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日本科学技术振兴机构数据库(JST)
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医药导报, 2017, 36(10): 1124-1129
doi: 10.3870/j.issn.1004-0781.2017.10.009
黄芩苷体外抗人巨细胞病毒活性及其对感染人胚肺成纤维细胞凋亡的影响*
Activity of Baicalin Against Human Cytomegalovirus In Vitro and Its Effect on the Apoptosis of Human Embryo Lung Fibroblasts Infected with Human Cytomegalovirus
刘杨, 黄媛, 廖毅, 秦文卿, 刘玲玲, 李革, 舒赛男, 方峰

摘要:

目的 观察黄芩苷体外抗人巨细胞病毒(HCMV)的活性;探讨黄芩苷对HCMV感染人胚肺成纤维细胞(HEL)凋亡的影响。方法 采用CCK-8法测定HEL细胞对黄芩苷药物的最大耐受剂量(MTC);标准空斑抑制法测定黄芩苷抗HCMV的半数有效浓度(EC50);分别用流式细胞术及Western blotting法检测不同浓度黄芩苷对高、低感染复数组不同时间点(感染后24,48,72,96 h)凋亡率及凋亡关键蛋白pro-caspase-3表达的影响。结果 HEL细胞对黄芩苷的MTC为20.6 μg·mL-1;黄芩苷抗HCMV的EC50为16.13 μg·mL-1;大小剂量黄芩苷处理高低感染复数HCMV感染的细胞后,均表现出较强的凋亡抑制效应。大剂量黄芩苷处理高感染复数HCMV感染细胞后pro-caspase-3表达量较未处理前增多(P<0.05)。结论 黄芩苷体外具有直接抗HCMV效应,其抑制感染细胞凋亡和拮抗pro-caspase-3活化可能是其机制之一。

关键词: 黄芩苷 ; 人巨细胞病毒 ; 人胚肺成纤维细胞 ; 细胞凋亡 ; pro-caspase-3表达

Abstract:

Objective To elucidate activity of baicalin against human cytomegalovirus (HCMV) in vitro, and explore its effect on apoptosis of human embryo lung fibroblasts (HEL) infected with HCMV. Methods CCK-8 kit was used to determine the maximum tolerated dose (MTC) of HEL cell to baicalin while the anti-HCMV median efficacious concentration (EC50) of baicalin was determined by standard plaque reduction method. After treatment with baicalin of different concentrations for 24, 48, 72 and 96 h, cell apoptosis and pro-caspase-3 expression was detected by flow cytometry and Western blotting, respectively. Results The MTC of baicalin was 20.6 μg·mL-1; The EC50 of anti-HCMV of baicalin was 16.13 μg·mL-1; The apoptosis rate increased gradually in the groups with low and high multiplicity HCMV infection at early time, showing significant dose-dependent manner. While the ratio of apoptotic cells was going to decrease in high multiplicity infection group 96 h after the infection. The expression of pro-caspase-3 was significantly higher in high-dose baicalin treatment group than in the infection control group (P<0.05). Conclusion Baicalin has a direct anti-HCMV effect in vitro. One of the mechanisms might be related with it inhibiting cell apoptosis and antagonizing activation of pro-caspase-3.

Key words: Baicalin ; Human cytomegalovirus ; Human embryo lung fibroblast ; Apoptosis ; Pro-caspase-3 expression

人巨细胞病毒(human cytomegalovirus,HCMV)是一种世界范围内普遍感染的双链DNA病毒,一旦感染将终身携带。我国是HCMV感染的高发地区,人群抗HCMV-IgG阳性率高,为86%~96%[1]。HCMV感染对胎儿、婴幼儿、器官移植患者、艾滋病患者等免疫低下人群可造成严重损害,甚至危及生命。但是,一直以来,抗HCMV感染药物的研究进展缓慢,能用于临床的抗HCMV药物寥寥无几,故寻找和开发安全、副作用小而且疗效佳的抗HCMV药物一直是医药学界的努力方向和研究任务。黄芩具有泻火解毒、清热燥湿等功效。国内外学者已经发现,黄芩提取物黄芩苷可以抑制流感病毒、登革热病毒、单纯疱疹病毒-Ⅰ型(herpes simplex virus type 1,HSV-Ⅰ)、HSV-Ⅱ等多种病毒的复制[2-5]。本课题组前期在黄芩苷抗小鼠巨细胞病毒(murine cytomegalovirus,MCMV)的整体研究中发现,黄芩苷对MCMV具有直接抑制作用,可显著下调MCMV感染小鼠唾液腺病毒滴度,减轻MCMV感染小鼠的肝脏病变[6]。因巨细胞病毒具有严格的宿主种属特异性,故考虑黄芩苷是否对HCMV也具有同样的抗病毒活性,抗病毒作用机制如何,笔者尚未见研究报道。调控细胞凋亡是HCMV致病的重要机制之一。本课题组前期研究发现,HCMV肝炎患儿,肝组织细胞凋亡率为53.72%,显著高于正常儿童的肝细胞凋亡率7.19%[7]。DOU等[8]发现,HCMV感染造成的血小板减少症也是通过诱导产板巨核细胞祖细胞的过度凋亡造成。本课题以HCMV感染的人胚肺成纤维细胞(human embryo lung fibroblast,HEL)为体外模型,检测黄芩苷体外抗HCMV的活性,并从细胞凋亡的角度探讨黄芩苷抗HCMV可能作用机制。

