Tryptophan alleviates lipopolysaccharide-induced liver injury and inflammation by modulating necroptosis and pyroptosis signaling pathways in piglets

Figures & data

Table 1. Composition and nutrient levels of basal diet (DM basis).

Ingredient	Content (%)
	7-11kg	11-25kg
Corn	27.37	30.6
Extruded Corn	30.84	32
Soybean oil	2.5	1.4
Glucose	2	2
Whey powder	5	5
Dehulled Soybean meal (46% CP)	13.24	13.04
Soybean protein concentrate	5	5
Extruded soybean	7	5
Fish meal (67% CP)	3	2.5
L-Lysine-HCl (78.8%)	0.52	0.44
DL-Methionine (99%)	0.11	0.08
L-Threonine (98.5%)	0.2	0.15
L-Tryptophan (98%)	0.03	0.01
L-Alanine (99%)	0.46	0.32
Choline chloride (50%)	0.15	0.15
Limestone	0.68	0.41
Monocalcium phosphate	1.35	1.35
NaCl	0.25	0.25
Vitamin premix*	0.05	0.05
Mineral premix^⋄	0.25	0.25
Total	100	100
Nutrient level^♦	Content (%)
Digestible energy Mcal/kg	3.55	3.49
Crude protein %	19.72	18.65
Calcium %	0.80	0.68
Total phosphorus %	0.66	0.64
Available phosphorus %	0.48	0.46
SID-Lysine %	1.36	1.24
SID-Methionine %	0.40	0.36
SID-Threonine %	0.80	0.73
SID-Tryptophan %	0.23	0.20

*The vitamin premix provides the following per kilogram of diet: VA 15000 IU; VD₃ 5,000 IU; VE 40 IU; VK₃ 5 mg; VB₁ 5 mg; VB₂ 12.5 mg; VB₆ 6 mg; VB₁₂ 600 μg; D-pantothenic acid 25 mg; nicotinic acid 50 mg; folic acid 2.5 mg; biotin 2.5 mg.

^⋄The mineral premix provides the following per kilogram of diet: copper (CuSO₄ •5H₂O) 6 mg; iron (FeSO₄•H₂O) 100 mg; zinc (ZnSO₄ •H₂O) 100 mg; manganese (MnSO₄•H₂O) 4 mg; iodine (KI) 0.14 mg; selenium (Na₂SeO₃) 0.3 mg.

^♦Nutrient levels are analyzed values.

Table 2. Primer sequences used for Real-time PCR.

Target Genes	Forward primer	Reverse primer	Accession number	Temperature (°C)	Product size (bp)
TNF-α	CGACTCAGTGCCGAGATCAA	GACCTGCCCAGATTCAGCAA	JF831365.1	58	60
IL-1β	AAGGCCGCCAAGATATAACTGA	GCCCTCTGGGTATGGCTTTC	NM_001302388.1	58	71
IL-6	ATGCTTCCAATCTGGGTTCAA	CACAAGACCGGTGGTGATTCT	AF518322.1	58	61
IL-8	ACATCCATGAGGAAGACAGTTTGA	CGGGAACTCCACGCTAGATTC	AB057440.1	58	70
NLRP3	TCCCCTGGTCTGCTGGATT	ACTCTTGCCGCTATCCATCTG	NM_001256770.2	58	61
ASC	CCAAGCCAGCTGGAATCAA	TGCAGTGCTGGTTTGTTGTCT	MK302492.1	58	58
Caspase-1	CGACCCCCACCTTGCA	AAGGCATTTTCCAGATTGTGAAC	NM_214162.1	58	61
GSDMD	GGCAGCGTCATTGCATTTC	TGAAGGTTCGCTGCTTCTTGT	XM_021090504.1	58	91
Claudin-1	GCTCCTGCCCCCGAAA	AAGGCGAAGGTTTTGGATAGG	NM_001244539.1	58	63
ZO-1	CCCAACCTCACAAATAGAAAGTGA	GCGAATAATGCCAGAGCTACGT	XM_013993251.1	58	70
Occludin	CCTCAGGCeAGCCTCATTACAG	GGGAGCCCGTTTTGAAGAC	NM_001163647.2	58	61
RIP1	ATGAGGAAGCGGGTTCGAT	CGCTGACACCAGATCCTCAA	XM_005665538.3	58	58
RIP3	GCCCATCGAGGACTGGAA	ACCGTGCCAAATCCTCCTT	XM_001927424.3	58	55
MLKL	CAAATGCTCCGGGACCAA	CAGGGCGGTGGTTATCTCA	XM_013998184.1	58	57
FADD	TTTGATATCATCTGCGACAACGT	CCTCGATGGCGTCGATCT	XM_013987237.1	58	91
PGAM5	ACATCCACCGGGCTGATG	GGCGTGGCAGATGAAGATCT	XM_013992365.1	58	62
HSP70	TTGGGCGCAAGTTTGCA	GGAAAGGCCAGTGCTTCAAG	NM_001123127.1	58	57

TNF-α: tumor necrosis factor-alpha. IL-1β: interleukin-1 beta. FGF21: fibroblast NLRP3: NOD-like receptor family pyrin domain containing 3. ASC: apoptosis-associated speck-like protein containing a CARD. GSDMD: gasdermin-D. ZO-1: zonula occludens-1. RIP1: the kinases receptor-interacting protein 1. RIP3: the kinases receptor-interacting 3. MLKL: the mixed lineage kinase domain-like protein. FADD: fas-associated death domain. PGAM5: phosphoglycerate mutase 5. HSP70: the 70-kDa heat shock proteins.

