Dietary dimethylglycine supplementation alleviates redox status imbalance

2.1. Experimental design and sample collection

This experiment was conducted on a trial pig farm that was owned by the Yangzhou Fangling Agricultural and Pastoral Co., Ltd. (Jiangsu, China). A total of 80 healthy pregnant multiparous sows (Landrace × Yorkshire) with similar expected farrowing dates (less than 3 d) and parity (the second or third) were preselected during gestation. The sows were fertilized by the pool of Duroc boars and fed the same gestating diet that met the National Research Council (NRC, 2012) nutrient requirements. 

At farrowing, 10 sows that had 11 to 13 live-born piglets and met the selection criteria for IUGR were chosen. In this study, 10 normal birth weight (NBW) newborn piglets (1.53 ± 0.04 kg) and 20 IUGR newborn piglets (0.76 ± 0.06 kg) were selected from 10 sows according to the method described by Wang et al. (2005), respectively. All newborn piglets were naturally weaned at 21 d of age, and one NBW weaned piglet and 2 IUGR weaned piglets were collected from each sow. They were allocated to 3 groups with 10 piglets per group. NBWC: NBW weaned piglets fed standard basal diets (Appendix Table 1); IUGRC: one IUGR weaned piglets fed standard basal diets; IUGRD: another IUGR weaned piglets from the same sow fed standard basal diets plus 0.1% DMG-Na (99.9% of purity, obtained from Qilu Sheng Hua Pharmaceutical Co., Ltd., Shandong, China). 

They were housed individually in plastic floored pens (1 m × 0.6 m) in an environmentally controlled room (temperature of 28 °C and free access to water). At 49 d of age, all the piglets were weighed after feed deprivation for 12 h to measure average daily weight gain (ADG), average daily feed intake (ADFI), and weight gain-to-feed intake (G:F) ratio to evaluate the feed conversion. 

The blood was withdrawn from the precaval vein, and the serum was separated by centrifugation at 3,500 × g for 15 min at a temperature of 4 °C, and then stored at a temperature of − 80 °C for further study. After that, the piglets were anesthetized via electrical stunning and sacrificed by exsanguination. Subsequently, the jejunum samples were harvested immediately, one (1 cm × 1 cm × 1 cm) sample embedded into 1% (vol/vol) glutaraldehyde solution and another (1 cm × 1 cm × 1 cm) sample embedded into 4% buffered formaldehyde for the morphological measurements, the rest sample storied at −80 °C for further study.

2.2. Evaluation of histomorphology of the jejunum

This assay was carried out according to the previous method described by Dong et al. (2014). Jejunum samples that fixed in 1% (vol/vol) glutaraldehyde solution were examined using a Philips 420 transmission electron microscope (Philips, Amsterdam, The Netherlands) at 80 kV.

The jejunum samples fixed in 4% buffered formaldehyde were dried using a graded series of xylene and ethanol and then embedded into paraffin for histological processing according to the method described before (Dong et al., 2014). Ten slides of the middle site of each sample were obtained, and the images were acquired. Villus length, villus width, and crypt depth were metered, and the villus area was calculated using the following formula:＋Villus area=π×(Villuswidth2)(Villuswidth2)2＋Villuslength2

2.3. Measurement of immunoglobulin in serum and jejunum

Serum immune globulin A (IgA), immune globulin G (IgG), and immune globulin M (IgM) concentrations were tested by corresponding ELISA assay kits bought from Nanjing Jiancheng Bioengineering Institute according to a previously described method (Lebacq-Verheyden et al., 1972).

Jejunum samples were homogenized in 0.9% sodium chloride buffer on ice and then centrifuged at 2,800 × g for 15 min at a temperature of 4 °C. The supernatant was used to measure the secreted immunoglobulin A (sIgA) concentration using an ELISA assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) (Lebacq-Verheyden et al., 1972).

2.4. Concentrations of jejunum inflammatory cytokine

The concentrations of the inflammatory cytokines in the jejunum, including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor α (TNF-α), were calculated by ELISA assay kits according to their manufacturer’s instructions (Elabscience Biotechnology Co. Ltd, Wuhan, China).

2.5. Concentrations of jejunum digestive enzyme

The jejunum samples were homogenized with a hand-held homogenizer in 1 mL of cold PBS (pH = 7.4, 0.01 mol/L). The homogenate was centrifuged at 500 × g for 10 min at a temperature of 4 °C, and the supernatants were collected. The activity of the digestive enzymes sucrase, maltase, lactase, amylase, lipase, and chymotrypsin were determined according to the manufacturer’s instructions of Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

2.6. Measurement of redox status

Jejunum samples were homogenized in 0.9% sodium chloride solution on ice and then centrifuged at 3,500 × g for 15 min at a temperature of 4 °C. Both serum and jejunum supernatant solution were used to measure the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), and malondialdehyde (MDA) according to the manufacturer’s instructions of corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein content was tested with a bicinchoninic acid (BCA) protein assay kit purchased from Nanjing Jiancheng Bioengineering Institute.

