金欣口服液调控心磷脂代谢抗呼吸道合胞病毒感染的机制研究

Study on Mechanism of Jinxin Oral Liquid Regulating Cardiolipin Metabolism against Respiratory Syncytial Virus Infection

    摘要
  • 摘要:
      目的  研究金欣口服液及其主要成分黄芩苷对呼吸道合胞病毒(Respiratory syncytial virus, RSV)诱导心磷脂代谢异常的影响及可能的作用机制。
      方法  将BALB/c小鼠随机分为空白组、模型组和金欣口服液给药组。前3 d以RSV混悬液滴鼻建立RSV感染模型, 从第3天下午开始, 金欣口服液组予金欣口服液(27.6 g·kg-1·d-1)连续灌胃给药3 d。体外实验采用RSV感染Raw264.7细胞的方式建立病毒感染模型，黄芩苷组给予黄芩苷(100 mg·kg-1·d-1)干预。采用色谱质谱联用的方式分别检测小鼠肺组织及Raw264.7细胞心磷脂代谢谱的变化。qPCR法检测RSV表面蛋白 RSV-F、RSV-G, 炎症因子IL-6、IL-1β、Tnf-α, 心磷脂代谢酶Tafazzin(TAZ)、心磷脂合成酶1( Crls1)、心磷脂转运酶(PLSCR3)以及自噬受体蛋白SQSTM1/P62(p62) 的转录水平。Western blot法检测p62蛋白表达水平，分子对接检测p62与CL14∶0-16∶1-16∶1-18∶2的结合能力。
      结果  RSV感染小鼠及细胞模型中 IL-6、IL-1β、Tnf-α mRNA表达增加(P < 0.05，P < 0.01，P < 0.001), 金欣口服液及黄芩苷可以起到一定的回调作用; RSV感染后心磷脂代谢谱发生了变化，金欣口服液及黄芩苷可起到一定的调节作用；模型组小鼠肺组织中的Crls1、TAZ mRNA表达显著上调(P < 0.01), 给予金欣口服液之后Crls1、TAZ mRNA表达明显降低下调(P < 0.01)，细胞模型组中Crls1 mRNA表达明显增加(P < 0.001), 黄芩苷干预后Crls1 mRNA表达显著降低(P < 0.000 1)；RSV感染小鼠肺组织p62蛋白表达明显下降(P < 0.001), 给予金欣口服液之后p62蛋白表达显著增加(P < 0.01)。Raw264.7细胞模型组p62蛋白表达明显增加(P < 0.05), 给予黄芩苷后p62蛋白水平进一步显著升高(P < 0.000 1)；分子对接结果表明心磷脂CL14∶0-16∶1-16∶1-18∶2可与p62的UBA结构发生阈结合。
      结论  金欣口服液以及黄芩苷可改善RSV诱导的心磷脂代谢紊乱, 调节线粒体功能，进而发挥了抗RSV感染作用。

     

    Abstract:
      OBJECTIVE  To study the possible effects and mechanism of Jinxin oral liquid and its main ingredient baicalin on Respiratory syncytial virus (RSV)-induced abnormal cardiolipin metabolism.
      METHODS  BALB/c mice were randomly divided into control group, RSV group and Jinxin oral liquid treatment group. Mice were nasally administrated of RSV suspension to establish RSV infection model at the first 3 days. Then Jinxin oral liquid 27. 6 g·kg-1·d-1 was administrated intragastrically for the next 3 days from the afternoon of the third day. Raw264.7 cells were infected by RSV to establish an in vitro infection model and baicalin (100 mg·kg-1·d-1) was given for intervention. The changes of cardiolipin metabolism profiles in mice lung tissue and Raw264. 7 cells were detected by chromatography-mass spectrometry. Transcriptional mRNA levels of RSV surface proteins RSV-F and RSV-G, inflammatory cytokines IL-6, IL-1β and Tnf-α, cardiolipin metabolic enzymes Tafazzin (TAZ), Cardiolipin Synthase 1 (Crls1), Phospholipid Scramblase 3 (PLSCR3) and autophagy-related protein p62 were detected by qPCR. The protein level of p62 was detected by Western blot. Molecular docking was used to detect the binding ability of p62 to CL14∶0-16∶1-16∶1-18∶2.
      RESULTS  The expression of IL-6, IL-1β, and Tnf-α mRNA increased in RSV-infected mice and cell models (P < 0.05, P < 0.01, P < 0.001). Jinxin oral liquid and baicalin plays a certain restoration effect. The cardiolipin metabolism profile changes after RSV infection, and Jinxin oral liquid and baicalin can play a certain regulatory role. The expression of Crls1 and TAZ mRNA in the lung tissue of mice in the model group was significantly up-regulated (P < 0. 01), after administration of Jinxin oral liquid, the expression of Crls1 and TAZ mRNA was significantly reduced (P < 0. 01); the expression of Crls1 mRNA in the cell model group was significantly increased (P < 0. 001), and after baicalin intervention, the expression of Crls1 mRNA was significantly reduced (P < 0.000 1). The expression of p62 protein in the lung tissue of RSV-infected mice was significantly reduced (P < 0.001), and the expression of p62 protein was significantly increased after administration of Jinxin oral liquid (P < 0.01). The p62 protein expression in the Raw264.7 cell model group increased significantly (P < 0.05), and the p62 protein level further increased significantly after administration of baicalin (P < 0.000 1). Molecular docking results showed that cardiolipin CL14∶0-16∶1-16∶1-18∶2 can threshold-bind with the UBA structure of p62.
      CONCLUSION  Jinxin oral liquid and baicalin can improve RSV-induced cardiolipin metabolism disorder, regulate mitochondrial function, and thus exert anti-RSV infection effects.

     

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