小艾飞蜜膏乙醇提取物及其活性成分胡椒碱对胃癌的作用和机制研究

Study on the Effects and Mechanisms of the Ethanol Extract of Xiaoaifei Honey Ointment and It’s Active Component Piperine on Gastric Cancer

关键词:  胃癌  小艾飞蜜膏乙醇提取物  石油醚萃取部位  胡椒碱

Key words:  gastric cancer  ethanol extract of compound Xiao-ai-fei honey ointment  petroleum-ether fraction  piperine

摘要:

摘要：目的 研究小艾飞蜜膏乙醇提取物(Ethanol extract of Xiao-ai-fei honey ointment，EXHO)及其活性成分胡椒碱对胃癌的作用和机制研究。方法 建立ICR小鼠胃癌(Mouse Forestomach Carcinoma, MFC)皮下荷瘤模型,成模后给予不同剂量EXHO，连续给药14 d，计算抑瘤率；利用溶剂萃取法获得EXHO不同剂型部位，噻唑蓝比色法(Methylthiazolyldiphenyl-tetrazolium bromide, MTT)对其进行活性筛选；气相色谱-质谱联用法(Gas Chromatography-Mass Spectrometry，GC-MS)对活性最佳的EXHO石油醚部位进行成分分析；利用网络药理学方法进行活性成分与作用靶点预测；再次建立ICR小鼠MFC胃癌荷瘤模型，胡椒碱不同剂量连续给药14 d，计算抑瘤率；HE染色法观察各组小鼠的主要脏器组织病理学变化，免疫组化法观察肿瘤组织中增殖细胞核抗原(Proliferating Cell Nuclear Antigen,PCNA)、血管内皮生长因子(Vascular Endothelial Growth Factor，VEGF)表达；细胞计数试剂盒-8(CCK-8)法检测胡椒碱对BGC-823细胞增殖的影响；酶联免疫吸附试验?法（Enzyme-Linked ImmunoSorbent Assay,ELISA）法检测细胞凋亡酶活性；免疫印迹(Western-blotting)法检检测细胞凋亡和细胞周期蛋白表达。结果 体内实验表明，与模型组相比，EXHO低、中、高剂量组瘤重显著降低(P＜0.05，P＜0.01)；体外实验表明，与空白对照组相比，EXHO石油醚萃取部位对细胞抑制作用最强(P＜0.01)；石油醚萃取部位GC-MS分析共检测出23个化合物，经化合物靶点预测筛选出作用最佳单体化合物胡椒碱；体内实验表明，与模型组相比，胡椒碱低、中、高剂量组瘤重显著降低(P＜0.05，P＜0.01)；组织病理学结果显示，胡椒碱高剂量组小鼠肺部出现轻微病理变化；与模型组相比，胡椒碱中、高剂量组动物肿瘤组织中PCNA、VEGF表达明显降低（P＜0.01)；体外实验表明，胡椒碱各剂量组均能抑制BGC-823细胞增殖，与空白对照组相比，胡椒碱各浓度下处理的BGC-823细胞内半胱天冬酶-3(Cysteine-dependent aspartate-specific protease-3,Caspase-3)、半胱天冬酶-8(Cysteine-dependent aspartate-specific protease-8,Caspase-8)、半胱天冬酶-9(Cysteine-dependent aspartate-specific protease-9,Caspase-9)酶活性均显著增加;Caspase-3、Caspase-8、Caspase-9、Bcl-2相关X蛋白(Bcl-2-associated X protein, Bax)表达显著升高，信号转导和转录激活因子3?(Signal Transducer and Activator of Transcription 3?，STAT3)、细胞周期蛋白B1(CyclinB1)、细胞周期蛋白D1(CyclinD1)、B细胞淋巴瘤-2 (B-cell lymphoma/leukemia-2,Bcl-2)表达显著降低(P＜0.05，P＜0.01)。结论 EXHO及其单体成分胡椒碱能抑制MFC胃癌小鼠肿瘤生长，其机制可能与抑制肿瘤细胞增殖、以及调节Caspase、Bax/Bcl-2信号通路和细胞周期蛋白CyclinB1、CyclinD1的表达有关。

Abstract:

ABSTRACT: OBJECTIVE To investigate the effects and mechanisms of the ethanol extract of compound Xiao-ai-fei honey ointment (EXHO) and it’s active component piperine on gastric cancer. METHODS ICR mice bearing subcutaneous Mouse Forestomach Carcinoma (MFC) were treated with graded doses of EXHO for 14 days and tumor inhibition rates were calculated. EXHO was sequentially partitioned to yield different fractions; activity-guided screening was performed by MTT assay. The most potent petroleum-ether fraction was analyzed by GC-MS, and network pharmacology was employed to predict active compounds and their targets. A second MFC-bearing mouse model received piperine at different doses for 14 days and tumor inhibition was again assessed. Major organs were examined by H&E staining, while PCNA and VEGF expression in tumors was evaluated immunohistochemically. In vitro, CCK-8 assay measured piperine-induced growth inhibition of BGC-823 cells; caspase activities were quantified by ELISA and apoptosis- and cell-cycle-related proteins were analyzed by Western blotting. RESULTS In vivo: Tumor weight was markedly reduced in EXHO low-, medium- and high-dose groups versus the model group (P < 0.05, P < 0.01).In vitro: The petroleum-ether fraction of EXHO exhibited the strongest inhibition on BGC-823 cells compared with blank control (P < 0.01). GC-MS identified 23 compounds; target prediction singled out piperine as the most active constituent. Second in vivo study: In vivo: Tumor weight was significantly reduced in the piperine low-, medium-, and high-dose groups compared with the model group (P < 0.05, P < 0.01). Histopathology showed pulmonary changes in the high-dose piperine group. Immunohistochemistry revealed pronounced down-regulation of PCNA and VEGF in tumors from the medium- and high-dose piperine groups (P < 0.01).Cell assays: Piperine suppressed BGC-823 proliferation in a dose-dependent manner. Relative to blank control, piperine-treated cells displayed significantly elevated activities of caspase-3, -8 and -9 (P < 0.05, P < 0.01) and increased protein levels of caspase-3, -8, -9 and Bax, whereas STAT3, Cyclin B1, Cyclin D1 and Bcl-2 were markedly decreased (P < 0.05, P < 0.01). CONCLUSION EXHO and its active monomer piperine inhibit MFC tumor growth in mice, presumably by suppressing tumor-cell proliferation and modulating the caspase and Bax/Bcl-2 pathways together with down-regulation of Cyclin B1 and Cyclin D1.