2.7. Measurement of jejunum mitochondrial redox status

Jejunum mitochondria was isolated using the mitochondria isolation kit (Solarbio, Beijing, China). The levels of protein content, manganese superoxide dismutase (MnSOD), GSH-Px, GSH, and γ-glutamylcysteine ligase (γ-GCL) were calculated according to the manufacturer’s instructions with the corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The ROS concentration was measured using a ROS assay kit bought from the Nanjing Jiancheng Institute of Bioengineering. Briefly, the mitochondria were incubated with 10 μmol/L of dichlorodihydrofluorescein diacetate (DCFH-DA) and 10 mmol/L of DNA stain Hoechst 33,342 at 37 °C for 30 min. Then the DCFH fluorescence of the mitochondria was measured at an emission wavelength of 500 nm and an excitation wavelength of 525 nm with a fluorescence reader (SpectraMax Gemini EM; Molecular Devices, Franklin Lakes, NJ, USA). The results were expressed as the mean DCFH-DA fluorescence intensity over that of the control. 

The mitochondrial membrane potential (MMP) level was calculated using an MMP assay kit bought from the Nanjing Jiancheng Institute of Bioengineering. Briefly, the mitochondria were loaded with 1 × JC-1 dye at 37 °C for 20 min, and then analyzed, after washing, by a fluorescence microscope. The MMP was calculated as the increase in ratio of green and red fluorescence. The results were calculated as the ratio of the fluorescence of aggregates (red) to that of the monomers (green). The number of apoptotic cells and necrotic cells was tested by an Alexa Fluor 488 Annexin V/dead Cell Apoptosis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and carried out according to their manufacturer’s instructions. Protein carbonyls (PC) and 8-hydroxy-2-deoxyguanosine (8-OHdG) in the jejunum sample were calculated using their respective ELISA assay kits and carried out following the manufacturer’s instructions (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China) (Berlett and Stadtman, 1997; Valavanidis et al., 2009; Bai et al., 2011).

The adenosine triphosphate (ATP) content in the jejunum sample was determined using an ATP Assay Kit (Solarbio, Beijing, China) following the manufacturer’s instructions. The mitochondrial DNA (mtDNA) copy number of the jejunum sample was determined using a real-time fluorescence quantitative polymerase chain reaction (PCR) kit (Tli RNaseH Plus; Takara Bio, Inc., Otsu, Japan). In brief, the 20 μL PCR mixture consisted of 10 μL of SYBR Premix Ex Taq (2 × ), 0.4 μL of upstream primer, 0.4 μL of downstream primer, 0.4 μL of ROX dye (50 × ), 6.8 μL of ultra-pure water, and 2 μL of cDNA template. 

The sequence of the Mt D-loop gene upstream primer was 5′-AGGACTACGGCTTGAAAAGC-3′, and that of the downstream primer was 5′-CATCTTGGCATCTTCAGTGCC-3′; the length of the target fragment was 198 bp. The sequence of the β-actin upstream primer was 5′-TTCTTGGGTATGGAGTCCTG-3′ and that of the downstream primer was 5′-TAGAAGCATTTGCGGTGG-3′; the length of the target fragment was 150 bp. The amplification of each jejunum sample was performed in triplicate. The fold-expression of each gene was calculated according to the 2^-ΔΔCt method (Mohamed et al., 2010), in which β-actin was used as an internal standard.

2.8. Quantitative real-time PCR analysis

Quantitative real-time PCR (qPCR) was conducted according to the method described before (Mohamed et al., 2010). Total RNA from each jejunum samples was extracted using a Trizol Reagent kit (TaKaRa, Dalian, China) and then reverse-transcribed with a kit (Perfect Real Time, SYBR PrimeScript, TaKaRa) following the manufacturer’s instructions. 

The mRNA expression levels of genes listed in Appendix Table 2 were quantified via real-time qPCR using SYBR Premix Ex Taq II (Tli RNaseH Plus, TaKaRa) with an ABI 7300 Fast Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA). The β-actin was acted as an internal standard. The qRT-PCR amplification efficiency was calculated according to specific gene standard curves generated from serial dilutions. The gene expression levels of the target genes were analyzed by the 2^−ΔΔCt method (Mohamed et al., 2010) after verifying that the primers were amplified with an efficiency of approximately 100%, and data for IUGRC and IUGRD groups were compared with the data for the NBWC group.

2.9. Western blot analysis

Total protein was isolated from the 3 jejunum samples per group with a radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail (Beyotime Institute of Biotechnology, Jiangsu, China). The nuclear protein in the jejunum samples was carried out using a Nuclear Protein Extraction Kit (Beyotime Institute of Biotechnology, Jiangsu, China). The concentrations of total cellular protein and nuclear protein in the jejunum samples were measured by the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). 

Antibodies against related proteins were all purchased from Cell Signaling Technology (Danvers, MA, USA). Thereafter, equal quantities of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene difluoride membranes. After that, the membranes were incubated with blocking buffer (5% bovine serum albumin in Tris-buffered saline containing 1% Tween 20) for 1 h at room temperature and probed with the primary antibody (1:1,000) against nuclear factor erythroid 2-related factor 2 (Nrf2, # 12721 S), heme oxygenase 1 (HO1, # 82206 S), superoxide dismutase (SOD, # 37385 S), glutathione peroxidase (GSH-Px, # 3286 S), Sirt1 (# 9475 S), PGC1α (# 2178 S), cytochrome C (Cyt C, # 11940S), estrogen-related receptor α (ERRα, # 13826 S), mitochondrial transcription factor A (mtTFA, # 8076 S), nuclear respiratory factor 1 (NRF1, # 46743 S), mitochondrial mitofusin 2 (Mfn2, # 9482 S), dynamin-related protein 1 (Drp1, # 8570 S), mitochondrial fission 1 (Fis1, # 84580 S), Tubulin (# 2125 S) overnight at 4 °C. 