1 材料与方法
1.1 材料

1.1.1 细胞 本课题组自制HEL细胞,第10~20代细胞用于本实验。

1.1.2 药物 黄芩苷粉末,四川百利药业公司提供,高效液相色谱法鉴定含量为90%,以二甲亚砜配制成10 mg·mL-1浓缩液,-20 ℃分装包存,用时稀释成相应浓度。根据检测HEL细胞对黄芩苷最大耐受浓度,将黄芩苷给药浓度设置为:大剂量(20 μg·mL-1),小剂量(10 μg·mL-1)。

1.1.3 病毒 HCMV标准实验毒株AD169,由湖北省预防医学科学院病毒所提供。空斑法测定HCMV AD169株的感染性毒力为5.4×106 PFU ·mL-1

1.1.4 试剂 达尔伯克改良伊格尔培养基(dulbecco's modified eagle's medium,DMEM)(Gibco公司,批号:12800-017)按说明书方法配制。生长液:在配制好DMEM液中加入小牛血清(Gibco公司)至终浓度为20%(原代培养)、10%(传代培养)。维持液:在配制好DMEM液中加入小牛血清至终浓度为4%。兔抗人pro-caspase-3多克隆抗体(美国Cell Signaling Technology公司,批号:L0104)。小鼠抗人肌动蛋白(β-actin)多克隆抗体(美国Santa Cruz 公司,批号:26618)。 Annexin-V-FITC/PI 细胞凋亡检测试剂盒(上海贝博生物有限公司,批号:401002)。cell counting kit-8(CCK-8)试剂盒(日本同仁试剂有限公司,批号:C0037)。

1.1.5 仪器 DYY-7C型电泳仪(北京六一仪器厂);Mini P-4垂直电泳槽(北京六一仪器厂);DYCP-40E型湿转仪槽(北京六一仪器厂);PHOmo型全自动酶标仪(河南安图有限公司);超净工作台(Hfsafe-760S ClassⅡTYPE A/B3);二氧化碳(CO2)培养箱(heal force HF90/HF240);倒置显微镜(Olympus IX50型)。

1.2 HCMV细胞感染模型的建立

HCMV AD169感染HEL细胞。病毒感染复数(multiply of infection,MOI),MOI=感染性病毒量/细胞数,现国际公认MOI<1为低感染复数,本实验设低MOI组和高MOI组,MOI分别为0.25和2.5。

1.3 药物细胞毒性实验

将指数生长期的细胞接种入96孔板,待细胞长成单层后,加入倍比稀释的黄芩苷,同时设正常细胞作为阴性对照,入细胞培养箱培养72 h,用CCK-8法,测定药物的半数致死浓度(median cytotoxic concentration,IC50);同步观察细胞形态学变化,得出HEL细胞对黄芩苷的最大耐受浓度(maximum tolerated concentration,MTC)。每组均设复孔5个。

1.4 黄芩苷体外抗HCMV活性

采用空斑抑制法检测黄芩苷对HCMV的半数有效浓度(median efficacious concentration,EC50),得到系列浓度的空斑抑制率,进一步采用对数单位法(Logit法)求出黄芩苷抗HCMV的EC50值。黄芩苷抗HCMV病毒的药物选择指数(selective index,SI)按如下公式计算:SI=IC50 /EC50

1.5 分组

根据病毒感染量和药物剂量设置9个组,分别为:①模拟感染组;②低感染复数组;③高感染复数组;④大剂量黄芩苷组;⑤小剂量黄芩苷组;⑥低感染+小剂量黄芩苷组;⑦低感染+大剂量黄芩苷组;⑧高感染+小剂量黄芩苷组;⑨高感染+大剂量黄芩苷组。于感染后24,48,72,96 h收集细胞。

1.6 流式细胞术检测凋亡细胞比例

按Annexin-V-FITC/PI细胞凋亡检测试剂盒说明书方法处理各组细胞,采用流式细胞术检测。在双变量流式细胞仪的散点图上,左下象限为活细胞;左上象限为坏死细胞;右下象限为凋亡早期细胞;右上象限为凋亡晚期细胞。凋亡细胞比例=右下象限 +右上象限之和。

1.7 Western blotting法检测pro-caspase-3蛋白表达

根据流式细胞术检测结果,选择细胞凋亡率最高的时间点,即感染后72 h收获细胞。设模拟感染组、高感染复数组、高感染+小剂量黄芩苷组、高感染+大剂量黄芩苷组,共4组。常规提取各组细胞总蛋白,测定蛋白浓度,Western blotting法检测pro-caspase-3蛋白表达,其中兔抗人pro-caspase-3工作浓度1:1 000,鼠抗人β-actin工作浓度1:200,ECL发光试剂进行荧光显色。

