Regulative Effects Of Ala-Gln For Weaned Pigs Challenged With Oxidative Stress And Preliminary Study On The Mechanisms

This study was based on researches before and conducted to master the relationshipAla-Gln and intestinal epithelial cell autophagy of piglets and investigate the influence ofAla-Gln on antioxidative capacity of weaned piglets from cellular and molecular level,through the application of modern nutrition, pathology, oxidative stress model, cell linemodels, real-time quantitative PCR (RT-PCR), immunofluorescence techniques, in vivoand vitro test, combined with the current progress of clinical studies,which may supportevidence as well as new study direction for direction for relieving oxida tive stress thatpiglets had suffered.Experiment I: Effect of Ala-Gln on antioxidative capacity of weanedpiglets under oxidative stressTwenty four similar genetic background and body weight,21day old weaned pigletswere selected and completely randomly divided into2groups: The control group (basicdiet), Group B (basic diet+0.30%Ala-Gln) with twelve repeats in every group and onepiglets in every repeat. After7d, half of piglets in each group were challenged with8mg/kg diquat, the others were injected with the same volume of sterilized physiologicalsaline. Digestion trial was carried out from7to14d, and the whole experiment lasted for14d. To gain insights into the effects of Ala-Gln on antioxidative capacity of weanedpiglets under oxidative stress, an oxidative stress model was established byintraperitoneally injecting with diquat of weaned piglets. The results showed that:(1)underoxidative stress, supplemented Ala-Gln could increase serum Gln, GSHconcentration (P<0.01) and SOD activities (P<0.01) and T-AOC (P<0.01), reduced serumMDA concentration (P>0.05).(2) Ala-Gln could increase jejunum SOD(P<0.05), GSH-Px(P<0.01) activities and T-AOC (P<0.01), reduced serum MDA concentration (P<0.01).(3)Ala-Gln could increase liver GSH-Px (P<0.01), SOD, T-AOC (P<0.05) activities andT-AOC (P<0.01), reduced serum MDA concentration (P<0.01).(4) Ala-Gln significantlyimproved7-14d jejunum villus height (P<0.01). And crypt depth was significantly lower(P<0.05), significantly increased the ratio of the jejunum villus height and crypt depth(P<0.05).(5) Ala-Gln through improved antioxidant activity, oxidative stress on improvingthe jejunum, liver and spleen damage.(6) Ala-Gln through improved antioxidant activity,oxidative stress on improving the jejunum, liver and spleen damage. Those data indicatedthat Ala-Gln can improve the body’s tissues Gln and GSH content, and enhance antioxidantenzyme GSH-Px, SOD activity and its corresponding expression levels, improve organizational antioxidant capacity, reduced oxidation products over the content of MDA,slowing oxidative stress on weaning body injury.Experiment II: Anti-oxidative stress of Ala-Gln on pigletslymphocytes under oxidative stressIn the experiment, using the in vitro oxidative stress model, the effects of Ala-Gln onantioxidant enzyme activith and immune function of piglets lymphocytes under oxidativestress were determined. Test using single factor experiment design, peripheral bloodlymphocytes were isolated and cultured piglets, add6Ala-Gln levels (0,0.25,0.5,1.0,2.0,4.0mM) to the cell culture medium, and then added to a final concentration of400μMH2O2induced oxidative stress, test a total of six treatments with at least eight repeats. Bydetecting lymphocyte transformation rate, the culture supernatant GSH-Px, SOD activityand MDA, cytokines IL-2, IL-4, TNF-α, IFN-γ levels, proven Ala-Gln to oxidative stressstate weaned lymphocyte antioxidant effects. The results showed that:(1) under oxidativestress conditions, adding Ala-Gln can significantly improve lymphocyte GSH-Px andT-SOD enzyme activity (P<0.01), which when added level of2.0mM, T-SOD has thehighest activity level; while MDA content was significantly decreased (P<0.01).