L-Arginine protects ovine intestinal epithelial cells from lipopolysaccharide-induced intestinal barrier injury

Figures & data

Table 1. The primer sequences used in the real-time PCR.

Genes^a	Sequences (5′–3′)^b	Gene Bank No
MyD88	F: ATGGTGGTGGTTGTCTCTGAC R: GGAACTCTTTCTTCATTGGCTTGT	GQ221044.1
TLR-2	F: CAAGAGGAAGCCCAGGAAG R: TGGACCATGAGGTTCTCCA	DQ890157.1
TLR-4	F: TGCTGGCTGCAAAAAGTATG R: CCCTGTAGTGAAGGCAGAGC	HQ343416.1
TLR-9	F: ATGGGCCCCTACTGTG R: CTATTCGGCTGTCGTGG	HQ263217.1
TRAF-6	F: TCAGAGAACAGATGCCCAAT R: GCGTGCCAAGTGATTCCT	XM_012134166.2
IL-6	F: AGGAAAAAGATGGATGCTTCCA R: GACCAGCAGTGGTTTTGATCAA	NM_001009392
IL-1β	F: CGTCTTCCTGGGACGTTTTAG R: CTGCGTATGGCTTCTTTAGGG	NM_001009465
NF-κB	F: ATACGTCGGCCGTGTCTAT R: GGAACTGTGATCCGTGTAG	XM_005226864.2
TNF-a	F: ACACCATGAGCACCAAAAGC R: AGGCACAAGCAACTTCTGGA	NM_001024860.1
SLC7A1	F: ACCTAATGTCCATCGGCACTCT R: ATCGCTGCTGCTCACCAACT	XM_012184646.2
SLC1A1	F: TTGGTCTGCGTGCTGTCGT R: CATGGCATCCACTGTGCTAACT	XM_004004350.3
SLC7A7	F: CAGTTGCATTATCCTGCTTCGGT R: GCCTATCGGGCTCCTTCCAG	XM_004010352.3
SLC1A5	F: GATCGTGGAGATGGAGGAT R: TGACTGCTTCGAGGATGATG	AF334818
SLC15A1	F: GGGCACCGCCATCTATCAC R: CAGACACGCAAGGCTTTATCC	NM_001009758.1
ZO-1	F: AGAAGATAGCCCTGCAGCCAA R: CCT CTC CTT TGT TAA AAC TAA GTC	AJ313188
Occludin	F: CTGGATCAGGGAATATCCACC R: ACTCTTCACTTTCTTCTCTATAGT	AJ313191
Claudin-1	F: CACCCTTGGCATGAAGTGTA R: AGCCAATGAAGAGAGCCTGA	HM117762.1
iNOS	F: TGAGAATGGCAGCTACTGGGTCAA R: GGTGATGTCCAGGAAATGGGTGA	AF223942.1
eNOS	F: AGCATCACCTACGACACTCTG R: ACTGGTTGATGAAGTCCCTGG	NM_001129901.1
β-actin	F: GCTCTTCCAGCCGTCCTT R: TGAAGGTGGTCTCGTGAATGC	NM_001009784.1

^aMyD88, myeloid differentiation factor 88; TRAF-6, TNF receptor-associated factor 6; TLR, Toll-like receptor; IL, interleukin, TNF-α, tumour necrosis factor alpha; SLC7A1, solute carrier family 7 (cationic amino acid transporter, y⁺ system), member 1; SLC1A1, solute carrier family 1 (neuronal/epithelial high-affinity glutamate transporter, system XAG), member 1; SLC7A7, solute carrier family 7 (amino acid transporter light chain, y^+L system), member 7; SLC1A5, solute carrier family 1 (alanine–serine–cysteine amino acid transporter 2), member 5; SLC15A1, solute carrier family 15, (peptide transporter 1) member 1; ZO-1, zonula occludens-1.

^bF, forward; R, reverse.

