金丝桃素体外抗传染性支气管炎病毒的研究
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摘要
将不同浓度的金丝桃素和鸡传染性支气管炎病毒(IBV)以三种方式加入鸡胚肾细胞(CEK)培养体系中,采用细胞病变效应(CPE)法、MTT法、病毒滴度法、实时荧光定量PCR以及间接免疫荧光技术检测金丝桃素体外抗IBV作用,同时检测其对IBV诱发细胞凋亡的影响。结果显示,金丝桃素在CEK细胞感染IBV前、后及同时加入,都有明显抑制CPE的作用;随着添加药物浓度的增加,CEK细胞活力明显增高,病毒TCID50滴度、病毒核衣壳蛋白RNA相对表达量和CEK细胞中病毒阳性绿色荧光信号显著降低或减少(P<0.01),并且呈现明显的剂量依赖性。三种给药方式以药物和病毒互作后同时加入效果最好。IBV可以引起CEK细胞凋亡的发生,添加金丝桃素能够抑制病毒引起的凋亡。上述试验结果表明,金丝桃素具有很好的抗IBV作用,其作用可能是通过抑制感染细胞凋亡产生的,这可能是金丝桃素抗IBV的作用机制之一。
Hypericin of different concentrations and IBV were added to the culture system of chicken embryo kidney(CEK) cells in three ways,cytopathic effect(CPE) observation,virus titer assay,MTT method,real-time fluorescence quantitative PCR and indirect immunofluorescence were used to detect the anti-IBV effect of hypericin in vitro.At the same time,the apoptotic morphological changes of CEK cells post-addition of hypericin to IBV were detected with apoptosis kit.In result,hypericin could significantly inhibit the CPE in all three ways of adding hypericin before,after or simultaneously with IBV into CEK cells.With the increase of hypericin concentration,the viability of CEK cells increased remarkably,the virus titer of TCID50,the relative expression of IBV N gene,and the positive fluorescence signal of the virus in CEK cells decreased significantly(P<0.01).The anti-viral effect of hypericin was obviously concentration dependent.Among three ways of addition of the drug,the most effective way was addition of hypericin into IBV simultaneously post-incubation.Hypericin could inhibit the apoptosis caused by IBV.In conclusion,hypericin had good anti-IBV effect and its function may be mediated by inhibiting the apoptosis of infected cells,which may be one of its anti-IBV mechanisms.
Hypericin of different concentrations and IBV were added to the culture system of chicken embryo kidney(CEK) cells in three ways,cytopathic effect(CPE) observation,virus titer assay,MTT method,real-time fluorescence quantitative PCR and indirect immunofluorescence were used to detect the anti-IBV effect of hypericin in vitro.At the same time,the apoptotic morphological changes of CEK cells post-addition of hypericin to IBV were detected with apoptosis kit.In result,hypericin could significantly inhibit the CPE in all three ways of adding hypericin before,after or simultaneously with IBV into CEK cells.With the increase of hypericin concentration,the viability of CEK cells increased remarkably,the virus titer of TCID50,the relative expression of IBV N gene,and the positive fluorescence signal of the virus in CEK cells decreased significantly(P<0.01).The anti-viral effect of hypericin was obviously concentration dependent.Among three ways of addition of the drug,the most effective way was addition of hypericin into IBV simultaneously post-incubation.Hypericin could inhibit the apoptosis caused by IBV.In conclusion,hypericin had good anti-IBV effect and its function may be mediated by inhibiting the apoptosis of infected cells,which may be one of its anti-IBV mechanisms.
引文
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[16]金修哲,何雷,程相朝,等.6种中药成分对猪传染性胃肠炎病毒的体外抑制作用[J].中国农业科学,2016,49(11):2234-2244.JIN Xiu-zhe,HE Lei,CHENG Xiang-chao,et al.Effect of six Chinese traditional medicine components on inhibiting swine transmissible gastroenteritis virus in vitro[J].Scientia Agricultura Sinica,2016,49(11):2234-2244.(in Chinese)
[17]沈飚,王忠发,胡兴娟,等.实时荧光定量PCR及RT-PCR与常规PCR及RT-PCR检测对虾病毒效果的比较[J].微生物学杂志,2012,32(3):30-36.SHEN Biao,WANG Zhong-fa,HU Xing-juan,et al.Comparison of the effects among real-time fluorescent quantitative PCR and RT-PCR with routine PCR or RT-PCR to detect prawn viruses[J].Journal of Microbiology,2012,32(3):30-36.(in Chinese)
[18] CHHABRA R,KUCHIPUDI S V,CHANTREY J,et al.Pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses[J].Virology,2016,488:232-241.
[2] LI H,WU J,ZHANG Z,et al.Forsythoside a inhibits the avian infectious bronchitis virus in cell culture[J].Phytotherapy Research,2011,25(3):338-342.
[3] OLIVEIRA A I,PINHO C,SARMENTO B,et al.Neuroprotective activity of Hypericum perforatum and its major components[J].Frontiers in Plant Science,2016,7:1004.
