七味白术散对轮状病毒感染乳鼠小肠iEL CD3~+T细胞核内抗病毒蛋白表达的影响
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摘要
目的探讨七味白术散对轮状病毒(human rotarirus, HRV)感染乳鼠小肠黏膜上皮内淋巴细胞(iEL) CD3~+T细胞核内抗病毒蛋白表达的影响,进一步阐明该方抗HRV机制。方法选取5日龄NIH乳鼠168只,随机分为7组,正常对照组(N组)常规喂养,其余各组建立HRV腹泻模型,并分别灌服生理盐水(M组)、七味白术散(B组)、病毒唑(Z组)、抑制剂(Y组)、七味白术散加抑制剂(BY组),病毒唑加抑制剂(ZY组);分别于用药后32、40、48 h取各组乳鼠小肠黏膜,采用Western blotting方法测定小肠iELCD3~+T细胞核中双链RVA依赖的蛋白质激酶(PKR)、真核翻译起始子2的α亚基(eIF-2α)、2',5'-寡腺苷酸合成酶(OAS),RNA酶L(RNaseL)蛋白的表达。结果 B组乳鼠小肠粘膜iEL胞核中PKR、eIF-2α、OAS、RNaseL蛋白表达48 h时明显高于N组、M组、Y组、BY组、ZY组以及Z组,差异有统计学意义(P<0.05)。结论七味白术散能促进感染乳鼠小肠黏膜iEL CD3~+T细胞核中抗病毒蛋白PKR、eIF-2α、OAS、RNaseL的表达,从而实现对HRV的清除作用。
Objective To investigate the effect of Qiwei Baizhu Powder on the intranuclear expression of antiviral proteins in small intestinal intraepithelial lymphocyte(iEL) CD3~+T cells in human rotavirus(HRV)-infected neonatal mice, and to further elucidate its anti-HRV mechanism. Methods A total of 168 neonatal NIH mice aged 5 days were selected and randomly divided into seven groups. The normal control group(group N) was fed normally, while the mice in the other groups were treated to establish a model of HRV-induced diarrhea, and then were gavaged with normal saline(group M), Qiwei Baizhu Powder(group B),virazole(group Z), inhibitor(group Y), Qiwei Baizhu Powder plus inhibitor(group BY), and virazole plus inhibitor(group ZY),respectively. The small intestinal mucosa was taken from the neonatal mice in each group at 32, 40, and 48 hours after treatment,and the intranuclear expression of double-stranded RNA-dependent protein kinase R(PKR), α subunit of eukaryotic initiation factor2(eIF-2α), 2',5'-oligoadenylate synthetase(OAS), and ribonuclease L(RNaseL) in small intestinal iEL CD3~+T cells was determined by Western blot. Results The intranuclear expression of PKR, eIF-2α, OAS, RnaseL in small intestinal iELs was obviously higher than those nin groups N, M, Y, BY, ZY and Z at 48 h, and the differences were statistically significant(P<0.05). Conclusion Qiwei Baizhu Powder can enhance the intranuclear expression of antiviral proteins, PKR, eIF-2α, OAS, and RNaseL, in small intestinal iEL CD3~+T cells in HRV-infected neonatal mice, thus eliminating the HRV.
Objective To investigate the effect of Qiwei Baizhu Powder on the intranuclear expression of antiviral proteins in small intestinal intraepithelial lymphocyte(iEL) CD3~+T cells in human rotavirus(HRV)-infected neonatal mice, and to further elucidate its anti-HRV mechanism. Methods A total of 168 neonatal NIH mice aged 5 days were selected and randomly divided into seven groups. The normal control group(group N) was fed normally, while the mice in the other groups were treated to establish a model of HRV-induced diarrhea, and then were gavaged with normal saline(group M), Qiwei Baizhu Powder(group B),virazole(group Z), inhibitor(group Y), Qiwei Baizhu Powder plus inhibitor(group BY), and virazole plus inhibitor(group ZY),respectively. The small intestinal mucosa was taken from the neonatal mice in each group at 32, 40, and 48 hours after treatment,and the intranuclear expression of double-stranded RNA-dependent protein kinase R(PKR), α subunit of eukaryotic initiation factor2(eIF-2α), 2',5'-oligoadenylate synthetase(OAS), and ribonuclease L(RNaseL) in small intestinal iEL CD3~+T cells was determined by Western blot. Results The intranuclear expression of PKR, eIF-2α, OAS, RnaseL in small intestinal iELs was obviously higher than those nin groups N, M, Y, BY, ZY and Z at 48 h, and the differences were statistically significant(P<0.05). Conclusion Qiwei Baizhu Powder can enhance the intranuclear expression of antiviral proteins, PKR, eIF-2α, OAS, and RNaseL, in small intestinal iEL CD3~+T cells in HRV-infected neonatal mice, thus eliminating the HRV.