1.8 图像分析及结果处理

图片用Gel-PRO Analyzer软件测定各个条带积分吸光度值。校正值κ=目的蛋白条带积分吸光度值/β-actin条带的积分吸光度值,以校正值κ对目的蛋白pro-caspase-3表达水平进行半定量分析。

1.9 统计学方法

采用SPSS18.0 for windows软件进行统计学分析,计数资料以均数±标准差( x ¯ ±s)表示。多组间均数比较采用方差分析法,方差齐性时两组间均数比较采用t检验,而方差不齐时采用秩和检验,以P<0.05为差异有统计学意义。

2 结果
2.1 黄芩苷抗HCMV的选择指数

黄芩苷对HEL细胞的IC50为225.23 μg·mL-1;HEL对黄芩苷MTC为 20.6 μg·mL-1。加入黄芩苷后,病毒空斑形成数较病毒对照组减少,且随黄芩苷浓度的增大逐渐减少,表现为剂量依赖性。通过Logit法分析黄芩苷对HCMV AD169的EC50为16.13 μg·mL-1,故黄芩苷抗HCMV的选择指数(SI)为13.96。

2.2 HCMV对HEL细胞凋亡率的影响

结果见表1。模拟感染组细胞凋亡率保持相对稳定的低水平,各时相之间差异无统计学意义。低感染复数组与高感染复数组分别于感染HCMV 48及24 h后凋亡率明显增高,于感染后96及72 h达峰值,高感染复数组增幅较大。高感染复数组在感染HCMV 96 h后凋亡率降低,但仍远高于模拟感染组。

表1 HCMV对HEL细胞凋亡率的影响
Tab.1 Effect of HCMV on apoptosis ratio of HEL %,x¯±s,n=5
组别 24 h 48 h
模拟感染组 7.42±0.52 7.88±0.29
低感染复数组 11.46±5.70 14.63±4.26*1
高感染复数组 15.15±1.27*1 21.44±5.54*2*3
组别 72 h 96 h
模拟感染组 7.31±0.73 7.87±0.84
低感染复数组 21.13±4.68*2 28.75±5.42*2
高感染复数组 36.36±3.44*2*3 31.17±6.99*2

Compared with mimetic infection group, *1P<0.05,*2P<0.01; Compared with low multiplicity infection group, *3P<0.01

与模拟感染组比较,*1P<0.05,*2P<0.01;与低感染复数组比较,*3P<0.01

表1 HCMV对HEL细胞凋亡率的影响

Tab.1 Effect of HCMV on apoptosis ratio of HEL %,x¯±s,n=5

2.3 黄芩苷对HCMV感染细胞凋亡的影响

2.3.1 黄芩苷对正常HEL 细胞凋亡比率的影响 两种剂量黄芩苷处理的HEL凋亡细胞比率尽管都增高(表2),但与模拟感染组比较均差异无统计学意义(均P>0.05)。

表2 黄芩苷处理对正常HEL细胞凋亡率的影响
Tab.2 Effect of baicalin on apoptosis ratio of HEL %,x¯±s,n=5
组别 24 h 48 h
模拟感染组 7.42±0.52 7.88±0.29
黄芩苷
小剂量组 7.75±0.48 7.95±0.15
大剂量组 7.96±0.62 7.99±0.21
组别 72 h 96 h
模拟感染组 7.31±0.73 7.87±0.84
黄芩苷
小剂量组 7.11±0.34 8.24±0.87
大剂量组 7.42±0.57 8.45±0.68

表2 黄芩苷处理对正常HEL细胞凋亡率的影响

Tab.2 Effect of baicalin on apoptosis ratio of HEL %,x¯±s,n=5

2.3.2 黄芩苷对HCMV感染HEL细胞凋亡比率的影响 低感染+小剂量黄芩苷组和低感染+大剂量黄芩苷组:感染细胞凋亡比率均出现降低。低感染+大剂量黄芩苷组在给药后24 h即出现有意义的凋亡率降低,在72 h时达到抑制细胞凋亡的最高峰,使细胞凋亡率降低63.98%;而低感染+小剂量黄芩苷组凋亡抑制效应相对延后,在给药后48 h出现有意义的凋亡细胞比率降低。见图1。

高感染+大剂量黄芩苷组在给药后24 h凋亡细胞比率出现有意义降低,此效应一直持续至给药后72 h;高感染+小剂量黄芩苷组凋亡抑制效应发挥较晚,于处理后72 h才出现有意义的凋亡比率下调。与高感染复数组比较,两组在96 h均出现凋亡细胞比率增高。见图2。

2.3.3 黄芩苷对HCMV感染HEL细胞pro-caspase-3表达的影响 如图3所示。实验组和模拟感染组在 42 000处β-actin 条带,各泳道的显色基本均一,说明各泳道上样量基本一致。在感染后72 h,小剂量黄芩苷处理,pro-caspase-3表达与高感染复数组比较,上调差异无统计学意义(P>0.05),而大剂量黄芩苷处理后pro-caspase-3表达量较高感染复数组增多,两者比较差异有统计学意义(P<0.05),但仍远低于模拟感染组(P<0.01)。通过采用Gel-PRO Analyzer软件计算出各组的校正值κ,结果见图4。

