Table 1.
Primer sequences of human gut tight junctions and apoptosis-related genes for qPCR.
Fig 1.
T. spiralis IIL ESP effect on Caco-2 cell viability.
A: 0, 25, 50, 100 and 200 μg/ml of IIL ESP were incubated with Caco-2 cells for 24 h and 48 h. B: Caco-2 cells were incubated with 200 μg/ml of IIL ESP for different time. The data are presented as mean ± standard deviation (SD) of three independent tests. *P < 0.05 compared to the blank control (0 μg/ml or 0 h) group.
Fig 2.
T. spiralis IIL ESP reduced TJ protein expression and destroyed the integrity of Caco-2 monolayer.
A: IIL ESP decreased the TEER of Caco-2 monolayer. B: IIL ESP increased FITC-dextran (4 kDa) flux through the monolayer. C: qPCR analysis of the mRNA expression level of TJs in IIL ESP-treated Caco-2 monolayer. D: IFT analysis of the TJs protein expression in IIL ESP-treated Caco-2 monolayer, scale bar: 20 μm. E: Quantitative analysis of the TJs fluorescence intensity. F: Western blot analysis of the TJs protein expression in IIL ESP-treated Caco-2 cells. Data are presented as mean ± SD of three independent assays. *P < 0.05 compared to PBS group.
Fig 3.
T. spiralis IIL ESP induced Caco-2 cell apoptosis stained by DAPI, Hoechst 33258 and TUNEL staining.
Caco-2 cells were incubated with 200 μg/ml IIL ESP for 18 h. A: The nuclear morphology stained by DAPI (blue), white arrows indicating the broken nuclei, scale bar: 20 μm; B: Hoechst 33258 staining, the apoptotic cells exhibited deep blue staining and nuclear condensation (white arrows), scale bar: 50 μm; C: TUNEL staining of Caco-2 cells, the apoptotic cells were stained green, scale bar: 50 μm.
Fig 4.
T. spiralis IIL ESP-induced apoptosis stained by Annexin V-AbFlour488/PI staining.
Annexin V-AbFlour488 and PI staining was performed to distinguish the apoptosis cells. Annexin V-AbFlour488 positive cells were apoptosis cells and PI positive cells were death cells, while the positive cells of both Annexin V-AbFlour488 and PI were late apoptosis cells. In addition, Annexin V-AbFlour488 and PI negative cells were normal cells. A: Annexin V-AbFlour488 (green) and PI (red) staining for apoptosis detection via a fluorescence microscope. B: Annexin V and PI staining for apoptosis detection via flow cytometry, and the percentage of Annexin V-AbFlour488 positive cells was shown in right bar graph. Scale bar: 50 μm. Data are presented as mean ± SD of three independent tests. *P < 0.05 compared to the PBS group.
Fig 5.
Changes of mRNA and protein expression levels of apoptotic genes of Caco-2 cells treated with T. spiralis IIL ESP.
A: qPCR analysis of the mRNA expression levels of apoptotic genes of IIL ESP-treated Caco-2 cells. B: Western blot analysis of apoptotic proteins expression levels of IIL ESP-treated Caco-2 cells. Data are presented as mean ± SD of three independent experiments, *P < 0.05 compared to the PBS group.
Fig 6.
Apoptosis inhibitor prevented the decrease of IIL ESP-induced TJs expression and alleviated barrier disruption of Caco-2 monolayer.
A: Apoptosis inhibitor Z-VAD-FMK pretreatment increased the IIL ESP-decreased TEER. B: Z-VAD-FMK pretreatment reduced the IIL ESP-increased FITC-dextran flux. C: qPCR analysis of the Z-VAD-FMK pretreatment increasing mRNA expression level of TJs in IIL ESP-treated Caco-2 monolayer. D and E: fluorescence microscopy of Z-VAD-FMK pretreatment recovering the expression of TJs proteins in IIL ESP-treated Caco-2 monolayer (scale bars: 20 μm) and quantitative analysis of the TJs fluorescence intensity. F: Western blot analysis of TJs expression in Z-VAD-FMK pretreated- Caco-2 monolayer after incubation with IIL ESP. Data are presented as mean ± SD of three independent experiments. *P < 0.05 compared to PBS group, #P < 0.05 compared to the individual IIL ESP group.
Fig 7.
Apoptosis inhibitor impeded larval invasion of Caco-2 monolayer.
Caco-2 monolayers were pretreated with Z-VAD-FMK and then incubated with T. spiralis IIL ESP for 18 h, one hundred IIL were added and incubated at 37°C for another 2 h. The larval invasion was observed under a microscope. A and B: larva invaded Caco-2 monolayer, and the larval migratory traces were indicated by arrows. C: non-invaded larva exhibited a spiral coil. D: IIL ESP facilitated the larval invasion while apoptosis inhibitor Z-VAD-FMK pretreatment suppressed the larval invasion. Data are presented as mean ± SD of three independent assays. Scale bars: 100 μm. *P < 0.05 compared to PBS group, #P < 0.05 compared to the only IIL ESP group.