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1.
Impairment of placental growth is a major factor contributing to intrauterine growth retardation (IUGR) in both human pregnancy and animal production. Results of recent studies indicate that administration of L-arginine (Arg) to gestating pigs or sheep with IUGR fetuses can enhance fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that Arg stimulates the mammalian target of rapamycin (mTOR) signaling pathway and protein synthesis in porcine conceptus trophectoderm (pTr2) cells. The cells were cultured for 4 days in Arg-free Dulbecco's modified Eagle's Ham medium containing 10, 50, 100, 200, 350 or 500 μM Arg. Cell numbers, protein synthesis and degradation, as well as total and phosphorylated levels of mTOR, ribosomal protein S6 kinase 1 (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. The pTr2 cells exhibited time (0-6 days)- and Arg concentration (10-350 μM)-dependent increases in proliferation. Addition of 100 and 350 μM Arg to culture medium dose-dependently increased (a) protein synthesis and decreased protein degradation and (b) the abundance of total and phosphorylated mTOR, p70S6K and 4EBP1 proteins. Effects of 350 μM Arg on intracellular protein turnover were only modestly affected when nitric oxide synthesis was inhibited. Collectively, these results indicate a novel and important role for Arg in promoting growth of porcine placental cells largely via a nitric-oxide-independent pathway. Additionally, these findings help to explain beneficial effects of Arg supplementation on improving survival and growth of embryos/fetuses in mammals.  相似文献   

2.
Yao K  Yin Y  Li X  Xi P  Wang J  Lei J  Hou Y  Wu G 《Amino acids》2012,42(6):2491-2500
α-Ketoglutarate (AKG) is a key intermediate in glutamine metabolism. Emerging evidence shows beneficial effects of AKG on clinical and experimental nutrition, particularly with respect to intestinal growth and integrity. However, the underlying mechanisms are unknown. Intestinal porcine epithelial cells (IPEC-1) were used to test the hypothesis that AKG inhibits glutamine degradation and enhances protein synthesis. IPEC-1 cells were cultured for 3 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 0, 0.2, 0.5 or 2 mM of AKG. At the end of the 3-day culture, cells were used to determine L-[U-14C]glutamine utilization, protein concentration, protein synthesis, and the total and phosphorylated levels of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1). Compared with 0 mM of AKG (control), 0.2 and 0.5 mM of AKG dose-dependently reduced (P<0.05) glutamine degradation and the production of glutamate, alanine and aspartate in IPEC-1 cells. Addition of 0.5 and 2 mM of AKG to culture medium enhanced protein synthesis (P<0.05) by 78 and 101% without affecting protein degradation, compared to the control group. Rapamycin (50 nM; a potent inhibitor of mTOR) attenuated the stimulatory effect of AKG on protein synthesis. Consistent with these metabolic data, the addition of 0.5 or 2 mM of AKG to culture medium increased (P<0.05) the phosphorylated levels of mTOR, S6k1 and 4E-BP1 proteins. Collectively, these results indicate that AKG can spare glutamine and activate the mTOR signaling pathway to stimulate protein synthesis in intestinal epithelial cells.  相似文献   