Figure 1. Effects of tryptophan on liver Morphology in piglets after 4h lipopolysaccharide (LPS) challenge in piglets. (A) CONTR group, piglets were fed basal diet and injected with normal saline. Normal liver histology. (B) LPS group, piglets were fed a basal diet and injected intraperitoneally with LPS. Piglet liver showed significant pathological changes, including (a) heptatocyte caryolysis, (b) karyopycnosis, (c) inflammatory cell infiltration, hyperemia in hepatic sinusoids and hepatic cell cords arrangement in disorder were found. (C) Trp + LPS group, piglets were fed a basal diet supplemented with 0.2% tryptophan and injected intraperitoneally with LPS. n = 6 (1 pig/pen). original magnifications 400×. scale bars = 22.4 µm.

Figure 2. Tryptophan decreases serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities after 4h lipopolysaccharide (LPS) challenge in piglets. P values < 0.05 were considered significant. P values between 0.05 and 0.10 were regarded as a tendency. Values are presented as mean ± standard error, n = 6. CONTR: control group; LPS: piglets challenged with LPS; Trp + LPS: piglets fed with 0.2% tryptophan and challenged with LPS. * denotes P < 0.05, ** means P < 0.01.

Figure 3. Effects of tryptophan on antioxidant indicators in the liver in piglets after 4h lipopolysaccharide (LPS) challenge in piglets. (A) the content of malondialdehyde (MDA). (B) the activity of glutathione peroxidase (GSH-Px). (C) the activity of catalase (CAT). (D) the activity of superoxide dismutase (T-SOD). (E) the content of hydrogen peroxide (H₂O₂). P values < 0.05 were considered significant. P values between 0.05 and 0.10 were regarded as a tendency. Values are showed as mean ± standard error, n = 6. CONTR: control group; LPS: piglets challenged with LPS; Trp + LPS: piglets fed with 0.2% tryptophan and challenged with LPS. *denotes P < 0.05, **means P < 0.01.

Figure 4. Effects of tryptophan on content and mRNA expression of liver pro-inflammatory cytokines in piglets after 4h lipopolysaccharide (LPS) challenge in piglets. (A) the content of liver pro-inflammatory cytokines (n = 5). (B) mRNA expression of liver pro-inflammatory cytokines (n = 6). P values < 0.05 were considered significant. P values between 0.05 and 0.10 were regarded as a tendency. Values are showed as mean ± standard error. CONTR: control group; LPS: piglets challenged with LPS; Trp + LPS: piglets fed with 0.2% tryptophan and challenged with LPS. TNF-α: tumor necrosis factor-α. IL-1β: interleukin-1 β. * denotes P < 0.05, ** means P < 0.01.

Figure 5. Effects of tryptophan on mRNA expressions of tight junction (A), HSP70 gene (B), necroptosis (C), and pyroptosis (D) in liver after 4h lipopolysaccharide (LPS) challenge in piglets. P values < 0.05 were considered significant. P values between 0.05 and 0.10 were regarded as a tendency. Values are showed as mean ± standard error, n = 6. CONTR: control group; LPS: piglets challenged with LPS; Trp + LPS: piglets fed with 0.2% tryptophan and challenged with LPS. NLRP3: NOD-like receptor family pyrin domain containing 3. ASC: apoptosis-associated speck-like protein containing a CARD. GSDMD: gasdermin-D. ZO-1: zonula occludens-1. RIP1: the kinases receptor-interacting protein 1. RIP3: the kinases receptor-interacting 3. MLKL: the mixed lineage kinase domain-like protein. FADD: fas-associated death domain. PGAM5: phosphoglycerate mutase 5. HSP70: the 70-kDa heat shock proteins. * denotes P < 0.05, ** means P < 0.01.

Figure 6. Effects of tryptophan on protein expressions of tight junction in liver after 4h lipopolysaccharide (LPS) challenge in piglets. P values < 0.05 were regarded as significant. P values between 0.05 and 0.10 were regarded as a tendency. Values are presented as mean ± standard error, n = 4. CONTR control group; LPS piglets challenged with LPS; Trp + LPS piglets fed with 0.2% tryptophan and challenged with LPS. ZO-1: zonula occludens-1. ^ns denotes not significant, * means P < 0.05, ** denotes P < 0.01.

Figure 7. Effects of tryptophan on expression pyroptosis-related proteins in liver after 4h lipopolysaccharide (LPS) challenge in piglets. P values < 0.05 were considered significant. P values between 0.05 and 0.10 were regarded as a tendency. Values are presented as mean ± standard error, n = 4. CONTR: control group; LPS: piglets challenged with LPS; Trp + LPS: piglets fed with 0.2% tryptophan and challenged with LPS. NLRP3: NOD-like receptor family pyrin domain containing 3. GSDMD: gasdermin-D. MLKL: the mixed lineage kinase domain-like protein. HSP70: the 70-kDa heat shock proteins. * denotes P < 0.05, ** means P < 0.01.