Figure 1. Effects of Arg on cell viability. (A) Toxicity of Arg on cell viability; (B) toxicity of LPS on cell viability; (C) time-dependent effects of Arg on cell viability; (D) time-dependent effects of LPS on cell viability; (E) effects of Arg on cell viability of IOECs challenged by LPS. Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine. Values are expressed as means ± SEM, n = 6/group. Mean values in columns without a common letter differ (P < 0.05).

Figure 2. Effects of Arg on intestinal barrier function in LPS-treated IOECs for 24h. TEER (A) and paracellular permeability (B) were determined. TEER, transepithelial electrical resistance; Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine. Values are expressed as means ± SEM, n = 6/group. Mean values in columns without a common letter differ (P < 0.05).

Table 2. Effects of Arg on concentrations of cytokines in LPS-treated IOECs for 24h.

Items	Con	LPS	Arg + LPS	Arg	SEM	P-value
IL-1β (pg/mL)	100.35^c	332.19^a	216.23^b	108.34^c	4.87	.006
IL-6 (pg/mL)	40.12^c	110.09^a	76.09^b	38.18^c	2.12	.009
TNF-α (pg/mL)	44.98^c	85.99^a	60.12^b	45.17^c	2.08	.007

Note: Data are means ± pooled SEMs, n = 6/group. TNF-α, tumour necrosis factor α; IL, interleukin; Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine.

^a,b,cWithin a row, means without a common superscript letter differ (P < 0.05).

Table 3. Effect of Arg on the content of NO and activity of NOS in LPS-treated IOECs for 24 h^a.

Items^b	Con	LPS	Arg + LPS	Arg	SEM	P-value
NO (μmol/g of protein)	3.28^b	1.75^c	3.01^b	4.66^a	0.79	.008
NOS (U/g of protein)	569^b	487^c	558^b	623^a	32	.007

Note: ^a,b,cWithin a row, means without a common letter differ (P < 0.05).

^aMean values with their SEM (n = 6/group).

^bNO, nitric oxide; NOS, NO synthase; Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine.

Table 4. Effects of Arg on the mRNA abundance of genes in LPS-treated IOECs for 24 h.

Items^a	Con	LPS	Arg + LPS	Arg	SEM	P-value
MyD88	1.00^b	1.72^a	0.88^b	0.98^b	0.12	.021
TLR-9	1.00^b	1.63^a	0.78^b	0.89^b	0.13	.005
TLR-2	1.00	1.09	1.02	1.01	0.07	.109
TRAF-6	1.00	0.97	0.93	0.96	0.15	.094
TLR-4	1.00^c	1.85^a	1.46^b	0.94^c	0.10	.009
IL-6	1.00^b	1.64^a	1.01^b	0.99^b	0.08	.011
NF-κB	1.00^c	1.71^a	1.39^b	1.02^c	0.11	.009
IL-1β	1.00^c	1.73^a	1.39^b	0.99^c	0.08	.007
TNF-α	1.00^c	1.69^a	1.32^b	0.96^c	0.17	.008
SLC7A1	1.00^b	0.66^c	0.97^b	1.55^a	0.18	.009
SLC1A1	1.00^b	0.49^d	0.71^c	1.49^a	0.11	.005
SLC7A7	1.00	0.97	0.91	0.94	0.18	.109
SLC1A5	1.00^a	0.53^c	0.76^b	1.02^a	0.12	.006
SLC15A1	1.00^b	0.56^c	0.91^b	1.37^a	0.09	.008
ZO-1	1.00^b	0.53^c	0.89^b	1.43^a	0.14	.007
occludin	1.00^b	0.61^c	0.96^b	1.52^a	0.07	.009
claudin-1	1.00^b	0.64^c	1.02^b	1.49^a	0.12	.012
iNOS	1.00^b	0.52^d	0.75^c	1.62^a	0.15	.008
eNOS	1.00^b	0.60^c	0.95^b	1.54^a	0.16	.006

Note: Data are means ± pooled SEMs, n = 6/group.

