| The immune system serves as an important barrier for organisms to prevent the invasion of pathogenic microorganisms.The livestock and poultry are easily subjected to lower immunity and cause various diseases,which are affected by some factors such as feeding management,nutrition,diseases and so on.Toll-like receptors(TLRs)plays an important role in immune regulation by recognizing pathogen molecules on the immune cells surface and inducing the expression of immunomodulation related proteins.In order to investigate the effect of sheep bone collagen peptide(SBCP)and calcium chelated with collagen peptide(CCCP)on immune function in rats,the immune regulation mechanism was investigated based on TLRs signal transduction pathway,which would provide a basis for the development and utilization of SBCP and CCCP.Female Sprague-Dawley rats were randomly divided into five groups(n = 10).Ten rats were included in the Sham operation(Sham)group,and 40 were bilaterally ovariectomized and divided into the ovariectomized(OVX)group as an immunosuppressive model and SBCP-H(3.40 g/(kg·d)),SBCP-M(2.05 g/(kg·d)),and SBCP-L(0.68 g/(kg·d))groups for 8 weeks.OVX and Sham groups were given equal volume of distilled water daily.The immunological indexes,morphological and pathological changes of spleen and the m RNA expression levels of genes in the Toll-like receptors pathway were evaluated.The results showed that compared with Sham group,the phagocytic capacity of phagocytes decreased,the rosette rate of RBC-C3 b R decreased significantly,and the ear swelling increased significantly(P < 0.05)in OVX group.The above-mentioned indicators could be reversed after intervention of various doses of SBCP.The contents of TNF-α and IFN-γ in the OVX group were significantly higher,and while IL-2 and lysozyme were significantly lower than those of Sham group(P < 0.01).Once intervention of SBCP,the changes of serological indexes could be significantly inhibited in a dose-dependent pattern.After OVX,the spleen index was significantly decreased(P < 0.05)compared with Sham group.Those indexes could be improved to varying degrees after administering different doses of SBCP and the middle dose group showed the best effect.Compared with Sham group,the morphology of spleen tissue in the OVX group was more red pulp and less white pulp.Various doses of SBCP improved the spleen injury caused by OVX.The q RT-PCR results showed that the relative expression levels of SIGIRR in spleen tissue was significantly lower and key genes in TLRs signaling pathway was significantly higher than that of Sham group(P < 0.01)after OVX.Different doses of SBCP negatively regulated the expression of the above-mentioned related genes in the TLRs pathway.Rat peritoneal macrophages were treated by various doses of SBCP or CCCP with or without stimulation with LPS.The metabolic activity and phagocytic ability of macrophages were measured by MTT assay and the neutral red method,respectively.The production of NO was measured with the Griess reaction,and then TNF-α and IL-6 were detected by enzyme-linked immunosorbent assay(ELISA).The m RNA expression levels of related genes in the Toll-like receptors pathway were measured by q RT-PCR.The results showed that treatment of normal macrophages with SBCP or CCCP increased their metabolic activity,phagocytosis,NO,TNF-α,and IL-6.Relative level of m RNA expression of key genes in the TLRs pathway were up-regulated and the m RNA expression of SIGIRR was down-regulated.At a concentration of 105 μg/m L,there was no significant difference among SBCP or CCCP and LPS groups.SBCP or CCCP treatment of LPS-stimulated macrophages could inhibit their metabolic activity,with the greatest effect at a concentration of 102 μg/m L.While the concentration of 103 μg/m L,mophagocytosis and the secretion of NO,TNF-α and IL-6 strongly inhibited.Relative level of m RNA expression of key genes in the TLRs pathway were up-regulated and the m RNA expression of SIGIRR was down-regulated in this treatment.In conclusion,SBCP can significantly improve the immune deficiency caused by the reduction of estrogen,and regulate the immune system by affecting the expression of related genes in the TLRs pathway.When SBCP or CCCP treatment of normal macrophages,it enhances the immune activity of macrophages at a low of concentration and induces macrophages to release various inflammatory mediators at a high of concentration.SBCP or CCCP could inhibit the inflammatory reaction LPS induced.Furthermore,SBCP or CCCP showed a dual regulatory effect on the inflammatory and immune response through the TLRs signaling pathway. |
