| Intrauterine growth retardation (IUGR) is a serious problem in human medicine. Since high incidence rate of IUGR in pigs, it will also suffer heavy losses in animal production. This study investigated that effect of IUGR on immune function and expression of hsp70mRNA and protein in neonatal piglets. In addition, using RNA interference technology, this study was conducted to investigate that the mechanisms of immune deficiency mediated by Hsp70. Furthermore, this study was conducted to test that effect of glutamine supplementation on immune function of neonatal piglets after weaning and expression of Hsp70in the intestine of neonatal piglets. This study will provide a new framework for regulation of growth and development in swine production and theoretical basis for studying in human medicine.1. Effects of IUGR on immune function and expression of Hsp70and FoxO3a in neonatal pigletsFifteen NBW piglets and fifteen IUGR piglets were selected to slaughter and sample at0and7day of age, respectively. The body weight, intestine weight, villus height, crypt depth, concentrations of cytokine in serum and mucosa, expression of Hsp70and FoxO3a were tested in this study. Results showed that:(1) IUGR piglets had lower body weight and absolute intestine weight than NBW piglets (P<0.01) at0and7day of age. The relative weight of proximal jejunum and ileum were significantly lower in IUGR piglets than in NBW piglets. In addition, villus height of all intestinal segments and villus/crypt ratio were significantly decreased in IUGR piglets (P<0.05). Compared to NBW piglets, blood T and B-lymphocytes proliferation in IUGR piglets was significantly decreased and IUGR piglets had20.8%lower IgG concentrations than NBW piglets (P<0.05). There was tendency to compare NBW piglets in concentrations of IgM (P>0.05). Furthermore, concentrations of IFN-y and IL-10in serum and ileum of IUGR piglets were significantly lower than those in NBW piglets, but there was no effect of IUGR on the concentrations of IL-8. Concentrations of serum IL-4were reduced in IUGR piglets compared with NBW piglets (P<0.01), however, concentrations of IL-4in intestine were increased in IUGR piglets (P<0.05).(2) At0day of age, expression of hsp70mRNA in proximal and distal jejunum of IUGR piglets was higher than those in NBW piglets. At7day of age, IUGR piglets had13.2%and20.3%higher hsp70mRNA in proximal jejunum and colon compared with NBW piglets, respectively (P<0.05). Agreed with its corresponding hsp70mRNA expression in the same pattern, expression of Hsp70protein in proximal jejunum, ileum and colon of IUGR piglets was increased compared with NBW piglets (P<0.05). IHC showed that Hsp70was predominantly localized in outside of cell and the cytoplasm of epithelial cell. The signals of Hsp70were distributed diffusely in the epithelial cells of the whole villi and intestinal gland rather than in the lamina propria and myenteron, suggesting that Hsp70is associated with protection of cell and regulation of immune function.2. Study on the mechanism of impairment of NF-κB signaling mediated by Hsp70IEC-6cells were cultured and were treatmented as five groups:control group; LPS group; LPS+heat group; LPS+siRNA group; LPS+siRNA+heat group. In order to achieve optimum condition of siRNA transfection, different concentrations of LPS, heat shock temperature and transfection time were tested. The expression of Hsp70, NF-κB signaling pathway and expression of FoxO3a were determined by Western blot. Apoptosis cells were measured by flow cytometry. Results showed that optimum condition for RNA interference Hsp70gene expression was1000ng/mL LPS, heat shock at41℃for1h and transfection for48h. Activity of NF-κB was induced significantly by LPS in IEC-6cells, however, it was inhibited by heat shock. Compared with other three groups, activity of NF-κB was significantly increased after transfection Hsp70siRNA. Contrasted to NF-κB, degradation of I-κBα was significantly stimulated by LPS in IEC-6. Heat shock can significantly decrease LPS mediated degradation of I-κBα in IEC-6. Degradation of I-κBα in IEC-6was significantly increased after Hsp70gene silence. In addition, activity of IKKα/β was significantly higher in LPS group than those in control group, but activity of IKKα/β mediated by LPS was inhibited by heat shock in IEC-6cells. Compared to control group, activity of IKKa/(3was significantly increased after transfection Hsp70-siRNA. Furthermore, the expression of FoxO3a in IEC-6cells was significantly higher in LPS group than those in control group, but it was significantly inhibited after RNA interference Hsp70gene expression. Interestingly, flow cytometry testing showed that41.8%IEC-6cells were induced to apoptosis by LPS (P<0.01), and heat shock can decrease cells apoptosis induced by LPS. Compared to other three groups, apoptosis cells were significantly decrease after Hsp70gene silence (P<0.01).3. Effects of Gln supplementation on immune function and expression of Hsp70in weaning IUGR pigletsTwenty IUGR piglets (body weight3.5±0.5kg) were selected and randomly assigned to two treatments:control group and Gln group at21day of age. The piglets received0g (control) or1g of Gln per kg BW every12h. Alanine was used to make isonitrogenous in control piglets. All of piglets were killed to sample at14day after weaning. Using ELISA, RT-PCR and Western blot, concentrations of cytokine, hsp70mRNA and protein were tested. Results showed that average daily gain and Feed/Gain were increased by27.1%(P<0.05) and15.2%(P>0.05) in weaning IUGR piglets of Gln group, respectively. Compared with control group, Gln supplementation increased significantly villus height and villus/crypt ratio in jejunum (P<0.05). Moreover, concentrations of inflammatory cytokine IL-1and IL-8in serum and intestinal mucosa were significantly decreased by Gln supplementation, whereas concentrations of anti-inflammatory cytokine IL-4were increased. Compared with control group, expression of hsp70mRNA in duodenum and jejunum of Gln group were increased by16.1%(P<0.05) and8.4%(P>0.05), respectively. At the same time, expression of Hsp70protein was improved by12.4%(P<0.05) and11.6%(P<0.05) in duodenum and jejunum, respectively.In conclusion(1) Body weight and intestine weight in IUGR piglets were decreased. Intestinal function and structure, lymphocyte transformation rate and secretion of cytokine were impaired by IUGR, thus leading to immune deficiency in neonatal IUGR piglets.(2) Expression of hsp70mRNA and protein induced by IUGR was significantly increased in neonatal piglets. On one hand, over-expression of Hsp70may impair NF-κB signaling pathway, and decrease lymphocyte transformation rate and secretion of cytokine; On the other hand, over-expression of Hsp70may stimulate expression of FoxO3a, then increase of FoxO3a can induce cells apoptosis, thus leading to impair intestinal immune function, digestion and absorption.(3) Hsp70gene silence increases activity of IKK in IEC-6cells and promotes degradation of I-KBa, increases activity of NF-κB, and also inhibits expression of Fox03a, thus decreases IEC-6cells apoptosis. In contrast, over-expression of Hsp70in IEC-6can inhibit activity of IKK, decrease degradation of I-KBa, inhibit activity of NF-κB, and improve expression of FoxO3a, thus promote cells apoptosis.(4) Gln supplementation enhances growth performance of weaning piglets, protects intestinal structure and function, inhibits secretion of pro-inflammatory cytokine and increases secretion of anti-inflammatory cytokine. At the same time, Gln induces expression of Hsp70to protect epithelial cells from lethal stress. So Gln supplementation plays a critical role in catching up growth in neonatal IUGR piglets. |
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