栀子提取物ZG对副流感病毒1型感染后宿主细胞膜的影响

Effect of Extracted ZG from Gardenia on Hep-2 Cell Membrane Post Infected with Parainfluenza Virus Type 1(PIV-1)

为了探讨栀子提取物ZG抗病毒作用的生物学机制,观察了栀子提取物ZG对副流感病毒1型(PIV-1)感染后宿主细胞膜电位、膜Na+-K+-ATP酶活性和膜流动性的影响。以氯化乙酰胆碱为阳性对照,采用荧光探针Di-BAC4(3)标记Hep-2细胞膜电位,借助流式细胞仪检测膜电位;定磷法,分光光度计检测Na+-K+-ATP酶活性;荧光探针NBD-C6-HPC标记细胞膜磷脂,以荧光漂白恢复法和激光扫描共聚焦显微镜检测膜流动性。结果显示:PIV-1感染后宿主细胞膜电位下降,处于超极化状态;膜Na+-K+-ATP酶活性显著增加,膜流动性显著降低。栀子提取物ZG作用后,对宿主细胞膜的超极化状态没有明显影响;对膜Na+-K+-ATP酶活性没有明显影响;而对膜流动性则有明显的恢复作用。阳性对照药乙酰胆碱能明显改善病毒感染后膜电位的超极化状态。PIV-1感染后膜电位、Na+-K+-ATP酶活性和膜流动性等细胞膜能态和功能的改变,可能为病毒感染的生物学机制之一;栀子提取物ZG可能是通过改善细胞膜流动性,维持细胞膜的正常功能来发挥抗病毒感染的作用,而与膜电位和膜Na+-K+-ATP酶活性等能态来源的环节可能无关。

In order to study the anti-viral mechanism of extracted ZG from Gardenia , the effect of extracted ZG on Hep-2 cell membrane potential, Na^＋-K^＋-ATPase activity and membrane fluidity post infected with parainfluenza virus type 1 （PIV-1）was observed. Acetylcholine which was fluorescent labeled with DiBAC4 （3）was taken as positive control to observe the changes of membrane potential and was measured by flow cytometer. The phosphorus determination method and spectrophotometer were used to measure the Na^＋-K^＋-ATPase activity of Hep-2 cell membrane post PIV-1 infection. Hep-2 cell membrane phospholipids was labeled with fluorescent NBD-C6-HPC and membrane fluidity was measured by confocal laser scanning microscope. The results demonstated that after PIV-1 infection the Hep-2 cell membrane potential decreased significantly and the membrane was in the state of hyperpolarization, Na^＋-K^＋-ATPase activity increased and membrane fluidity decreased significantly. There was no apparent interferring effect of extracted ZG on the changes of membrane potential and Na^＋-K^＋-ATPase activity post PIV-1 infection, while membrane fluidity was improved significantly. Acetylcholine improved the state of hyperpolarization. The changes of membrane potential, Na^＋-K^＋-ATPase activity and membrane fluidity might be the biomechanism of PIV- 1 infectoin. The extracted ZG improved membrane fluidity to prevent from PIV-1 infection by protecting the cell membrane,which was probably the mechanism of anti-PIV-1 activity of the extracted ZG, but ZG probably had nothing to do with membrane potential and Na^＋-K^＋-ATPase activity.