Then, the membranes were washed by Tris-buffered saline with 0.05% Tween-20 and incubated with a suitable secondary antibody (goat anti-rabbit IgG, 1:2,000, # 7074 S) for 1 h at room temperature. Finally, the blots were detected using enhanced chemiluminescence reagents (ECL-Kit, Beyotime, Jiangsu, China), followed by autoradiography. Photographs of the membranes were taken using the Luminescent Image Analyzer LAS-4000 system (Fujifilm Co.) and quantified with ImageJ 1.42 q software (NIH, Bethesda, MD, USA).

2.10. Statistical analysis

Data are shown as the mean ± SEM and were analyzed by a one-way analysis of variance procedure using SAS (version 9.1; SAS Institute, Inc., Cary, NC, USA). This was followed by Tukey’s test when significant differences were expressed. Statistical significance was considered at P < 0.05.

3.1. Growth performance

The piglets in the IUGRC group showed lower (P < 0.05) initial body weight (IBW), final body weight (FBW), ADG, and ADFI in comparison with the NBWC group. The FBW, ADG, and G:F ratio were improved (P < 0.05) in the piglets in the IUGRD group than those in the IUGRC group (Table 1).

Table 1. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the growth performance in intrauterine growth restricted (IUGR) weaned piglets ¹.

ADG = average daily weight gain; ADFI = average daily feed intake; IBW = initial body weight; FBW = final body weight; G:F ratio = weight gain-to-feed intake ratio.

^{a, b, c} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

3.2. Evaluation of histomorphology of the jejunum

The jejunum microvilli in the IUGRC group were shorter and less frequent than those in the NBWC group. In addition, autophagosomes and mitochondrial swelling were observed in the IUGRC group but not in the NBWC group (Fig. 1A). Compared with the NBWC group, the jejunum villi in IUGRC group were shorter and of different lengths (Fig. 1B). The IUGRD group’s jejunum villi, internal microvilli, and internal structure were improved relative to those in the IUGRC group (Fig. 1A and B).

1. Download : Download high-res image (2MB)
2. Download : Download full-size image

Fig. 1. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the jejunum microvilli integrity and their structure in intrauterine growth restricted (IUGR) weaned piglets. (A) Jejunum mitochondrial swelling and microvilli. Scale bars represent 1 μm; (B) jejunum histological morphology (villus length, villus width, crypt depth, and villus area). Scale bars represent 100 μm, n = 10 piglets per group. NBWC, NBW weaned piglets fed standard basal diets; IUGRC, one IUGR weaned piglets fed standard basal diets; IUGRD, another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

Histomorphological parameters (villus length, villus width, crypt depth, and villus area) of the jejunum were lower (P < 0.05) in the IUGRC group than those in the NBWC group. Compared with the IUGRC group, the IUGRD group revealed improvement (P < 0.05) in the histomorphological parameters of the jejunum (Table 2).

Table 2. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the jejunum villi length in intrauterine growth restricted (IUGR) weaned piglets ¹.

^{a, b, c} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

3.3. Concentrations of jejunum inflammatory cytokines

The IUGRC group revealed a decrease (P < 0.05) of serum IgA, IgG, IgM, and jejunum sIgA levels compared with the NBWC group. Serum IgA, IgG, IgM, and jejunum sIgA levels were all improved (P < 0.05) in the IUGRD group compared to these in the IUGRC group (Table 3).

Table 3. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the serum immunoglobulin and jejunum sIgA levels in intrauterine growth restricted (IUGR) weaned piglets ¹.

IgA = immune globulin A; IgG = immune globulin G; IgM = immune globulin M; sIgA = secreted immunoglobulin A.

^{a, b} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

3.4. Concentrations of jejunum inflammatory cytokine

Increased (P < 0.05) concentrations of IL-1β, IL-6, and TNF-α, and decreased (P < 0.05) concentrations of IL-8 were observed in group IUGRC compared with those in group NBWC. The IUGRD group showed an improvement (P < 0.05) in IL-1β, IL-6, IL-8, and TNF-α levels in comparison with those in the IUGRC group (Table 4).

Table 4. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the concentrations of jejunum inflammatory cytokines in intrauterine growth restricted (IUGR) weaned piglets (ng/g protein) ¹.

IL-1β = interleukin 1β; IL-6 = interleukin 6; IL-8 = interleukin 8; TNF-α = tumor necrosis factor α.

^{a, b} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

3.5. Concentrations of digestive enzyme

Intestinal digestive enzyme (amylase, lipase, trypsin, maltase, and lactase) activity of the jejunum showed a decrease (P < 0.05) in the IUGRC group compared with those in the NBWC group. The IUGRD group showed increased (P < 0.05) activity of the jejunum digestive enzymes (amylase, lipase, trypsin, maltase, and lactase) compared with that in the IUGRC group (Table 5).

Table 5. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the digestive enzyme activity in intrauterine growth restricted (IUGR) weaned piglets (U/mg protein) ¹.

