基于AMPK/ULK1介导的自噬探讨牡荆素对鼻咽癌CNE-2细胞裸鼠移植瘤生长抑制作用及其机制

Inhibitory effect and mechanism of vitexin on the growth of transplanted nasopharyngeal carcinoma CNE-2 cells in nude mice based on AMPK/ULK1 mediated autophagy

目的探究牡荆素对鼻咽癌CNE-2细胞裸鼠移植瘤生长的抑制作用及其可能的调控机制。方法将BALB/c裸鼠通过左侧前肢腋窝下接种CNE-2细胞构建鼻咽癌裸鼠成瘤模型,待模型构建成功后随机分为模型组、牡荆素组(5、10 mg/kg)、腺苷酸活化蛋白激酶(AMPK)激动剂组(AICAR,200 mg/kg)、牡荆素+AMPK抑制剂组(Compound C,20 mg/kg),每组各10只。牡荆素组分别按照相应剂量ig;AICAR组ip 200 mg/kg AICAR;牡荆素+Compound C组采取ig 10 mg/kg牡荆素的同时ip 20 mg/kg Compound C,以上各组连续给药21 d。给药结束后测定裸鼠移植瘤体积和质量;苏木素–伊红(HE)染色观察移植瘤组织病理学改变;TUNEL染色检测肿瘤组织细胞凋亡情况;免疫组织化学检测肿瘤组织微管相关蛋白轻链3(LC3)-II、泛素结合蛋白(p62)蛋白表达;免疫荧光检测肿瘤组织LC3-II和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)表达;Western blotting检测肿瘤组织Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、Casapse-3蛋白及AMPK/UNC-51样激酶1(ULK1)通路相关蛋白相对表达。结果与模型组相比,牡荆素各剂量组、AICAR组肿瘤生长缓慢,肿瘤体积和质量均显著降低(P<0.05);肿瘤细胞凋亡率,Caspase-3、LC3-II、Bax/Bcl-2、p-AMPK/AMPK、p-ULK1/ULK1蛋白表达均显著升高,p62蛋白表达均显著降低(P<0.05)。与牡荆素10 mg/kg组相比,牡荆素+Compound C组肿瘤生长良好,肿瘤体积和质量均增加(P<0.05),肿瘤细胞凋亡率,Caspase-3、LC3-II、Bax/Bcl-2、p-AMPK/AMPK、P-ULK1/ULK1蛋白表达均显著降低,p62蛋白表达均显著升高(P<0.05)。结论牡荆素能够抑制鼻咽癌CNE-2细胞裸鼠移植瘤生长,其机制与激活AMPK/ULK1通路介导的自噬,促进肿瘤细胞凋亡相关。

Objective To investigate the inhibitory effect of vitexin on the growth of nasopharyngeal carcinoma CNE-2 cells transplanted into nude mice and its possible regulatory mechanism.Methods BALB/c nude mice were inoculated with CNE-2 cells under the left forearm armpit to construct a nasopharyngeal carcinoma nude mice tumor model.After the model was successfully constructed,they were randomly divided into model group,vitexin group(5 and 10 mg/kg),AMPK agonist group(AICAR,200 mg/kg),and vitexin+AMPK inhibitor group(Compound C,20 mg/kg),with 10 mice in each group.Vitexin group was ig administered at corresponding doses,AICAR group received ip injection of 200 mg/kg AICAR,vitexin+Compound C group received 10 mg/kg vitexin by ig and ip injection of 20 mg/kg Compound C,and the above groups were continuously administered for 21 d.Measure the volume and mass of transplanted tumor in nude mice after administration.HE staining was used to observe the pathological changes of the transplanted tumor tissue,TUNEL staining was used to detect apoptosis in tumor tissue.Detection of LC3-II and p62 protein expression by immunohistochemical in tumor tissue,immunofluorescence detection of LC3-II and Caspase-3 expression in tumor tissue,Western blotting was used to detect the relative expression of Bax,Bcl-2,Casapse-3 proteins,and AMPK/ULK1 pathway related proteins in tumor tissue.Results Compared with the model group,the tumor growth was slow,tumor volume and mass were significantly reduced(P<0.05),tumor cell apoptosis rate,the protein expression of Caspase-3,LC3-II,Bax/Bcl-2,p-AMPK/AMPK,p-ULK1/ULK1 were significantly increased,and p62 protein expression were significantly reduced(P<0.05)in each dose group of vitexin.Compared with the 10 mg/kg group of vitexin,the vitexin+Compound C group showed good tumor growth,increased tumor volume and mass(P<0.05),increased tumor cell apoptosis rate,the protein expression of Caspase-3,LC3-II,Bax/Bcl-2,p-AMPK/AMPK,p-ULK1/ULK1 were significantly decreased,while p62 protein expression were significantly increased(P<0.05).Conclusion Vitexin can inhibit the growth of nasopharyngeal carcinoma CNE-2 cells transplanted into nude mice,and its mechanism is related to activating AMPK/ULK1 pathway mediated autophagy and promoting tumor cell apoptosis.