大片形吸虫感染及其分泌排泄产物对小鼠Toll样受体mRNA表达量的影响
|
摘要
目的研究大片形吸虫感染及其分泌排泄产物对小鼠Toll样受体(TLRs) m RNA表达量的影响。方法 40只雌性BALB/c小鼠随机分为感染组和未感染组,每组20只。感染组每鼠经口灌喂大片形吸虫嚢蚴20个,未感染组经口灌喂等体积PBS,分别于感染后1、 4、 7、 14和28 d,每组各取4只小鼠进行剖杀。取小鼠肝脏组织,提取总RNA,逆转录为cDNA, qRT-PCR检测TLR1～9 mRNA的相对表达量。于感染的水牛肝脏中收集大片形吸虫虫体, PBS培养6 h,收集自然活性的大片形吸虫分泌排泄产物(n Fg ESP)。18只雌性BALB/c小鼠随机分为n Fg ESP组和PBS组,每组9只。n Fg ESP组每鼠经腹腔注射1 mg/ml n Fg ESP,对照组注射等量PBS,分别于注射后1、 4、 7 d,每组各取3只小鼠进行剖杀。取小鼠肝脏组织,提取总RNA,逆转录为cDNA, qRT-PCR检测TLR1～9 m RNA的相对表达量。将n Fg ESP溶液95℃热处理15 min,制备热灭活的大片形吸虫分泌排泄产物(hi Fg ESP);分别用含25、50、 100和200μg/ml n Fg ESP或hi Fg ESP的PBS体外刺激小鼠RAW264.7细胞24 h (细胞浓度为2×106个细胞/孔),设2个重复孔, 3次重复实验,以等体积PBS为空白对照;收集各组细胞,提取细胞总RNA,逆转录为cDNA, q RTPCR检测TLR1～9 mRNA的相对表达量。结果大片形吸虫感染小鼠实验结果显示,感染组小鼠7 d后肝脏组织中TLR6和TLR7的mRNA相对表达量分别为2.9±0.3和2.5±0.5,均高于未感染组(1.0±0.2和1.3±0.5)(P <0.01); 14 d后TLR1和TLR9的mRNA相对表达量分别为3.4±0.8和2.8±0.9,均高于未感染组(1.1±0.2和1.1±0.3)(P <0.05或P <0.01); 28 d后TLR1、 TLR8和TLR9的mRNA相对表达量分别为3.8±1.3、 4.2±1.5和3.1±0.9,均高于未感染组(1.0±0.2、 1.2±0.4和1.1±0.3)(P <0.01或P <0.05)。Fg ESP体内免疫小鼠实验结果显示, n Fg ESP注射1 d后,小鼠肝脏组织中TLR1和TLR2的mRNA相对表达量分别为2.5±0.4和1.9±0.4,均高于PBS组(1.0±0.1)(P <0.05或P <0.01)。Fg ESP体外刺激RAW264.7细胞实验结果显示, 100μg/ml n Fg ESP刺激下的TLR1、 TLR3、 TLR7和TLR8的mRNA相对表达量分别为1.6±0.0、 1.4±0.0、 1.6±0.0和1.9±0.0,均高于PBS组(1.0±0.0)(P <0.05或P <0.01); 200μg/ml n Fg ESP和hi Fg ESP刺激下的TLR8 mRNA相对表达量分别为3.4±0.0和4.0±0.5,均高于PBS组(1.0±0.0)(P <0.01); 100μg/ml hi Fg ESP刺激下的TLR2和TLR8的mRNA相对表达量分别为1.5±0.1和2.0±0.0,均高于PBS组(1.0±0.0)(P <0.01); 25μg/ml hi Fg ESP刺激下的TLR3m RNA相对表达量为0.6±0.0,低于PBS组(1.0±0.0)(P <0.01)。结论大片形吸虫可能通过其分泌排泄产物影响小鼠TLR1、 TLR7和TLR8的转录,对宿主免疫应答进行调控。
Objective This work aims to investigate the effects of Fasciola gigantica infection and the released excretory-secretory products(Fg ESP) on the mRNA expression of mice Toll-like receptors(TLRs).Methods To determine the effect of F. gigantica infection on the TLR1-9 mRNA expression, 20 female BALB/c mice were orally infected with 20 metacercariae of F. gigantica, another 20 mice were given with PBS as non-infection control. Four mice from each group were sacrificed 1, 4, 7, 14 or 28 days post infection. The liver tissue of mice was obtained, total RNA was extracted, reverse transcribed into cDNA, and the relative expression of TLR1-9 mRNA was detected by qRT-PCR. To prepare the Fg ESP, the flukes from liver of buffalo infected with F. gigantica was collected from slaughterhouse and incubated in PBS for 6 hours, the incubation supernatant were collected as native Fg ESP(n Fg ESP). To determine the effect of Fg ESP on the TLR1-9 mRNA expression in mouse liver, 9 mice were treated with 1 mg/ml n Fg ESP by intraperitoneal injection. The same number of mice were given PBS only as control. Three mice from each group