情志应激激活潜伏单纯疱疹病毒1型建立“肝郁化火”动物模型的研究  

Establishing an Animal Model of Emotional Stress Activated Latent HSV-1 for TCM Theory“Liver Qi Stagnation Forming Huo”

作　　者：姜珊 罗卓 梁磊[1,2] 牛杰 龚海标 李娥 李怡芳 栗原博[1,2] 何蓉蓉[1,2,3] JIANG Shan;LUO Zhuo;LIANG Lei;NIU Jie;GONG Haibiao;LI E;LI Yifang;LI Yuanbo;HE Rongrong(Jinan University Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility,Guangzhou 510632,China;Institute of Traditional Chinese Medicine and Natural Products,College of Pharmacy,Jinan University,Guangzhou 510632,China;School of Traditional Chinese Medicine,Jinan University,Guangzhou 510632,China)

机构地区：[1]暨南大学广东省疾病易感性及中医药研发工程技术研究中心,广州510632 [2]暨南大学药学院中药及天然药物研究所,广州510632 [3]暨南大学中医学院,广州510632

出　　处：《世界中医药》2022年第4期466-471,476,共7页World Chinese Medicine

基　　金：国家自然科学基金项目(81873209,U1801284,81622050);珠江创新团队项目(2017BT01Y036);珠江学者计划项目(GDUPS2019);广州市科技计划-基础与应用基础研究资助项目(907158833068,201903010062)。

摘　　要：目的:建立情志应激引起潜伏单纯疱疹病毒1型(HSV-1)复发的“肝郁化火”动物模型。方法:小鼠通过鼻滴感染HSV-14周后,采用定量反转录聚合酶链式反应(qRT-PCR)和空斑法分别检测三叉神经节(TG)中HSV-1潜伏相关转录体(LAT)的表达和病毒颗粒,确定单纯疱疹病毒1型(HSV-1)潜伏模型成功建立。小鼠每天拘束应激12 h,持续应激4 d,采用致细胞病变效应(CPE)法和免疫荧光法检测三叉神经节中HSV-1的gB蛋白的表达,检测情志应激诱导HSV-1激活情况。HSV-1潜伏小鼠于拘束应激前3 d给予“清肝泻火”经典方丹栀逍遥散或清肺热的经典方泻白散,以方测证判定模型的中医证型。采用眼球摘除法对小鼠血浆进行取样,液质色谱-质谱法(LC-MS)检测各激素的含量。用Western Blotting法检测TG中脂质氧化指标4-HNE和固有免疫指标干扰素-β(IFN-β),肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)蛋白的表达情况。结果:小鼠鼻滴感染HSV-14周后,可在其三叉神经节中建立潜伏,小鼠拘束4 d后可再次激活。与激活组比较,丹栀逍遥散比泻白散更能有效降低HSV-1的激活率,并能减轻复发性眼部感染。激活组以及丹栀逍遥散组的血浆中皮质酮水平都明显增加,2组差异无统计学意义。此外,丹栀逍遥散能降低TG中4-HNE和IFN-β的表达,并能减少HSV-1复发引起的TNF-α和IL-6炎症介质蛋白的表达。结论:丹栀逍遥散可增强固有免疫,降低情志应激介导的HSV-1重激活,并能减轻HSV-1激活复发而引起的氧化应激以及炎症反应。Objective:To establish an animal model of emotional stress activated latent HSV-1,which is suitable for studying the TCM theory of“Liver qi stagnation forming Huo”.Methods:The expression of HSV-1 latent associated transcripts(LAT)and virus particles in trigeminal ganglion(TG)were detected respectively by RT-qPCR and plaque assay,to confirm the successful establishment of HSV-1 latent model in mice after 14 days of nasal infection.To detect the HSV-1 activation induced by emotional stress,the mice were daily subjected to restraint stress for 12 hours for 4 days.Following restraint stress,the TG of the mice was collected and the expression of gB protein in TG was measured by cytopathic effect(CPE)and immunofluorescence method.The mice in the latent phase of HSV-1 were treated with Danzhi Xiaoyao Powder which was a classic recipe for clearing liver heat or Xiebai Powder that had the effects of clearing lung heat three days before the restraint stress.And the TCM syndromes type of the model was determined by the method of“Syndrome Differentiation through Formula Effect Assessment”.At the same time,the plasma of the mice was sampled by enucleation method,and the content of hormones was detected by LC-MS.The expression of lipid oxidation indicator 4-HNE and innate immune index IFN-β,TNF-αand IL-6 proteins in TG were measured by Western Blotting method.Results:HSV-1 latent infection could be established in the TG of mice after 4 weeks nasal infection and it could be reactivated after being restrained for 4 days.Compared with the activated group,the Danzhi Xiaoyao Powder could significantly reduce the activation rate of HSV-1 and recurrent eye infections than Xiebai Powder.The plasma corticosterone(CORT)level in the reactivated group and the Danzhi Xiaoyao Powder group were increased significantly.However,no significant difference between the two groups was observed.Moreover,the expression of 4-HNE and IFN-βin TG was significantly reduced by Danzhi Xiaoyao Powder.Danzhi Xiaoyao Powder can also reduce TNF-αand

关 键 词：丹栀逍遥散 情志应激 单纯疱疹病毒1型 潜伏激活 肝郁化火 脂质过氧化 中医动物模型 

分 类 号：R275[医药卫生—中医皮科]