关键词: 大片形吸虫, 分泌排泄产物, Toll样受体, 实时荧光定量PCR

Abstract:

Objective This work aims to investigate the effects of Fasciola gigantica infection and the released excretory-secretory products (FgESP) 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 FgESP, 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 FgESP(nFgESP). To determine the effect of FgESP on the TLR1-9 mRNA expression in mouse liver, 9 mice were treated with 1 mg/ml nFgESP 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 nFgESP was inactivated by heating at 95 ℃ for 15 min to make the heat-inactivated ESP(hiFgESP). To determine the effect of nFgESP and hiFgESP 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 nFgESP or hiFgESP, 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 mRNA 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 and 3.1 ± 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 FgESP treatment experiment, the mRNA levels of TLR1 and TLR2 in the livers of mice treated with nFgESP 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 FgESP 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 nFgESP 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 200 μg/ml of nFgESP and hiFgESP (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 hiFgESP (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 mRNA level was significantly lower in cells stimulated with 25 μg/ml hiFgESP (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.

Key words: Fasciola gigantica, Excretory-secretory products, Toll-like receptor, Real time PCR