昆虫学报 ›› 2021, Vol. 64 ›› Issue (9): 1041-1049.doi: 10.16380/j.kcxb.2021.09.004

• 研究论文 • 上一篇    下一篇

黑水虻抗菌肽HI-3对人结肠癌HCT-8细胞 谷氨酰胺和谷氨酸代谢通路的影响

高嘉敏, 许晓燕, 胡紫媛, 陈映丹, 孙虹霞, 杨愈丰, 夏嫱*   

  1.  (遵义医科大学珠海校区, 广东珠海 519041)
  • 出版日期:2021-09-20 发布日期:2021-09-03

Effects of antimicrobial peptide HI-3 from Hermetia illucens (Diptera: Stratiomyidae) on the glutamine and glutamic acid metabolic pathway of human colon carcinoma HCT-8 cells

 GAO Jia-Min, XU Xiao-Yan, HU Zi-Yuan, CHEN Ying-Dan, SUN Hong-Xia, YANG Yu-Feng, XIA Qiang*   

  1.  (Zhuhai Campus of Zunyi Medical University, Zhuhai, Guangdong 519041, China)
  • Online:2021-09-20 Published:2021-09-03

摘要:

 【目的】研究黑水虻Hermetia illucens抗菌肽HI-3对人结肠癌HCT-8细胞氨基酸代谢的影响,以丰富对其抑癌机理的认识。【方法】采用CCK-8法测定不同浓度(80, 160和320 μg/mL)抗菌肽HI-3对HCT-8细胞的抑制率;利用GC-MS进行HCT-8细胞代谢物测定,通过基于R软件的通路分析找出氨基酸含量差异最显著的氨基酸代谢通路并筛选出该通路靶标酶。320 μg/mL HI-3处理HCT-8细胞后,利用酶活性检测试剂盒测定靶标酶谷氨酰胺酶(GLS)活性;利用RT-qPCR和Western blot技术分别对HCT-8细胞的GLS基因进行mRNA及蛋白表达水平的测定;利用生化试剂盒和ELISA试剂盒检测HCT-8细胞内谷氨酰胺和谷氨酸代谢通路涉及的重要代谢物谷氨酰胺(Gln)、谷氨酸(Glu)、谷胱甘肽(GSH)、α-酮戊二酸(α-KG)和ATP含量的变化。【结果】浓度为80, 160和20 μg/mLHI-3对HCT-8细胞的抑制率分别为33.85%±3.50%, 46.26%±0.90%和55.53%±1.70%,且抑制率随HI3浓度升高而增大。320 μg/mL HI-3处理对谷氨酰胺和谷氨酸代谢通路的影响最大,其中氨基酸代谢物含量与阴性对照组(0 μg/mL HI-3)相比差异最为显著;这一通路中的靶标酶GLS活性及其GLS的mRNA和蛋白表达水平均极显著低于阴性对照组;另外与此通路相关的重要代谢物Gln, Glu, GSH, α-KG和ATP含量与阴性对照组相比亦显著减少。【结论】浓度为320 μg/mL黑水虻抗菌肽HI-3对HCT-8细胞谷氨酰胺和谷氨酸代谢通路影响最为显著,并能通过阻碍该通路来显著抑制HCT-8细胞的增殖。

关键词: 黑水虻, 抗菌肽, 人结肠癌, HCT-8细胞, 细胞代谢组学, 谷氨酰胺, 谷氨酸, 代谢通路

Abstract:

【Aim】 To enrich the knowledge of the anticancer mechanism of antimicrobial peptide HI-3 extracted from Hermetia illucens by studying the effects of HI-3 on the amino acid metabolism of human colon carcinoma HCT-8 cells. 【Methods】 CCK-8 assay was used to determine the inhibition rates of different concentrations (80, 160, and 320 μg/mL) of antimicrobial peptide HI-3 on HCT-8 cells. The metabolites in HCT-8 cells were determined by GS-MS, and then the metabolic pathway with the most significant difference in amino acid contents and the target enzyme in this pathway were screened by pathway analysis based on R software. After the HCT-8 cells were treated with 320 μg/mL HI-3, the activity of the selected target enzyme glutaminase (GLS) was assayed by enzyme activity kit, its mRNA and protein expression levels were detected by RT-qPCR and Western blot, respectively, and the changes in the contents of important metabolites including glutamine (Gln), glutamic acid (Glu), glutathione (GSH), α-ketoglutaric acid (α-KG) and ATP involved in the glutamine and glutamic acid metabolic pathway in HCT-8 cells were detected by biochemical kits and ELISA kit. 【Results】 The inhibition rates of HCT-8 cells treated by HI-3 at the concentrations of 80, 160, and 320 μg/mL were 33.85%  ±3.50%, 46.26%±0.90%, and 55.53%±1.70%, respectively, and the inhibition rates increased with the increase of the HI-3 concentration. HI-3 at the concentration of 320 μg/mL showed the greatest effects on the glutamine and glutamic acid pathway with the contents of amino acid metabolites in this pathway most significantly different from those in the negative control group (0 μg/mL HI-3). The activity of GLS (the target enzyme in this pathway) and the mRNA and protein expression levels of its gene GLS in the 320 μg/mL HI-3 treatment group were extremely significantly lower than those in the negative control group. Moreover, the contents of important metabolites in this pathway, including Gln, Glu, GSH, α-KG and ATP, in the 320 μg/mL HI-3 treatment group were also significantly reduced as compared with those in the negative control group. 【Conclusion】 Antimicrobial peptide HI-3 from H. illucens at the concentration of 320 μg/mL has the most significant effect on the glutamine and glutamic acid metabolic pathway in HCT-8 cells, and can significantly inhibit the proliferation of HCT-8 cells by blocking this pathway.

Key words: Hermetia illucens, antimicrobial peptide, human colon carcinoma, HCT-8 cell, cell metabolomics, glutamine, glutamic acid, metabolic pathway