α-神经毒素的分离纯化及其经小鼠直肠给药后肠道体外荧光成像分析

Isolation and purification ofα-neurotoxin and fluorescence imaging analysis of intestinal tract after rectum administration in mice

目的从眼镜蛇粗毒中分离纯化短链α-神经毒素活性成分,观察荧光染料标记的α-神经毒素经小鼠直肠给药后的肠道内分布和动态过程。方法使用CM Sepharose Fast Flow阳离子交换层析和Sephadex G-50凝胶过滤层析分离纯化电泳纯的α-神经毒素。将4只C57小鼠分为对照组和实验组,对照组采用荧光染料CY7-SE标记,实验组采用荧光染料CY7-SE与α-神经毒素相互结合。使用成像仪检测CY7-SE标记的α-神经毒素在直肠给药1、3 h后肠道内的荧光强度分布情况。结果分离纯化得到电泳纯的α-神经毒素。α-神经毒素直肠给药1 h后,实验组相较于对照组的小肠段和胃的荧光强度较强,盲肠、结肠段荧光强度较弱。α-神经毒素直肠给药3 h后,实验组相较于对照组的小肠、盲肠、结肠段和胃的荧光强度均较弱。结论从眼镜蛇粗毒中分离纯化得到的α-神经毒素经直肠给药可先富集于小鼠小肠段及胃,3 h后被肠道黏膜吸收代谢。本研究为开发神经毒素的直肠递送制剂提供了实验依据。

Objective To isolate and purify the short-chainα-neurotoxin active ingredient from the crude venom of cobra,and to observe the distribution and dynamic process of fluorescent dye-labeledα-neurotoxin in the intestinal tract after rectal administration to mice.Methods Electrophoretically pureα-neurotoxin was isolated and purified using CM Sepharose Fast Flow cation exchange chromatography and Sephadex G-50 gel filtration chromatography.Four C57 mice were divided into two groups:control and experimental.In the experimental group,the fluorescent dye CY7-SE was conjugated withα-neurotoxin to each other.The fluorescence intensity distribution of CY7-SE-labeledα-neurotoxin in the intestine was detected by using an imager after 1h and 3h of rectal administration,respectively.Results Electrophoretically pureα-neurotoxin was obtained by isolation and purification.α-neurotoxin administered rectally for 1h showed stronger fluorescence intensity in the small intestine and stomach,and weaker fluorescence intensity in the cecum and colon than that in the control group.α-neurotoxin administered rectally for 3h showed weaker fluorescence intensity in the small intestine,cecum,colon,and stomach than that in the control group.Conclusion Theα-neurotoxin isolated and purified from the crude venom of cobra was first enriched in the small intestine and stomach of mice,and then absorbed and metabolized by the intestinal mucosa after 3h.This study provides an experimental basis for the development of a neurotoxin formulation for rectal delivery.