【作者】 张媛媛；

【导师】 丁健； 黄成钢；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2006， 博士

【摘要】 病毒性心肌炎(Viral myocarditis,以下简称VMC)主要由柯萨奇病毒(Coxsackievirus)等引起,是以心肌细胞变性坏死、间质单个核细胞浸润为主要病变的一种临床常见病,严重危害人类健康。柯萨奇病毒(Coxsackievirus)属于小RNA病毒(Picornavirus)科,肠道病毒(Enterovirus)属,根据在新生鼠中引起的病理状态不同而分为A、B两族,其中A族共有24个亚型,B族包括6个亚型。柯萨奇B3病毒(Coxsackievirus B3,以下简称CVB3)是引起VMC的最主要病原体。目前临床尚无针对VMC的特异性的疫苗和治疗药物,多采用综合及支持疗法,包括抗病毒、营养心肌、免疫调节及对症治疗等。近年来,中医药在治疗VMC方面显示出很大潜力。一些中药,如黄芪,在临床上常常以单味药或复方粗提物形式应用,如黄芪口服液、芪冬颐心口服液等,收到一定治疗效果。但至今仍缺乏有效成分和作用机理明确、药效显著、剂型先进的现代中药。经多年研究,本课题组从黄芪和苦豆子中提取分离出对VMC有显著治疗作用的有效部位和单体有效成分。本文采用动物模型、细胞模型和血清药理学、药代动力学及分子生物学等方法,分别对黄芪中的黄芪总苷、黄芪甲苷、毛蕊异黄酮葡萄糖苷和苦豆子中的槐定碱等成分进行研究,观察其对CVB3的体内外抑制作用,对VMC动物的免疫调节作用,药物的体内过程,以及治疗VMC的药更多还原