引文
[1] BROQUET A H, HIRATA Y, McAllister C S, et al. RIG-I/MDA5/MAVS are required to signal a protective IFN response in ro-tavirus-infected intestinal epithelium[J]. Journal of Immunology,2011,186(3):1618-1626.
[2] PARASHAR UD AND GLASS RI. Rotavirus vaccines-early suc-cess, remaining questions[J]. New England Journal of Medicine, 2009,360:1063-1065.
[3]钱乙.小儿药证直诀(实用中医古籍丛书)[M].天津:天津科学技术出版社,2000:43.
[4] HE S T, HE F Z, WU C R, et al. Treatment of rotaviral gastroenteritiswith Qiwei Baizhu powder[J]. World Journal of Gastroenterology,2001, 7(5):735-740.
[5]贺双腾,何飞舟,伍参荣,等.七味白术散对人轮状病毒在培养细胞内复制的抑制作用[J].中国中西医结合杂志.1995,15(11):669-671.
[6]贺双腾,何飞舟,伍参荣,等.七味白术散治疗轮状病毒肠炎的临床与实验研究[J].中国中西医结合杂志,1996,16(3):132-135.
[7]伍参荣,杨波,胡建中,等.七味白术散对人轮状病毒感染乳鼠胸腺细胞的程序性死亡和IL-2、IL-2R表达的影响[J].湖南中医学院学报,2001,21(3):8-9.
[8]伍参荣,谢朝晖,贺双腾.七味白术散对HRV感染乳鼠NK、IFN-γ、IL-4的影响[J].中国中医药信息杂志,2002,9(4):23-24.
[9] WU C R, XIAO J G, HE S T, et al. Effects of QWBZP on T-cell subsets and their cytokines in intestinal mucosa of HRVinfection suckling mice[J]. Journal of Ethnopharmacology, 2010, 131(1):130-134.
[10] SU Q, WANG S, BALTZIS D, et al. Tyrosine phosphorylationacts as a molecular switch to full-scale activation of theeIF2alpha RNAdependent protein kinase[J]. Proceedings of theNational Academy of Sciences of the United States of America,2006, 103(1):63-68.
[11] KAZEMI S, PAPADOPOULOU S, LI S, et al. Control of alphasubunit of eukaryotic translation initiation factor2(eIF2 alpha)phosphorylation by the human papillomavirus type 18 E6 onco-protein:implicationsfor eIF2 alpha-dependent gene expressionand cell death[J]. Molecular Biology of the Cell, 2004, 24(8):3415-3429.
[12] GOODMAN A G, SMITH J A, BALACHANDRAN S, et al. TheCellular Protein P58IPK Regulates Influenza Virus mRNATranslation and Replication through a PKR-Mediated Mecha-nism[J]. Journal of Virology, 2007, 81(5):2221-2230.
[13] ZHU R, ZHANG Y B, ZHANG Q Y, et al. Functional domainsand the antiviral effect of the double-stranded RNA-dependentprotein kinase PKR from paralichthys olivaceus[J]. Journal ofVirology, 2008, 82(14):6889-6901.
[14] Sànchez R and Mohr I. Inhibition of Cellular 2'-5'Oligoad-enylate Synthetase by the Herpes Simplex Virus Type 1 Us11Protein[J]. Virology, 2007, 81(7):3455-3464.
[15]周正任,叶晓卉.小鼠小肠上皮内淋巴细胞的提取与抗原表型测定和功能的初步研究[J].中国免疫学杂志,2001,17(3):135-137.
[16] BURNETT E, YEN C, TATE J E, et al. Rotavirus vaccines:current global impact and future perspectives[J]. Future Virolo-gy, 2016, 11(10):699-708.
[17] YANG X, TWITCHELL E, LI G, et al. High protective effica-cy of rice bran against human rotavirus diarrhea via enhancingprobiotic growth, gut barrier function, and innate immunity[J].Scientific Reports, 2015, 5:15004.
[18] WU C R, ZUO S N, SUN B Q, et al. Qi-Wei-Bai-Zhu pow-der improves inflammatory response via the myd88 pathwayin neonatal mice infected with human rotavirus[J]. BiomedicalResearch, 2017, 28(20):9701-9706.