^{a, b} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

3.6. Measurement of redox status

The IUGRC group showed a lower (P < 0.05) antioxidant enzymes activity (SOD, GSH-Px, GSH, GR, and CAT) and higher (P < 0.05) MDA content in the serum (Table 6) and jejunum (Table 7) compared with those in the NBWC group. The IUGRD group revealed an increased (P < 0.05) antioxidant enzymes activity (SOD, GSH-Px, GSH, GR, and CAT) and decreased (P < 0.05) MDA content in the serum (Table 6) and jejunum (Table 7) compared with these in the IUGRC group.

Table 6. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the serum redox status in intrauterine growth restricted (IUGR) weaned piglets ¹.

SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; GSH = glutathione; GR = glutathione reductase; CAT = catalase; MDA = methane dicarboxylic aldehyde.

^{a, b} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

Table 7. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the jejunum redox status in intrauterine growth restricted (IUGR) weaned piglets ¹.

SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; GSH = glutathione; GR = glutathione reductase; CAT = catalase; MDA = methane dicarboxylic aldehyde.

^{a, b} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

3.7. Measurement of mitochondrial redox status

The IUGRC group showed a lower (P < 0.05) level of jejunum mitochondrial MnSOD, GSH-Px, GSH, GR, and γ-GCL activity relative to their levels in the NBWC group. The IUGRD group showed higher (P < 0.05) levels of jejunum mitochondrial MnSOD, GSH-Px, GSH, GR, and γ-GCL activity in comparison with the IUGRC group (Table 8).

Table 8. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the jejunum mitochondrial redox status in intrauterine growth restricted (IUGR) weaned piglets ¹.

MnSOD = manganese superoxide dismutase; GSH-Px = glutathione peroxidase; GSH = glutathione; GR = glutathione reductase; γ-GCL = γ-glutamylcysteine ligase.

^{a, b} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

Compared with the NBWC group, higher (P < 0.05) levels of ROS, PC, 8-OHdG, apoptosis cells, and necrosis cells, and lower (P < 0.05) levels of MMP, ATP, and mtDNA were shown in group IUGRC. Decreased (P < 0.05) levels of ROS, PC, 8-OHdG, apoptosis cells, and necrosis cells, and increased (P < 0.05) levels of MMP, ATP, and mtDNA were revealed in the IUGRD group than those in the IUGRC group (Table 9).

Table 9. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the jejunum oxidative damage in intrauterine growth restricted (IUGR) weaned piglets ¹.

ROS = reactive oxygen species; PC = protein carbonyls; 8-OHdG = 8-hydroxy-2- deoxyguanosine; MMP = mitochondrial membrane potential; ATP = adenosine triphosphate; mtDNA = mitochondria DNA.

^{a, b} Within a row, means with different superscripts were significantly different (P < 0.05).

1

Data are shown as mean ± standard error of mean, n = 10 piglets per group.

2

NBWC = normal birth weight weaned piglets fed standard basal diets.

3

IUGRC = one IUGR weaned piglets fed standard basal diets.

4

IUGRD = another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na.

3.8. Quantitative real-time PCR analysis

The redox status-related gene expression (Nrf2, HO1, Cu/ZnSOD, GSH-Px, MnSOD, γ-glutamylcysteine ligase c [γ-GCLc], γ-glutamylcysteine ligase m [γ-GCLm], thioredoxin 2 [Trx2], thioredoxin reductase 2 [Trx-R2], peroxiredoxin 3 [Prx3], Sirt1, PGC1α), cell adhesion-related (occluding [OCLN], cloudin2 [CLDN2], cloudin3 [CLDN3], zonula occludens-1 [ZO1]) genes expression (Fig. 2A), and mitochondrial function-related gene expression (lipid oxidation enzymes malonyl-CoA decarboxylase [MCD], medium-chain acyl-CoA dehydrogenase [MCAD], mitochondrial proteins succinate dehydrogenase [SDH], uncoupling protein 2 [UCP2], cyclooxygenase 2 [COX2], citrate synthase [CS], cyclooxygenase 1 [COX1], Cyt C, ERRα, major histocompatibility complex I [MHC1], mtTFA, NADH dehydrogenase (ubiquinone) iron-sulfur protein 2 [Ndufa2], NRF1, uncoupling protein 1 [UCP1], γ DNA polymerases catalytic subunit [POLG1], γ DNA polymerases accessory subunit [POLG2], single-strand DNA binding protein 1 [SSBP1], Drp1, Fis1, Mfn2) (Fig. 2B and C) levels of the jejunum deteriorated (P < 0.05) in IUGRC group relative to the NBWC group. Compared with the IUGRC group, redox status-related gene expression, cell adhesion-related gene expression, and mitochondrial function-related gene expression levels of the jejunum were improved (P < 0.05) in IUGRD group (Fig. 2A–C).