【Abstract】 Viral myocarditis can be caused by many viruses, such as coxsackievirus,adenovirus, and echovirus. Viral myocarditis is characterized with mononuclear cellinfiltration and necrosis in myocardium, and can develop to dilated cardiomyopathy(DCM). Coxsackievirus is a member of the family Picornaviridae and lies in theEnterovirus genus, together with polioviruses, echoviruses and unclassified viruses.Coxsackieviruses are classified into group A and B according to their pathogenecityin new born mice. Type B Coxsackievirus (CVB) is a common human pathogen andhas been implicated in acute and chronic myocarditis. Coxsackievirus B3 (CVB3)can cause fatal viral myocarditis, hepatitis, and meningo-encephalitis in children andadults, which is an important human pathogen responsible for acute and chronic viralmyocarditis in children and young adults.Although much efforts has been devoted to the development of vaccines andtherapeutic agents against CVB3, no effective clinical therapeutic methods have beenavailable so far. Traditional Chinese medicines have been widely used in clinicallytreating viral myocarditis for a long time. Some herbs or their compositions weredemonstrated to have significant antivirus effects against coxsackievirus B3 andtherapeutic effects for viral myocarditis in clinical. But what compounds in them orhow they play antiviral roles still remains unclear. It is of great urgency to investigateand develop new reagents with clear composition and mechanisms for viralmyocarditis induced by coxsackievirus B3.Scores of active components extracted from traditional Chinese medicines hadbeen screened by us. Several active components had been demonstrated to havesignificant antiviral effects against coxsackievirus B3 and therapeutic effects for viralmyocarditis by in vitro (primarily cultured myocardial cells), in vivo (Balb/cmice),serum pharmacological and pharmacokinetics experiments. They are totalastragalosides, astragaloside IV, calycosin-7-β-D-glucoside, and sophoridine.1. Total astragalosides had been demonstrated to have significant antiviruseffects against coxsackievirus B3 by in vitro and in vivo experiments. It wasextracted from Radix Astragali which had been used in China for a long history. Ithad been demonstrated to have significant antivirus effects against coxsackievirus B3and therapeutic effects for viral myocarditis. And it had been shown to have noobvious influence on cardiovascular system, respiratory system, and nerve system ofexperimental animals. To investigate the mechanisms of total astragalosides againstcoxsackievirus B3, NO concentrations in serum of infected Balb/c mice weremeasured by enzyme-linked immunosorbent assay (ELISA) method. IFN-γ andTNF-α expressions in serum of infected Balb/c mice were also determined by elisamethod. It was demonstrated that total astragalosides could significantly decreaseNO concentrations in serum. IFN-γ protein concentrations in serum were obviouslyenhanced after total astragalosides treatment. TNF-α protein concentrations inserum were obviously decreased compared with that in infected controls.2. Astragaloside IV was extracted from triditional Chinese medicine RadixAstragali. It had been demonstrated that astragaloside IV could decrease virus titersof infected and primarily cultured myocardial cells. Balb/c mice wereintraperitoneally inoculated with CVB3. Then, mice were orally administered with120 mg/kg or 60 mg/kg astragaloside IV consecutively for 7 days. Astragaloside IVwas demonstrated to be able to significantly decrease morbidity, mortality, HW/BWratios, and virus titers of hearts. Mononuclear cell infiltration and necrosis of heartslices were obviously alleviated compared with that of infected controls. IFN-γmRNA expression in hearts of infected Balb/c mice were measured by RT-PCRmethod to investigate how astragaloside IV played its antivirus roles. The level ofmRNA expressions of IFN-γwas significantly enhanced in CVB3 infected micewith astragaloside IV treatment, compared with those of infected control group.3. Calycosin-7-β-D-glucoside was a compound extracted from traditionalChinese medicine Radix Astragali, which had been demonstrated to be effective todecrease LAD and AST activities in serum of infected Balb/c mice. IFN-γ andTNF-α expressions in serum of infected Balb/c mice were also determined by elisamethod. Balb/c mice were orally administered with Calycosin-7-β-D-glucoside atthe dosages of 40 mg/kg and 20 mg/kg for 12 days after intraperitoneally inoculatedwith CVB3. Animals were sacrificed at 15 days after inoculation. Mortality, HW/BWratios, and activities of AST and LDH were significantlt decreased after Calycosin-7-β-D-glucoside treatment.4. Sophoridine had been demonstrated to have significant antivirus effectsagainst coxsackievirus B3 by in vitro and in vivo experiments. It had beendemonstrated that sophoridine could decrease virus titers of infected primarilycultured myocardial cells. Balb/c mice were intraperitoneally inoculated with CVB3.Then, mice were orally administered with 40 mg/kg or 20 mg/kg sophoridineconsecutively for 7 days. Sophoridine was demonstrated to be able to significantlydecrease mortality, HW/BW ratios, and virus titers of hearts. Mononuclear cellinfiltration and necrosis of heart slices were obviously alleviated compared with thatof infected controls. To investigate the relation between its pharmacology effects andmetabolism, its pharmacokinetics were investigated in SD rats serum. Matrine waschosen as an internal standard because it had a structure similar to that of sophoridine.SD rats were orally administered with 150 mg/kg sophoridine after a 24 h fastingperiod. Animals were sacrificed at 5 min, 15 min, 30 min, 1, 2, 3, 4, 8, 12, and 24 hafter administration. After appropriate preparation, serum samples were analyzed bythe high-performance liquid chromatography (HPLC) method. Andconcentration-time profile in serum was constructed. After oral administration ofsophoridine, all rats showed measurable amounts of the compound in serum at thetime of the first post-dose blood sampling. The tmax was 0.5 h, and cmax was 133ug/ml. Since we got positive results in serum pharmacological experiments, we triedto measure antiviral activities of serum at timed points to draw an antiviralactivity-time profile and to investigate association between the concentration-timeprofile and the antiviral activity-time profile. Serum samples obtained at differenttime points were diluted to determine the antiviral effects by serum pharmacologicalexperiments. The inhibitory rate, which is (the average of virus titers in infectedcontrols-average of virus titers in serum samples)/average of virus titers in infectedcontrols × 100%, was used as an indicator for antiviral activities of serum samplesobtained at timed points and depicted as an antiviral activity-time profile. The twoprofiles were analyzed and compared. After oral administration of sophoridine, alltested serum samples had antiviral activity. The sample obtained at 0.5h had thegreatest antiviral activity, which was correlated with the concentration-time profile.Then, cytokines expressions in hearts of Balb/c mice infected with CVB3 weremeasured by reverse transcription polymerase chain reaction (RT-PCR). Expressionsof IFN-γ, TNF-α and IL-10 mRNA were determined and compared with controls.mRNA expressions of IL-10 and IFN-γ was significantly enhanced, butexpressions of TNF-α mRNA significantly decreased in mice with sophoridinetreatment, compared with those of infected control group.In summary, we systematically investigated antivirus activities and therapeuticeffects of total astragalosides, astragaloside IV, calycosin-7-O-β-D-glucoside, andsophoridine. Total astragalosides was demonstrated to be effective components ofRadix Astragali. Astragaloside IV and calycosin-7-O-β -D-glucoside weredemonstrated to be effective compounds of Radix Astragali. They were suggested toplay their roles by regulating of cytokines expression, such as IL-10, IFN-γ, andTNF-α. These findings played a foundation for development of new traditionalChinese medicines for viral myocarditis.更多还原