重楼皂苷Ⅱ溶血作用机制的研究(1)
[摘要] 该文旨在探讨阴离子通道蛋白和葡萄糖转运蛋白1介导的重楼皂苷Ⅱ(polyphyllin Ⅱ，PPⅡ)的溶血作用，初步揭示PPⅡ体外溶血作用机制。实验采用分光光度法检测PPⅡ体外溶血，设计了与阴离子通道相关的溶液与阻断剂、葡萄糖通道相关的抑制剂和多靶点药物脱氢表雄酮与安定对PPⅡ溶血的影响3个方面的主要内容;通过扫描电镜和透射电镜观察PPⅡ对红细胞形态的影响。电镜观察显示PPⅡ处理的红细胞变形严重，出现大量球形红细胞和棘形红细胞以及囊泡。PPⅡ体外溶血试验结果显示，阴离子盐渗液LiCl，NaHCO3，Na2SO4和PBS均显著抑制PPⅡ溶血(P<0.05)，阻断剂NPPB和DIDIS显著促进PPⅡ溶血(P<0.01);一定浓度下的高渗液氯化钠、果糖和葡萄糖显著拮抗PPⅡ溶血(P<0.05);葡萄糖通道抑制剂细胞松弛素B和维拉帕米能显著拮抗PPⅡ溶血(P<0.01);预处理1 min的1 mg·L-1安定和100 μmol·L-1 DHEA能完全拮抗PPⅡ溶血(P<0.01)。以上结果表明PPⅡ体外溶血的机制可能是以GLUT1为主要的竞争性作用位点，通过改变阴离子通道转运活性和构象，最终引起胞内渗透压升高，红细胞破裂溶血。
[关键词] 阴离子通道蛋白;葡萄糖转运体1;重楼皂苷Ⅱ;溶血;红细胞;机制
Study on hemolytic mechanism of polyphyllin Ⅱ
NING Li-hua， ZHOU Bo， ZHANG Yao-xiang， LI Xin-ping*
(School of Veterinary Medicine， Northwest Agriculture and Forest University， Yangling 712100， China)
[Abstract] To study the hemolytic effect of polyphyllin Ⅱ(PP Ⅱ) mediated by anion channel protein and glucose transporter 1 (GLUT1)， in order to initially reveal its hemolytic mechanism in vitro. In the experiment， the spectrophotometric method was adopted to detect the hemolysis of PP Ⅱ in vitro and the effect of anion channel-related solution and blocker， glucose channel-related inhibitor and multi-target drugs dehydroepiandrosterone (DHEA) and diazepam on the hemolysis of PP Ⅱ. The scanning electron microscope and transmission electron microscope were used to observe the effect of PP Ⅱ on erythrocyte (RBC) morphology. The results showed that PP Ⅱ-processed blood cells were severely deformed into spherocytes， acanthocyturia and vesicae. According to the results of the PP Ⅱ hemolysis experiment in vitro， the anion hypertonic solution LiCl， NaHCO3， Na2SO4 and PBS significantly inhibited the hemolysis induced by PPⅡ (P<0.05)， while blockers NPPB and DIDS remarkably promoted it (P<0.01). Hyperosmotic sodium chloride， fructose and glucose at specific concentrations notably antagonized the hemolysis induced by PP Ⅱ (P<0.05). The glucose channel inhibitor Cytochalasin B and verapamil remarkably antagonized the hemolysis induced by PP Ⅱ(P<0.01). The hemolysis induced by PPⅡ could also be antagonized by 1 μmol·L-1 diazepam and 100 μmol·L-1 DHEA pretreated for 1 min (P<0.01). In conclusion， the hemolytic mechanism of PP Ⅱ in vitro may be related to the increase in intracellular osmotic pressure and rupture of erythrocytes by changing the anion channel transport activity， with GLUT1 as the major competitive interaction site. (宁利华 周博 张耀相 李新平)
[关键词] 阴离子通道蛋白;葡萄糖转运体1;重楼皂苷Ⅱ;溶血;红细胞;机制
Study on hemolytic mechanism of polyphyllin Ⅱ
NING Li-hua， ZHOU Bo， ZHANG Yao-xiang， LI Xin-ping*
(School of Veterinary Medicine， Northwest Agriculture and Forest University， Yangling 712100， China)
[Abstract] To study the hemolytic effect of polyphyllin Ⅱ(PP Ⅱ) mediated by anion channel protein and glucose transporter 1 (GLUT1)， in order to initially reveal its hemolytic mechanism in vitro. In the experiment， the spectrophotometric method was adopted to detect the hemolysis of PP Ⅱ in vitro and the effect of anion channel-related solution and blocker， glucose channel-related inhibitor and multi-target drugs dehydroepiandrosterone (DHEA) and diazepam on the hemolysis of PP Ⅱ. The scanning electron microscope and transmission electron microscope were used to observe the effect of PP Ⅱ on erythrocyte (RBC) morphology. The results showed that PP Ⅱ-processed blood cells were severely deformed into spherocytes， acanthocyturia and vesicae. According to the results of the PP Ⅱ hemolysis experiment in vitro， the anion hypertonic solution LiCl， NaHCO3， Na2SO4 and PBS significantly inhibited the hemolysis induced by PPⅡ (P<0.05)， while blockers NPPB and DIDS remarkably promoted it (P<0.01). Hyperosmotic sodium chloride， fructose and glucose at specific concentrations notably antagonized the hemolysis induced by PP Ⅱ (P<0.05). The glucose channel inhibitor Cytochalasin B and verapamil remarkably antagonized the hemolysis induced by PP Ⅱ(P<0.01). The hemolysis induced by PPⅡ could also be antagonized by 1 μmol·L-1 diazepam and 100 μmol·L-1 DHEA pretreated for 1 min (P<0.01). In conclusion， the hemolytic mechanism of PP Ⅱ in vitro may be related to the increase in intracellular osmotic pressure and rupture of erythrocytes by changing the anion channel transport activity， with GLUT1 as the major competitive interaction site. (宁利华 周博 张耀相 李新平)