1. Download : Download high-res image (453KB)
2. Download : Download full-size image

Fig. 2. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the jejunum redox status-related and mitochondrial function-related gene expression in intrauterine growth restricted (IUGR) weaned piglets. Data are shown as mean ± standard error of mean, n = 10 piglets per group. Data on bars with different letters (a, b) were significantly different (P < 0.05). All the experiment was repeated 3 times. NBWC, normal birth weight weaned piglets fed standard basal diets; IUGRC, one IUGR weaned piglets fed standard basal diets; IUGRD, another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na. Nrf2 = nuclear factor erythroid 2-related factor 2; HO1 = heme oxygenase 1; SOD = copper and zinc superoxide dismutase; GSH-Px = glutathione peroxidase; MnSOD = manganese superoxide dismutase; γ-GCLc = γ-glutamylcysteine ligase c; γ-GCLm = γ-glutamylcysteine ligase m; Trx2 = thioredoxin 2; Trx-R2 = thioredoxin reductase 2; Prx3 = peroxiredoxin 3; Sirt1 = sirtuin 1; PGC1α = peroxisome proliferator-activated receptorγcoactivator-1α; OCLN = occluding; CLDN2 = cloudin2; CLDN3 = cloudin3; ZO1 = zonula occludens-1; MCD = lipid oxidation enzymes malonyl-CoA decarboxylase; MCAD = medium-chain acyl-CoA dehydrogenase; SDH = mitochondrial proteins succinate dehydrogenase; UCP2 = uncoupling protein 2; COX2 = cyclooxygenase 2; CS = citrate synthase; COX1 = cyclooxygenase 1; Cyt C = Cytochrome C; ERRα = estrogen-related receptor a; MHC1 = major histocompatibility complex I; mtTFA = mitochondrial transcription factor A; Ndufa2 = NADH dehydrogenase (ubiquinone) iron-sulfur protein 2; NRF1 = nuclear respiratory factor 1; UCP1 = uncoupling protein 1; POLG1 = γ DNA polymerases catalytic subunit; POLG2 = γ DNA polymerases accessory subunit; SSBP1 = single-strand DNA binding protein 1; Drp1 = dynamin-related protein 1; Fis1 = mitochondrial fission 1; Mfn2 = mitochondrial mitofusin 2.

3.9. Western blot analysis

Compared with the NBWC group, the IUGRC group showed lower (P < 0.05) protein levels of SOD, GSH-Px, Sirt1, PGC1α, Cyt C, ERRα, mtTFA, NRF1, Mfn2, Drp1, and Fis1, along with higher (P < 0.05) protein levels of Nrf2 and HO1 (Fig. 3A and B). The IUGRD group presented higher (P < 0.05) protein levels of SOD, GSH-Px, Sirt1, Cyt C, ERRα, mtTFA, NRF1, Mfn2, Drp1, and Fis1, and lower (P < 0.05) protein levels of Nrf2 and HO1 compared with IUGRC group (Fig. 3A and B).

1. Download : Download high-res image (762KB)
2. Download : Download full-size image

Fig. 3. Dietary dimethylglycine sodium salt (DMG-Na) supplementation improves the jejunum redox status-related and mitochondrial function-related protein expression in intrauterine growth restricted (IUGR) weaned piglets. Data are shown as mean ± standard error of mean n = 3 piglets per group. Data on bars with different letters (a, b) were significantly different (P < 0.05). All the experiment was repeated 3 times. NBWC, normal birth weight weaned piglets fed standard basal diets; IUGRC, one IUGR weaned piglets fed standard basal diets; IUGRD, another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na. Nrf2 = nuclear factor erythroid 2-related factor 2; HO1 = heme oxygenase 1; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; Sirt1 = sirtuin 1; PGC1α = peroxisome proliferator-activated receptorγcoactivator-1α; Cyt C = Cytochrome C; ERRα = estrogen-related receptor α; mtTFA = mitochondrial transcription factor A; NRF1 = nuclear respiratory factor 1; Mfn2 = mitochondrial mitofusin2; Drp1 = dynamin-related protein 1; Fis1 = mitochondrial fission 1.

Bai et al., 2019

K. Bai, L. Jiang, S. Zhu, C. Feng, Y. Zhao, L. Zhang, et al.

Dimethylglycine sodium salt protects against oxidative damage and mitochondrial dysfunction in the small intestines of miceInt J Mol Med, 43 (2019), pp. 2199-2211 

View Record in ScopusGoogle Scholar

Bai et al., 2016

K. Bai, W. Xu, J. Zhang, T. Kou, Y. Niu, X. Wan, et al.

Assessment of free radical scavenging activity of dimethylglycine sodium salt and its role in providing protection against lipopolysaccharide-induced oxidative stress in micePLoS One, 12 (11) (2016), p. e0155393 View PDF

CrossRefView Record in ScopusGoogle Scholar

Bai et al., 2011

P. Bai, C. Cantó, H. Oudart, A. Brunyánszki, Y. Cen, C. Thomas, et al.

Houtkooper. PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activationCell Metabol, 6 (13) (2011), pp. 461-468ArticleDownload PDFView Record in ScopusGoogle Scholar

Bandyopadhyay, 1999

U. Bandyopadhyay

Reactive oxygen species: oxidative damage pathogenesisCurr Sci, 5 (1999), pp. 658-666Google Scholar

Bartel, 2004

D.P. Bartel

MicroRNAs: genomics, biogenesis, mechanism, and functionCell, 23 (116) (2004), pp. 281-297ArticleDownload PDFGoogle Scholar

