丹参复合有效部位抗HIV-1活性的实验研究

作者	单位
彭宗根	中国医学科学院中国协和医科大学医药生物技术研究所
秦德华	中国医学科学院中国协和医科大学医药生物技术研究所
滕立	中国医学科学院中国协和医科大学医药生物技术研究所
高雷	中国医学科学院中国协和医科大学医药生物技术研究所
蒋建东	中国医学科学院中国协和医科大学医药生物技术研究所
陈鸿珊	中国医学科学院中国协和医科大学医药生物技术研究所

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中文摘要:

目的研究和评价云南丹参复合有效部位(DS-MEF)的抗艾滋病病毒1型(human immunodefi-ciency virus type1,HIV-1)活性。方法采用放射性核素3H掺入法、荧光方法和酶联免疫吸附实验分别测定其对HIV-1逆转录酶、蛋白酶和整合酶的抑制作用;采用传代人T淋巴细胞MT-4细胞、慢性HIV-1ⅢB感染传代人T淋巴细胞H9细胞和健康人新鲜外周血单核细胞(peripheral blood mononuclear cell,PBMC),用HIV-1实验室传代的ⅢB病毒株和临床分离的齐多夫定(zidovudine,AZT)敏感株018a及AZT耐药株018c感染细胞测定其对细胞的毒性和在细胞培养内对HIV-1P24的抑制作用;灌胃或腹腔注射1次不同剂量DS-MEF后测定其对昆明种小鼠的急性毒性。结果DS-MEF抑制HIV-1整合酶、逆转录酶和蛋白酶的半数抑制浓度(50%inhibiting concentration,IC50)分别为(2.59±0.50)mg/L、(27.39±11.18)mg/L和(9.38±2.45)mg/L;对MT-4细胞毒性的半数有毒浓度(50%toxic concentration,TC50)为(13.19±6.07)mg/L,抗HIV-1活性的IC50为(0.224±0.163)mg/L,选择指数(selected index,SI)为58.7;在HIV-1ⅢB慢性感染的H9细胞培养内对细胞毒性的TC50为(18.11±9.84)mg/L,抑制HIV-1P24抗原的IC50为(17.230±21.114)mg/L,SI为1.1。对PBMC细胞毒性的TC50为(288.70±0.08)mg/L,抑制AZT敏感株HIV-1018a的IC50为(26.42±11.16)mg/L,SI为10.9,抑制AZT耐药株HIV-1018c的IC50为(27.87±5.35)mg/L,SI为10.4;与AZT和奈韦拉平均有协同抗HIV-1的作用,联合指数分别为0.78和0.67。灌胃20g/kg DS-MEF对小鼠无明显毒性,无毒剂量>20g/kg,腹腔注射后对小鼠的半数致死剂量为1.18g/kg。结论DS-MEF毒性小,具有多环节多功能地抑制HIV-1的作用。

英文摘要:

Objective To explore and evaluate the activities of composite extract from Salvia Yunnanensis and in cell cultures(DS-MEF)for inhibition of human immuno-deficiency virus type 1(HIV-1)in vitro and in cell cultures.Methods The inhibitory activity of DS-MEF on HIV-1 reverse transcriptase(RT),protease(PR)and integrase(IN)were detected in vitro with radionuclide 3H incorporation,fluorescence assay and enzyme-linked immunosorbent assay respectively.The human T-lymphocyte MT-4 cell line,human T-lymphocyte H 9 cell line chronically infected with HIV-1 ⅢB,and the fresh peripheral blood mononuclear cell(PBMC)of healthy persons as well as the laboratory passed HIV-1 ⅢB and the clinically isolated HIV-1 AZT sensitive 018a or resistant 018c infected cell cultures were used for evaluating the cytotoxicities and inhibitory activities of DS-MEF on HIV-1 P 24 antigen.The acute toxicities of DS-MEF on KM mice were determined by gastric gavages and intraperitoneal injections with various dosages.Results The IC50 of DS-MEF for inhibiting HIV-1 IN,RT and PR were 2.59±0.50 mg/L,27.39±11.18 mg/L and 9.38±2.45 mg/L respectively.In MT-4 cell cultures infected with HIV-1 Ⅲ,TC50 were 13.19±6.07 mg/L,IC50 and SI of anti-HIV-1 activity were 0.224±0.163 mg/L and 58.7;in chronically infected H 9 cell cultures,TC50 were 18.11±9.84 mg/L,IC50 on HIV-1 P 24 antigen and SI were17.230±21.114 mg/L and 1.1 respectively;TC50 in HIV-1 infected PBMC cultures were 288.70±0.08 mg/L;IC50 on AZT sensitive HIV-1 018a:26.42±11.16 mg/L,and SI:10.9;On AZT resistant HIV-1 018c,IC50:27.87±5.35 mg/L,and SI:10.4.Moreover,DS-MEF showed synergistic effect with AZT or nevirapine(NVP)on HIV-1 ⅢB in MT-4 cell cultures,the respective combination index was 0.78 or 0.67.DS-MEF showed no acute toxicity in KM mice with the dosage up to 20 g/kg via gastrogavage,and the 50% lethal dose(LD50)via intraperitoneal injection was 1.18 g/kg.Conclusion DS-MEF is a promising anti-HIV-1 agent with low toxicity in mice and possesses multi-targets and effective activities.

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