Effect of Dietary Amylose/amylopectin Ratio on Antioxidant Ability and Amino Acid Metabolism of Liver in Weaned Pigs Challenged with Lipopolysaccharide

Background: Antioxidant function could be regulated by resistant starch supplementation in rats with kidney disease. It is controversial whether antioxidant ability could be regulated by changing dietary amylose/amylopectin ratio (DAR) when weaned piglets under lipopolysaccharide (LPS) stress. Methods: Sixty male weaned piglets (Duroc × Landrace × Yorkshire, 21 day old, 6.51 ± 0.64 kg) were allotted to 5 dietary treatments with 12 cages per treatment, and fed ad libitum with diets different in DAR (0.00, 0.20, 0.40, 0.60 and 0.80). Feed transformation happened at day 15. On day 29, 12 h before slaughter, pigs were intraperitoneal injected with 100 μg/kg body weight LPS or sterile saline. Results: Results showed that LPS stress caused increase in serum urea nitrogen and glucose concentration (P < 0.05). Serum immunoglobulin G concentration increased after LPS stress in diet 0.80 but decreased in other groups (P<0.05). Serum serine concentrations decreased after LPS stress in diets 0.20 and 0.80 but increased in other groups (P<0.05). All tested hepatic amino acids (AA) increased after LPS stress (P<0.05) but hepatic isoleucine, leucine, threonine, aspartate, serine, glutamate, glycine concentrations decreased after LPS stress in diet 0.20 (P<0.05). Piglets supplemented diet with DAR 0.00 had highest superoxide dismutase (SOD1), nuclear factor 2 (Nrf2) and glutathione peroxidase 1 (GPX1) mRNA expression in liver (P<0.05). Total superoxide dismutase (T-SOD) activity of liver increased in diet 0.00 and 0.80 but decreased in diet 0.40 (P<0.05). Total antioxidant capacity (T-AOC) activity of longissimus dorsi muscle (LM) increased in diets 0.20 and 0.80 but decreased in diet 0.40 peroxidase Conclusions: be improved by with low or high amylose/amylopectin In conclude, antioxidant ability of liver was affected by dietary amylose/amylopectin ratio when weaned piglets suffered LPS stress. More acids would be mobilized to synthesis glutathione peroxidase to defend LPS stress when weaned piglets supplemented diet with DAR 0.20. More Cu and Zn deposited in the liver and more Mn deposited in the muscle, and then antioxidant ability of liver and muscle increased after LPS stress when weaned piglets consuming diet with DAR0.80. Dietary amylose/amylopectin ratio (DAR) for most of the diet used in swine production is 0.40. Our study conrmed that diet with DAR 0.40 had disadvantageous when weaned piglets had experienced feed transition and then LPS stress. Supplemented amylose or amylopectin would be better for piglets under LPS stress through improving antioxidant function or amino acid metabolism.

dorsal muscles (approximately 2 cm×2 cm) and chyme from cecal and colon were collected using the sterile scissors and tweezers. All of the samples were immediately stored at -80℃ for the further study.
Analysis of AA contents in serum, liver, and muscle The serum samples were thawed on 4℃. Taking 600 μL of serum and 8% sulfosalicylic acid in 1.5 ML were mixed thoroughly, which stored at 4℃ for 12 h.
Then the mixed uids were centrifuged at 10,000 rpm and 4℃ for 10 min. The supernatant was ltered through a 0.22 μm membrane and analyzed for AA using an AA analyzer (Hitachi L-8900, Japan). When AA contents in liver and muscle were tested, about 0.1g ground freeze-dried sample was hydrolyzed in 10 mL of 6mol/L HCL at 110℃ for 22 h. The solution was then adjusted to a volume of 100 mL and 1 mL of the settled solution was used for further analysis after 10-fold dilution. The diluent solution analyzed using an amino acid analyzer (L-8900, Hitachi, Japan) after ltered by a 0.22-μm membrane.
Assay of hepatic, muscular antioxidant enzyme activities Hepatic and muscular tissue samples, were grounded in liquid nitrogen and homogenized in saline, and centrifuged at 3000×g, 4℃ for 10 min. The activities of malonaldehyde (MDA), superoxide dismutase (SOD) which includes cooper-zinc SOD (CuZn-SOD) and manganese SOD (Mn-SOD), glutathione peroxidase (GSH-PX/GPx), and total antioxidant capacity (T-AOC) were analyzed using commercial kits (Jiancheng Bioengineering Institue, Nanjing, China) according to the manufacturer's instructions.

