清热消炎宁抑制潜伏Ⅰ型单纯疱疹病毒激活的作用研究  

Qingre Xiaoyanning inhibits emotional stress-induced reactivation of latent herpes simplex virus type 1 in mice

作　　者：姜珊 牛杰 欧阳淑桦 江涛 彭红英 罗卓 栗原博[1,2] 李怡芳[1,2] 何蓉蓉[1,2,3] JIANG Shan;NIU Jie;OUYANG Shu-hua;JIANG Tao;PENG Hong-ying;LUO Zhuo;HIROSHI Kurihara;LI Yi-fang;HE Rong-rong(Guangdong Engineering Research Center of Chinese Medicine and Disease Susceptibility,Jinan University,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;Guangzhou Baiyunshan Jingxiutang Pharmaceutical Co.,Ltd.,Guangzhou 510130,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,广东广州510632 [2]暨南大学药学院中药及天然药物研究所,广东广州510632 [3]暨南大学中医学院,广东广州510632 [4]广州白云山敬修堂药业股份有限公司,广东广州510130

出　　处：《药学学报》2022年第6期1641-1648,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(U1801284);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(201903010062)。

摘　　要：本研究采用情志应激诱导的潜伏Ⅰ型单纯疱疹病毒(HSV-1)复发的“肝郁化火”动物模型,研究清热消炎宁对HSV-1激活复发的作用及机制。实验方案经暨南大学动物实验伦理委员会批准,所有程序均严格按照动物使用和护理的伦理原则进行。实验分为正常组、潜伏组、激活组、清热消炎宁低剂量组(0.658 g·kg^(-1)·day^(-1))、高剂量组(1.316 g·kg^(-1)·day^(-1))和阳性药阿昔洛韦组(0.206 g·kg^(-1)·day^(-1))。除正常组和潜伏组外,其他组小鼠在给药的第4天开始每天拘束12 h,连续拘束4天,在给药7天结束以后,记录小鼠的体质量变化和眼部炎症复发情况。乙醚麻醉小鼠后,取脑组织进行以下指标的检测:免疫组化实验方法检测HSV-1抗原分布情况;空斑法检测病毒滴度;RT-PCR法检测HSV-1相关病毒基因感染细胞多肽27(ICP27)和糖蛋白B(gB)的表达水平;Western blot法检测HSV-1相关病毒蛋白ICP27和gB的表达、脂质过氧化产物4-羟基壬烯醛(4-HNE)水平和铁死亡相关通路中花生四烯酸-15-脂加氧酶(ALOX15)、铁蛋白转运受体1(TFR1)、二价金属转运蛋白1(DMT1)、谷胱甘肽过氧化酶4(GPX4)和钙非依赖磷脂酶A2β(iPLA2β)蛋白表达水平;丙二醛(MDA)检测试剂盒检测另一脂质过氧化产物MDA的含量。实验结果显示,清热消炎宁能抑制情志应激诱导HSV-1激活复发小鼠体质量比的下降,降低眼部炎症复发率和脑组织HSV-1抗原分布和病毒滴度,显著下调HSV-1病毒基因ICP27和gB、病毒蛋白ICP27和gB的表达水平。此外,清热消炎宁还能明显抑制HSV-1激活复发小鼠脑组织中铁死亡通路正调节相关蛋白DMT1、TFR1和ALOX15的表达,并能减少脂质过氧化物产物4-HNE和MDA的水平。以上实验结果表明,清热消炎宁能抑制情志应激介导的HSV-1激活复发,其作用机制可能与干预铁死亡有关。本研究可为清肝泻火中药防治HSV-1激活复发感染导致的相关疾病的中医药In this study,according to TCM theory of"liver qi stagnation forming fire",emotional stress mice model was employed to evaluate the protective effects of Qingre Xiaoyanning on herpes simplex virus type 1(HSV-1)induced reactivation.The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Jinan University,in compliance with the Institutional Animal Care Guidelines.BALB/c mice were divided into six groups,including mock group,HSV-1 latency group,HSV-1 reactivation group(HSV-1 latency+stress),low(0.658 g·kg^(-1)·day^(-1))and high dose(1.316 g·kg^(-1)·day^(-1))of Qingre Xiaoyanning groups and positive control group(acyclovir,0.206 g·kg^(-1)·day^(-1)).Except for the normal group and HSV-1 latency group,all mice in other groups received a daily 12-h restraint stress for 4 days.After 7-day treatment of drugs,body weight and recurrent eye infections of mice were recorded.Brain tissues were harvested to monitor HSV-1 antigen distribution by immunohistochemical staining and detect virus titer by plaque assay.In the meantime,the mRNA and protein levels of infected cell polypeptide(ICP27)and glycoprotein B(gB)in the brain tissues were detected by RT-PCR and Western blot,respectively.The level of 4-hydroxynonenal(4-HNE)and expressions of ferroptosis-related proteins were measured by Western blot.The evaluation of malondialdehyde(MDA)content in the brain tissues was conducted by MDA assay commercial kit.The results showed that Qingre Xiaoyanning significantly retarded the decline of body weight of mice induced by HSV-1 reactivation,reduced the activation rate of HSV-1 and recurrent eye infections,declined virus titer of HSV-1,down-regulated gene and protein expressions of ICP27 and gB,and hindered the distribution of HSV-1 antigen in the brain of mice.Meanwhile,Qingre Xiaoyanning also decreased the protein expression of ferroptosis-related proteins,including DMT1,TFR1 and ALOX15 in the brain tissue of HSV-1 reactivated mice.The levels of lipid peroxidation products,4-HNE and

关 键 词：情志应激 清热消炎宁 九节茶 肝郁化火 Ⅰ型单纯疱疹病毒激活 铁死亡 

分 类 号：R966[医药卫生—药理学]