[4] ERIKSSON E S,SANTOS D J,GUEDES R C,et al.Properties and permeability of hypericin and brominated hypericin in lipid membranes[J].Journal of Chemical Theory and Computation,2009,5(12):3139-3149.
[5] QU X B,SU Z M,HU D H,et al.Studies on molecular structure of hypericin and its interactions with HIV-1 protease by molecular modeling[J].Chemical Journal of Chinese Universities-Chinese,2009,30(7):1402-1405.
[6]贾永蕊,胡然,张永鹤,等.金丝桃素和盐酸赖氨酸抗疱疹病毒作用体外试验研究[J].中国临床康复,2004,8(5):996-997.JIA Yong-rui,HU Ran,ZHANG Yong-he,et al.In vitro study on the effects of hypericin and lysine hydrochloride against herpes virus[J].Chinese Journal of Clinical Rehabilitation,2004,8(5):996-997.(in Chinese)
[7]王曙阳,梁剑平,陈积红,等.金丝桃素体外抗口蹄疫病毒与宿主细胞吸附作用研究[J].中兽医医药杂志,2009,28(1):5-8.WANG Shu-yang,LIANG Jian-ping,CHEN Ji-hong,et al.Studies on the inhibitory effects of hypericin on the adsorption ability of foot-and-mouth virus to host cells in vitro[J].Journal of Traditional Chinese Veterinary Medicine,2009,28(1):5-8.(in Chinese)
[8]蒲秀英,梁剑平,许涛,等.金丝桃素体外抗高致病性猪生殖与呼吸综合征病毒活性的研究[J].中国兽医科学,2008,38(9):810-815.PU Xiu-ying,LIANG Jian-ping,XU Tao,et al.Study on activity in vitro of hypericin against highly pathogenic PRRSV[J].Chinese Veterinary Science,2008,38(9):810-815.(in Chinese)
[9]郭志廷,陈士恩,梁剑平,等.金丝桃素可溶性粉体外抗犬瘟热病毒的试验研究[J].中国兽医科学,2010,40(2):201-204.GUO Zhi-ting,CHEN Shi-en,LIANG Jian-ping,et al.Study on hypericin soluble powder against canine distemper virus in vitro[J].Chinese Veterinary Science,2010,40(2):201-204.(in Chinese)
[10] PU X Y,LIANG J P,SHANG R F,et al.Therapeutic efficacy of Hypericum perforatum L.extract for mice infected with an influenza A virus[J].Canadian Journal of Physiology and Pharmacology,2012,90(2):123-130.
[11]尚若锋,刘宇,陈炅然,等.金丝桃素粉剂对人工感染传染性囊病病毒鸡的疗效试验[J].中国兽医杂志,2012,48(1):12-15.SHANG Ruo-feng,LIU Yu,CHEN Jiong-ran,et al.Therapeutic efficacy of hypericin powder for chickens infected with infectious bursal disease virus[J].Chinese Journal of Veterinary Medicine,2012,48(1):12-15.(in Chinese)
[12] MURTHY H N,KIM Y S,PARK S Y,et al.Hypericins:biotechnological production from cell and organ cultures[J].Applied Microbiology and Biotechnology,2014,98(22):9187-9198.
[13] JACKSON A,D’AVOLIO A,MOYLE G,et al.Pharmacokinetics of the co-administration of boceprevir and St John's wort to male and female healthy volunteers[J].Journal of Antimicrobial Chemotherapy,2014,69(7):1911-1915.
[14] VAN MEERLOO J,KASPERS G J,CLOOS J.Cell sensitivity assays:the MTT assay[J].Methods in Molecular Biology,2011,731(88):237-245.
[15] ANJANA A K,FRANCIS P,GOMATHY S,et al.In vitro antiviral screening of Psidium Gujava L.against HSV-1 and HSV-2 by CPE inhibition assay[J].Asian Journal of Pharmaceutical and Clinical Research,2014,7(1):102-104.
[16]金修哲,何雷,程相朝,等.6种中药成分对猪传染性胃肠炎病毒的体外抑制作用[J].中国农业科学,2016,49(11):2234-2244.JIN Xiu-zhe,HE Lei,CHENG Xiang-chao,et al.Effect of six Chinese traditional medicine components on inhibiting swine transmissible gastroenteritis virus in vitro[J].Scientia Agricultura Sinica,2016,49(11):2234-2244.(in Chinese)
[17]沈飚,王忠发,胡兴娟,等.实时荧光定量PCR及RT-PCR与常规PCR及RT-PCR检测对虾病毒效果的比较[J].微生物学杂志,2012,32(3):30-36.SHEN Biao,WANG Zhong-fa,HU Xing-juan,et al.Comparison of the effects among real-time fluorescent quantitative PCR and RT-PCR with routine PCR or RT-PCR to detect prawn viruses[J].Journal of Microbiology,2012,32(3):30-36.(in Chinese)
[18] CHHABRA R,KUCHIPUDI S V,CHANTREY J,et al.Pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses[J].Virology,2016,488:232-241.
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