RNA Extraction and real-time quantitative PCR
Total RNA was isolated from liver using RNAiso Plus (TaKaPa) and then reverse transcription reactions were performed using RT reagent kit (TaKaPa). All the procedures were carried out as described by the manufacturer's protocol. The quantity and quality of RNA were determined using NanoDrop ND-2000 spectrophotometer system (Thermo Fisher Scienti c). Real-time RT-PCR primers were designed to assay genes related to antioxidant function (supplemented Table 2). 18S were used as reference gene. Real-time RT-PCR for target genes were performed on MyIQ instrument (Bio-Rad, Hercules, California) using SYBR Green quantitative PCR mix (TaKaRa).

Analysis of mineral element contents in liver and muscle
Macro and micro elements (Ca, S, Mg, Cu, Fe, Mn, Zn) were analyzed using analytical method as described earlier [14]. Brie y, liver and muscle samples (5.00 ±0.20 g) were weighed in triplicate and digested using a mixture acid (25 mL of HNO3: HCLO4 in volume ratio of 4:1) following heating (80 C for 60 min; 120 C for 30 min; and 180 C for 30 min). Samples were dried at 260 C and redissolved in 5 mL of 1% HNO3. Solution was then transferred to a 25-mL volumetric ask and lled with 1% HNO3. Finally, target elements (Cu, Fe, Mn and Zn) in samples were determined on ICP-OES (Varian 720ES Agilent, Santa Clara, CA, USA) for con rmation with standard references.

Analysis of indole and skatole contents in chyme
Indole and skatole (3-methylindole) of luminal contents in cecal and colon were analysed as described previously on high-performance liquid chromatography (HPLC, Agilent 1260, Agilent Technologies Inc., USA) [15]. Brie y, luminal contents in cecal and colon were weighted (1.000 g) and extracted by 8 mL 0.4 mol/L perchloric acid overnight. Then the suspension was acquired by centrifuge 10 min at 8000 r/min at 4℃. 1 mL of supernatant was being homogenate with 1 mL sodium hydroxide (2 mol/l, pH 10.6)-saturated sodium bicarbonate buffer solution, then added 1 ml of dansulfonyl chloride (10 mg/ml acetone solution). The solution incubated at 40℃ water in a dark place for 30 min, and then add 1 mL 5% of ammonia water. Finally, 3 mL of anhydrous ether was used to extract the solution. The anhydrous ether layer was dried and dissolved in 1 mL methanol, ltered by 0.22 μm syringe lter membrane and analyzed on the machine.

Statistics analysis
Gene expression data from replicate measurement within the same RNA extraction were averaged and analyzed using Livak's (2001) [16] method to measure the difference between the ve DAR diets. All data were analyzed using the General Linear Model (GLM) procedure of SAS 8.0. Duncan differences were determined to compare differences among the groups. The variability in data was showed as standard error of mean (SEM), and the P < 0.05 was considered statistical signi cance. and free amino acids at day 15   Serum TP, ALB, ALT, AST, BUN, TG, AMS at day 15 were not affected by DAR (Table 1). Serum CREA was higher in diet 0.80 than in other groups (P < 0.05).

Serum biochemical variables
Serum GLU was higher in group 0.20 than in groups 0.00 and 0.40 (P < 0.05), with no signi cant difference compared with groups 0.60 and 0.80 (P > 0.05). Serum CHOL was higher in groups 0.00 and 0.60 than in group 0.40 (P < 0.05).
Absolute and relative organ weight Absolute weight of organ was not affected by DAR (Table 3, P>0.05). LPS stress caused increase in absolute weight of liver and decrease in large intestine (P<0.05). Absolute weight of kidney tended to increase after LPS stress (P=0.051). Relative organ weight such as stomach, liver, spleen, kidney and length of small intestine were not affected by DAR (P>0.05). Relative weight of small intestine increased after LPS stress in diet 0.20 and 0.80 but decreased in other groups (P<0.05). Relative weight of large intestine decreased after LPS stress (P<0.05).