were sacrificed 1, 4 and 7 days post treatment. The liver was collected from each mouse for RNA extraction, the relative mRNA expression of TLR1-9 in the liver was detected by qRT-PCR.The n Fg ESP was inactivated by heating at 95 ℃ for 15 min to make the heat-inactivated ESP(hi Fg ESP). To determine the effect of n Fg ESP and hi Fg ESP on the TLR1-9 mRNA expression in macrophage cell line RAW264.7,2 × 106 cells were incubated with 25, 50, 100 or 200 μg/ml of n Fg ESP or hi Fg ESP, or PBS in duplicates for 24 h.The cell experiment was repeated three times. The cells were harvested for RNA extraction. The TLR1-9 mRNA expression in the cells were detected by qRT-PCR. Results In the F. gigantica infection experiment, qRT-PCR results demonstrated that the relative mRNA expression of TLR6 and TLR7 in the livers of mice infected for 7 days(2.9 ± 0.3 and 2.5 ± 0.5) were significantly higher than that in the control mice(1.0 ± 0.2 and 1.3 ± 0.5)(P < 0.01).The m RNA expression of TLR1 and TLR9 in the infected mice at 14 days(3.4 ± 0.8 and 2.8 ± 0.9) were significantly higher than that in the control mice(1.1 ± 0.2 and 1.1 ± 0.3)(P < 0.05, P < 0.01). After being infected for 28 days, the TLR1, TLR8 and TLR9 mRNA expression levels in the livers(3.8 ± 1.3, 4.2 ± 1.5 and3.1 ± 0.9) were significantly higher than that in the control mice(1.0 ± 0.2, 1.2 ± 0.4 and 1.1 ± 0.3)(both P <0.01, P < 0.05). In the Fg ESP treatment experiment, the mRNA levels of TLR1 and TLR2 in the livers of mice treated with n Fg ESP for 1 day(2.5 ± 0.4 and 1.9 ± 0.4) were significantly higher than that in the PBS-treated mice(1.0 ± 0.1)(P < 0.05, P < 0.01). For in vitro Fg ESP stimulation experiment on RAW264.7 cell line, the TLR1,TLR3, TLR7 and TLR8 mRNA expression levels in the cells stimulated with 100 μg/ml of n Fg ESP were significantly upregulated(1.6 ± 0.0, 1.4 ± 0.0, 1.6 ± 0.0 and 1.9 ± 0.0, respectively) than the cells treated with PBS(1.0 ± 0.0)(P < 0.05, all P < 0.01). The TLR-8 mRNA expression level was significantly higher in cells stimulated with 200μg/ml of n Fg ESP and hi Fg ESP(3.4 ± 0.0 and 4.0 ± 0.5) than that in cells treated with PBS(1.0 ± 0.0)(P < 0.01).The TLR2 and TLR8 mRNA expression levels in cells stimulated with 100 μg/ml hi Fg ESP(1.5 ± 0.1 and 2.0 ± 0.0)were significantly higher than that in PBS treated cells(1.0 ± 0.0)(P < 0.01). However, the TLR3 m RNA level was significantly lower in cells stimulated with 25 μg/ml hi Fg ESP(0.6 ± 0.0) than that in PBS treated cells(1.0 ± 0.0)(P < 0.01). Conclusion F. gignatica may regulate host immune response by affecting the transcriptional expression of TLR1, TLR7 and TLR8 in mice through releasing ESP.
