Inflammatory Response in Patients With Ulcerative Colitis
Inflammatory Response in Patients With Ulcerative Colitis
Firstly, we investigated whether there was any difference in survival of colonic epithelial cells isolated from UC patients and control individuals in cell culture. As shown in Figure 1, after 24 h in culture the vast majority of the control cells were viable and accounted for 72%, necrosis was observed in 18% and apoptosis in 10% of cells. The viability of cells isolated from UC patients and maintained in cell culture for 24 h was significantly lower than in control group (Figure 1A and B). Treatment of cells with NADPH oxidase inhibitor apocynin or with catalase (which selectively removes H2O2) increased the viability of the UC group cells reaching the survival level of untreated cells. In contrast, apocynin as well as catalase had no effect on cell survival in the control group.
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Figure 1.
Assessment of viability of human colonic epithelial cells. Where indicated, cells were incubated with 20 μg/ml LPS, 1 mM apocynin (apoc), 20 μg/ml LPS + 1 mM apocynin, 200 units/ml catalase (Cat), and control without stimulation for 24 h. A - control group, B - UC group. Statistically significant difference between control and another subgroups in the control or UC groups. Statistically significant difference between LPS and LPS + Apocynin subgroups in the control or UC groups. Statistically significant difference between control and UC groups. Statistically significant, as p <0.05.
Stimulation of colonic epithelial cells with LPS significantly reduced the number of live cells from 72% to 57% and increased necrotic cell death from 18% to 37% in the control group; whereas the viability of cells in UC group remained unchanged upon LPS treatment (Figure 1B). The cultivation of cells with LPS and apocynin increased the viability of the cells in the control and UC groups (to 76% and 78%, respectively) by decreasing the number of necrotic cells (Figure 1A and B). The percentage of apoptotic cells was 5–8% in all study groups.
Further, we analysed the ROS generation in primary colonic epithelial cells. As shown in Figure 2, the level of extracellular hydrogen peroxide production was approximately two times higher in epithelial cells of UC patients compared to the control group. Similar results were obtained when hydrogen peroxide production was measured directly in the colonic biopsies (Figure 2B). The treatment of colonic epithelial cells with catalase significantly decreased the level of extracellular hydrogen peroxide production in both experimental groups.
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Figure 2.
Assessment of hydrogen peroxide production. A - colonic epithelial cells were incubated for 30 min with 5 units/ml catalase (Cat), 10 μg/ml LPS, 10 μM DPI, 10 μg/ml LPS + 10 μM DPI or control without stimulation (where indicated). B - colonic biopsies (10 mg) of UC patients and control subjects were incubated without stimulation for 30 min. Statistically significant difference between control and another subgroups in the control group. Statistically significant difference between control and another subgroups in the UC group. & Statistically significant difference between LPS and LPS + DPI subgroups in the UC group. Statistically significant difference between control and UC groups. Statistically significant, as p <0.05.
DPI slightly decreased the level of ROS production in both UC and control groups, however, the differences were not statistically significant. The treatment of cells with LPS significantly increased the production of hydrogen peroxide in cells of both study groups (Figure 2A). The addition of DPI to LPS-treated colonic epithelial cells significantly decreased (by 1.7 fold) the level of ROS production in the UC group. In control group, DPI also diminished hydrogen peroxide production in LPS-treated cells, though the effect of DPI per se was not statistically significant due to higher variability of results (Figure 2A).
In addition, we analysed the influence of NADPH oxidase on the production of pro-inflammatory cytokine TNF-α. As shown in Figure 3, the highest concentrations of TNF-α were determined in cells of UC patients after stimulation with LPS. The level of cytokine production was approximately three-fold higher in LPS treated UC cells when compared to UC cells without stimulation and control group cells treated with LPS. In the UC group, treatment of cells with LPS and NADPH oxidase inhibitor apocynin decreased the levels of TNF-α production approximately 2.5-fold as compared with the LPS treated colonic epithelial cells. The differences of TNF-α concentration between untreated and treated cells in the control group were insignificant.
(Enlarge Image)
Figure 3.
Assessment of TNF-α production by human colonic epithelial cells. Cells were incubated for 24 h with 20 μg/ml of LPS, 1 mM of apocynin (Apoc), 20 μg/ml LPS + 1 mM apocynin or control without stimulation (as indicated). After incubation cell media were collected and TNF-α concentration was measured as described in Methods. Statistically significant difference between control and LPS subgroups in the UC group. Statistically significant difference between LPS and LPS + apocynin subgroups in the UC group. Statistically significant difference between LPS subgroup in the UC and control groups. Statistically significant, as p <0.05.