图1 黄芩苷对低感染复数HEL细胞凋亡率的影响(x¯±s,n=5)
与低感染复数组比较,*1P<0.05;与低感染+小剂量黄芩苷组比较,*2P<0.05

Fig.1 Effect of baicalin on apoptosis ratio of HEL with low multiplicity infection(x¯±s,n=5)
Compared with low multiplicity infection group, *1P<0.05; Compared with low multiplicity infection and low dose baicalin group, *2P<0.05

图2 黄芩苷对高感染复数HEL细胞凋亡比率的影响(x¯±s,n=5)
与高感染复数组比较,*1P<0.05

Fig.2 Effect of baicalin on apoptosis ratio of HEL with high multiplicity infection(x¯±s,n=5)
Compared with high multiplicity infection group, *1P<0.05

笔者发现HEL予低感染复数的HCMV感染时,凋亡率随感染时间延长逐渐增大,96 h达峰值,为正常细胞的4倍;予高感染复数的HCMV感染时,凋亡细胞比率增加更为明显,24 h就出现有意义的增高,72 h已经为模拟感染组5~6倍,达峰值,96 h却出现降低。检测结果与文献[12]结果相似。

图3 4组72 h pro-caspase-3表达量
A.模拟感染组;B.高感染复数组;C.高感染+小剂量黄芩苷组;D.高感染+大剂量黄芩苷组

Fig.3 Pro-caspase-3 expression of four groups at 72 h
A. mimetic infection group; B. high multiplicity infection group; C. high multiplicity infection and low dose baicalin group; D. high multiplicity infection and high dose baicalin group

图4 4组pro-caspase-3表达量的校正值κ(x¯±s,n=5)
A.模拟感染组;B.高感染复数组;C.高感染+小剂量黄芩苷组;D.高感染+大剂量黄芩苷组;与模拟感染组比较,*1P<0.01;与高感染复数组比较,*2P<0.05

Fig.4 Adjusted value κ of pro-caspase-3 level in four groups(x¯±s,n=5)
A. mimetic infection group; B. high multiplicity infection group; C. high multiplicity infection and low dose baicalin group; D. high multiplicity infection and high dose baicalin group. Compared with mimetic infection group, *1P<0.01; Compared with high multiplicity infection group, *2P<0.05

3 讨论

细胞凋亡是由基因控制的自主有序的生理性死亡,对维持机体发育和内环境稳定发挥重要作用。尽管病毒感染细胞发生凋亡,实为机体以牺牲少许感染细胞为代价换取整体生存和大环境稳态的重要机制。但近来研究发现,人类许多疾病和病理现象都与细胞凋亡机制的紊乱有关,其中CMV的致病作用也与感染细胞凋亡有着密切的关系 [7-11],而若有药物可以调控这一机制或许对于治疗HCMV感染性疾病产生重大意义。

综合分析发现,无论是低感染复数还是高感染复数的HCMV感染,均可诱导受染细胞凋亡,随感染时间延长凋亡逐渐增加;其机制可能为受染细胞通过启动凋亡机制以实现清除病毒的一种本能防御反应[13]

实验中还观察到尽管高感染复数组病毒剂量是低感染复数组的10倍,但处理后细胞并未出现凋亡细胞比率成倍增长,其原因可能与HCMV即刻早期基因UL36及UL37编码的凋亡抑制物vICA和vMIA有关,因vMIA与vICA表达量与病毒滴度呈正相关[14-15]

通过显微镜跟踪观察及CCK-8法检测,显示黄芩苷的细胞毒性较低,黄芩苷对HEL细胞的半数致死浓度为225.23 μg·mL-1,适合用于抗病毒药物的体外实验研究。通过空斑抑制实验,发现随着药物浓度增大,空斑抑制率逐渐增大。至药物浓度为20 μg·mL-1,接近MTC(20.6 μg·mL-1)时,空斑抑制率达到57.95%。通过计算得出黄芩苷体外抗HCMV的选择指数为13.96,其中选择指数大于1为有效,指数越大安全范围越大[16]。综上所述,黄芩苷体外环境下是一种安全有效的抗HCMV药物。

既往研究还发现黄芩苷可以通过影响凋亡调节蛋白Bcl-2和Bax的表达,降低肾脏细胞凋亡水平,延缓糖尿病肾病的发生[17-18];在流感病毒感染过程中,可通过调控细胞周期分布,抑制pro-caspase-8的激活,进一步抑制pro-caspase-3激活,降低流感病毒感染细胞凋亡,进而抑制流感病毒复制[3]

