 in the brain of the mice. Conclusion WCR combined with aumolertinib shows stronger inhibitory effects against tumor xenografts of EGFR-mutant NSCLC possibly due to the effect of WCR in facilitating the transmembrane transport of aumolertinib by downregulating ZO-1, claudin-5 and P-glycoprotein expression.

Key words: non-small cell lung cancer; Ligusticum chuanxiong Hort.; aumolertinib; blood-brain barrier