人参皂苷Rb1对PRRSV的体外抑制作用及对病毒N蛋白表达的影响
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摘要
为了在感染猪繁殖与呼吸综合征病毒(PRRSV)的Marc-145细胞模型上,探讨人参皂苷Rb1(Ginsenoside Rb1, GSR1)抑制PRRSV能力及其作用机制。采用MTT法测定GSR1在Marc-145细胞上的最大安全浓度(Maximum no-cytotoxic concentration, MNTC)和半数毒性浓度(50%Cytotoxic concentration, CC_(50))。建立PRRSV感染Marc-145细胞模型,设计阻断PRRSV复制和直接杀灭PRRSV试验,确定GSR1体外抗PRRSV的作用方式;利用FQ-PCR检测GSR1对病毒N基因的影响,间接免疫荧光试验(Indirect immunofluorescence assay,IFA)和Western Blot技术测定GSR1对PRRSVN蛋白表达的影响。结果表明,GSR1在直接杀灭PRRSV试验中最高抑制率为54.65%,在阻断PRRSV复制试验中前12 h起抗病毒作用,且最高抑制率达60.01%,推测GSR1主要通过直接使病毒灭活或干扰病毒的早中期复制来发挥其抗病毒作用。此外,在设定12～72 h试验时间内,阳性药物利巴韦林组,4 mg/mL、2 mg/mL GSR1组PRRSV N基因拷贝数显著低于病毒组(P<0.05)。在对N蛋白含量影响试验中4 mg/mL GSR1和阳性药物可以减少N蛋白载量。表明GSR1可以通过抑制病毒N蛋白的表达来抑制病毒的复制,并可作为抗PRRSV的候选药物或先导物。
The inhibitory effect of ginsenoside Rb1(GSR1) on PRRSV and its mechanism were investigated in Marc-145 cell model infected with porcine reproductive and respiratory syndrome virus.MTT method was used to determine the maximum no-cytotoxic concentration(MNTC) and the 50% cytotoxic concentration(CC_(50)) of GSR1 in Marc-145 cells. The model of Marc-145 cells infected with PRRSV was established and then the inhibition mode of action of ginsenoside Rb1 was determined though the blocking PRRSV replication assay and the direct inactivated PRRSV assay. The effect of GSR1 on viral N gene was detected by FQ-PCR. The indirect immunofluorescence assay and Western Blot were used to determine the PRRSV N protein expression.The maximum inhibition rate of ginsenoside Rb1 was up to 54.65% in the direct inactivation assay. The maximum inhibition rate of ginsenoside Rb1 in the time-of-addition assay was up to 60.01% and ginsenoside Rb1 possessed the antiviral activity before 12 h post infection. Its mechanism of antiviral actions could be due to inhibiting the virus the early or middle stage of replication or/and inactivating the virus directly. The copy number of N gene in the positive drug group or 4 mg/mL or 2 mg/mL of ginsenoside Rb1 group was significantly lower than the virus control group from 12 to 72 h(P<0.05). Moreover, 4 mg/mL ginsenoside Rb1 and positive drug could reduce virus N protein in the cell.GSR1 inhibits the replication of the virus by inhibiting the expression of the viral N protein and GSR1 could be used for developing a new drug or lead compound in treating and preventing PRRSV.
The inhibitory effect of ginsenoside Rb1(GSR1) on PRRSV and its mechanism were investigated in Marc-145 cell model infected with porcine reproductive and respiratory syndrome virus.MTT method was used to determine the maximum no-cytotoxic concentration(MNTC) and the 50% cytotoxic concentration(CC_(50)) of GSR1 in Marc-145 cells. The model of Marc-145 cells infected with PRRSV was established and then the inhibition mode of action of ginsenoside Rb1 was determined though the blocking PRRSV replication assay and the direct inactivated PRRSV assay. The effect of GSR1 on viral N gene was detected by FQ-PCR. The indirect immunofluorescence assay and Western Blot were used to determine the PRRSV N protein expression.The maximum inhibition rate of ginsenoside Rb1 was up to 54.65% in the direct inactivation assay. The maximum inhibition rate of ginsenoside Rb1 in the time-of-addition assay was up to 60.01% and ginsenoside Rb1 possessed the antiviral activity before 12 h post infection. Its mechanism of antiviral actions could be due to inhibiting the virus the early or middle stage of replication or/and inactivating the virus directly. The copy number of N gene in the positive drug group or 4 mg/mL or 2 mg/mL of ginsenoside Rb1 group was significantly lower than the virus control group from 12 to 72 h(P<0.05). Moreover, 4 mg/mL ginsenoside Rb1 and positive drug could reduce virus N protein in the cell.GSR1 inhibits the replication of the virus by inhibiting the expression of the viral N protein and GSR1 could be used for developing a new drug or lead compound in treating and preventing PRRSV.