^a,b,c,dWithin a row, means without a common superscript letter differ (P < 0.05).

^a MyD88, myeloid differentiation factor 88; TRAF-6, TNF receptor-associated factor 6; TLR, Toll-like receptor; IL interleukin, TNF-α, tumour necrosis factor alpha; SLC7A1, solute carrier family 7 (cationic amino acid transporter, y⁺ system), member 1; SLC1A1, solute carrier family 1 (neuronal/epithelial high-affinity glutamate transporter, system XAG), member 1; SLC7A7, solute carrier family 7 (amino acid transporter light chain, y^+L system), member 7; SLC1A5, solute carrier family 1 (alanine–serine–cysteine amino acid transporter 2), member 5; SLC15A1, solute carrier family 15, (peptide transporter 1) member 1; ZO-1, zonula occludens-1; iNOS, inducible NO synthase; eNOS, epithelial NO synthase; Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine.

Figure 3. The effect of Arg on TLR4 and NF-κB protein expression in LPS-treated IOECs for 24 h. Values are means, with standard errors represented by vertical bars. The protein expression value = densitometry units of selected protein/densitometry units of β-actin detected by western blotting. TLR4, Toll-like receptor 4; NF-κB Nuclear factor-κB; Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine. Values are expressed as means ± SEM, n = 6/group. Mean values in columns without a common letter differ (P < 0.05).

Figure 4. The effect of Arg on the relative protein (A) ZO-1 and (B) claudin-1 expression levels in LPS-treated IOECs for 24 h. Values are means, with standard errors represented by vertical bars. The protein expression value = densitometry units of selected protein/densitometry units of β-actin detected by western blotting. ZO-1, zonula occludens-1; Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine. Values are expressed as means ± SEM, n = 6/group. Mean values in columns without a common letter differ (P < 0.05).

Figure 5. Effects of Arg on the mTOR signalling pathways in LPS-treated IOECs for 24 h. Phosphorylation state of (A) Akt, (B) mammalian target of rapamycin (mTOR) and (C) p70 S6 kinase (S6K1) in LPS-treated IOECs. Akt phosphorylation on Ser⁴⁷³, mTOR phosphorylation on Ser²⁴⁴⁸ and S6K1 phosphorylation on Thr³⁸⁹ were measured by western blot analysis using antibodies that recognize these proteins only when that residue was phosphorylated. Western blots are shown above each graph. Values for phosphorylation of each protein were normalized for total protein. Values are means, with their standard errors represented by vertical bars. Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine. Values are expressed as means ± SEM, n = 6/group. Mean values in columns without a common letter differ (P < 0.05).

Figure 6. Effects of Arg on the protein abundance of amino acid and peptide transporters in LPS-treated IOECs for 24 h.Values are means, with standard errors represented by vertical bars. The protein expression value = densitometry units of selected protein/densitometry units of β-actin detected by western blotting. (A) CAT1, (B) EAAT3, (C) y⁺LAT2, (D) ASCT2, and (E) PEPT1 were encoded by SLC7A1, SLC1A1, SLC7A7, SLC1A5, and SLC15A1, respectively. CAT1, cationic amino acid transporter, y⁺ system, member 1; EAAT3, neuronal/epithelial high-affinity glutamate transporter, system X_AG, member 1; y⁺ LAT2, amino acid transporter light chain, y⁺ L system, member 7; ASCT2, alanine–serine–cysteine amino acid transporter 2; PEPT1, peptide transporter 1; Con, Arg-free DMEM; LPS, Arg-free DMEM supplemented with 10 μg/mL LPS; Arg + LPS, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM L-arginine; Arg, Arg-free DMEM supplemented with 100 μM L-arginine. Values are expressed as means ± SEM, n = 6/group. Mean values in columns without a common letter differ (P < 0.05).