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

Effects of Fasciola gigantica infection and released excretorysecretory products on the m RNA expression of Toll-like receptors in mice

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.