实验中发现黄芩苷处理可有效降低感染细胞凋亡比率,其抑制功效不仅与处理剂量有关,还与病毒滴度和作用时间密切相关。大小剂量黄芩苷处理正常HEL细胞后,凋亡率并未出现显著上调,可见在MTC浓度范围内黄芩苷本身并不会诱导细胞凋亡。黄芩苷的抗凋亡作用在低感染复数组表现较为显著,表现出明显剂量依赖性,即处理剂量越大,凋亡细胞比率下降越明显。而在高感染复数组,大、小剂量黄芩苷处理,分别于治疗后48,72 h凋亡细胞比率显著下调,然而,96 h却均出现凋亡细胞比率较感染对照组骤然增加,与低感染复数黄芩苷处理组表现不同。但这并不能武断得出黄芩苷在高感染组处理96 h已经丧失抗凋亡作用转为促凋亡的结论,因为存在这种可能性,即虽然黄芩苷依然持续发挥抗凋亡作用,但由于其拮抗高感染组子代病毒即刻早期基因UL36及UL37编码的凋亡抑制物 vICA和 vMIA的大量合成,使得处理组细胞并没有出现感染对照组凋亡细胞比率的下调,造成促凋亡的假象。这是因为有研究发现,黄芩苷的同系物黄芩素(在体内环境下二者可以相互转化[2,4])具有显著下调HCMV即刻早期基因表达水平的作用[18]

通过检测黄芩苷处理对pro-caspase-3的表达影响,发现大剂量黄芩苷处理72 h后pro-caspase-3表达量较高感染复数组增多,可见黄芩苷不仅在流感病毒感染中可以通过拮抗pro-caspase-3的活化发挥抑凋亡作用[3],而且在HCMV感染中也可能通过抑制pro-caspase-3的活化来减少细胞凋亡。高感染+小剂量组可能由于处理剂量较低,而仅出现pro-caspase-3表达数值上的上调,但与高感染复数组比较差异无统计学意义。由于本课题并未测定caspase-3的活性片段,无法确切判断黄芩苷是通过直接影响pro-caspase-3的剪切活化,还是通过促进pro-caspase-3的合成来实现上调pro-caspase-3表达水平,但结合流式细胞术凋亡细胞比率的检测结果,即大剂量黄芩苷显著下调高感染细胞凋亡比率,推测前一种机制的可能性更大。

综上所述,黄芩苷体外环境下通过拮抗HCMV诱导的受染细胞凋亡上调,表现出较强的细胞保护作用,该结果为临床使用黄芩苷治疗巨细胞感染性疾病提供了实验依据。但黄芩苷具体的抗凋亡作用机制,除与pro-caspase-3的表达水平相关外,是否也可能与影响其他在HCMV感染细胞凋亡过程中发挥重要作用的调控因子,如Bcl-2、Bax等表达相关,则需要更加深入的研究探讨。

The authors have declared that no competing interests exist.

