基于心血管芯片模型的4种深海毒素致伤评价与雷公藤甲素的保护作用研究

Evaluation of toxin-induced injury and protective effect of triptolide based on a cardiovascular chip model

目的构建一种心血管芯片模型,评估四种海洋毒素即大田软海绵酸(OA)、芋螺毒素(CTX)、河豚毒素(TTX)和环亚胺毒素(GYM)对血管糖萼组织的损伤,初步探索雷公藤甲素对毒素致伤的保护作用。方法将人脐静脉内皮细胞(HUVEC)接种于三通道结构的微流控芯片,并对所构建的心血管芯片模型进行表征。采用CCK-8法和免疫荧光染色法分析低、中、高三个浓度的海洋毒素对细胞活力和糖萼组织损伤情况,以及雷公藤甲素对毒素致伤的保护作用。结果所构建心血管器官芯片中的细胞生长状态良好,具有结构完整的糖萼组织,与对照组相比,OA中、高浓度以及GYM高浓度均对细胞活力具有一定抑制(P<0.05),CTX和TTX在所测浓度下对细胞没有明显活力抑制,但是四种毒素均引起细胞表面糖萼组织的严重损伤(P<0.01),且随着浓度增加,糖萼损伤率升高。经雷公藤甲素预保护后,四种毒素对HUVEC的毒性显著降低,糖萼组织损伤率下降。结论四种毒素对HUVEC细胞活力以及表面的糖萼组织具有损伤,且呈一定的剂量关系,而雷公藤甲素对毒素损伤后的HUVEC细胞具有保护作用。

Objective To construct a cardiovascular chip model for evaluating the damage of vascular glycocalyx induced by four marine toxins:okadaic acid(OA),conotoxin(CTX),tetrodotoxin(TTX)and gymnodimine(GYM),and explore the protective effect of triptolide on toxin-induced injury.Methods Human umbilical vein endothelial cells(HUVEC)were inoculated into a three-channel microfluidic chip.CCK-8 method and immunofluorescence staining were used to analyze the damage of cell viability and glycocalyx tissue induced by low,middle and high concentrations of marine toxin,as well as the protective effect of triptolide on toxin-induced injury.Results The cells in the cardiovascular chip grew well and had structurally intact glycocalyx.Compared with the control group,the activity of HUVEC cells were inhibited in group of the medium and high concentration of OA and high concentration of GYM(P<0.05).The activity of cells had not been inhibited by CTX and TTX significantly,but all the four toxins caused serious damage to the glycocalyx tissue(P<0.01).After pre-protection with triptolide,the toxicity of the four toxins to HUVEC cells and the damage rate of glycocalyx decreased significantly.Conclusion The four marine biotoxins could damage the activity and glycocalyx of HUVEC cells in a dose-dependent manner,while triptolide has a protective effect on HUVEC cells injured by toxin.