Objective This work aims to investigate the effects of Fasciola gigantica infection and the released excretory-secretory products(Fg ESP) on the mRNA expression of mice Toll-like receptors(TLRs).Methods To determine the effect of F. gigantica infection on the TLR1-9 mRNA expression, 20 female BALB/c mice were orally infected with 20 metacercariae of F. gigantica, another 20 mice were given with PBS as non-infection control. Four mice from each group were sacrificed 1, 4, 7, 14 or 28 days post infection. The liver tissue of mice was obtained, total RNA was extracted, reverse transcribed into cDNA, and the relative expression of TLR1-9 mRNA was detected by qRT-PCR. To prepare the Fg ESP, the flukes from liver of buffalo infected with F. gigantica was collected from slaughterhouse and incubated in PBS for 6 hours, the incubation supernatant were collected as native Fg ESP(n Fg ESP). To determine the effect of Fg ESP on the TLR1-9 mRNA expression in mouse liver, 9 mice were treated with 1 mg/ml n Fg ESP by intraperitoneal injection. The same number of mice were given PBS only as control. Three mice from each group were sacrificed 1, 4 and 7 days post treatment. The liver was collected from each mouse for RNA extraction, the relative mRNA expression of TLR1-9 in the liver was detected by qRT-PCR.The n Fg ESP was inactivated by heating at 95 ℃ for 15 min to make the heat-inactivated ESP(hi Fg ESP). To determine the effect of n Fg ESP and hi Fg ESP on the TLR1-9 mRNA expression in macrophage cell line RAW264.7,2 × 106 cells were incubated with 25, 50, 100 or 200 μg/ml of n Fg ESP or hi Fg ESP, or PBS in duplicates for 24 h.The cell experiment was repeated three times. The cells were harvested for RNA extraction. The TLR1-9 mRNA expression in the cells were detected by qRT-PCR. Results In the F. gigantica infection experiment, qRT-PCR results demonstrated that the relative mRNA expression of TLR6 and TLR7 in the livers of mice infected for 7 days(2.9 ± 0.3 and 2.5 ± 0.5) were significantly higher than that in the control mice(1.0 ± 0.2 and 1.3 ± 0.5)(P < 0.01).The m RNA expression of TLR1 and TLR9 in the infected mice at 14 days(3.4 ± 0.8 and 2.8 ± 0.9) were significantly higher than that in the control mice(1.1 ± 0.2 and 1.1 ± 0.3)(P < 0.05, P < 0.01). After being infected for 28 days, the TLR1, TLR8 and TLR9 mRNA expression levels in the livers(3.8 ± 1.3, 4.2 ± 1.5 and3.1 ± 0.9) were significantly higher than that in the control mice(1.0 ± 0.2, 1.2 ± 0.4 and 1.1 ± 0.3)(both P <0.01, P < 0.05). In the Fg ESP treatment experiment, the mRNA levels of TLR1 and TLR2 in the livers of mice treated with n Fg ESP for 1 day(2.5 ± 0.4 and 1.9 ± 0.4) were significantly higher than that in the PBS-treated mice(1.0 ± 0.1)(P < 0.05, P < 0.01). For in vitro Fg ESP stimulation experiment on RAW264.7 cell line, the TLR1,TLR3, TLR7 and TLR8 mRNA expression levels in the cells stimulated with 100 μg/ml of n Fg ESP were significantly upregulated(1.6 ± 0.0, 1.4 ± 0.0, 1.6 ± 0.0 and 1.9 ± 0.0, respectively) than the cells treated with PBS(1.0 ± 0.0)(P < 0.05, all P < 0.01). The TLR-8 mRNA expression level was significantly higher in cells stimulated with 200μg/ml of n Fg ESP and hi Fg ESP(3.4 ± 0.0 and 4.0 ± 0.5) than that in cells treated with PBS(1.0 ± 0.0)(P < 0.01).The TLR2 and TLR8 mRNA expression levels in cells stimulated with 100 μg/ml hi Fg ESP(1.5 ± 0.1 and 2.0 ± 0.0)were significantly higher than that in PBS treated cells(1.0 ± 0.0)(P < 0.01). However, the TLR3 m RNA level was significantly lower in cells stimulated with 25 μg/ml hi Fg ESP(0.6 ± 0.0) than that in PBS treated cells(1.0 ± 0.0)(P < 0.01). Conclusion F. gignatica may regulate host immune response by affecting the transcriptional expression of TLR1, TLR7 and TLR8 in mice through releasing ESP.