[19] GUERRERO C A, ACOSTA O. Inflammatory and oxidative stressin rotavirus infection. World Journal of Virology, 2016,5:38-62.
[20] BASS D M. Interferon gamma and interleukin 1, but not inter-feron alfa, inhibit rotavirus entry into human intestinal celllines[J]. Gastroenterology, 1997, 113(1):81-89.
[21] MCALLISTER C S, TOTH A M, ZHANG P, et al. Mechanismsof protein kinase PKR-mediated amplification of beta interferoninduction by C protein-deficient measles virus[J]. Journal of Vi-rology, 2010, 84(1):380-386.
[22] MINOR R A, LIMMON G V, MILLER-DEGRAFF L, et al.Double-stranded RNA-activated protein kinase regulates earlyinnate immune responses during respiratory syncytial virus infection[J]. Journal of Interferon and Cytokine Research, 2010,30(4):263-272.
[23] RYMAN K D, WHITE L J, JOHNSTON R E, et al. Effects ofPKR/RNase-dependent and alternative antiviral pathways on alphavirus replication and pathogenesis[J]. Viral Immunology, 2002,15:53-76.
[24] VATTEM K M, STASCHKE K A, WEK R C. Mechanism ofactivation of the double-stranded-RNA-dependent protein ki-nase, PKR:role of dimerization and celluar localization in thestimulation of PKR phosphorylation of eukaryotic initiation fac-tor-2(eIF2)[J]. European Journal of Biochemistry, 2001, 268(13):3674-3684.
[25] KAUFMAN R J. Double-stranded RNA-activated protein ki-nase mediates virus-induced apoptosis:a new role for an oldactor[J]. Proceedings of the National Academy of Sciences ofthe United States of America. 1999, 96(21):11693-11695.
[26] LI S D, GREGORY A P, DING K Y, et al. Molecular basisfor PKR activation by PACT or dsRNA[J]. Proceedings of the Na-tional Academy of Sciences of the United States of America,2006, 103(26):10005-10010.
[27] COUTURIER J, MOREL M, PONTCHARRAUD R. Interactionof double-stranded RNA-dependent protein kinase(PKR)withthe death receptor signaling pathway in amyloid beta(Abeta)-treated cells and in APPSLPS1 knock-in mice[J]. Journal Bio-logical Chemistry, 2010, 285(2):1272-1282.
[28] RICARDO SNCHEZ, IAN MOHR. Inhibition of cellular 2'-5'oligoadenylate synthetase by the herpes simplex virus type 1Us11 protein[J]. Journal of Virology, 2007, 81(7):3455-3464.
[2] PARASHAR UD AND GLASS RI. Rotavirus vaccines-early suc-cess, remaining questions[J]. New England Journal of Medicine, 2009,360:1063-1065.
[3]钱乙.小儿药证直诀(实用中医古籍丛书)[M].天津:天津科学技术出版社,2000:43.
[4] HE S T, HE F Z, WU C R, et al. Treatment of rotaviral gastroenteritiswith Qiwei Baizhu powder[J]. World Journal of Gastroenterology,2001, 7(5):735-740.
[5]贺双腾,何飞舟,伍参荣,等.七味白术散对人轮状病毒在培养细胞内复制的抑制作用[J].中国中西医结合杂志.1995,15(11):669-671.
[6]贺双腾,何飞舟,伍参荣,等.七味白术散治疗轮状病毒肠炎的临床与实验研究[J].中国中西医结合杂志,1996,16(3):132-135.
[7]伍参荣,杨波,胡建中,等.七味白术散对人轮状病毒感染乳鼠胸腺细胞的程序性死亡和IL-2、IL-2R表达的影响[J].湖南中医学院学报,2001,21(3):8-9.
[8]伍参荣,谢朝晖,贺双腾.七味白术散对HRV感染乳鼠NK、IFN-γ、IL-4的影响[J].中国中医药信息杂志,2002,9(4):23-24.
[9] WU C R, XIAO J G, HE S T, et al. Effects of QWBZP on T-cell subsets and their cytokines in intestinal mucosa of HRVinfection suckling mice[J]. Journal of Ethnopharmacology, 2010, 131(1):130-134.
[10] SU Q, WANG S, BALTZIS D, et al. Tyrosine phosphorylationacts as a molecular switch to full-scale activation of theeIF2alpha RNAdependent protein kinase[J]. Proceedings of theNational Academy of Sciences of the United States of America,2006, 103(1):63-68.