参考文献

[1] 中华医学会儿科学分会感染学组,全国儿科临床病毒感染协作组,《中华儿科杂志》编辑委员会.儿童巨细胞病毒性疾病诊断和防治的建议[J].中华儿科杂志,2012,50(4):290-292.
人巨细胞病毒( human cytomegalovirus,HCMV),正式命名为人疱疹病毒5型(human herpes virus 5,HHV-5),其感染在我国极其广泛,一般人群HCMV抗体阳性率为86%~96%,孕妇95%左右,婴幼儿期为60%~80%,原发感染多发生于婴幼儿时期.HCMV具有潜伏-活化的生物学特性,一旦感染,将持续终身.虽然HCMV是弱致病因子,对免疫功能正常个体并不具有明显致病性,绝大多数表现为无症状性感染;但是,HCMV是引起病理性和生理性免疫低下人群,包括发育性免疫缺陷的胎儿和新生儿发生疾病的常见病原,亦是导致艾滋病和器官、骨髓移植患者严重疾病和增加病死率的重要病因之一. 二十余年来,我国儿科对儿童HCMV疾病进行了大量研究,取得丰富经验[ 1].中华医学会儿科学分会感染消化学组于1995年拟定《小儿巨细胞病毒感染诊断标准》(试行稿),1999年修订为《巨细胞病毒感染诊断方案》[2],为指导临床医生正确认识HCMV感染、深入其临床研究和开展防治工作做出积极贡献.现就儿童HCMV相关性疾病的诊断与防治提出如下建议.
[本文引用:1]
[2] XU G,DOU J,ZHANG L,et al.Inhibitory effects of baica-lein on the influenza virus in vivo is determined by baicalin in the serum[J].Biol Pharm Bull,2010,33(2):238-243.
Baicalein, an extract from Scutellaria baicalensis, was evaluated for its ability to inhibit the influenza virus in vivo. Oral administration of baicalein to BALB/c mice infected with the influenza A/FM1/1/47(H1N1) virus showed significant effects in preventing death, increasing the mean time to death, inhibiting lung consolidation, and reducing the lung virus titer in a dose-dependent manner. These effects are believed to be due to baicalin, the metabolite of baicalein in the serum. At a concentration of baicalin 2 microg/ml in overlay medium, it showed significant inhibition in the plaque assay, and the mean IC(50) value of baicalin was calculated as 1.2 microg/ml in the cytopathic effect assay. Our results showed that baicalein warrants further research as a potential antiinfluenza viral agent.
DOI:10.1248/bpb.33.238      PMID:20118546      URL    
[本文引用:2]
[3] 张春晶,顾立刚,于海涛.黄芩苷干预甲型H1N1流感病毒感染诱导的A549细胞周期分布及凋亡[J].病毒学报,2011,27(2):108-116.
以甲型H1N1流感病毒作为刺激因素,在人肺腺癌A549细胞培养内采用MTT比色法和细胞病变(CPE)法观察黄芩苷不同作用时间的抗病毒效率;以碘化丙啶(Propidium iodide,PI)单染流式细胞仪分析细胞周期中各时期的细胞百分数和对细胞增殖的影响,以Hoechst33258染色荧光显微镜观察细胞凋亡的形态学变化,并采用免疫荧光实验测定膜受体通路Caspase 8和线粒体通路Caspase 9及作为细胞凋亡标志的Caspase 3的活性。结果显示:流感病毒感染36h后宿主细胞周期阻滞于S期(P〈0.05),并在G0期细胞峰前出现一个亚二倍体细胞峰(凋亡细胞峰)。A549细胞中Caspase 8/3活性明显升高,但标记Caspase 9活性的荧光亮度增强不明显。黄芩苷对甲型流感病毒感染诱导的细胞损伤有较好的保护作用,最大剂量的黄芩苷100μg/mL无毒,可抑制病毒细胞病变的产生,50100μg/mL治疗组可明显调节流感病毒感染诱导的细胞周期分布(P〈0.05),细胞凋亡现象明显减少,100μg/mL黄芩苷治疗组Caspase 8/3活性显著降低,接近正常对照组细胞水平,有剂量依赖性。实验说明:黄芩苷可拮抗甲型流感病毒H1N1细胞病变,调控细胞周期分布,并通过抑制Caspase 8的激活,进一步抑制Caspase 3活性,从而发挥抗病毒作用。
URL    
[本文引用:2]
[4] DOU J,CHEN L,XU G,et al.Effects of baicalein on Sendai virus in vivo are linked to serum baicalin and its inhibition of hemagglutinin-neuraminidase[J].Arch Virol,2011,156(5):793-801.
Parainfluenza viruses are significant respiratory-tract pathogens that are notorious for infecting children. However, there are no clinical drugs to control the infection caused by these viruses. Sendai virus (SeV) belongs to the family Paramyxoviridae and causes fatal pneumonia in mice, its natural host. Baicalein is a flavonoid derived from the root of Scutellaria baicalensis, which is a traditional Chinese medicine that has been used for hundreds of years and has demonstrated a variety of biological activities. Our findings reveal that oral administration of baicalein to mice infected with Sendai virus results in a significant reduction in virus titers in the lungs and protection from death. The in vivo inhibitory effects of baicalein on Sendai virus are determined by baicalin in the serum. The mean IC(50) of baicalin was 0.71 μg/ml in an HA inhibition assay and 3.22 μg/ml in an NA inhibition assay. The mean IC(50) of baicalin in a CPE assay was measured to be 0.70 μg/ml, and significant inhibition was observed in a plaque assay at a concentration of 1.6 μg/ml baicalin in overlay medium, which suggests that baicalein is a potential anti-parainfluenzaviral agent in vivo.
DOI:10.1007/s00705-011-0917-z      PMID:21286764      URL    
[本文引用:1]
[5] LYU S Y,RHIM J Y,PARK W B.Antiherpetic activities of flavonoids against herpes simplex virus type 1(HSV-1) and type 2(HSV-2) in vitro[J].Arch Pharm Res,2005,28(11):1293-1301.
DOI:10.1007/BF02978215      URL    
[本文引用:1]
[6] 黄永建. 黄芩苷抗小鼠巨细胞病毒肝炎的治疗作用及其机制研究[D].武汉:华中科技大学,2011:17-21.
[本文引用:1]
[7] 李红,董永绥,方峰,.大蒜素和更昔洛韦抑制巨细胞病毒所致细胞凋亡的实验研究[J].中华儿科杂志,1999,37(4):26-30.
URL    
[本文引用:2]
[8] DOU J,LI X,CAI Y,et al.Human cytomegalovirus induces caspase-dependent apoptosis of megakaryocytic CHRF-288-11 cells by activating the JNK pathway[J].Int J Hematol,2010,91(4):620-629.