引文
[1] World Health Organization. Accelerating work to overcome the global impact of neglected tropical diseases:a roadmap for implementation:executive summary[J]. Prev Med,2012,1(4):499-512.
[2] Zhang WY,Moreau E,Yang BZ,et al. Humoral and cellular immune responses to Fasciola gigantica experimental infection in buffaloes[J]. Res Vet Sci,2006,80(3):299-307.
[3] Ingale SL,Singh P,Raina OK,et al. Interleukin-2 and interleukin-10 gene expression in calves experimentally infected with Fasciola gigantica[J]. Livest Sci,2010,131(1):141-143.
[4] Zhang FK,Guo AJ,Hou JL,et al. Serum levels of cytokines in water buffaloes experimentally infected with Fasciola gigantica[J]. Vet Parasitol,2017,244:97-101.
[5] Luo HL,Zhang YY,Sheng ZA,et al. Long noncoding RNA profiling from Fasciola gigantica excretory/secretory product-induced M2 to M1 macrophage polarization[J]. Cell Physiol Biochem,2018,47(2):505-522.
[6]何邵平,贺建华,陈佳亿,等.固有免疫相关模式识别受体研究进展[J].动物营养学报,2017,29(11):3844-3851.
[7] Ulevitch RJ. Therapeutics targeting the innate immune system[J]. Nat Rev Immunol,2004,4(7):512-520.
[8] Ueno K,Koga T,Kato K,et al. MUC1 mucin is a negative regulator of Toll-like receptor signaling[J]. Am J Respir Cell Mol Biol,2008,38(3):263-268.
[9] Zhang FK,Hou JL,Guo AJ,et al. Expression profiles of genes involved in TLRs and NLRs signaling pathways of water buffaloes infected with Fasciola gigantica[J]. Mol Immunol,2018,94:18-26.
[10] Davis MJ,Tsang TM,Qiu YF,et al. Macrophage M1/M2polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection[J].Mbio,2013,4(3):e00264-e00213.
[11]徐田云. Toll样受体(TLR)2、 TLR3、 TLR4和TLR9在华支睾吸虫感染小鼠肝脏中动态表达的初步研究[D].徐州:徐州医学院,2011.
[12]万青. Toll样受体(TLR)2、 TLR4和TLR5在华支睾吸虫感染小鼠中的初步研究[D].徐州:徐州医学院,2012.
[13]刘瀛,颜超,华慧,等. TLR3和IRF3在华支睾吸虫感染C3H/HeN小鼠中表达的初步分析[J].徐州:徐州医学院学报,2013,33(9):602-605.
[14]张文娟,付琳琳,刘宜升,等. TLR4在华支睾吸虫感染致小鼠肝纤维化作用的研究[J].中国热带医学,2014,14(5):541-544.
[15]汤娟娟,季小芳,朱郇悯,等.日本血吸虫感染小鼠肝脏Toll样受体2和6的表达变化[J].中国寄生虫学与寄生虫病杂志,2014,32(2):101-105.
[16] Dawes B. On the growth and maturation of Fasciola hepatica L. in the mouse[J]. J Helminthol,1962,36:11-38.
[17]全琛宇.小鼠模型感染大片形吸虫研究平台初探[D].南宁:广西大学,2015.
[18] Piccinini AM,Midwood KS. DAMPening inflammation by modulating TLR signalling[J]. Mediat Inflamm,2010,2010:672395.
[19]艾敏.日本血吸虫热休克蛋白70诱导小鼠先天免疫的初步研究[D].北京:中国农业科学院,2017.
[20] Zhang R,Sun Q,Chen Y,et al. Ts-Hsp70 induces protective immunity against Trichinella spiralis infection in mouse by activating dendritic cells through TLR2 and TLR4[J]. PLoS Negl Trop Dis,2018,12(5):e0006502.
[21] Takeda K. Toll-like receptors in innate immunity[J]. Int Immunol,2004,17(1):1-14.
