MC-LR致肠细胞毒性的初步研究

Preliminary Study on Intestinal Cytotoxicity of Microcystin-LR

摘要: 随着工农业的发展，越来越多含氮磷的污水排放至淡水河流湖泊当中，导致水体富营养化，进而导致蓝藻爆发。藻毒素是蓝藻细菌代谢的次级产物，是水体中含量最多、危害最大的一类藻毒素。藻毒素是一种环状7肽毒素，目前已经被报道的有100多种异构体，其中微囊藻毒素-LR (Microcystin-LR, MC-LR)是分布最广泛、毒性最强的一种。本研究利用正常结直肠上皮细胞NCM460及结直肠癌细胞HT29为模型初步探究了MC-LR致肠毒性。我们通过对肠道细胞不同剂量(0 µM、5 µM及10 µM) MC-LR染毒，结果表明在处理的两个剂量组中，细胞数量及形态均无变化；利用荧光探针DCFH-DA法检测活性氧，结果表明不管是NCM460细胞还是HT29细胞，在染毒之后活性氧水平都显著增加(5 µM, HT29, p = 0.03, NCM460, p = 0.0003; 10 µM, HT29, p = 0.003, NCM460, p = 0.02)，并且呈剂量依赖效应；通过彗星实验，初步探讨了MC-LR对肠道细胞的毒性机制。研究结果表明，在5 µM和10 µM剂量组中，出现明显的DNA拖尾现象，且5 µM剂量组拖尾现象的发生明显低于10 µM剂量组，说明DNA损伤与MC-LR浓度存在剂量依赖关系。因此，本研究结果初步证明了MC-LR能致肠细胞毒性，为进一步了解微囊藻毒素致肠毒性的研究，以及预防、诊断和治疗微囊藻毒素引起的肠道疾病提供一定的参考。

Abstract: The rapid development of industries and agriculture, has led to the increasing discharge of wastewater containing nitrogen and phosphorus into freshwater, rivers and lakes. This activity results to eutrophication and development of cyanobacteria bloom. The monocyclic heptapep-tidemicrocystin (MC) is the secondary product of cyanobacteria metabolism and most harmful cyanotoxin found in water. More than 100 isomers of MCs have been reported, with MC-LR being the most widely distributed, abundant and toxic. The aim of this study was to investigate the intestinal toxicity caused by MC-LR. NCM460 and HT29 cell models were used to explore the intestinal toxicity of MC-LR. Intestinal cells were treated with different doses (0 μM, 5 μM and 10 μM) of MC-LR, and the number and morphology of the cells were observed. Fluorescence probe DCFH-DA was used to detect reactive oxygen species (ROS). In addition, the toxic mechanism of MC-LR on intestinal cells was performed via comet assay. Data indicated that there was no change in the number and morphology of the cells between the test and control groups. Both NCM460 and HT29 cells showed a significant (5 μM, HT29, p = 0.03, NCM460, p = 0.0003; 10 μM, HT29, p = 0.003, NCM460, p = 0.02) increase in ROS level after MC-LR exposure, and revealed a dose/time-effect relationship. The DNA tail-trailing phenomenon in 5 μM dose group was significantly lower than that in 10 μM dose group, indicating a dose-dependent relationship between DNA damage and MC-LR concentration. Hence, our data suggested MC-LR has the potential of intestine toxicity. The results of this study provide a reference for further understanding of the intestinal toxicity induced by MC-LR, prevention, diagnosis and treatment of intestinal diseases induced by MC-LR.