野大黄含药血清体外抑制猪繁殖与呼吸综合征病毒活性试验
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摘要
为了研究中药野大黄(中药提取物)含药血清体外抑制猪繁殖与呼吸综合征病毒活性,探讨其抗猪繁殖与呼吸综合征病毒作用机理。取"野大黄"药液,生药含量为2 g/mL,对照药物利巴韦林终浓度为0.025 g/mL,分别灌胃给药,饲喂SD大鼠,制备各组含药血清。采用MTT法和细胞形态观察法相结合,测定含药血清对Marc-145细胞的最大无毒稀释度。将含药血清倍比稀释至1∶4、1∶8、1∶16三种浓度,采用三种加药方式,以细胞病变效应(cytopathic effect,CPE)观察与MTT染色检测OD492值,计算含药血清病毒抑制率为评价指标,观察野大黄含药血清体外抑制猪繁殖与呼吸综合征病毒活性。结果显示,野大黄含药血清体外抑制猪繁殖与呼吸综合征病毒活性最显著的是含药血清与病毒混合后加入方式,在1∶4稀释度时,野大黄含药血清组与利巴韦林对照含药血清组OD492均值差异显著(P<0.05),两者对猪繁殖与呼吸综合征病毒抑制率分别为83.51%、73.07%,相差约10%。野大黄含药血清组三种稀释度镜检观察Marc-145细胞均无CPE出现,细胞单层完好,而利巴韦林对照含药血清在1∶16稀释度时病毒抑制率为63.55%,且出现CPE,细胞单层遭到破坏、部分细胞变圆、脱落。结果表明,野大黄含药血清体外具有显著的抑制猪繁殖与呼吸综合征病毒活性,以同时给药时具有直接杀灭猪繁殖与呼吸综合征病毒作用,有望作为抗猪繁殖与呼吸综合征病毒药物进行开发。
In order to study the inhibitory effect of Rhubarb(extract of traditional Chinese medicine) serum on Porcine reproductive and respiratory syndrome virus(PRRSV)in vitro, and explore its mechanism of anti-PRRSV, so as to provide basis for screening anti-PRRSV. The crude drug content of Rhubarb was 2 g/mL and the final concentration of ribavirin was 0.025 g/mL. The SD rats were fed intragastrically to prepare serum containing drugs. The maximum non-toxic dilution of drug-containing serum to Marc-145 cells was determined by MTT assay and cell morphology observation. The drug-containing serum was diluted to 1∶4, 1∶8 and 1∶16 concentration. The OD492 value was detected by CPE observation and MTT staining. The virus inhibition rate of the drug-containing serum was calculated as an evaluation index. The antiviral activity of the drug-containing serum of Rhubarb against PRRSV in vitro was observed. The results showed that the most significant inhibitory effect of rhubarb serum on PRRSV in vitro was the way of adding the drug serum mixed with the virus. At 1∶4 dilution, the difference of OD492 value between rhubarb serum group and ribavirin control serum group was significant(P< 0.05). The inhibitory rates of Rhubarb serum group and ribavirin control serum Group on PRRS virus were 83.51%, 73.07%, the difference is about 10 percentage points. The cytopathic effect(CPE) of Marc-145 cells was not observed by three dilutions microscopy in the Rhubarb-containing serum group, and the single layer of cells was intact. The viral inhibition rate of ribavirincontaining serum was 63.55% at 1∶16 dilution, and CPE appeared. The single layer of cells was destroyed, some cells became round and degraded. The results showed that Rhubarb serum had significant inhibitory activity against PRRSV in vitro, and it could kill PRRSV directly when administered simultaneously. It was expected to be developed as an anti-PRRSV drug.
In order to study the inhibitory effect of Rhubarb(extract of traditional Chinese medicine) serum on Porcine reproductive and respiratory syndrome virus(PRRSV)in vitro, and explore its mechanism of anti-PRRSV, so as to provide basis for screening anti-PRRSV. The crude drug content of Rhubarb was 2 g/mL and the final concentration of ribavirin was 0.025 g/mL. The SD rats were fed intragastrically to prepare serum containing drugs. The maximum non-toxic dilution of drug-containing serum to Marc-145 cells was determined by MTT assay and cell morphology observation. The drug-containing serum was diluted to 1∶4, 1∶8 and 1∶16 concentration. The OD492 value was detected by CPE observation and MTT staining. The virus inhibition rate of the drug-containing serum was calculated as an evaluation index. The antiviral activity of the drug-containing serum of Rhubarb against PRRSV in vitro was observed. The results showed that the most significant inhibitory effect of rhubarb serum on PRRSV in vitro was the way of adding the drug serum mixed with the virus. At 1∶4 dilution, the difference of OD492 value between rhubarb serum group and ribavirin control serum group was significant(P< 0.05). The inhibitory rates of Rhubarb serum group and ribavirin control serum Group on PRRS virus were 83.51%, 73.07%, the difference is about 10 percentage points. The cytopathic effect(CPE) of Marc-145 cells was not observed by three dilutions microscopy in the Rhubarb-containing serum group, and the single layer of cells was intact. The viral inhibition rate of ribavirincontaining serum was 63.55% at 1∶16 dilution, and CPE appeared. The single layer of cells was destroyed, some cells became round and degraded. The results showed that Rhubarb serum had significant inhibitory activity against PRRSV in vitro, and it could kill PRRSV directly when administered simultaneously. It was expected to be developed as an anti-PRRSV drug.