[22] Nakao Y,Funami K,Kikkawa S,et al. Surface-expressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells[J]. J Immunol,2005,174(3):1566-1573.
[23]张焱皓,龙润莹,刘旭恒,等.病原菌影响巨噬细胞极化的研究进展[J].中国免疫学杂志,2018,34(11):1748-1752,封3.
[24]董育玮,陆伦根.巨噬细胞:肝脏纤维化形成和降解的中心调控者[J].肝脏,2013,18(1):45.
[25]许丹,律颖,朱小语,等.小鼠单核巨噬细胞白血病细胞(RAW264.7)的培养及其在诱导破骨细胞中的应用[J].中国骨质疏松杂志,2016,22(10):1355-1360.
[26] Lee SY,Chen PY,Lin JC,et al. Melaleuca alternifolia induces heme oxygenase-1 expression in murine RAW264.7cells through activation of the Nrf2-ARE pathway[J]. Am J Chin Med,2017,45(8):1631-1648.
[27]杨庆利,蒋智华,申继清,等.华支睾吸虫排泄-分泌产物对小鼠巨噬细胞一氧化氮产生和核转录因子κB活化的影响[J].中国寄生虫学与寄生虫病杂志,2015,33(2):96-100.
[28]禹洋,徐佳,吕兴锋,等.旋毛虫ES抗原对小鼠巨噬细胞TLR2/4mRNA表达的影响[J].中国兽医科学,2012,42(6):562-565.
[29] Han CX,Yu J,Zhang ZQ,et al. Immunomodulatory effects of Trichinella spiralis excretory-secretory antigens on macrophages[J].Exp Parasitol,2019,196:68-72.
[30] Figueroa-Santiago O,Espino AM. Fasciola hepatica ESPs could indistinctly activate or block multiple Toll-like receptors in a human monocyte cell line[J]. Ann Clin Pathol,2017,5(3):1112.
[31] Flynn RJ,Mulcahy G. Possible role for Toll-like receptors in interaction of Fasciola hepatica excretory/secretory products with bovine macrophages[J]. Infect Immun,2008,76(2):678-684.
[32] Rodríguez E,Noya V,Cervi L,et al. Glycans from Fasciola hepatica modulate the host immune response and TLR-induced maturation of dendritic cells[J]. PLoS Negl Trop Dis,2015,9(12):e0004234.
[2] Zhang WY,Moreau E,Yang BZ,et al. Humoral and cellular immune responses to Fasciola gigantica experimental infection in buffaloes[J]. Res Vet Sci,2006,80(3):299-307.
[3] Ingale SL,Singh P,Raina OK,et al. Interleukin-2 and interleukin-10 gene expression in calves experimentally infected with Fasciola gigantica[J]. Livest Sci,2010,131(1):141-143.
[4] Zhang FK,Guo AJ,Hou JL,et al. Serum levels of cytokines in water buffaloes experimentally infected with Fasciola gigantica[J]. Vet Parasitol,2017,244:97-101.
[5] Luo HL,Zhang YY,Sheng ZA,et al. Long noncoding RNA profiling from Fasciola gigantica excretory/secretory product-induced M2 to M1 macrophage polarization[J]. Cell Physiol Biochem,2018,47(2):505-522.
[6]何邵平,贺建华,陈佳亿,等.固有免疫相关模式识别受体研究进展[J].动物营养学报,2017,29(11):3844-3851.
[7] Ulevitch RJ. Therapeutics targeting the innate immune system[J]. Nat Rev Immunol,2004,4(7):512-520.
[8] Ueno K,Koga T,Kato K,et al. MUC1 mucin is a negative regulator of Toll-like receptor signaling[J]. Am J Respir Cell Mol Biol,2008,38(3):263-268.
[9] Zhang FK,Hou JL,Guo AJ,et al. Expression profiles of genes involved in TLRs and NLRs signaling pathways of water buffaloes infected with Fasciola gigantica[J]. Mol Immunol,2018,94:18-26.
[10] Davis MJ,Tsang TM,Qiu YF,et al. Macrophage M1/M2polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection[J].Mbio,2013,4(3):e00264-e00213.
