| Toll-like receptors is a kind of Pattern recognition receptor (PPR), and the relationship between TLRs and nutritions is not clear. The study was conducted to ascertain the influence of Arg on gene expression of TLRs onthe cell membrane surface and its signal pathway of weaned piglets under immune stress in vivo and to preliminarily reveal the mechanisms in vitro.Exp.l The influence of Arg on the TLRs gene expression on the cell membrane surface of weaned piglets under immune stressThis study was conducted to evaluate whether dietary Arg supplementation could affect the expression of TLRs and some key genes in its signal pathway on the cell membrane surface of piglets under immune stress. Thirty-six healthy DurocxLandracexLarge White castrated piglets weaned at24±1d with initial mean BW of7.19±0.63kg were randomly assigned to6treatments according to body weight, with6replicates per treatment and1piglet per replicate. In the first week, piglets in treatment1and4,2and5,3and6were fed diets added with0,0.5%,1.0%Arg, respectively, and the piglets fed the same diets were injected intramuscularly4mL S. C500or4mL sterile saline by half on the8d, and then the piglets were supplied with the same diet as before for10days. Blood sample were collected on d8(before injection),9,11and18, respectively, and to assay serum salmonella antibody, CRP and cytokine levels. On the18d, the piglets were slaughtered and liver, spleen, lung, kidney, ileum, jejunum, mesenteric and inguinal lymph nodes were collected after blood collection to evaluate the mRNA expression levels of TLRs and key genes in its signal pathway. Results showed that:S.C500injection increased serum salmonella antibody level on d9,11, and18(P＜0.05), and significantly improved the concentrations of serum CRP and IL-6(d9, d11), IFN-y (dll, d18) and IL-12(d11)(P＜0.05). Arg supplementation decreased the concentrations of serum IL-6(d9, d11and d18), CRP (d9and d11) and IFN-y (d11and d18)(P<0.05). Meanwhile, Arg supplementation reversed the observably up-regulated mRNA expressions levels of TLR4, TLR5and MyD88, p65NF-kB, TNF-a (except the ileum and jejunum) in liver, spleen, lung, ileum, jejunum, mesenteric and inguinal lymph nodes (P<0.05) caused by S.C500stress. The results showed that dietary Arg supplementation down-regulated the excessive gene expressions of TLR4, TLR5and its key downstreamsignal molecules in tissues caused by immune stress and further ameliorated the immune stress in piglets.Exp.2The primary exploration of the effect of Arg on TLR4expression in IPEC-J2cell lines under LPS stressThe aim of this experiment was to initially reveal the possible mechanism by which Arg affected the TLR4-MyD88signal pathway. The pig jejunal epithelial cell line (IPEC-J2) were chosen as experiment model, and the iNOS inhibitors (SMT), Arg and LPS were added into cell culture solutions, the concentrations of NO, Arg, IL-1(3, IL-6, IL-12, IFN-y and TNF-a in cell culture solutions and the mRNA expression levels of TLR4, TLR5, iNOS, MyD88, p65NF-kB, p38MAPK and TNF-a in cell were evaluated to reveal the possible mechanism between Arg and TLR4signal pathway.Test1The determination of effect concentration and the relative optimal action time of ArgTo determine the effect concentration and the relative optimal action time of Arg affects TLR4, the IPEC-J2cell line was chosen as experiment model,5μg/mL LPS and different levels of Arg (0,50,100and200μg/mL) were added into all cell culture solutions, the cells were collected at different cultured time (0,6,12,24h) and the mRNA expressions level of TLR4and iNOS were evaluated. The results showed that: the mRNA expressions of TLR4and iNOS in IPEC-J2cell were significantly up-regulated after24h cocultivation with LPS (0μg/mL Arg)(P＜0.05). Adding Arg into the cell culture solution for6h and12h had no significant influence on the relative mRNA expressions of TLR4and iNOS. But the mRNA abundance were decreaseed along with the increased concentration of Arg after24h cocultivation (P＜0.05). The results revealed that Arg supplementation could inhibit the over expressions of TLR4and iNOS in cells caused by LPS. Moreover, the optimal effect concentration of Arg was200μg/mL and the optimal action time was24h.Test2The determination of the concentration of iNOS inhibitorTo determine the optimal inhibition concentration by which iNOS inhibitor SMT (S-Methylisothiourea Sulfate) inhibited NO synthesis of IPEC-J2, different levels of SMT (0,0.1.0.3,1mM) were supplemented into serum-free culture solutions (containing200μg/mL Arg and5μg/mL LPS) for culturing, respectively. There were4treatments with3replicates per treatment and1culture hole per replicate. The results indicated that the NO concentration in culture solutions treated by0.3mM and1mM SMT were significantly lower than that in0.1mM and0mM SMT treatments (P<0.05, P<0.01, respectively) after culturing for24h. But there was no significant difference between0.3mM and1mM SMT treatments. Therefore,0.3mM SMT was chosen as the optimal dosage.Teat3Interaction mechanisms between Arg and TLR4in vitroThe experiment was conducted to primarily reveal the mechanism by which Arg affected the TLR4gene expression in IPEC-J2. The IPEC-J2cell line were chosen as model, and5μg/mL LPS,0.3mM SMT,200μg/mL Cit and200μg/mL Arg were respectively added into cell culture solutions by a single-factor test design with3replicates per treatment and1culture hole per replicate. The6treatments were:basal group, LPS group, LPS+Arg group, LPS+Arg+SMT group, SMT group, LPS+SMT+Cit group, respectively,.The results showed that the expressions of TLR4, TLR5, MyD88, p65NF-κB, p38MAPK, TNF-a and iNOS in IPEC-J2and the secretion of IL-1β，IL-6, IFN-γ and TNF-α were significantly improved by LPS when compared with the basal group (P<0.05). Increased consumption of Arg and production of NO and Cit (P<0.01), and decreased genes expressions and cytokines levels (P<0.05) were observed after Arg supplementing when compared with LPS group. Compared to LPS+Arg group, the supplementation of SMT decreased Arg consumption and the productions of NO and Cit (P<0.01), wheares increased the relative mRNA expressions of TLR4, TLR5, MyD88, p65NF-kB, p38MAPK, TNF-a, iNOS and the levels of IL-1βIL-6, IFN-y, TNF-a (P<0.05). The results suggested that the relative mRNA expressions of TLR4, TLR5, MyD88, p65NF-kB, TNF-a and iNOS were down-regulated by NO generated from increased Arg consumption in IPEC-J2, and the productions of inflammatory factors (IL-1β, IL-6, TNF-a and IFN-y) were accordingly declined.In conclusion, Arg supplemtation in diet and cell culture system could significantly down-regulate the over expression of TLR4, TLR5and the downstream signal molecules such as MyD88and p65NF-kB in tissues and cells of piglets, inhibit the over activation of the TLR4-MyD88signal pathway, decrease the generation of inflammatory factors (IL-6, IFN-γ and TNF-a, et al), and then ameliorated the immune stress. Furthermore, NO was the key signal molecule that mediat the action by which Arg affected the signal pathway of TLR4under immune stress. |
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