基于ROS/HIF-1通路探讨白花蛇舌草多糖对鼻咽癌裸小鼠肿瘤的抑制作用及机制

目的:基于活性氧（ROS）/缺氧诱导因子-1（HIF-1）通路探讨白花蛇舌草多糖对鼻咽癌裸小鼠肿瘤的抑制作用及其可能作用机制。方法:56只BALB/c裸小鼠于右侧腋下接种CNE-2细胞悬液诱导鼻咽癌成瘤，成瘤后的裸小鼠随机分为:模型组、顺铂组（DDP，5 mg/kg）、白花蛇舌草多糖低剂量组（50 mg/kg）、白花蛇舌草多糖中剂量组（100 mg/kg）、白花蛇舌草多糖高剂量组（200 mg/kg）、阴性对照组（NC-siRNA）、si-HIF-1α组、si-HIF-1α+白花蛇舌草多糖组，每组6只，剩余6只裸小鼠作为空白组。各组按相应剂量分别腹腔注射给药，连续14天。测定裸小鼠瘤重及肿瘤体积［苏木素-伊红（HE）染色观察肿瘤组织病理学变化［流式细胞术检测肿瘤组织Treg细胞数量［免疫组化染色检测肿瘤组织Foxp3表达［荧光TUNEL染色检测肿瘤组织细胞凋亡［酶联免疫吸附测定法（ELISA）检测肿瘤组织中ROS含量［实时荧光定量聚合酶链式反应（real-time PCR）和蛋白免疫印迹法（Western blot）分别检测肿瘤组织HIF-1α、GLUT1、HK2 mRNA及蛋白表达。结果:与模型组比较，白花蛇舌草多糖高剂量组可明显降低肿瘤重量，降低Foxp3、HIF-1α、GLUT1、HK2表达，明显缩小瘤体积，降低Treg细胞数量，明显增加细胞凋亡及ROS含量（P＜0.01）。与si-HIF-1α组比较，si-HIF-1α+白花蛇舌草多糖组HIF-1α、GLUT1、HK2表达明显降低（P＜0.05）。与模型组比较，si-HIF-1α+白花蛇舌草多糖组ROS含量明显升高，瘤重及肿瘤体积明显减小（P＜0.05）。结论:白花蛇舌草多糖对鼻咽癌裸小鼠肿瘤具有抑制作用，其作用机制可能与降低HIF-1α、GLUT1、HK2表达有关。

Exploring the tumor suppressive effect and mechanism of polysaccharides extracts from hedyotis diffusa on nasopharyngeal carcinoma nude mice based on ROS/HIF-1 pathway

Objective:To investigate the tumor suppressive effect of polysaccharides extracts from hedyotis diffusa on nasopharyngeal carcinoma mice based on reactive oxygen species (ROS)/hypoxia-inducible factor-1 (HIF-1) pathway and to elucidate its possible mechanism of action.Methods:Fifty-six BALB/c nude mice were inoculated with CNE-2 cell suspension in the right axilla to induce nasopharyngeal carcinoma tumor formation.The tumor-forming nude mice were randomly divided into model group,cisplatin group (DDP,5 mg/kg),polysaccharides extracts from hedyotis diffusa low-dose group (50 mg/kg),polysaccharides extracts from hedyotis diffusa medium-dose group (100 mg/kg),polysaccharides extracts from hedyotis diffusa high-dose group (200 mg/kg),negative control group (NC-siRNA),si-HIF-1α group,si-HIF-1α+polysaccharide extracts from hedyotis diffusa group,6 mice in each group,and the remaining 6 nude mice as a control group.Each group was administered intraperitoneally at the corresponding doses for 14 consecutive days.The tumor weight and tumor volume of nude mice were measured.The histopathological changes of tumor were observed by hematoxylin-eosin (HE) staining.The number of Treg cells in tumor tissues was detected by flow cytometry.The expression of Foxp3 in tumor tissues was detected by immunohistochemical staining.The apoptosis in tumor tissues was detected by fluorescent TUNEL staining.The ROS content in tumor tissues was detected by ELISA.The real-time fluorescence real-time PCR and Western blot were used to detect HIF-1α,GLUT1 and HK2 mRNA and protein expression in tumor tissues,respectively.Results:Compared with the model group,the high-dose group of polysaccharides extracts from hedyotis diffusa significantly reduced tumor weight,decreased Foxp3,HIF-1α,GLUT1,and HK2 expression,significantly reduced tumor volume,decreased Treg cells,and significantly increased apoptosis and ROS content (P＜0.01).Compared with si-HIF-1α group,the expression of HIF-1α,GLUT1 and HK2 was significantly decreased in si-HIF-1α+polysaccharides extracts from hedyotis diffusa group (P＜0.05).Compared with the model group,the ROS content of si-HIF-1α+polysaccharides extracts from hedyotis diffusa group was significantly increased,and the tumor weight and tumor volume were significantly reduced (P＜0.05).Conclusion:Polysaccharides extracts from hedyotis diffusa has inhibitory effect on nasopharyngeal carcinoma tumors in nude mice,and its mechanism of action may be related to the reduction of HIF-1α,GLUT1 and HK2 expression.