System y⁺ CAT1 siRNA knocked down L-Arg transport activity, CAT1 mRNA and System y⁺ protein.

<p>Caco-2 cells were transfected system y⁺ CAT1 siRNA for 24 hour as according to manufacturer’s instruction. L-Arg transport activity was measured as described in <i>Methods</i>. Cell lysates were assayed for CAT1 mRNA and protein. A: CAT1 mRNA. The levels of CAT1 mRNA were determined by Northern Blot analysis, then normalized to β-actin. System y⁺ CAT1 siRNA knocked down CAT mRNA by 60%. B: System y⁺ protein. Western blot analysis was used to detect System y⁺ protein and normalized to β-actin. C: L-Arg transport activity. Transport activity was expressed as picomoles L-Arg per milligram protein per 1 min of uptake. Data are means ± SE, * <i>P</i><0.05. (n = 6 /group).</p

Effects of L-Arg and IL-1β on iNOS expression.

<p>Caco-2 cells were treated with IL-1β (4 ng/ml) for 4 hours after an incubation of L-Arg (5 mM) for 4 hours (Fresh medium with L-Arg was applied after removing medium). Cells were harvested for the isolation of total RNA and protein. A: iNOS mRNA was determined by qRT-PCR and normalized to β-actin. B: iNOS protein was measured by Western blot analysis normalized to β-actin C: NO levels in cell culture media were measured as described in Methods. Data are means ± SE, * <i>P</i><0.05. (n = 6 /group).</p

Effects of system y⁺ CAT1 siRNA and L-Arg’s ability on NF-κB promoter activity and NF-κB expression.

<p>Caco-2 transfected with system y⁺ CAT1 siRNA for 24 hour prior to the transfection of pNF-κB Luc vector for 18 hours were treated with IL-1β (4ng/ml) for 4 hours after pretreatment of 4 hours of L-Arg (5 mM) (Fresh medium with L-Arg was applied after removing medium for pre-treatment). Cells were collected for luciferase activity and isolations of protein and total RNA as described in <i>Methods</i>. A: NF-κB luciferase activity. Data are expressed as fold-induction of normalized luciferase activity. B: NF-κB mRNA levels were determined by qRT-PCR, then normalized to β-actin. C: NF-κB p65 protein levels were measured by Western blot analysis and normalized to β-actin. Data are means ± SE. * <i>P</i><0.05 (n = 6 /group).</p

Effects of iκB-α and IL-1β-inducible NF-κB activation.

<p>Caco-2 cells were transfected with pNF-κB-Luc for 18 hours, then treated±IL-1β (4 ng/ml) for 4 hours. Cell lysates were assayed for luciferase activity and protein concentration as described in <i>Methods</i>. Data are expressed as fold induction of normalized luciferase activity ± SE. * <i>P</i><0.05 (n = 6 /group).</p

Effects of L-Argon IL-1β-induced NF-κB promoter activity, expression and IL-6 production.

<p>Caco-2 cells transfected with pNF-κB-Luc vector were treated with or without IL-1β (4 ng/ml) for 4 hours after pretreatment in the absence or presence of L-Arg (5 mM) for 4 hours (Fresh medium with L-Arg was applied after removing medium for pre-treatment). Cells were harvested for luciferase activity, isolations of total RNA and protein. A: NF-κB luciferase activity data are expressed as fold induction of normalized luciferase activity. B: NF-κB mRNA levels were determined by qRT-PCR, then normalized to β-actin. C: NF-κB p65 protein levels were measured by Western blot analysis and normalized to β-actin. D: IL-6 levels in the cell culture media were measured by ELISA, normalized to total protein and expressed as fold-induction. All data are means± SE, * <i>P</i><0.05. (n = 5–6 /group).</p

The activation of NF-κB in FHs 74 Int cells.

<p>FHS 74 int cells were transfected with pNF-κB-Luc vector, then treated with filtered fermented formula (100 μl/ml) or propionic acid (2.5 mM) in serum-free media for 18 h. Cells were harvested for luciferase activity. NF-κB luciferase activity data are expressed as fold induction of normalized luciferase activity. The levels of NF-κB luciferase activity in FF and propionic acid groups were significantly increased compared to control group. The data are represented as mean ± SE (* P<0.05, **P<0.01, N = 4 / group).</p

p65 NF-κB and TLR4 activation and expression.

<p>Intestinal p65 NF-κB activation in each group was assayed by EMSA. NF-κB mRNA was determined by qRT-PCR. P65 NF-κB and TLR4 proteins were measured by Western blot. Fig 3A: Image and bar graphs showed TLR4 protein expressions. Significant increase in TLR4 protein were seen in FF and BO groups. Fig 3B: Intestinal NF-κB mRNA expression levels. NF-κB mRNA levels in FF group were higher than BO and FO groups. Fig 3C: Intestinal p65 NF-κB protein levels. The levels of p65 NF-κB protein from FF group were higher than BO and FO groups. Fig 3D: Bar graphs, obtained by densitometric analysis of EMSA (the images not shown). The DNA binding activities in FF and BO groups were significantly increased compared to FO group. The data are represented as mean ± SE (* P<0.05, **P<0.01, N = 5–8 / group).</p

Claudin-2 and occludin protein expressions.

<p>Western blot was used to measure claudin-2 (Fig 6A) and occludin (Fig 6B) protein expressions in intestine. The levels of claudin-2 and occludin in FF and BO groups were significantly decreased compared to the FO group. Claudin-2 and occludin levels were also lower in the FF group compared to the BO group. The data are presented as mean ± SE (* P<0.05, **p<0.01, N = 5–8 / group).</p

Enteral administration of bacteria fermented formula in newborn piglets: A high fidelity model for necrotizing enterocolitis (NEC) - Fig 2

<p>Fig 2A: Histology from FF group. Representative hematoxylin and eosin stained section of small intestine with classic features of NEC: Regional villus disruption & destruction of villus architecture, mucosal sloughing, blood congestion, separation of the sub-mucosa and lamina propria (pneumatosis intestinalis). Fig 2B: Histology from BO group. Representative section of small bowel showing no inflammation, normal villus anatomy and architecture. Fig 2C: Histology from FO group. Representative section of small bowel showing no inflammation, normal villus anatomy and architecture. Fig 2D: Histological Scoring in FF, BO and FO groups. Histogram of quantitative histological scoring of small intestine for each experimental group. Blinded quantitative scoring was performed as described in the methods. The histopathologic features of NEC (villous destruction, inflammatory infiltrates, and pneumatosis intestinalis) were assigned a quantitative score of zero if absent and 1 if present in a single microscopic field of 10x magnification. All animals in the FF group scored 4 in all sections analyzed, representing severe disease. BO & FO groups had normal intestinal histology. Histological scores from FF group were significantly higher than BO and FO groups. The data are represented as median and IQR (*P<0.05, **P<0.01, N = 5–8 / group). Fig 2E: Villus length in FF, BO and FO groups. The measurement of villus length was performed as described in the methods. The significant reduction in FF group were observed compared to BO and FO groups. The data are represented as median and IQR (* P<0.05, N = 5–8 / group).</p

Data analysis.

<p>Data analysis.</p