Serum biochemical variables and free amino acids at day 29
Serum TP, ALB, ALT, AST, BUN, CREA, GLU, CHOL and IGM was not affect by DAR (Table 4). Serum TG was lower in diet 0.40 than in groups 0.60 and 0.80(P < 0.05). Serum AMS was lower in diets 0.00 than in diet 0.20, which lower than in diet 0.60 (P < 0.05). Serum IGG was lower in diet 0.00 than in diet 0.20, which was lower than in diet 0.40 and diet 0.80 had the highest IGG concentration (P < 0.05). Serum ALT, CREA, AMS and LIPC decreased but serum GLU, BUN, AST increased after LPS stress (P < 0.05). Serum IGG increased after LPS stress in diets 0.80 but decreased after LPS stress in other groups (P < 0.05).
Serum essential amino acid was not affected by DAR (P > 0.05) except that diet 0.80 had the highest serum arginine, higher than in groups 0.00, 0.20 and 0.40 (Table 5, P < 0.05). Serum non-essential amino acid was not affected by DAR (P > 0.05) except proline, sarcosine, citrulline, anserine, hydroxylysine, βaminoisobutyric acid and hydroxy-L-proline (P < 0.05). Serum proline was higher in diet 0.60 than in 0.80, 0.40 and 0.00 (P < 0.05). Piglets consumping diet with DAR 0.80 had the highest concentration of serum sarcosine, citrulline, anserine, hydroxylysine, b-aminoisobutyric acid. Serum hydroxy-L-proline was higher in diets 0.20 and 0.60 than in diet 0.00 (P < 0.05). Serum essential amino acid decreased after LPS stress (P < 0.05) except histidine which was not affected by LPS stress (P > 0.05). Serum tyrosine, alanine, aspartate, glutamate, cysteine, proline, urea, citrulline, carnosine, ornithine, a-Amino-n-butyric acid, Hydroxy-L-proline were affected by LPS stress (P < 0.05). There is signi cant effect of interaction between AAR and LPS on serum methionine, phenylalanine, valine, arginine, tyrosine, serine, hydroxy-L-proline (P < 0.05). Serum phenylalanine increased after LPS stress in diet 0.40 but decreased in other groups (P < 0.05). Serum valine decreased after LPS stress but maintained stable in other groups (P < 0.05). Serum arginine and tyrosine maintain stable in diet 0.00 after LPS stress but decreased in other groups (P < 0.05). Serum serine decreased after LPS stress in diets 0.20 and 0.80 but increased in other groups (P < 0.05). Serum hydorxy-L-proline increased in diets 0.00 and 0.60 after LPS stress but maintained stable in other groups (P < 0.05).

Amino acid contents in liver and dorsal muscle
Amino acid concentration of longissimus dorsi muscle was not affected by DAR and LPS stress ( Table 6, P > 0.05). Hepatic cysteine was lower in diet 0.80 than in diets 0.00 and 0.20 (P < 0.05). There was signi cant effect of interaction between DAR and LPS stress on hepatic isoleucine, leucine, phenylalanine, threonine, aspartate, serine, glutamate, glycine concentrations (P < 0.05). All these amino acids of liver decreased after LPS stress in diet 0.20 but increased in other groups (P < 0.05).

Antioxidant function of liver and muscle
Concentration of MDA in muscle was not affected by DAR or LPS stress (Table 7, P>0.05). Activity of T-AOC in muscle was higher in diets 0.60 and 0.80 than in diets 0.00, 0.20 and 0.40 (P < 0.05), it decreased after LPS stress in diet 0.40 but increased in other groups (P < 0.05). Activity of T-AOC in liver was not affected by DAR or LPS stress (P > 0.05). Activities of GSH-PX was higher in diet 0.20 than in diets 0.40, 0.60 and 0.80(P < 0.05), they increased after LPS stress (P < 0.05). Concentration of MDA was higher in diet 0.20 than in diets 0.00, 0.40 and 0.80(P < 0.05). Hepatic T-SOD activity decreased after LPS stress in diet 0.40 but increased in diets 0.00 and 0.80(P < 0.05). Activity of Cu-SOD was lower in diet 0.20 than in other groups (P < 0.05).
Gene related to antioxidant function such as SOD1, Nrf2, GPX1 mRNA expression was affected by DARA, which was higher in group 0.00 than in other groups (P < 0.05). LPS stress caused increased in abundance of gene SOD1 and GPX1(P < 0.05). mRNA expression of SOD1 and Nrf2 decreased after LPS stress in diets 0.40 and 0.60 but increased in other groups (P < 0.05). Gene of GCLC expression decreased after LPS stress in diets 0.40, 0.60, 0.80 but increased after LPS stress in diets 0.00 and 0.20(P < 0.05).