3.
15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory prostaglandin that plays a role in promoting the resolution of inflammation. We investigated the effects of 15d-PGJ2 on the production of IL-8 and on the expression of Toll-like receptors (TLRs) 2 in human primary keratinocytes stimulated with lipopolysaccharide (LPS). Cell proliferation was analyzed using the MTT assay, TLR2 and -4 mRNA expression was detected by RT–PCR, and IL-8 production and NF-κB p65 activities were determined by ELISA. LPS and 15d-PGJ2 did not influence the proliferation rate at low concentrations (0.5 and 2.0 μM) in keratinocytes, and showed toxicity at high concentrations (5.0 μM). LPS, compared with control, induced the expression of TLR2 mRNA, increased IL-8 production, and enhanced NF-κB activity. 15d-PGJ2 decreased TLR2 mRNA, increased IL-8 production, and suppressed NF-κB activity. Costimulation with LPS and 15d-PGJ2, compared with LPS stimulation alone, decreased TLR2 mRNA (1.8-fold), increased IL-8 production (1.8-fold at 0.5 μM and 3.7-fold at 2.0 μM), and inhibited NF-κB activity (3.3-fold at 0.5 μM and 5.1-fold at 2.0 μM). TLR4 mRNA was not expressed in primary keratinocytes. These results suggest that 15d-PGJ2 suppresses TLR2 expression and that it up-regulates the production of IL-8 by inhibiting the NF-κB signaling pathway in primary keratinocytes. Thus, 15d-PGJ2 can have both anti- and pro-inflammatory effects, and 15d-PGJ2-mediated IL-8 up-regulation is related to the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways.  相似文献   

4.
Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerotic lesions. It was confirmed that the presence of functional TLR4 promotes a proinflammatory phenotype and proliferation of vascular smooth muscle cells (VSMCs). Here we tested whether designed TLR4 small interfering RNAs (TLR4 siRNAs) is capable of inducing TLR4 deficient and simultaneously regulating the expression of matrix metalloproteinase-9 (MMP-9) in human aortic smooth muscle cells (HASMCs). Human aortic smooth muscle cells were obtained from Cascade Biologics (Portland, USA). The siRNAs used in this study were chemically synthesized by Ambion, diluted in RNase free water at concentration of 2 μg/ml. The TLR4 siRNAs were complexed with LipofectamineTM2000 in transfection buffer. After 30 min incubation at room temperature, the complexes were added to the cells. Subsequent to 5 h incubation, cells were treated with 10 ng/ml LPS for 24 h. RT–PCR analysis was used to detect mRNA expression of GAPDH, TLR4 and MMP-9; Western blot analysis was used to examine GAPDH, TLR4 and MMP-9 protein expression. It was shown that all three designed TLR4 siRNAs inhibited the expression of TLR4 in HASMCs as compared to nontargeting siRNA. Notably, TLR4 siRNA-1 exhibited the strongest inhibition effect. Transfection of HASMCs with TLR4 siRNA-1 resulted in down-regulation of LPS-induced expression of MMP-9. It was concluded that TLR4 siRNA-transfected HASMCs were capable for regulating the expression of MMP-9, providing support for the rational design of siRNAs as atherosclerotic therapy.  相似文献   

5.
Increasing evidence indicates that carbon monoxide (CO) may protect against several diseases including sepsis. The ability of CO pre-treatment to provide good pre-conditioning against lipopolysaccharide (LPS)-induced injury was tested using an in vitro model of primary culture of porcine aortic endothelial cells (pAEC). pAEC were exposed to CO (250 ppm) or air for 1 h prior to the addition of LPS (10 μg/ml). Hsp70, HO-1, and Egr-1 protein levels were determined as well as vascular endothelial growth factor (VEGF) secretion after 4, 7, and 15 h. The effect of CO on LPS-induced apoptosis was also detected at 15 h. CO pre-treatment before the addition of LPS, significantly reduced LPS-induced apoptosis. LPS induced an increase in the level of VEGF in culture media after 7 and 15 h, and a larger increase was detected in CO pre-treated cells. In addition, CO pre-treatment reduced LPS-induced Hsp70, HO-1, and Egr-1 protein expression. In conclusion, CO treatment seems to provide a good pre-conditioning for the prevention of LPS-induced endothelial injury.  相似文献   

