【摘 要】
:
[目的]探讨红景天苷对黏菌素所致神经毒性的拮抗作用.[方法]采用MTT法构建黏菌素体外损伤雪旺细胞的模型,在此基础上筛选能有效拮抗黏菌素致雪旺细胞毒性的红景天苷浓度.采用Annexin V-FITC/PI双染流式细胞仪检测观察对细胞凋亡率的影响;倒置显微镜、Hoechst33258荧光染色、透射电镜观察对细胞形态、细胞核形态及细胞内超微结构的影响;LDH水平测试检测对细胞膜完整性的影响;ROS含量
【机 构】
:
东北农业大学动物医学学院,哈尔滨,黑龙江,150030
【出 处】
:
中国畜牧兽医学会兽医药理毒理学分会第十一届会员代表大会暨第十三次学术讨论会与中国毒理学会兽医毒理专业委员会第五次学术研讨
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[目的]探讨红景天苷对黏菌素所致神经毒性的拮抗作用.[方法]采用MTT法构建黏菌素体外损伤雪旺细胞的模型,在此基础上筛选能有效拮抗黏菌素致雪旺细胞毒性的红景天苷浓度.采用Annexin V-FITC/PI双染流式细胞仪检测观察对细胞凋亡率的影响;倒置显微镜、Hoechst33258荧光染色、透射电镜观察对细胞形态、细胞核形态及细胞内超微结构的影响;LDH水平测试检测对细胞膜完整性的影响;ROS含量测定、SOD活性测定、MDA含量检测来探讨对氧化应激的影响;DNA Ladder观察对DNA完整性的影响;Rh123染色流式细胞仪检测对线粒体膜电位的影响.[结果]MTT法检测结果显示,250μg·mL-1硫酸黏菌素作用于雪旺细胞24 h时,细胞活力为63.78%(对照组为100%),损伤程度适宜.因此,后续试验选用250μg·mL-1硫酸黏菌素作用雪旺细胞24 h来构建黏菌素体外损伤雪旺细胞模型.选用12.5、25、50μg·mL-1浓度作为红景天苷保护硫酸黏菌素致雪旺细胞损伤的工作浓度.Annexin V-FITC/PI双染流式检测结果显示,红景天苷能有效降低硫酸黏菌素诱导雪旺细胞的凋亡率.降低损伤细胞培养液内LDH含量,使细胞内ROS、MDA含量显著降低,SOD活性升高,说明红景天苷能保护硫酸黏菌素损伤细胞膜完整性,稳固黏菌素引起细胞内氧化-抗氧化失衡.倒置显微镜、Hoechst33258荧光染色、电镜超微结构观察发现,红景天苷能有效缓解硫酸黏菌素诱导的雪旺细胞胞体变圆,轴突变短,细胞核变小、固缩,染色质凝聚、边缘化,线粒体结构不清、肿胀、空泡化等凋亡特征.DNA Ladder试验结果提示,红景天苷能有效缓解硫酸黏菌素诱导的雪旺细胞DNA断裂.Rh123染色流式细胞仪检测发现,红景天苷能有效缓解硫酸黏菌素引起的线粒体膜电位降低.[结论]硫酸黏菌素能引起雪旺细胞凋亡,诱发神经毒性,引起细胞内氧化应激损伤.红景天苷能有效拮抗其损伤.
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