基于单细胞三维形态学体外分析重楼皂苷Ⅰ的人红细胞毒性
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摘要
目的从单细胞三维形态学角度上体外分析重楼皂苷I(PPI)的人红细胞毒性。方法将浓度为0.78—50μg/mL的PPI和人红细胞在37℃下震荡孵育3h作为实验组,以未经PPI处理的人红细胞作为实验对照;通过相对红细胞溶血率和红细胞沉降实验分析PPI红细胞溶血活性;通过激光三维显微成像技术检测红细胞三维形态学图像,并分析相关三维形态学参数。结果 PPI红细胞溶血活性存在浓度依赖性,EC_(50)值为3.92μg/mL;随着PPI浓度增大,红细胞形态先后经历轻微膨胀、出芽状结构形成、膜缺失到薄片状细胞膜结构的改变过程,PPI浓度达到6.25μg/mL时红细胞结构完全被破坏;与对照组相比,0.78μg/mL和1.56μg/mL PPI处理后红细胞平均高度、最大高度、表面积和体积均显著增大(P<0.01),3.125μg/mL PPI处理后上述参数显著降低(P<0.01),PPI处理后红细胞周长(1.56μg/mL PPI处理除外)和水平费雷特直径变化则无统计学意义(P>0.05)。结论本研究建立了单细胞三维形态学可视化分析方法将有利于加深PPI和人红细胞间的相互作用认知,促进PPI临床开发和应用。
Objective To analysis the cytotoxicity of polyphyllin I(PPI) on human erythrocytes in-vitro from the view of single cell three-dimensional(3 D) morphology. Methods The human erythrocytes treated with PPI solutions, whose concentrations ranging from 0.78 μg/mL to 50 μg/mL, at 37℃ for 3 h were referred as experimental groups, while the human erythrocyte without PPI treatment was considered as control group. The hemolytic activities of PPI were analyzed by relative erythrocyte hemolytic rate and erythrocyte sedimentation experiment. The 3 D morphological images of erythrocyte were acquired by a laser 3 D microscopic imaging technology, and the related 3 D morphological parameters were calculated later. Results The hemolytic activities of PPI was concentration-dependent and its EC50 value was 3.92 μg/mL. With the increase of PPI concentration, the morphology of erythrocyte successively changed form mild inflation, budding structure form, lack of membrane structure and to thin sheet liked membrane structure at last. The membrane structure was completely destroyed when the concentration of PPI reached to 6.25 μg/mL. Compared with control group, the average height, maximum height, surface area and volume of erythrocytes treated by PPI with the concentrations of 0.78 μg/mL and 1.56 μg/mL were increased significantly(P<0.01), and these parameters mentioned above for the erythrocyte treated by 3.125 μg/mL PPI were decreased significantly(P<0.01). However, the perimeter and horizontal Ferrette diameter of erythrocytes treated with PPI did not change significantly(P>0.05), except for the perimeter of erythrocytes treated with 1.56 μg/mL PPI(P<0.05). Conclusion This study established a single-cell 3 D morphological visualization analysis method, which can be benefit for deepening the knowledge of interaction between PPI and human erythrocytes and promoting the clinical development and application of PPI.
Objective To analysis the cytotoxicity of polyphyllin I(PPI) on human erythrocytes in-vitro from the view of single cell three-dimensional(3 D) morphology. Methods The human erythrocytes treated with PPI solutions, whose concentrations ranging from 0.78 μg/mL to 50 μg/mL, at 37℃ for 3 h were referred as experimental groups, while the human erythrocyte without PPI treatment was considered as control group. The hemolytic activities of PPI were analyzed by relative erythrocyte hemolytic rate and erythrocyte sedimentation experiment. The 3 D morphological images of erythrocyte were acquired by a laser 3 D microscopic imaging technology, and the related 3 D morphological parameters were calculated later. Results The hemolytic activities of PPI was concentration-dependent and its EC50 value was 3.92 μg/mL. With the increase of PPI concentration, the morphology of erythrocyte successively changed form mild inflation, budding structure form, lack of membrane structure and to thin sheet liked membrane structure at last. The membrane structure was completely destroyed when the concentration of PPI reached to 6.25 μg/mL. Compared with control group, the average height, maximum height, surface area and volume of erythrocytes treated by PPI with the concentrations of 0.78 μg/mL and 1.56 μg/mL were increased significantly(P<0.01), and these parameters mentioned above for the erythrocyte treated by 3.125 μg/mL PPI were decreased significantly(P<0.01). However, the perimeter and horizontal Ferrette diameter of erythrocytes treated with PPI did not change significantly(P>0.05), except for the perimeter of erythrocytes treated with 1.56 μg/mL PPI(P<0.05). Conclusion This study established a single-cell 3 D morphological visualization analysis method, which can be benefit for deepening the knowledge of interaction between PPI and human erythrocytes and promoting the clinical development and application of PPI.
