Effects Of Chitosan On Immune Function In Weaned Piglets And The Underlying Regulation Mechanism

The current research was conducted to investigate the effects of chitosan on immune function in weaned piglets and the underlying regulation mechanisms. The first four experiments were done in vivo. One hundred and eighty piglets weaned at28day (Duroc×Large white×Landrace) were assigned randomly to5dietary treatments (3pens of males and3pens of females) with6repetitions in each treatment. The piglets in the5treatments were fed on the basal diet supplemented with0(control),100,500,1000and2000mg chitosan/kg feed, respectively. The experimental period was28days. Feed and water were freely available. In trial1, we studied the effects of dietary supplementation of chitosan on humoral and cellular immune function in weaned piglets. The effects of dietary supplementation of chitosan on stress hormones and antioxidative enzymes in weaned piglets were investigated in trial2. We also investigated the effects of chitosan on immune function via nitric oxide (NO) pathway and the underlying molecular mechanisms in weaned piglets in trial3. The effects of chitosan on immune function via arachidonic acid (AA) pathway and the underlying molecular mechanisms in weaned piglets were elucidated in trial4. The trial5and trial6were carried out to explore the effects of chitosan on immune function via NO and AA pathways in the peripheral blood mononuclear cells (PBMCs) of weaned piglets.The results were showed as follows:1. Chitosan improved serum immunoglobulin G (IgG) and specific ovalbumin (OVA) IgG concentrations of piglets (P<0.05). With increasing chitosan, the secretory immunoglobulin A (sIgA) was enhanced in ileum or jejunum mucosal surfaces in a linear or quadratic manner (P<0.05). Chitosan quadratically enhanced serum concentrations of interleukin-1(IL-1), interleukin-2(IL-2) and tumor necrosis factor-alpha (TNF-a)(P<0.05). In addition, chitosan decreased serum concentrations of soluble CD4(sCD4) and soluble CD8(sCD8) in a linear or quadratic dose-dependent manner (P<0.05). These results implied that dietary supplement with chitosan improved humoral and cellular immune responses of weaned piglets.2. Dietary supplementation of chitosan decreased serum adrenocorticotropic hormone (ACTH) and cortisol (COR) concentrations in a linear or quadratic dose-dependent manner (P<0.05). With increasing chitosan, serum glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) were improved linearly or quadratically (P<0.05). These results suggested that dietary supplementation of chitosan mitigated weaning stress and improved the activity of antioxidative enzymes, thereby improving immune function in weaned piglets.3. Serum NO concentration and iNOS activity were linearly or quadratically improved with increasing chitosan (P<0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the small intestine (P<0.05). In addition, piglets in500and1000mg/kg chitosan treatments had higher serum NO concentrations, iNOS activity and iNOS mRNA expressions compared with other treatments. However, positive effects of chitosan tended to be suppressed when chitosan was increased to2000mg/kg. The results indicated that the NO concentrations, iNOS activity and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner.4. Serum AA, prostaglandin E2(PGE2) and leukotriene B4(LTB4) contents of weaned piglets were improved in a linear or quadratic dose-dependent manner with increasing chitosan (P<0.05). Chitosan increased serum cytosolic phospholipase (cPLA2),5-Lipoxygenase (5-LOX) and cyclooxygenase-2(COX-2) activity in a linear or quadratic manner (P<0.05). Moreover, chitosan elevated gene expression of cPLA2, COX-2and5-LOX mRNA in the small intestine in a linear or quadratic dose-dependent manner (P<0.05). In addition, most of the above indexes of500or1000mg/kg chitosan treatment were higher than those of the control treatment. However, the positive effects of chitosan tended to be suppressed when chitosan was increased to2000mg/kg. These results implied that the metabolism of AA was regulated by chitosan in a dose-dependent manner, which may be one reason that affected immune function in weaned piglets.5. In vitro,1400W, a highly selective iNOS inhibitor, was used to confirm whether NO was generated by iNOS. Results showed that the NO concentration, iNOS activity and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentration and iNOS activity by chitosan were markedly inhibited by1400w (P<0.05). Moreover, the increase of NO concentration, iNOS activity and mRNA expression in PBMCs induced by LPS were suppressed significantly by chitosan (P<0.05). The results indicated that the NO concentration, iNOS activity and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which suggested dietary chitosan had a bidirectional regulatory action on the NO production via modulating iNOS activity and its mRNA expression.6. Chitosan increased the activity of cPLA2, COX-2and5-LOX as well as content of AA, PGE2and LTB4in supernatants in a quadratic dose-dependent manner (P<0.05). With increasing chitosan, the mRNA expression of cPLA2, COX-2and5-LOX in PBMCs was enhanced in a quadratic manner (P<0.05). In addition, the supplementation of80or160μg/mL chitosan to the medium had higher values of all above indexes compared with those in other treatments. However, positive effects of chitosan tended to be suppressed when its additional dose was increased to320μg/mL. These results implied that appropriate dose of chitosan could increase the contents of AA, PGE2and LTB4, and improve the activity of cPLA2, COX-2and5-LOX in medium as well as the gene expression of cPLA2, COX-2and5-LOX mRNA in PBMCs, which might be one of the reasons for chitosan to regulate the immune functions of weaned piglets.From the results, it could be concluded that chitosan elevated immune function via mitigating the weaning stress, improving the activity of antioxidative enzymes, regulating the NO and AA pathway in weaned piglets, and the appropriate adding dosage of chtiosan in diets was500～1000mg/kg.