Mineral element contents in liver and muscle
All of the tested mineral element in muscle was not affected by LPS stress (Table 8, P > 0.05). Mineral element concentrations such as Ca, Fe, Mg, S were not affected by DAR (P > 0.05). Muscle from piglets in diet 0.80 had the lowest Cu and Zn concentrations compared with other groups (P < 0.05). Diet 0.80 tended to have the lowest Mn in muscle (P=0.052). Mn of muscle decreased after LPS stress in diets 0.20 and 0.60 but increased in other groups (P < 0.05).

Indole and skatole contents in chyme
Cecal indole concentration was higher in diet 0.00 than in diet 0.80 (P < 0.05), it was not affected by LPS stress (P > 0.05). Cecal skatole and colonic indole were not affected by DAR or LPS stress (P > 0.05). Diet 0.00 had the lowest colonic skatole than in diet 0.40, 0.60 and 0.80 (P < 0.05). Skatole in colon increased after LPS stress in diets 0.40 and 0.80 but decreased in other groups (P < 0.05).

Discussion
Oxidative stress is condition in which production of reactive oxygen species (ROS) exceeds the capacity of the antioxidant system, it caused tissue damage and dysfunction. Generally, the ROS including MDA which is a metabolite produced after lipid peroxidation [17]. Increases in indices of lipid peroxidation in rat were accompanied by reduced activity levels of liver GPX and CuZn-SOD. Serum AST activity is a key biochemical markers of liver health and function. LPS stress caused elevation of serum AST level, which could be considered to be a sensitive indicator of liver injury [18]. In order to overcome LPS stress, antioxidant activity represented by GSH-PX activity and gene expression of GPX1 and SOD1 increased in liver of weaned piglets. The SOD enzymes keep the intracellular steady-state concentration of O2low, while GPX remove the H2O2 generated from the dismutation [19]. Our results are inconsistent with [20], who summarized that the SOD1 and GPX1 activity of mice will down-regulate, and the expression levels of GCLC and Nrf2 were signi cantly decreased 2 h after 8 mg/kg LPS challenge [21]. This has to do with our sampling time. We sample 12h after LPS challenge, 12-hour recovery time was more effective in fully restored piglet liver function than 2 hours. 15 day after LPS stress, proteins involved in in ammatory response, oxidation-redox processes and defense reactions were enriched in a phagosome pathway in liver of weaning piglet [4].
Weaned piglets supplemented diet with DAR 0.40 showed weak response to LPS stress, T-SOD of liver and T-AOC concentration in muscle decreased after LPS stress in diet with DAR 0.40 but increased in group 0.80. Addition of resistant starch (RS) in the high fat diet will signi cantly increase the glutamate-cysteine ligase catalytic subunit (GCLC) gene expression, which involved in celluar antioxidant response to ROS, and then perform the better e ciently in suppressing stress by elevating T-AOC and GSH-PX levels [22]. Copper, zinc-superoxide dismutase (SOD1) is a major intracellular antioxidant enzyme in mammals, its activity is important for the catalysis of LPS-induced hepatic protein nitration in mice [23]. Eukaryotic cells have two forms of SOD: one is founded in the mitochondrial matrix (Mn-SOD) and another is founded in the cytosol (CuZn-SOD) [24]. Relative organ weight of intestine increased after LPS stress in diet 0.80, which indicates an increase in digestion and absorption. As a result of the hypertrophy of the cecal wall and of the cecal acidi cation, raw potato starch (RS2) fermentation increases intestinal absorption of Ca, Mg, Fe, Zn and Cu [25]. Hepatic Cu concentration and muscular Mn increased after LPS stress in diet 0.80 but not in other groups. Accordingly, SOD1 mRNA expression decreased after LPS stress in diet 0.40 but increased in diet 0.80. Indoxyl sulfate and Pcresole sulfate are among the major microbial derived pro-in ammatory and pro-oxidant uremic toxins. Production of indoxyl sulfate and P-cresole sulfate increased at higher pH [26]. By increasing production of SCFA, production of indoles of cecum decreased as a result of lowering the luminal pH in diet 0.80. Serum a-Amino-n-butyric acid was the highest in diet 0.80. Serum IGG decreased after LPS stress in other groups but increased in diet 0.80. Increase of immunoglobulin increases the body's resistance to in ammation, which could be bene t to antioxidant ability as oxidative stress and in ammation are inseparable linked as each trigger and ampli es the other.
Studies assumed that an increase RS would decrease the abundance of protein fermentation products [27,28]. After 24 h incubation of cecal and colonic digesta, production of ammonia-nitrogen and branched-chain fatty acids, which indicated protein fermentation, decreased when the corn resistant starch levels increased [27]. BCFA including isobutyrate and isovalerate concentrations signi cantly increased when the DAR was 0.20, suggesting that the DAR changed fermentation pattern and increased protein fermentation (data not shown). Protein fermentation products have been associated with toxic and proin ammatory impacts on the intestinal epithelium [29], which resulted in highest hepatic MDA concentration in diet 0.20. Normally, liver protein synthesis increased when animals undergoing immune challenge. Due to higher protein fermentation, hepatic isoleucine, leucine decreased after LPS stress in diet 0.20 because BCFA such as iso-butyrate, 2-methyl-butyrate, iso-valerate originate from deamination of valine, isoleucine and leucine. We should notice that serum serine decreased after LPS stress in diet 0.20 and 0.80 but increased in other groups. Serine participates in a metabolic network that interlinks the folate cycle and methionine cycle to support cell proliferation [30]. Serine is a precursor of glycine and cysteine that constitutes components of the cellular antioxidant glutathione (GSH). On the other side, hepatic amino acids such as phenylalanine, threonine, aspartate, serine, glutamate, glycine could be mobilized to build defences battle with MDA. Increase in the quantity of amino acid which enter the portal vein could be useful in improving tissue protein synthesis [31] . Higher hepatic cysteine in group 0.20 suggests that glutathione is in the process of synthesis in this group. Finally, it (diet 0.20) had highest GSH-PX activity in liver than in other groups. Gene expression of Nrf2 and glutamate-cysteine ligase catalytic subunit (GCLC) of liver increased after LPS stress in diet 0.00 and 0.20 but decreased in other groups. Nrf2 is the master regulator of close to 250 genes encoding antioxidant and cytoprotective enzymes and proteins [32].