引文
[1] 杨远贵,张霁,张金渝,等.重楼属植物化学成分及药理活性研究进展.中草药,2016,47(18):3301-3323.
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[3] 阳丹丹,齐琪,陈正礼,等.内质网应激调控重楼皂苷Ⅰ诱导肝癌HepG2细胞凋亡的研究.中国细胞生物学学报,2017,39(11):1397-1406.
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[10] 黄艳,聂偲,田玲玲,等.用原子力显微镜观察术中失血回收对红细胞形态的影响.中国病理生理杂志,2012,28(2):380-384.
[11] Feng R,Yu Y,Shen C,et al.Impact of graphene oxide on the structure and function of important multiple blood components by a dose-dependent pattern.J Biomed Mater Res A,2015,103(6):2006-2014.
[12] Farag MR,Alagawany M.Erythrocytes as a biological model for screening of xenobiotics toxicity.Chem Biol Interact,2018,279(1):73-83.
[13] Mukherjee R,Chaudhury K,Chakraborty C.Topological features of erythrocytes in thalassemic patients:quantitative characterization by scanning electron and atomic force microscopy.Anal Quant Cytopathol Histpathol,2014,36(2):91-99.
[14] Mukherjee R,Saha M,Routray A,et al.Nanoscale Surface Characterization of Human Erythrocytes by Atomic Force Microscopy:A Critical Review.IEEE Trans Nanobioscience,2015,14(6):625-633.
[15] 杨黎江,沈放,王德斌,等.抗重楼皂苷类化合物溶血作用的研究.天然产物研究与开发,2011,23(4):629-632,651.
[2] Dong R,Guo J,Zhang Z,et al.Polyphyllin I inhibits gastric cancer cell proliferation by downregulating the expression of fibroblast activation protein alpha (FAP) and hepatocyte growth factor (HGF) in cancer-associated fibroblasts.Biochem Biophys Res Commun,2018,497(4):1129-1134.
[3] 阳丹丹,齐琪,陈正礼,等.内质网应激调控重楼皂苷Ⅰ诱导肝癌HepG2细胞凋亡的研究.中国细胞生物学学报,2017,39(11):1397-1406.
[4] Gu L,Feng J,Zheng Z,et al.Polyphyllin I inhibits the growth of ovarian cancer cells in nude mice.Oncol Lett,2016,12(6):4969-4974.
[5] 杨蓉蓉,王跃虎,施敏,等.长柱重楼总皂苷体外抗肿瘤活性及毒性研究.中国临床药理学杂志,2018,34(4):439-442.
[6] 宁利华,周博,张耀相,等.重楼皂苷Ⅱ溶血作用机制的研究.中国中药杂志,2015,40(18):3623-3629.
[7] Gao M,Cheung KL,Lau IP,et al.Polyphyllin D induces apoptosis in human erythrocytes through Ca2+ rise and membrane permeabilization .Arch Toxicol,2012,86(5):741-752.
[8] 丁玲,赵广德,欧阳熊妍,等.利用新型激光三维成像显微技术分析静止条件下纯化血小板的聚集行为.中国输血杂志,2018,31(2):132-136.
[9] Han W,Hou G,Liu L.Polyphyllin I (PPI) increased the sensitivity of hepatocellular carcinoma HepG2 cells to chemotherapy.Int J Clin Exp Med,2015,8(11):20664-20669.
[10] 黄艳,聂偲,田玲玲,等.用原子力显微镜观察术中失血回收对红细胞形态的影响.中国病理生理杂志,2012,28(2):380-384.
[11] Feng R,Yu Y,Shen C,et al.Impact of graphene oxide on the structure and function of important multiple blood components by a dose-dependent pattern.J Biomed Mater Res A,2015,103(6):2006-2014.
[12] Farag MR,Alagawany M.Erythrocytes as a biological model for screening of xenobiotics toxicity.Chem Biol Interact,2018,279(1):73-83.
[13] Mukherjee R,Chaudhury K,Chakraborty C.Topological features of erythrocytes in thalassemic patients:quantitative characterization by scanning electron and atomic force microscopy.Anal Quant Cytopathol Histpathol,2014,36(2):91-99.
[14] Mukherjee R,Saha M,Routray A,et al.Nanoscale Surface Characterization of Human Erythrocytes by Atomic Force Microscopy:A Critical Review.IEEE Trans Nanobioscience,2015,14(6):625-633.
[15] 杨黎江,沈放,王德斌,等.抗重楼皂苷类化合物溶血作用的研究.天然产物研究与开发,2011,23(4):629-632,651.