《环境昆虫学报》

作者	单位
冯群，徐晨露，崔会程，孙虹霞，夏嫱	1. 遵义医科大学珠海校区，广东珠海 519041；2. 遵义医科大学第三附属医院，贵州遵义 563000

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中文摘要:

黑水虻作为一种全球广泛分布的昆虫，因生长环境决定了其丰富的抗菌肽来源，目前针对黑水虻抗菌肽的研究已逐渐从抗菌转向了抗癌。本文研究黑水虻抗菌肽HI-3对HCT-8细胞凋亡影响，探索其可能的抑癌机制，旨在为后续黑水虻抗菌肽抗肿瘤应用的研究提供作用靶点。采用流式细胞仪检测抗菌肽HI-3对HCT-8细胞凋亡率、线粒体膜电位、钙离子及活性氧（Reactive oxygen species, ROS）水平变化的影响；酶标仪检测抗菌肽HI-3对半胱天冬酶（Caspase）-3和Caspase-9活力的影响；并通过荧光定量PCR技术和蛋白芯片技术检测细胞凋亡相关基因和蛋白表达情况。结果表明，抗菌肽HI-3可以诱导HCT-8细胞凋亡，且随处理浓度增加，凋亡率亦增加，当浓度达到400 μg/mL时，HCT-8细胞凋亡率与阴性对照组及其他处理组相比均有显著性差异（P<0.05）；HI-3处理后HCT-8细胞内线粒体膜电位、钙离子浓度及ROS水平均随处理浓度增加而升高，当浓度达到400 μg/mL时达到最高，各处理浓度下3组数值与阴性对照组相比均存在显著差异（P<0.05）；随着HI-3处理浓度增加HCT-8细胞内Caspase-3、Caspase-9活力均呈现先增强后减弱的变化趋势，200 μg/mL抗菌肽HI-3对HCT-8细胞内Caspase-3、Caspase-9的活力影响均较强，与其他处理组和对照组相比差异显著（P＜0.01）。200 μg/mL抗菌肽HI-3可以上调凋亡因子Caspase-3、Caspase-8、Caspase-9和Fas的基因表达；下调凋亡因子P53、P21、XIAP、Bcl-2、Bax和FasL的基因表达。而蛋白芯片结果显示：Caspase-3、P21的蛋白表达上调，P53、XIAP、Bcl-2、Bax、Fas、FasL的蛋白表达下调。因此，黑水虻抗菌肽HI-3可以诱导HCT-8细胞发生凋亡，其凋亡可能通过线粒体途径和钙离子信号途径进行，与线粒体膜电位降低、钙离子内流、ROS产生和Caspase-3、Caspase-9活力的增强及凋亡相关因子XIAP、Caspase-3、Caspase-9的表达相关。

英文摘要:

Hermetia illucens as a kind of insect widely distributed all over the world, the growth environment determines the source of its abundant antibacterial peptides. At present, the research on the antibacterial peptides of the H.illucens has gradually shifted from antibacterial to anti-cancer. This article to study the effect of antibacterial peptide HI-3 of H.illucens on apoptosis of HCT-8 cells and explore its possible anti-tumor mechanism, to provide a target for the subsequent anti-tumor application research of H.illucens antimicrobial peptides. Flow cytometry was used to detect the effect of antimicrobial peptide HI-3 on the apoptosis rate, mitochondrial membrane potential, calciumion and reactive oxygen species (ROS) levels in HCT-8 cells; the effect of antimicrobial peptide HI-3 on the activity of Caspase-3 and Caspase-9 was detected by microplate reader; the expression of apoptosis-related genes and proteins was detected by fluorescence quantitative PCR technology and protein chip technology. The results showed that antibacterial peptide HI-3 could induce the apoptosis of HCT-8 cells, and the apoptosis rate enhanced with the increase of treatment concentration, the apoptosis rate of HCT-8 cells was significantly different from that of the negative control group and other treatment groups (P<0.05) in 400 μg/mL. The mitochondrial membrane potential, calciumion concentration and ROS levels in HCT-8 cells improved with the increase of the HI-3 treatment concentration, and reached the highest level in 400 μg/mL, and the values of the three treatment groups were significantly different with the negative control (P<0.05). With the increase of HI-3 concentration, the activity of Caspase-3 and Caspase-9 in HCT-8 cells showed a trend of increased first and then reduced, and it was strongly affected when the antimicrobial peptide HI-3 at 200 μg/mL, the value was obviously different with other groups (P<0.01). The genes expression of Caspase-3, Caspase-8, Caspase-9 and Fas were up-regulated by 200 μg/mL, while the genes expression of P53, P21, XIAP, Bcl-2, Bax and FasL were down-regulated. The results of protein microarray showed that the proteins expression of Caspase-3 and P21 were up-regulated, and the proteins expression of P53, XIAP, Bcl-2, Bax, Fas and FasL were down-regulated. Therefore,H.illucens antibacterial peptide HI-3 induces the apoptosis of HCT-8 cells, and may promote the apoptosis through the mitochondrial pathway and calcium signaling pathway, it is associated with the decrease of mitochondrial membrane potential, calcium influx, ROS generation, the enhancement of Caspase-3 and Caspase-9 activity, and the expression of apoptosis-related factors XIAP, Caspase-3 and Caspase-9.

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