Human cytomegalovirus (HCMV) infection is usually implicated in thrombocytopenia occurring in newborns and immunocompromised patients. However, the underlying mechanisms remain elusive. This study was conducted to investigate the effects of HCMV infection on the viability of megakaryocytic CHRF-288-11 cells and the underlying mechanisms involved. RT-PCR for determining mRNA expression of HCMV immediate early gene 1 and Western blot for measuring protein expression of late HCMV gene pp65 showed that CHRF-288-11 cells were susceptible to HCMV infection. HCMV infection reduced the viability of CHRF-288-11 cells via apoptosis in a dose- and time-dependent manner. Both caspase 3 and c-Jun terminal kinase (JNK) signaling pathway were activated in the HCMV-treated CHRF-288-11 cells. z-DEVD-fmk (a caspase inhibitor) and SP600125 (a JNK inhibitor) significantly prevented the death of CHRF-288-11 cells induced by HCMV, respectively. Furthermore, inhibition of JNK activity could reduce the formation of active caspase 3 induced by HCMV. Interestingly, the co-application of antivirus drug ganciclovir and SP600125 synergistically prevented the death of CHRF-288-11 cells induced by HCMV. Collectively, these findings suggest that HCMV infection may induce the caspase-dependent apoptosis of megakaryocytic CHRF-288-11 cells by the activation of JNK signaling pathway.
DOI:10.1007/s12185-010-0560-6      PMID:20376580      URL    
[本文引用:1]
[9] 熊建文,肖化,张镇西.MTT法和CCK-8法检测细胞活性之测试条件比较[J].激光生物学报,2007,16(5):559-562.
利用HL60作为研究对象,对比了MTT法和CCK-8法在最佳检测时刻和检测波长等参数的差异.对于CCK-8法,最佳测试时刻在细胞液培养4 h后,最佳测试波长为490 nm.实验结果表明用CCK-8法检测的OD值与活细胞数的线性相关度要优于MTT法,是一种灵敏度较高、重复性好的的细胞活性检测方法.
URL    
[本文引用:0]
[10] 甄宏,方峰,舒赛男,.大蒜新素抑制人巨细胞病毒即刻早期基因表达在抗人巨细胞病毒机制中的作用[J].中国循证儿科杂志,2006,1(1):26-32.
<p>目的:研究大蒜新素抑制HCMV复制的作用环节和机制。方法:采用时间依赖的药物添加和移除实验分析大蒜新素在HCMV病毒复制周期中的作用环节。用Southern blot检测大蒜新素处理后感染细胞中HCMV DNA负荷量变化。用Northern blot和Western blot检测大蒜新素对HCMV即刻早期(ie)基因转录和翻译的影响,并与相应浓度更昔洛韦的作用效应相比较。结果:时间依赖的药物添加和移除实验发现大蒜新素只有在HCMV感染复制周期的早期存在时才能发挥对病毒增殖的抑制作用。大蒜新素对ie 1 mRNA的抑制率达30.38%~46.61%。在感染后24 h时,对IE72表达的抑制率为42.6%~64.9%,对IE86表达的抑制率为50.7%~70.6%。到感染后72 h,大蒜新素对IE蛋白仍然有抑制作用,对IE72的抑制率为36.4%~49.3%,对IE86的抑制率为77.9%~87.7%。而更昔洛韦对ie 1 mRNA和感染后24 h的即刻早期蛋白表达均无抑制作用。Southern blot结果显示大蒜新素和更昔洛韦均能减少HCMV DNA负荷量,两药相当剂量的抑制效应之间相比,差异无统计学意义(P &gt;0.05)。结论:大蒜新素抑制HCMV即刻早期基因的转录和翻译,进而抑制病毒的复制增殖,是其抗HCMV的重要作用机制。</p>
DOI:10.3969/j.issn.1673-5501.2006.01.007      Magsci     URL    
[本文引用:0]
[11] YOSHIDA H,SUMICHIKA H,HAMANO S,et al.Induction of apoptosis of T cells by infecting mice with murine cytomegalovirus[J].J Virol,1995,69(8):4769-4775.
Abstract Cytomegalovirus (CMV) is associated with several lymphocyte dysfunctions, but the precise mechanisms of the dysfunctions are still unclear. To elucidate the mechanisms, a cell cycle-DNA content analysis was performed on splenic T cells of murine CMV (MCMV)-infected BALB/c mice. T cells from mice infected with 3 x 10(3) PFU of MCMV contained a higher percentage of hypodiploid nuclei after 12 or 24 h of culture than those from naive mice. T cells from infected mice also contained a larger amount of fragmented DNA. Taken together, these results suggested that infection with MCMV induced the apoptotic cell death of T cells. This induction of apoptosis accounted for the dysfunction of lymphocytes, at least partially. Flow cytometric analysis showed that T cells as well as B cells from MCMV-infected mice expressed an augmented level of Fas antigen, an apoptosis-associated cell surface molecule, which might be the cause of the apoptosis of cells. T cells from MCMV-infected C57BL/6-lpr/lpr mice with mutations at the lpr/fas locus, however, also showed a substantial level of apoptosis, which was reproducibly lower than that seen in C57BL/6 mice. Therefore, it was suggested that the Fas-mediated pathway contributed to but was not sufficient for the induction of apoptosis and that mechanisms other than the Fas-associated pathway were also involved in the induction of apoptosis.
DOI:10.1016/0166-0934(95)00037-U      PMID:7609043      URL    
[本文引用:1]
[12] 聂兴草,方峰,李红,.大蒜新素对人巨细胞病毒感染的人胚肺成纤维细胞凋亡的影响[J].中草药,2004,35(9):60-63.
URL    
[本文引用:0]
[13] BRUNE W.Inhibition of programmed cell death by cytome-galoviruses[J].Virus Res,2011,157(2):144-150.
The elimination of infected cells by programmed cell death (PCD) is one of the most ancestral defense mechanisms against infectious agents. This mechanism should be most effective against intracellular parasites, such as viruses, which depend on the host cell for their replication. However, even large and slowly replicating viruses like the cytomegaloviruses (CMVs) can prevail and persist in face of cellular suicide programs and other innate defense mechanisms. During evolution, these viruses have developed an impressive set of countermeasures against premature demise of the host cell. In the last decade, several genes encoding suppressors of apoptosis and necrosis have been identified in the genomes of human and murine CMV (HCMV and MCMV). Curiously, most of the gene products are not homologous to cellular antiapoptotic proteins, suggesting that the CMVs did not capture the genes from the host cell genome. This review summarizes our current understanding of how the CMVs suppress PCD and which signaling pathways they target.
