| The study was consisted of 5 experiments by which the effect of chitosan(CHI)on immune function in dairy cows and the underlying regulating mechanism were studied.In experiments 1~3,a total of forty heathy mid-lactating Holstein cows with similar parturition date,days in lactation and milk yield were randomly divided into five dietary treatment groups,eight replicates per treatment,with one cow per replicate.The five experimental diets contained,respectively,0(control),500,1000,1500 or 2000 mg/kg CHI.Feeding trial lasted for 60 days,and was divided equally into two phases,namely d 0 to 30 and d 30 to 60.Cows had free access to feed and water,and the environment and management of experimental cows were consistent during the trial.Experiment 1 studied the effects of adding CHI to feed on immune and antioxidant functions in dairy cows.Experiment 2 and 3 explored molecular mechanism of CHI affecting immune function in cows from NO and AA immune regulating pathways.Experiment 4 studied the impact of CHI on the immune function of LPS-stimulated peripheral blood lymphocyte(PBL)in vitro.Experiment 5 explored the effects of CHI on immune activity of LPS-stimulated PBL with the existence of inhibitor in vitro.The two in vitro experiments aimed at finding the molecular mechanism by which CHI modulated immune function in dairy cows.Experiment 4 adopted 2×5 factor design,with 2 LPS treatments(0,10 μg/m L LPS)and 5 CHI treatments(0,40,80,160,320 μg/m L).There were 10 treatments in total,and each treatment had 6 replicates.Experiment 5 adopted 2×2×2 factor design,with 2 LPS treatment(0,10 μg/m L LPS),2 CHI treatments(0,160 μg/m L),and 2 inhibitors(adding inhibitor,without inhibitor).There were 8 treatments in total,with 6 replicates per treatment.The results were showed as follows:(1)With the increasing of the CHI addition in the feed,serum concentration of immunoglobulin G(Ig G)and interleukin-2(IL-2)showed extremely significant quadratic increasing effects(P<0.01)on d 60.Serum concentration of immunoglobulin A(Ig A)and the activity of catalase(CAT)showed quadratic increasing trends(0.05<P<0.1),concentration of IL-2 and the activity of glutathione and peroxidase(GSH-PX)showed linear increasing trends(0.05<P<0.1)on d 60.The activity of GSH-PX showed significant quadratic increasing effects(P<0.05)on d 30.the contents of soluble CD4(s CD4)and malondialdehyde(MDA)linearly or quadratically decreased(P<0.01).The results suggested that CHI could improve immune and antioxidant functions in dairy cows and the effects were dose-dependent.(2)With the increasing of the CHI addition in the diet,nitric oxide(NO)content showed extremely significant linear(P<0.01)or quadratic increasing effects(P<0.01)on d 60,inducible nitric oxide synthase(i NOS)activity showed significant quadratic increasing effects(P<0.05)on d 60.With the increasing of addition level,gene expression of i NOS and nuclear factor κB(NF-κB)showed linear increasing trends(0.05<P<0.1)or significant quadratic increasing effects(P<0.05),with 1500 mg/kg being the optimum dosage.This showed that CHI could dose-dependently affect immune function of dairy cows by increasing gene expression level of i NOS and NF-κB,improving the activity of i NOS,stimulating the releasing of NO from immunological cells.(3)With the increasing of CHI addition in the diet,the serum contents of arachidonic acid(AA)and leukotriene B4(LTB4),activity of lipoxygenase(5-LOX)showed significant linear increasing effects(P<0.05)on d 60,contents of LTB4 and prostaglandin E2(PGE2),activities of phospholipase A2(c PLA2)and cyclooxygenase(COX-2)showed extremely significant linear(P<0.01)or significant quadratic increasing effects(P<0.05)on d 60,respectively.Content of c PLA2 showed significant linear increasing effects(P<0.05)on d 30.As the adding level of CHI increased,gene expressions of c PLA2 and 5-LOX showed linear or quadratic increasing effects on d 30 and 60(P<0.05),and COX-2 gene expression exhibited quadratic increasing effects on d 60(P<0.05).The dosage of 1500 mg/kg was considered as the optimum level of CHI.The results suggested that CHI could dose-dependently affect immune function of dairy cows by increasing gene expression level of c PLA,COX-2,and 5-LOX,improving the activities of c PLA,COX-2,and 5-LOX,stimulating the releasing of PGE2,LTB4,and AA from immunological cells.(4)CHI significantly improved the concentrations of IL-1,IL-6,NO and AA,the activities of i NOS,c PLA2,and COX-2,and the gene expressions of i NOS,NF-κB,c PLA2 and COX-2 in the inoculum of PBLs(P<0.05).Under LPS-stimulated conditions,adding CHI significantly decreased the contents of IL-1,IL-2,IL-6,tumor necrosis factor(TNF-α),NO,AA,the activities of i NOS,COX-2,and PGE2,and the gene expressions of i NOS,NF-κB,c PLA2,and COX-2(P<0.05),with 160 μg/m L CHI group showing the highest values.The above results suggested that CHI could improve immune function through both NO and AA pathways under normal psychological status,and ameliorate the inflammation to some extent.(5)With the stimulation of LPS,LPS could increase NO concentration,i NOS activity and i NOS expression(P<0.05),and the NO concentration and i NOS activity were significantly decreased(P<0.05)after the CHI was added,while in the presence of nitric oxide synthase inhibitor(1400W)or NF-κB inhibitor(PDTC),addition of CHI had no significant effect on the above indicators.These results indicated that CHI-induced improvement of NO contents in PBLs was attributed to improvement of i NOS activity.In addition,CHI could increase AA concentrations(P<0.05)in the absence of LPS,and CHI did not have a significant effect on the AA concentration after adding the PLA2 inhibitor(AACOCF3).With the stimulation of LPS,LPS could increase AA and PGE2 concentrations and COX-2 activity(P<0.05),and AA and PGE2 concentrations and COX-2 activity were significantly decreased(P<0.05)after the CHI was added,while in the presence of PLA2 inhibitor(AACOCF3),addition of CHI had no significant effect on the above indicators.These results suggested that improving of activities of c PLA2 and COX-2 could result in the increasing of AA and PGE2 contents in CHI-induced PBLs.it also shows that CHI could dual-directionally regulate key factors in the pathways of NO and AA in PBLs.In conclusion,CHI not only exhibited antioxidant action,but also improved immune function of dairy cows,and the optimum dosage of CHI was 1500 mg/kg. |