Baserga et al., 2004

M. Baserga, C. Bertolotto, N.K. Maclennan, J.L. Hsu, T. Pham, G.S. Laksana, et al.

Uteroplacental insufficiency decreases small intestine growth and alters apoptotic homeostasis in term intrauterine growth retarded ratsEarly Hum Dev, 79 (2004), pp. 93-105ArticleDownload PDFView Record in ScopusGoogle Scholar

Bayrak et al., 2008

O. Bayrak, E. Uz, R. Bayrak, F. Turgut, A.F. Atmaca, S. Sahin, et al.

Curcumin protects against ischemia/reperfusion injury in rat kidneysWorld J Urol, 26 (2008), pp. 285-291 View PDF

CrossRefView Record in ScopusGoogle Scholar

Berlett and Stadtman, 1997

B.S. Berlett, E.R. Stadtman

Protein oxidation in aging, disease, and oxidative stressJ Biol Chem, 15 (272) (1997), pp. 20313-20316ArticleDownload PDFView Record in ScopusGoogle Scholar

Bernstein et al., 2000

I.M. Bernstein, J.D. Horbar, G.J. Badger, A. Ohlsson, A. Golan

Morbidity and mortality among very-low-birth-weight neonates with intrauterine growth restrictionAm J Obstet Gynecol, 182 (1 Pt 1) (2000), pp. 198-206ArticleDownload PDFView Record in ScopusGoogle Scholar

Biri et al., 2007

A. Biri, N. Bozkurt, A. Turp, M. Kavutcu, O. Himmetoglu, I. Durak

Role of oxidative stress in intrauterine growth restrictionGynecol Obstet Invest, 64 (2007), pp. 187-192 View PDF

CrossRefView Record in ScopusGoogle Scholar

Brandtzaeg, 2002

P.E. Brandtzaeg

Current understanding of gastrointestinal immunoregulation and its relation to food allergyAnn N Y Acad Sci, 964 (2002), pp. 13-45 

View Record in ScopusGoogle Scholar

Cheng et al., 2020

K. Cheng, T. Wang, S. Li, Z. Song, H. Zhang, L. Zhang, et al.

Effects of early resveratrol intervention on skeletal muscle mitochondrial function and redox status in neonatal piglets with or without intrauterine growth retardationOxid Med Cell Longev, 22 (2020) (2020), p. 4858975 

View Record in ScopusGoogle Scholar

Chiu et al., 2002

H. Chiu, J.A. Brittingham, D.L. Laskin

Differential induction of heme oxygenase-1 in macrophages and hepatocytes during acetaminophen-induced hepatotoxicity in the rat: effects of hemin and biliverdinToxicol Appl Pharmacol, 1 (181) (2002), pp. 106-115ArticleDownload PDFView Record in ScopusGoogle Scholar

D’Inca et al., 2011

R. D’Inca, C. Gras-Le Guen, L. Che, P.T. Sangild, I. Le Huërou-Luron

Intrauterine growth restriction delays feeding-induced gut adaptation in term newborn pigsNeonatology, 99 (2011), pp. 208-216 View PDF

CrossRefView Record in ScopusGoogle Scholar

Dillin et al., 2002

A. Dillin, A.L. Hsu, N. Arantes-Oliveira, J. Lehrer-Graiwer, H. Hsin, A.G. Fraser, et al.

Rates of behavior and aging specified by mitochondrial function during developmentScience, 20 (298) (2002), pp. 2398-2401 

View Record in ScopusGoogle Scholar

Dong et al., 2014

L. Dong, X. Zhong, H. Ahmad, W. Li, Y. Wang, L. Zhang, et al.

Intrauterine growth restriction impairs small intestinal mucosal immunity in neonatal pigletsJ Histochem Cytochem, 62 (2014), pp. 510-518 View PDF

CrossRefView Record in ScopusGoogle Scholar

Feng et al., 2018

C. Feng, K. Bai, A. Wang, X. Ge, Y. Zhao, L. Zhang, et al.

Effects of dimethylglycine sodium salt supplementation on growth performance, hepatic antioxidant capacity, and mitochondria-related gene expression in weanling piglets born with low birth weightJ Anim Sci, 7 (96) (2018), pp. 3791-3803 View PDF

CrossRefView Record in ScopusGoogle Scholar

Friesen et al., 2007

R.W. Friesen, E.M. Novak, D. Hasman, S.M. Innis

Relationship of dimethylglycine, choline, and betaine with oxoproline in plasma of pregnant women and their newborn infantsJ Nutr, 137 (2007), pp. 2641-2646 View PDF

CrossRefView Record in ScopusGoogle Scholar

Hariganesh and Prathiba, 2000

K. Hariganesh, J. Prathiba

Effect of dimethylglycine on gastric ulcers in ratsJ Pharm Pharmacol, 52 (2000), pp. 1519-1522 

View Record in ScopusGoogle Scholar

He et al., 2011

Q. He, P. Ren, X. Kong, W. Xu, H. Tang, Y. Yin, et al.

Intrauterine growth restriction alters the metabonome of the serum and jejunum in pigletsMol Biosyst, 7 (2011), pp. 2147-2155 View PDF

CrossRefView Record in ScopusGoogle Scholar

Hracsko et al., 2007

Z. Hracsko, Z. Safar, H. Orvos, Z. Novak, A. Pal, I.S. Varga

Evaluation of oxidative stress markers after vaginal delivery or caesarean sectionVivo, 21 (2007), pp. 703-706 