引文
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[25]冼琼珍,王丙云,伍盛鋆,等.16种中药体外抗猪繁殖与呼吸综合征病毒作用的研究[J].中国畜牧兽医,2014,41(2):245-249.
[26]邢娜.抗猪繁殖与呼吸综合征病毒(PRRSV)中药高效成分的体外筛选及其抗病毒机制的研究[D].晋中:山西农业大学,2015:8-10.
[27]Li X,Galliher-Beckley A,Wang L,et al.Comparison of immune responses in pigs infected with Chinese highly pathogenic PRRS virus strain HV and North American strain NADC-20[J].Open Virol J,2017,11:73-82.
[28]郭亚男,何生虎,雷元元,等.抗病毒中草药在兽医临床中的应用研究进展[J].中兽医医药杂志,2015(6):75-77.
[2]Liu C,Ning Y,Xu B,et al.Analysis of genetic variation of porcine reproductive and respiratory syndrome virus(PRRSV)isolates in Central China[J].J Vet Med Sci,2016,78(4):641-648.
[3]Jeong J,Park C,Choi K,et al.Evaluation of the new commercial recombinant chimeric subunit vaccine PRRSFREE in challenge with heterologous types 1 and 2porcine reproductive and respiratory syndrome virus[J].Can J Vet Res,2017,81(1):12-21.
[4]Yu X,Zhou Z,Cao Z,et al.Assessment of the safety and efficacy of an attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus[J].Clin Vaccine Immunol,2015,22(5):493-502.
[5]欧阳伟星,李美娣,武力,等.替米考星防控怀孕母猪蓝耳病的效果[J],福建畜牧兽医,2016,38(5):7-9.
[6]李桃梅.泰万菌素防控猪繁殖与呼吸综合征病毒的体外和体内研究[D].上海:上海交通大学,2015:7-8.
[7]马钦海,邢学锋,罗佳波.清热解毒类中药抗呼吸道病毒研究进展[J].广东药学院学报,2016,32(5):658-661.
[8]孙加节,蒋勇,习欠云,等.人参多糖体外抗猪生殖与呼吸综合征病毒活性[J].中国兽医学报,2017,37(4):697-703,716.
[9]赵昕,孙娜,白喜云,等.苦参碱对PRRSV的体外抑制作用及其机制[J].中国兽医学报,2013,33(6):808-812.
[10]刘樱,丁度伟,高求炜,等.3种中药及其提取物体外抗猪繁殖与呼吸综合征病毒作用的研究[J].中国畜牧兽医,2016,43(10):2730-2735.
[11]赵勇,李庆春,赵焱,等.野拔子的化学成分研究[J].中国中药杂志,2004,29(12):1144-1146.
[12]刘红淼,李艳玲,杨继章.土大黄的药理作用及临床应用研究进展[J].中国药房,2013(15):1422-1425.
[13]孙启文,吴松,柳航,等.荔枝草的化学成分及药理作用研究新进展[J].中国药师,2014,17(3):481-483.
[14]张志勇,徐洪,陈玲,等.苎麻根在贵州6个民族中的临床应用研究[J].中国民族医药杂志,2015,21(5):44-45.
[15]丁晓刚.高致病性猪蓝耳病的中药辨证施治[J].北方牧业,2009(14):20.
[16]童骄,葛资宇,王小玉,等.不同煎煮方法麻杏石甘汤含药血清体外抗A型流感病毒实验研究[J].辽宁中医药大学学报,2017,19(4):14-17.
[17]田朝晖,张彪.板蓝根、黄芪和青蒿提取物抗PRRSV作用比较[J].中国饲料,2016(17):16-19.
[18]易方.抗猪繁殖与呼吸综合征病毒中药多糖及多糖复方的体外筛选[D].南京:南京农业大学,2014:20-23.
[19]陈志胜,王丙云,冼琼珍,等.三种中药提取物体外抗猪繁殖与呼吸综合征病毒的作用[J].中国兽药杂志,2014,48(8):40-43.
[20]孙娜,赵昕,白元生,等.甘草酸二钾体外抗猪繁殖与呼吸综合征病毒(PRRSV)的活性[J].中国兽医学报,2013,33(2):282-286.
[21]国锦,高燕,赵渤年.中药复方血清药理学研究方法进展[J].中华中医药杂志,2017,32(4):1656-1658.
[22]胡宇莉,刘汉儒,黎建华.中药血清药理学方法及其在中兽药研究中的应用展望[J].广东畜牧兽医科技,2003,28(6):14-16.
[23]沈克姑,田淑琴.中药血清药理学方法及其在中兽药研究中的展望[J].四川畜牧兽医,2007,34(11):34-35.
[24]彭志,卢芳国,屈金艳,等.银黄清肺胶囊含药血清体外抗呼吸道合胞病毒实验研究[J].湖南中医药大学学报,2016,36(2):40-43.
[25]冼琼珍,王丙云,伍盛鋆,等.16种中药体外抗猪繁殖与呼吸综合征病毒作用的研究[J].中国畜牧兽医,2014,41(2):245-249.
[26]邢娜.抗猪繁殖与呼吸综合征病毒(PRRSV)中药高效成分的体外筛选及其抗病毒机制的研究[D].晋中:山西农业大学,2015:8-10.
[27]Li X,Galliher-Beckley A,Wang L,et al.Comparison of immune responses in pigs infected with Chinese highly pathogenic PRRS virus strain HV and North American strain NADC-20[J].Open Virol J,2017,11:73-82.
[28]郭亚男,何生虎,雷元元,等.抗病毒中草药在兽医临床中的应用研究进展[J].中兽医医药杂志,2015(6):75-77.