茶皂素对PRRSV感染Marc-145细胞受体表达及Caspase-9活化的影响
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摘要
以茶皂素(Teasaponin,TS)作为候选药物,研究茶皂素对Marc-145细胞受体CD163和波形蛋白(Vimentin)基因合成和蛋白表达的影响,以及茶皂素是否能通过细胞凋亡内源性通路影响PRRSV感染细胞,探究茶皂素抗PRRSV的作用机制。通过qRT-PCR和Western blot检测TS对细胞受体CD163和Vimentin的基因合成和蛋白表达的影响。运用Western blot技术检测TS对细胞内源性凋亡通路启动子caspase-9活化的影响,初探TS的抗PRRSV机制。qRT-PCR结果表明TS能显著抑制感染PRRSV的Marc-145细胞受体CD163和Vimentin基因的合成。Western blot结果表明TS能显著抑制细胞受体CD163和Vimentin的蛋白表达。TS能够引起细胞内源性凋亡通路启动子caspase-9的活化。研究表明,TS能抑制PRRSV在Marc-145细胞上的穿入过程,从而达到抗PRRSV的作用;亦可通过激活细胞凋亡内源性通路以早期促进细胞凋亡的方式产生抗PRRSV的作用。
Taking tea saponin( TS) as the candidate drug of anti-PRRSV,the antiviral mechanism of TS was explored through the determination of the effect on the Marc-145 receptor CD163 and Vimentin gene synthesis and protein expression,as well as whether could affect PRRSV infection by cell endogenous apoptotic pathway.qRT-PCR and Western blot technologies were used to detect the influence of TS on cell receptor CD163 and Vimentin gene synthesis and protein expression. In addition,the effect of TS on the caspase-9 activation which is cell endogenous apoptotic pathway promoter was determined by Western blot. qRT-PCR results showed that TS could significantly inhibit the gene synthesis of CD163 and Vimentin in the PRRSV infected Marc-145 cells.Moreover,Western blot results showed that TS could significantly inhibit the expression of CD163 and Vimentin and induce caspase-9 activation. TS could significantly inhibit PRRSV enter into Marc-145 cells and activate the cell endogenous apoptotic pathway to induce the early apoptosis to anti-PRRSV replication.
Taking tea saponin( TS) as the candidate drug of anti-PRRSV,the antiviral mechanism of TS was explored through the determination of the effect on the Marc-145 receptor CD163 and Vimentin gene synthesis and protein expression,as well as whether could affect PRRSV infection by cell endogenous apoptotic pathway.qRT-PCR and Western blot technologies were used to detect the influence of TS on cell receptor CD163 and Vimentin gene synthesis and protein expression. In addition,the effect of TS on the caspase-9 activation which is cell endogenous apoptotic pathway promoter was determined by Western blot. qRT-PCR results showed that TS could significantly inhibit the gene synthesis of CD163 and Vimentin in the PRRSV infected Marc-145 cells.Moreover,Western blot results showed that TS could significantly inhibit the expression of CD163 and Vimentin and induce caspase-9 activation. TS could significantly inhibit PRRSV enter into Marc-145 cells and activate the cell endogenous apoptotic pathway to induce the early apoptosis to anti-PRRSV replication.
引文
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[3]Sun N,Wang Z W,Wu C H,et al.Antiviral activity and underlying molecular mechanisms of Matrine against porcine reproductive and respiratory syndrome virus in vitro[J].Res Vet Sci,2014,96(2):323-327.
[4]Sun N,Yu T,Zhao J X,et al.Antiviral activities of natural compounds derived from traditional chinese medicines against porcine circovirus type 2(PCV2)[J].Biotechnol Bioproc E,2015,20(1):180-187.
[5]Sun N,Li E,Wang Z,et al.Sodium tanshinone IIA sulfonate inhibits porcine reproductive and respiratory syndrome virus via suppressing N gene expression and blocking virus-induced apoptosis[J].Antivir Ther,2014,19(1):89-95.
[6]Sun N,Cong X M,Jiang J B,et al.Sodium tanshinone IIA sulfonate inhibits the meq,ul49 and VP22 expression of Marek's disease virus[J].Antivir Ther,2014,19(8):793-8.
[7]Sun Y,Niu L,Song M,et al.Screening compounds of Chinese medicinal herbs anti-Marek's disease virus[J].Pharm Biol,2014,52(7):841-847.
[8]Li E,Sun N,Zhao J X,et al.In vitro evaluation of antiviral activity of tea seed saponins against porcine reproductive and respiratory syndrome virus[J].Antivir Ther,2015,20(7):743-752.
[9]Welch S K,Calvert J G.A brief review of CD163 and its role in PRRSV infection[J].Virus Res,2010,154(1-2):98-103.
[10]Kim J K,Fahad A M,Shanmukhappa K,et al.Defining the cellular target(s)of porcine reproductive and respiratory syndrome virus blocking monoclonal antibody 7G10[J].J Virol,2006,80(2):689-696.
[11]Katz D.International journal of experimental pathology.[M].Blackwell Science,1990.
[12]Gao L,Zhang W,Sun Y,et al.Cryptoporusvolvatus Extract Inhibits Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)In Vitro and In Vivo[J].Plos One,2013,8(5):e63767.
[13]李娥.抗猪繁殖与呼吸综合征病毒(PRRSV)天然化合物及其衍生物体外筛选与分子机制研究[D].山西农业大学,2014.Li E.In vitro screening natural compounds and derivatives with antiviral activities against porcine reproductive and respiratory syndrome virus(PRRSV)and investigating their molecular mechanisms[D].Shanxi Agricultural University,2014.
[14]Calvert J G,Slade D E,Shields S L,et al.CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses[J].J Virol,2007,81(14):7371-7379.
[15]Das P B,Dinh P X,Ansari I H,et al.The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor CD163[J].J Virol,2010,84(4):1731-1740.
[16]Wang X,Bai J,Zhang L,et al.Poly(A)-binding protein interacts with the nucleocapsid protein of porcine reproductive and respiratory syndrome virus and participates in viral replication.[J].Antiviral Res,2012,96(3):315-323.
[17]Beura L K,Dinh P X,Osorio F A,et al.Cellular poly(c)binding proteins 1 and 2 interact with porcine reproductive and respiratory syndrome virus nonstructural protein 1βand support viral replication[J].J Virol,2011,85(24):12939-12949.
[18]任静强.美洲型、欧洲型PRRSV及PCV2基因工程疫苗构建及实验免疫研究[D].吉林大学,2014.Ren J Q.Construction and immunogenicity of genetic engineer vaccines against EU-type/NA-type PRRSV and PCV2[D].Jilin University,2014.
[19]Lee S M,Kleiboeker S B.Porcine reproductive and respiratory syndrome virus induces apoptosis through a mitochondria-mediated pathway[J].Virology,2007,365(2):419-434.
[20]Costers S,Lefebvre D J,Delputte P L,et al.Porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages[J].Arch Virol,2008,153(8):1453-1465.
[21]李玉明.HP-PRRSV感染仔猪胸腺细胞凋亡的鉴定及凋亡相关因子mRNA表达水平的检测[D].东北农业大学,2013.Li Y M.Identification of apoptotic cells and analysis of apoptosisassociate factors in thymus of piglets infected with HP-PRRSV[D].Northeast Agricultural University,2013.
[22]Roy S,Nicholson D W.Cross-talk in cell death signaling[J].J Exp Med,2000,192(8):F21-25.