6.
L-Glutamine (Gln) plays an important role in sustaining the intestinal mucosal mass of humans and animals. However, the underlying mechanisms are largely unknown. This study tested the hypothesis that Gln regulates protein turnover in intestinal epithelial cells. Intestinal porcine epithelial cells (IPEC-1) were cultured for 3 h (short-term study) or 96 h (long-term study) in Gln-free Dulbecco's modified Eagle-F12 Ham medium containing 0, 0.5 or 2.0 mM Gln. To determine effects of ammonia (a metabolite of Gln, i.e., 0.18 mM ammonia produced from 2 mM Gln in 3 h) on protein turnover, additional experiments were conducted in which medium contained 0.5 mM Gln and 0, 0.2, 0.5 or 2.0 mM NH(4)Cl. Variables of analysis included cell growth, protein synthesis, proteolysis and mammalian target of rapamycin (mTOR) signaling. IPEC-1 cell growth increased with extracellular Gln concentrations. Compared with 0 mM Gln, the addition of 0.5 and 2 mM Gln to medium stimulated protein synthesis and inhibited protein degradation in those cells in both the short- and long-term studies. Ammonia (0.05 to 2.0 mM) did not affect protein synthesis, although higher levels of ammonia (0.5 and 2.0 mM) reduced protein degradation in IPEC-1 cells. Consistent with the data on protein turnover, 0.5 and 2 mM Gln increased abundance of phosphorylated eIF4E-binding protein-1 and phosphorylated S6 kinase-1 proteins. Collectively, these results demonstrate that physiological levels of Gln regulate protein turnover independent of ammonia production in intestinal cells through the mTOR signaling pathway.  相似文献   

7.
8.

Background  

Mesenchymal stem cells (MSCs)-based regenerative therapy is currently regarded as an alternative approach to salvage the acute myocardial infarcted hearts. However, the efficiency of MSCs transplantation is limited by lower survival rate of engrafted MSCs. In previous study, we found that 1.0 μg/ml Lipopolysaccharide (LPS) could protect MSCs against apoptosis induced by oxidative stress and meanwhile enhance the proliferation of MSCs. Therefore, in the present study, we firstly preconditioned MSCs with 1.0 μg/ml LPS, then transplanted MSCs into ischemic myocardium, and observed the survival and cardiac protective capacity of MSCs in a rat model of acute myocardial infarction. Furthermore, we tried to explore the underlying mechanisms and the role of Toll-like receptor-4 (TLR4) in the signal pathway of LPS-induced cardiac protection.  相似文献   

9.
Neonates are at increased risk for inflammatory bowel disease, but effective prevention and treatments are currently limited. This study was conducted with the lipopolysaccharide (LPS)-challenged piglet model to determine the effects of dietary supplementation with α-ketoglutarate (AKG) on the intestinal morphology and function. Eighteen 24-day-old pigs (weaned at 21 days of age) were assigned randomly to control, LPS, and LPS + AKG groups. The piglets in the control and LPS groups were fed a corn- and soybean meal-based diet, whereas the LPS + AKG group was fed the basal diet supplemented with 1% AKG. On days 10, 12, 14, and 16, piglets in the LPS and LPS + AKG groups received intraperitoneal administration of LPS (80 μg/kg BW), whereas piglets in the control group received the same volume of saline. On day 16, d-xylose was orally administrated to all pigs at the dose of 0.1 g/kg BW, 2 h after LPS or saline injection, and blood samples were collected 3 h thereafter. Twenty-four hours post-administration of LPS or saline, pigs were killed to obtain intestinal mucosae for analysis. Compared with the control group, LPS challenge reduced (P < 0.05) protein levels, the ratio of villus height to crypt depth, and the ratio of phosphorylated mTOR to total mTOR in duodenal, jejunal, and ileal mucosa. These adverse effects of LPS were attenuated (P < 0.05) by AKG supplementation. Moreover, AKG prevented the LPS-induced increase in intestinal HSP70 expression. Collectively, these novel results indicate that dietary supplementation with 1% AKG activates the mTOR signaling, alleviates the mucosal damage, and improves the absorptive function of the small intestine in LPS-challenged piglets. The findings not only help understand the mode of AKGs actions in the neonatal gut but also have important implications for infant nutrition under inflammatory conditions.  相似文献   

10.
An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.  相似文献   

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