View Record in ScopusGoogle Scholar

Huang et al., 2019

S. Huang, N. Li, C. Liu, T. Li, W. Wang, L. Jiang, et al.

Characteristics of the gut microbiota colonization, inflammatory profile, and plasma metabolome in intrauterine growth restricted piglets during the first 12 hours after birthJ Microbiol, 57 (2019), pp. 748-758 View PDF

CrossRefView Record in ScopusGoogle Scholar

Hwang et al., 2013

J.W. Hwang, H. Yao, S. Caito, I.K. Sundar, I. Rahman

Redox regulation of SIRT1 in inflammation and cellular senescenceFree Radic Biol Med, 61 (2013), pp. 95-110ArticleDownload PDFView Record in ScopusGoogle Scholar

Jozawa et al., 2019

H. Jozawa, A. Inoue-Yamauchi, S. Arimura, Y. Yamanashi

Loss of C/EBPδ enhances apoptosis of intestinal epithelial cells and exacerbates experimental colitis in miceGene Cell, 24 (2019), pp. 619-626 View PDF

CrossRefView Record in ScopusGoogle Scholar

Kowaltowski and Vercesi, 1999

A.J. Kowaltowski, A.E. Vercesi

Mitochondrial damage induced by conditions of oxidative stressFree Radic Biol Med, 26 (1999), pp. 463-471ArticleDownload PDFView Record in ScopusGoogle Scholar

Langston et al., 2011

J.W. Langston, W. Li, L. Harrison, T.Y. Aw

Activation of promoter activity of the catalytic subunit of γ-glutamylcysteine ligase (GCL) in brain endothelial cells by insulin requires antioxidant response element 4 and altered glycemic status: implication for GCL expression and GSH synthesisFree Radic Biol Med, 1 (51) (2011), pp. 1749-1757ArticleDownload PDFView Record in ScopusGoogle Scholar

Lebacq-Verheyden et al., 1972

A.M. Lebacq-Verheyden, J.P. Vaerman, J.F. Heremans

A possible homologue of mammalian IgA in chicken serum and secretionsImmunology, 22 (1972), pp. 165-175 

View Record in ScopusGoogle Scholar

Li et al., 2021

T. Li, S. Huang, L. Lei, S. Tao, Y. Xiong, G. Wu, et al.

Intrauterine growth restriction alters nutrient metabolism in the intestine of porcine offspringJ Anim Sci Biotechnol, 8 (12) (2021), p. 15 View PDF

CrossRefGoogle Scholar

Lim et al., 2005

L.P. Lim, N.C. Lau, P. Garrett-Engele, A. Grimson, J.M. Schelter, J. Castle, et al.

Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAsNature, 17 (433) (2005), pp. 769-773 View PDF

CrossRefView Record in ScopusGoogle Scholar

Lin et al., 2005

J. Lin, C. Handschin, B.M. Spiegelman

Metabolic control through the PGC-1 family of transcription coactivatorsCell Metabol, 1 (2005), pp. 361-370ArticleDownload PDFView Record in ScopusGoogle Scholar

Look et al., 2001

M.P. Look, R. Riezler, H.K. Berthold, S.P. Stabler, K. Schliefer, R.H. Allen, et al.

Decrease of elevated N,N-dimethylglycine and N-methylglycine in human immunodeficiency virus infection during short-term highly active antiretroviral therapyMetabolism, 50 (2001), pp. 1275-1281ArticleDownload PDFView Record in ScopusGoogle Scholar

Michelet et al., 2006

L. Michelet, M. Zaffagnini, V. Massot, E. Keryer, H. Vanacker, M. Miginiac-Maslow, et al.

Thioredoxins, glutaredoxins, and glutathionylation: new crosstalks to explorePhotosynth Res, 89 (2006), pp. 225-245 View PDF

CrossRefView Record in ScopusGoogle Scholar

Minamikawa et al., 1999

T. Minamikawa, D.A. Williams, D.N. Bowser, P. Nagley

Mitochondrial permeability transition and swelling can occur reversibly without inducing cell death in intact human cellsExp Cell Res, 10 (246) (1999), pp. 26-37ArticleDownload PDFView Record in ScopusGoogle Scholar

Mizushima, 2007

N. Mizushima

Autophagy: process and functionGenes Dev, 21 (2007), pp. 2861-2873 View PDF

CrossRefView Record in ScopusGoogle Scholar

Mohamed et al., 2010

J.S. Mohamed, M.A. Lopez, A.M. Boriek

Mechanical stretch up-regulates microRNA-26a and induces human airway smooth muscle hypertrophy by suppressing glycogen synthase kinase-3βJ Biol Chem, 17 (285) (2010), pp. 29336-29347ArticleDownload PDFView Record in ScopusGoogle Scholar

Mohamed et al., 2014

J.S. Mohamed, A. Hajira, P.S. Pardo, A.M. Boriek

MicroRNA-149 inhibits PARP-2 and promotes mitochondrial biogenesis via SIRT-1/PGC-1α network in skeletal muscleDiabetes, 63 (2014), pp. 1546-1559 View PDF

CrossRefView Record in ScopusGoogle Scholar

National Research Council, 2012

National Research Council

Nutrient requirements of swine(11th ed.), National Academy Press, Washington, DC (2012)Google Scholar

Nusrat et al., 2000

A. Nusrat, C.A. Parkos, P. Verkade, C.S. Foley, T.W. Liang, W. Innis-Whitehouse, et al.

Tight junctions are membrane microdomainsJ Cell Sci, 113 (2000), pp. 1771-1781 View PDF

CrossRefView Record in ScopusGoogle Scholar

Park et al., 2004

H.K. Park, C.J. Jin, Y.M. Cho, D.J. Park, C.S. Shin, K.S. Park, et al.