Results
Assessment of Viability of Human Colonic Epithelial Cells
Firstly, we investigated whether there was any difference in survival of colonic epithelial cells isolated from UC patients and control individuals in cell culture. As shown in Figure 1, after 24 h in culture the vast majority of the control cells were viable and accounted for 72%, necrosis was observed in 18% and apoptosis in 10% of cells. The viability of cells isolated from UC patients and maintained in cell culture for 24 h was significantly lower than in control group (Figure 1A and B). Treatment of cells with NADPH oxidase inhibitor apocynin or with catalase (which selectively removes H2O2) increased the viability of the UC group cells reaching the survival level of untreated cells. In contrast, apocynin as well as catalase had no effect on cell survival in the control group.
(Enlarge Image)
Figure 1.
Assessment of viability of human colonic epithelial cells. Where indicated, cells were incubated with 20 μg/ml LPS, 1 mM apocynin (apoc), 20 μg/ml LPS + 1 mM apocynin, 200 units/ml catalase (Cat), and control without stimulation for 24 h. A - control group, B - UC group. Statistically significant difference between control and another subgroups in the control or UC groups. Statistically significant difference between LPS and LPS + Apocynin subgroups in the control or UC groups. Statistically significant difference between control and UC groups. Statistically significant, as p <0.05.
Stimulation of colonic epithelial cells with LPS significantly reduced the number of live cells from 72% to 57% and increased necrotic cell death from 18% to 37% in the control group; whereas the viability of cells in UC group remained unchanged upon LPS treatment (Figure 1B). The cultivation of cells with LPS and apocynin increased the viability of the cells in the control and UC groups (to 76% and 78%, respectively) by decreasing the number of necrotic cells (Figure 1A and B). The percentage of apoptotic cells was 5–8% in all study groups.
Assessment of Hydrogen Peroxide Production in Cells
Further, we analysed the ROS generation in primary colonic epithelial cells. As shown in Figure 2, the level of extracellular hydrogen peroxide production was approximately two times higher in epithelial cells of UC patients compared to the control group. Similar results were obtained when hydrogen peroxide production was measured directly in the colonic biopsies (Figure 2B). The treatment of colonic epithelial cells with catalase significantly decreased the level of extracellular hydrogen peroxide production in both experimental groups.
(Enlarge Image)
Figure 2.
Assessment of hydrogen peroxide production. A - colonic epithelial cells were incubated for 30 min with 5 units/ml catalase (Cat), 10 μg/ml LPS, 10 μM DPI, 10 μg/ml LPS + 10 μM DPI or control without stimulation (where indicated). B - colonic biopsies (10 mg) of UC patients and control subjects were incubated without stimulation for 30 min. Statistically significant difference between control and another subgroups in the control group. Statistically significant difference between control and another subgroups in the UC group. & Statistically significant difference between LPS and LPS + DPI subgroups in the UC group. Statistically significant difference between control and UC groups. Statistically significant, as p <0.05.
DPI slightly decreased the level of ROS production in both UC and control groups, however, the differences were not statistically significant. The treatment of cells with LPS significantly increased the production of hydrogen peroxide in cells of both study groups (Figure 2A). The addition of DPI to LPS-treated colonic epithelial cells significantly decreased (by 1.7 fold) the level of ROS production in the UC group. In control group, DPI also diminished hydrogen peroxide production in LPS-treated cells, though the effect of DPI per se was not statistically significant due to higher variability of results (Figure 2A).
Assessment of TNF-α Concentration in the Colonic Epithelial Cells
In addition, we analysed the influence of NADPH oxidase on the production of pro-inflammatory cytokine TNF-α. As shown in Figure 3, the highest concentrations of TNF-α were determined in cells of UC patients after stimulation with LPS. The level of cytokine production was approximately three-fold higher in LPS treated UC cells when compared to UC cells without stimulation and control group cells treated with LPS. In the UC group, treatment of cells with LPS and NADPH oxidase inhibitor apocynin decreased the levels of TNF-α production approximately 2.5-fold as compared with the LPS treated colonic epithelial cells. The differences of TNF-α concentration between untreated and treated cells in the control group were insignificant.
(Enlarge Image)
Figure 3.
Assessment of TNF-α production by human colonic epithelial cells. Cells were incubated for 24 h with 20 μg/ml of LPS, 1 mM of apocynin (Apoc), 20 μg/ml LPS + 1 mM apocynin or control without stimulation (as indicated). After incubation cell media were collected and TNF-α concentration was measured as described in Methods. Statistically significant difference between control and LPS subgroups in the UC group. Statistically significant difference between LPS and LPS + apocynin subgroups in the UC group. Statistically significant difference between LPS subgroup in the UC and control groups. Statistically significant, as p <0.05.