引文
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[20] Auerochs S, Korn K, Marschall M. A reporter system for Epstein-Barr virus (EBV) lytic replication: anti-EBV activity of the broad anti-herpesviral drug artesunate[J]. Journal of Virological Methods, 2011, 173(2): 334.
[2] Lunney J K, Benfield D A, Rowland R R. Porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine[J]. Virus Research, 2010, 154(1- 2): 1- 6.
[3] Nieuwenhuis N, Duinhof T F, Van N A. Economic analysis of outbreaks of porcine reproductive and respiratory syndrome virus in nine sow herds[J]. Veterinary Record, 2012, 170(9): 225.
[4] Tong G Z, Zhou Y J, Hao X F, et al. Highly pathogenic porcine reproductive and respiratory syndrome, China[J]. Emerging Infectious Diseases, 2007, 13(9):1 434- 1 436.
[5] Guo Z, Chen X, Li R, et al. The prevalent status and genetic diversity of porcine reproductive and respiratory syndrome virus in China: a molecular epidemiological perspective[J]. Virology Journal, 2018, 15(1): 2.
[6] 王国丽, 李晓娇, 张力娜, 等. 人参皂苷Rb1、Rg1对中枢神经系统作用及相关机制的研究进展[J]. 上海中医药杂志, 2017, 3: 92- 95.
[7] Joh E H, Lee I A, Jung I H, et al. Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation-the key step of inflammation.[J]. Biochemical Pharmacology, 2011, 82(3): 278.
[8] Lee D, Lee D S, Jung K, et al. Protective effect of ginsenoside Rb1 against tacrolimus-induced apoptosis in renal proximal tubular LLC-PK1 cells[J]. Journal of Ginseng Research, 2018, 42(1): 75.
[9] 梁园园, 王斌, 李玲, 等. 人参皂苷Rbl抗单纯疱疹病毒1型感染及保护神经的实验研究[J].中国中西医结合杂志, 2012, 7: 975- 979.
[10] Jeong J J, Kim B, Kim D H. Ginsenoside Rb1 eliminates HIV-1 (D3)-transduced cytoprotective human macrophages by inhibiting the AKT pathway[J].Journal of Medicinal Food, 2014, 17(8): 849- 54.
[11] Kim Y, Lee C. Ribavirin efficiently suppresses porcine nidovirus replication[J]. Virus Research, 2013, 171(1):44- 53..
[12] 李丹. 蓝耳病毒RX株的分离鉴定和中药体外抗病毒的作用研究[D].泰安:山东农业大学, 2009.
[13] Mukhtar M, Arshad M, Ahmad M,et al. Antiviral potentials of medicinal plants[J]. Virus Research, 2008, 131(2): 111.
[14] Sun N, Zhao X, Bai X Y, et al. Anti-PRRSV effect and mechanism of sodium tanshinone IIA sulfonate in vitro[J]. Journal of Asian Natural Products Research, 2012, 14(8): 721.
[15] 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.
[16] Sun J, Song X, Hu S. Ginsenoside Rg1 and Aluminum Hydroxide Synergistically Promote Immune Responses to Ovalbumin in BALB/c Mice[J]. Clinical & Vaccine Immunology, 2008, 15(2): 303- 307.
[17] Yoojin L, Changhee L. Porcine reproductive and respiratory syndrome virus replication is suppressed by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway[J]. Virus Research, 2010, 152(1): 50- 58.
[18] Yoo D, Wootton S K, Li G, et al. Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin[J]. Journal of Virology, 2003, 77(22): 12 173- 12 183.
[19] 吴建兵. 小檗碱衍生物 HB-13 抗单纯疱疹病毒作用及其机制初探[D].北京:中国协和医科大学, 2006.
[20] Auerochs S, Korn K, Marschall M. A reporter system for Epstein-Barr virus (EBV) lytic replication: anti-EBV activity of the broad anti-herpesviral drug artesunate[J]. Journal of Virological Methods, 2011, 173(2): 334.