[11] KAZEMI S, PAPADOPOULOU S, LI S, et al. Control of alphasubunit of eukaryotic translation initiation factor2(eIF2 alpha)phosphorylation by the human papillomavirus type 18 E6 onco-protein:implicationsfor eIF2 alpha-dependent gene expressionand cell death[J]. Molecular Biology of the Cell, 2004, 24(8):3415-3429.
[12] GOODMAN A G, SMITH J A, BALACHANDRAN S, et al. TheCellular Protein P58IPK Regulates Influenza Virus mRNATranslation and Replication through a PKR-Mediated Mecha-nism[J]. Journal of Virology, 2007, 81(5):2221-2230.
[13] ZHU R, ZHANG Y B, ZHANG Q Y, et al. Functional domainsand the antiviral effect of the double-stranded RNA-dependentprotein kinase PKR from paralichthys olivaceus[J]. Journal ofVirology, 2008, 82(14):6889-6901.
[14] Sànchez R and Mohr I. Inhibition of Cellular 2'-5'Oligoad-enylate Synthetase by the Herpes Simplex Virus Type 1 Us11Protein[J]. Virology, 2007, 81(7):3455-3464.
[15]周正任,叶晓卉.小鼠小肠上皮内淋巴细胞的提取与抗原表型测定和功能的初步研究[J].中国免疫学杂志,2001,17(3):135-137.
[16] BURNETT E, YEN C, TATE J E, et al. Rotavirus vaccines:current global impact and future perspectives[J]. Future Virolo-gy, 2016, 11(10):699-708.
[17] YANG X, TWITCHELL E, LI G, et al. High protective effica-cy of rice bran against human rotavirus diarrhea via enhancingprobiotic growth, gut barrier function, and innate immunity[J].Scientific Reports, 2015, 5:15004.
[18] WU C R, ZUO S N, SUN B Q, et al. Qi-Wei-Bai-Zhu pow-der improves inflammatory response via the myd88 pathwayin neonatal mice infected with human rotavirus[J]. BiomedicalResearch, 2017, 28(20):9701-9706.
[19] GUERRERO C A, ACOSTA O. Inflammatory and oxidative stressin rotavirus infection. World Journal of Virology, 2016,5:38-62.
[20] BASS D M. Interferon gamma and interleukin 1, but not inter-feron alfa, inhibit rotavirus entry into human intestinal celllines[J]. Gastroenterology, 1997, 113(1):81-89.
[21] MCALLISTER C S, TOTH A M, ZHANG P, et al. Mechanismsof protein kinase PKR-mediated amplification of beta interferoninduction by C protein-deficient measles virus[J]. Journal of Vi-rology, 2010, 84(1):380-386.
[22] MINOR R A, LIMMON G V, MILLER-DEGRAFF L, et al.Double-stranded RNA-activated protein kinase regulates earlyinnate immune responses during respiratory syncytial virus infection[J]. Journal of Interferon and Cytokine Research, 2010,30(4):263-272.
[23] RYMAN K D, WHITE L J, JOHNSTON R E, et al. Effects ofPKR/RNase-dependent and alternative antiviral pathways on alphavirus replication and pathogenesis[J]. Viral Immunology, 2002,15:53-76.
[24] VATTEM K M, STASCHKE K A, WEK R C. Mechanism ofactivation of the double-stranded-RNA-dependent protein ki-nase, PKR:role of dimerization and celluar localization in thestimulation of PKR phosphorylation of eukaryotic initiation fac-tor-2(eIF2)[J]. European Journal of Biochemistry, 2001, 268(13):3674-3684.
[25] KAUFMAN R J. Double-stranded RNA-activated protein ki-nase mediates virus-induced apoptosis:a new role for an oldactor[J]. Proceedings of the National Academy of Sciences ofthe United States of America. 1999, 96(21):11693-11695.
[26] LI S D, GREGORY A P, DING K Y, et al. Molecular basisfor PKR activation by PACT or dsRNA[J]. Proceedings of the Na-tional Academy of Sciences of the United States of America,2006, 103(26):10005-10010.
[27] COUTURIER J, MOREL M, PONTCHARRAUD R. Interactionof double-stranded RNA-dependent protein kinase(PKR)withthe death receptor signaling pathway in amyloid beta(Abeta)-treated cells and in APPSLPS1 knock-in mice[J]. Journal Bio-logical Chemistry, 2010, 285(2):1272-1282.
[28] RICARDO SNCHEZ, IAN MOHR. Inhibition of cellular 2'-5'oligoadenylate synthetase by the herpes simplex virus type 1Us11 protein[J]. Journal of Virology, 2007, 81(7):3455-3464.
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