[11]徐田云. Toll样受体(TLR)2、 TLR3、 TLR4和TLR9在华支睾吸虫感染小鼠肝脏中动态表达的初步研究[D].徐州:徐州医学院,2011.
[12]万青. Toll样受体(TLR)2、 TLR4和TLR5在华支睾吸虫感染小鼠中的初步研究[D].徐州:徐州医学院,2012.
[13]刘瀛,颜超,华慧,等. TLR3和IRF3在华支睾吸虫感染C3H/HeN小鼠中表达的初步分析[J].徐州:徐州医学院学报,2013,33(9):602-605.
[14]张文娟,付琳琳,刘宜升,等. TLR4在华支睾吸虫感染致小鼠肝纤维化作用的研究[J].中国热带医学,2014,14(5):541-544.
[15]汤娟娟,季小芳,朱郇悯,等.日本血吸虫感染小鼠肝脏Toll样受体2和6的表达变化[J].中国寄生虫学与寄生虫病杂志,2014,32(2):101-105.
[16] Dawes B. On the growth and maturation of Fasciola hepatica L. in the mouse[J]. J Helminthol,1962,36:11-38.
[17]全琛宇.小鼠模型感染大片形吸虫研究平台初探[D].南宁:广西大学,2015.
[18] Piccinini AM,Midwood KS. DAMPening inflammation by modulating TLR signalling[J]. Mediat Inflamm,2010,2010:672395.
[19]艾敏.日本血吸虫热休克蛋白70诱导小鼠先天免疫的初步研究[D].北京:中国农业科学院,2017.
[20] Zhang R,Sun Q,Chen Y,et al. Ts-Hsp70 induces protective immunity against Trichinella spiralis infection in mouse by activating dendritic cells through TLR2 and TLR4[J]. PLoS Negl Trop Dis,2018,12(5):e0006502.
[21] Takeda K. Toll-like receptors in innate immunity[J]. Int Immunol,2004,17(1):1-14.
[22] Nakao Y,Funami K,Kikkawa S,et al. Surface-expressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells[J]. J Immunol,2005,174(3):1566-1573.
[23]张焱皓,龙润莹,刘旭恒,等.病原菌影响巨噬细胞极化的研究进展[J].中国免疫学杂志,2018,34(11):1748-1752,封3.
[24]董育玮,陆伦根.巨噬细胞:肝脏纤维化形成和降解的中心调控者[J].肝脏,2013,18(1):45.
[25]许丹,律颖,朱小语,等.小鼠单核巨噬细胞白血病细胞(RAW264.7)的培养及其在诱导破骨细胞中的应用[J].中国骨质疏松杂志,2016,22(10):1355-1360.
[26] Lee SY,Chen PY,Lin JC,et al. Melaleuca alternifolia induces heme oxygenase-1 expression in murine RAW264.7cells through activation of the Nrf2-ARE pathway[J]. Am J Chin Med,2017,45(8):1631-1648.
[27]杨庆利,蒋智华,申继清,等.华支睾吸虫排泄-分泌产物对小鼠巨噬细胞一氧化氮产生和核转录因子κB活化的影响[J].中国寄生虫学与寄生虫病杂志,2015,33(2):96-100.
[28]禹洋,徐佳,吕兴锋,等.旋毛虫ES抗原对小鼠巨噬细胞TLR2/4mRNA表达的影响[J].中国兽医科学,2012,42(6):562-565.
[29] Han CX,Yu J,Zhang ZQ,et al. Immunomodulatory effects of Trichinella spiralis excretory-secretory antigens on macrophages[J].Exp Parasitol,2019,196:68-72.
[30] Figueroa-Santiago O,Espino AM. Fasciola hepatica ESPs could indistinctly activate or block multiple Toll-like receptors in a human monocyte cell line[J]. Ann Clin Pathol,2017,5(3):1112.
[31] Flynn RJ,Mulcahy G. Possible role for Toll-like receptors in interaction of Fasciola hepatica excretory/secretory products with bovine macrophages[J]. Infect Immun,2008,76(2):678-684.
[32] Rodríguez E,Noya V,Cervi L,et al. Glycans from Fasciola hepatica modulate the host immune response and TLR-induced maturation of dendritic cells[J]. PLoS Negl Trop Dis,2015,9(12):e0004234.