DOI:10.1016/j.virusres.2010.10.012      PMID:20969904      URL    
[本文引用:1]
[14] PATTERSON C E,SHENK T.Human cytomegalovirus UL36 protein is dispensable for viral replication in cultured cells[J].J Virol,1999,73(9):7126-7131.
Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.
DOI:10.1016/S0166-0934(99)00088-9      PMID:104234      URL    
[本文引用:1]
[15] MAVINAKERE M S,COLBERG-POLEY A M.Dual target-ing of the human cytomegalovirus UL37 exon 1 protein during permissive infection[J].J Gen Virol,2004,85(Pt 2):323-329.
The human cytomegalovirus (HCMV) UL37 immediate-early (IE) gene minimally encodes three protein isoforms that share NH(2)-terminal sequences. The predominant UL37 isoform detected during HCMV infection was the UL37 exon 1 protein (pUL37x1), which was produced from IE and, more abundantly, through late times of infection. pUL37x1 was localized in both the endoplasmic reticulum (ER) and mitochondria in infected cells. To determine which UL37x1 NH(2)-terminal residues serve as ER and mitochondrial targeting signals, we examined the subcellular localization of two deletion mutants. pUL37x1Delta2-23, which lacks the hydrophobic leader, is neither translocated into the ER nor imported mitochondrially; conversely, pUL37x1Delta23-34, lacking the juxtaposed basic residues, was translocated into the ER but only imported weakly into mitochondria. These studies show for the first time the temporal production and localization of pUL37x1 during HCMV infection. The trafficking patterns of mutants suggest that the pUL37x1 targeting signal to ER and mitochondria is bipartite.
DOI:10.1099/vir.0.19589-0      PMID:14769889      URL    
[本文引用:1]
[16] 魏伟,吴希美,李元建,.药理实验方法学[M].4版.北京:人民卫生出版社,2010.
[本文引用:1]
[17] 孙洁,田林红,王收宝,.黄芩苷对糖尿病大鼠肾脏细胞凋亡及其Bcl-2和Bax表达的影响[J].实用医学杂志,2011,27(5) :757-760.
URL    
[本文引用:1]
[18] EVERS D L,CHAO C F,WANG X,et al.Human cytome-galovirus-inhibitory flavonoids:studies on antiviral activity and mechanism of action[J].Antiviral Res,2005,68(3):124-134.
We report antiviral activity against human cytomegalovirus for certain dietary flavonoids and their likely biochemical mechanisms of action. Nine out of ten evaluated flavonoids blocked HCMV replication at concentrations that were significantly lower than those producing cytotoxicity against growing or stationary phase host cells. Baicalein was the most potent inhibitor in this series (IC 50 02=020.4–1.202μM), including positive control ganciclovir. Baicalein and genistein were chosen as model compounds to study the antiviral mechanism(s) of action for this series. Both flavonoids significantly reduced the levels of HCMV early and late proteins, as well as viral DNA synthesis. Baicalein reduced the levels of HCMV immediate-early proteins to nearly background levels while genistein did not. The antiviral effects of genistein, but not baicalein, were fully reversible in cell culture. Pre-incubation of concentrated virus stocks with either flavonoid did not inhibit HCMV replication, suggesting that baicalein did not directly inactivate virus particles. Baicalein functionally blocked epidermal growth factor receptor tyrosine kinase activity and HCMV nuclear translocation, while genistein did not. At 2402h post infection HCMV-infected cells treated with genistein continued to express immediate-early proteins and efficiently phosphorylate IE1-72. However, HCMV induction of NF-κB and increases in the levels of cell cycle regulatory proteins—events that are associated with immediate-early protein functioning – were absent. The data suggested that the primary mechanism of action for baicalein may be to block HCMV infection at entry while the primary mechanism of action for genistein may be to block HCMV immediate-early protein functioning.
DOI:10.1016/j.antiviral.2005.08.002      PMID:16188329      URL    
[本文引用:2]
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关键词(key words)
黄芩苷
人巨细胞病毒
人胚肺成纤维细胞
细胞凋亡
pro-caspase-3表达

Baicalin
Human cytomegalovirus
Human embryo lung fibrobl...
Apoptosis
Pro-caspase-3 expression

作者
刘杨
黄媛
廖毅
秦文卿
刘玲玲
李革
舒赛男
方峰

LIU Yang
HUANG Yuan
LIAO Yi
QIN Wenqing
LIU Lingling
LI Ge
SHU Sainan
FANG Feng