Changes of mitochondrial DNA content in the male offspring of protein-malnourished ratsAnn N Y Acad Sci, 1011 (2004), pp. 205-216 

View Record in ScopusGoogle Scholar

Sastre et al., 2000

J. Sastre, F.V. Pallardó, J. Viña

Mitochondrial oxidative stress plays a key role in aging and apoptosisIUBMB Life, 49 (2000), pp. 427-435 

View Record in ScopusGoogle Scholar

Simmons, 2012

R.A. Simmons

Developmental origins of diabetes: the role of oxidative stressBest Pract Res Clin Endocrinol Metabol, 26 (2012), pp. 701-708ArticleDownload PDFView Record in ScopusGoogle Scholar

Sinclair, 2005

D.A. Sinclair

Toward a unified theory of caloric restriction and longevity regulationMech Ageing Dev, 126 (2005), pp. 987-1002ArticleDownload PDFView Record in ScopusGoogle Scholar

St-Pierre et al., 2006

J. St-Pierre, S. Drori, M. Uldry, J.M. Silvaggi, J. Rhee, S. Jäger, et al.

Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivatorsCell, 20 (127) (2006), pp. 397-408ArticleDownload PDFView Record in ScopusGoogle Scholar

Sun and Zemel, 2009

X. Sun, M.B. Zemel

Leucine modulation of mitochondrial mass and oxygen consumption in skeletal muscle cells and adipocytesNutr Metab (Lond), 5 (6) (2009), p. 26 View PDF

CrossRefGoogle Scholar

Tang et al., 2012

Y. Tang, C. Gao, M. Xing, Y. Li, L. Zhu, D. Wang, et al.

Quercetin prevents ethanol-induced dyslipidemia and mitochondrial oxidative damageFood Chem Toxicol, 50 (2012), pp. 1194-1200ArticleDownload PDFView Record in ScopusGoogle Scholar

Valavanidis et al., 2009

A. Valavanidis, T. Vlachogianni, C. Fiotakis

8-hydroxy-2′ -deoxyguanosine (8-OHdG): a critical biomarker of oxidative stress and carcinogenesisJ Environ Sci Health C Environ Carcinog Ecotoxicol Rev, 27 (2009), pp. 120-139 View PDF

CrossRefGoogle Scholar

Wang et al., 2008

J. Wang, L. Chen, D. Li, Y. Yin, X. Wang, P. Li, et al.

Intrauterine growth restriction affects the proteomes of the small intestine, liver, and skeletal muscle in newborn pigsJ Nutr, 138 (2008), pp. 60-66Google Scholar

Wang et al., 2005

T. Wang, Y.J. Huo, F. Shi, R.J. Xu, R.J. Hutz

Effects of intrauterine growth retardation on development of the gastrointestinal tract in neonatal pigsBiol Neonate, 88 (2005), pp. 66-72 View PDF

CrossRefView Record in ScopusGoogle Scholar

Wang et al., 2012

Y. Wang, L. Zhang, G. Zhou, Z. Liao, H. Ahmad, W. Liu, et al.

Dietary L-arginine supplementation improves the intestinal development through increasing mucosal Akt and mammalian target of rapamycin signals in intra-uterine growth retarded pigletsBr J Nutr, 28 (108) (2012), pp. 1371-1381 

View Record in ScopusGoogle Scholar

Xu et al., 2016

W. Xu, K. Bai, J. He, W. Su, L. Dong, L. Zhang, T. Wang

Leucine improves growth performance of intrauterine growth retardation piglets by modifying gene and protein expression related to protein synthesisNutrition, 32 (2016), pp. 114-1121 View PDF

CrossRefGoogle Scholar

Zhang et al., 2014

H. Zhang, Y. Chen, Y. Li, L. Yang, J. Wang, T. Wang

Medium-chain TAG attenuate hepatic oxidative damage in intra-uterine growth-retarded weanling piglets by improving the metabolic efficiency of the glutathione redox cycleBr J Nutr, 28 (112) (2014), pp. 876-885 

View Record in ScopusGoogle Scholar

Zhang et al., 2019

H. Zhang, Y. Li, Y. Chen, L. Zhang, T. Wang

N-Acetylcysteine protects against intrauterine growth retardation-induced intestinal injury via restoring redox status and mitochondrial function in neonatal pigletsEur J Nutr, 58 (2019), pp. 3335-3347 View PDF

CrossRefView Record in ScopusGoogle Scholar

Zhang et al., 2014

J. Zhang, L. Xu, L. Zhang, Z. Ying, W. Su, T. Wang

Curcumin attenuates D-galactosamine/lipopolysaccharide-induced liver injury and mitochondrial dysfunction in miceJ Nutr, 144 (2014), pp. 1211-1218 View PDF

CrossRefView Record in ScopusGoogle Scholar