(2) WithAla-Gln dipeptide added to raise the level of significantly increased lymphocytetransformation rate (P<0.01), and reached the maximum level at2.0mM; culture mediumIL-2, IL-4were significantly improved in (P<0.01), significantly reduced IFN-γ andTNF-α (P<0.01), wherein the IL-2has the highest level of2.0mM, IL-4has the highestvalue at1.0,2.0mM, IFN-γ the lowest level in1.0mM and subsequently with two peptidelevels are not increased with the increase, TNF-α levels with increased dipeptide addsignificantly increased (P<0.01). In summary, Ala-Gln can improve lymphocytetransformation rate of oxidative stress, while improving antioxidant enzyme in peripheralblood lymphocytes of GSH-Px, SOD activity, and enhance antioxidant capacity, reducedMDA produce, promote secretion of cytokines, slowing oxidative stress damage theperipheral lymphatic system, improve immunity.Experiment III: Oxidative stress induced by H2O2in IPEC-1in vitroThe present trial was conducted to study the effects of medium H2O2levels onantioxidant enzyme activith and MDA contents of IPEC-1in vitro, and to establish anoxidative stress IPEC-1model with H2O2as stressor. Single-factor test design, test a totalof seven treatment, namely (Intestinal Porcine Epithelial Cells, IPEC-1) in vitro culture ofporcine intestinal epithelial cells were added to a final concentration of0,50,100,200,400,800,1600μM of H2O2, each treatment was repeated6. By detecting IPEC-1cell viability, antioxidant enzyme activity and MDA content of different concentrations ofH2O2and IPEC-1relationship between oxidative damage, determine the optimal H2O2oxidation induction dose. The results showed that: when the H2O2concentration was400μM, cell viability was only49.2%in the control group (P<0.05), significantly lower thanthe control group, and when the concentration of H2O2was800,1600μM,400μM cellviability and no significant difference (P>0.05). Add H2O2significantly reduced theIPEC-1GSH-Px and SOD activity (P<0.01), significantly increased the MDA levels(P<0.01). Of which400μM H2O2levels were significantly reduced GSH-Px and SODactivity (P<0.01), significantly increased the MDA levels (P<0.01), and800,1600μMlevels of antioxidant enzyme activity and400μM no difference (P>0.05). H2O2cansignificantly reduce the IPEC-1activity, significantly decreased antioxidant enzymeactivity, significantly increased MDA content in cells obtained by this test.400μM ofH2O2oxidative stress can significantly effect on the IPCE-1production, oxidative stressmodel is the best dosage.Experiment IV: Anti-oxidative stress of Ala-Gln on IPEC-1underoxidative stressIt was the single factor test design that Ala-Gln was added6levels (0,0.25,0.5,1.0,2.0,4.0mM) to IPEC-1cell culture medium to determine the best use of the three testconcentration induced H2O2construct oxidative shock model, test a total of six treatmentswith at least eight repeats, and to study, under H2O2-induced oxidative stress, differentconcentrations of Ala-Gln IPEC-1’s protective effects. The results showed that:(1) withAla-Gln added to raise the level of, IPEC-1cell viability was significantly enhanced(P<0.01), and reached the maximum level at2.0mM,0.5mM level group under oxidativestress in normal cell viability level,4.0mM added level but to make the cell viabilitydecreased significantly (P<0.05), but still higher than the IPEC-1cell viability under0.5,1.0mM Gln dipeptide level.(2) Add Ala-Gln significantly increased oxidative stressIPCE-1GSH-Px and SOD activity (P<0.01), significantly decreased the content of MDA(P<0.01);4.0mM Gln dipeptide add group GSH when the content of MDA with thedipeptide added to raise the level decreases in the level of dipeptide reached1.0μM;GSH-Px activity has a maximum value; SOD at0.25and0.5mM dipeptide add the nexthighest level of activity, then added with a dipeptide raise the level of reduced, MDAconcentration levels become normal and stable.(3)Oxidative stress IPEC-1cell density toreduce short-term and severe damage cell membranes and nuclear membranes, cytoplasmaccelerated extravasation, the nucleus almost ablation, and rarely positive intracellular fluorescence signal; adding Ala-Gln later, with Gln dipeptide raise the level IPEC-1biofilm structure tends to complete, the cell density increased, their cytoplasm positivefluorescent signal is continuously enhanced, and at1.0mM and2.0mM level moreobvious;4.0mM Ala-Gln added level (F group) IPEC-1significantly increased densitycompared with the control group, and the structural integrity similar positive fluorescentsignal was no significant difference, but with2.0mM Ala-Gln added level (E group)compared to cell density and structural integrity of the biofilm increased, significantlyreduced intracellular fluorescence signal. The experiment found that the pre-add differentlevels of Ala-Gln can significantly increase under oxidative stress IPEC-1cells, GSH-Px,SOD activity was significantly reduced lipid content of MDA oxidation products,significantly enhanced autophagy, indicating Ala-Gln has strong antioxidant and cellulardefense role.