Conclusions
In conclude, antioxidant ability of liver was affected by dietary amylose/amylopectin ratio when weaned piglets suffered LPS stress. More hepatic amino acids would be mobilized to synthesis glutathione peroxidase to defend LPS stress when weaned piglets supplemented diet with DAR 0.20. More Cu and Zn deposited in the liver and more Mn deposited in the muscle, and then antioxidant ability of liver and muscle increased after LPS stress when weaned piglets consuming diet with DAR0.80. Dietary amylose/amylopectin ratio (DAR) for most of the diet used in swine production is 0.40. Our study con rmed that diet with DAR 0.40 had disadvantageous when weaned piglets had experienced feed transition and then LPS stress. Supplemented amylose or amylopectin would be better for piglets under LPS stress through improving antioxidant function or amino acid metabolism.

Declarations
Ethics approval and consent to participate Experimental procedure in this study was reviewed and approved by the Animal Care and Use Committee of the Hunan international joint laboratory of animal intestinal ecology and health, Hunan Normal University.

Consent for publication
Not applicable Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.       Table 6 Effects of dietary amylose/amylopectin ratio on amino acid composition in liver and muscle of weaned piglets 1 . MDA: malonaldehyde; T-SOD: total superoxide dismutase; CuZnSOD: cooper-zinc; Mn-SOD: manganese SOD; GSH-PX: glutathione peroxidase; T-AOC: total an capacity; GCLC: glutamate-cysteine ligase catalytic subunit; Nrf2: nuclear factor, erythroid 2 like 2; SOD1: superoxide dismutase; GPX1: and glutathione perox Table 8 Effects of dietary amylose/amylopectin ratio on mineral element concentrations in liver and muscle of weaned piglets 1 .  Table 9 Effects of dietary amylose/amylopectin ratio on indole and skatole concentrations in cecal and colonic chyme of weaned piglets 1 .