The Regulatory Effects Of Lariciresinol, A Chinese Medicine Monomer, On Function Of Dendritic Cells And Its Mechanisms

Over the past decades, a number of immunosuppressive drugs, such as corticosteroids, vitamin D3, cyclosporin A, rapamycin, tacrolimus and so on have been approved for clinical use and have been highly successful in preventing or delaying graft rejection and treatment of autoimmune diseases. Nevertheless, there is an incessant need for better and safer drugs to improve therapeutic effects in clinical applications. As a treasure of traditional Chinese medicine, Chinese herbal medicines function mainly through the immune system for disease prevention and treatment, especially for inflammation, infection, cancer, autoimmune et al. Chinese herbal medicines may contain ingredients with immunosuppression. Identification of these components and to study their functions have important theoretical and practical value. As dendritic cells (DCs) are crucial in immune response and might be target cells of immunosuppressive drugs, therefore, the immunosuppressive components might be identified as target cells in term of DCs.In view of this, we cooperated with Professor Weidong Zhang from Second Military Medical University and established a screening platform involved in development, differentiation and functions of DCs. We successfully screened out 31 kinds of effective components from 496 kinds of chemical constituents extracted from traditional Chinese medicine. Then we selected Lariciresinol (molecular formula C20H21O6) extracted from Silky Daphne as the object of study, because it could inhibit proliferation of pre-DCs with low toxicity, and could effectively promote the secretion of IL-10 from DCs. In the present study, the effects of Lariciresinol on function regulation of DCs and its mechanisms were investigated and the therapeutic effects of Lariciresinol on the experimental autoimmune encephalomyelitis (EAE) were also observed.PartⅠThe functional regulation of Lariciresinol on DCsThe regulatory effects of Lariciresinol on functions of DCs including apoptosis, phagocytic function, phenotypes, cytokine secretion and the capacity of antigen presentation were investigated in this section. The 5 day immature DCs from murine bone marrow cells cultured with GM-CSF and IL-4 were treated with gradient concentrations of drug ingredients for 24 hours, then stimulated with 100ng/ml of LPS for another 24 hours. The results showed that Lariciresinol has not affected the phagocytic function and the secretions of IL-24P70, TNF-αand TGF-βof DCs. The We found the 0 to 16nmol/ml of Lariciresinol is of non-toxic concentration range for immature DCs by annexinV-PI method. The results also indicated that Lariciresinol could significantly promote the secretion of IL-10. The abilities to stimulate allogeneic T cells and antigen-specific T cells of Lariciresinol treated DCs were also inhibited significantly.PartⅡThe mechanisms of Lariciresinol on secretion of IL-10 in DCsIL-10 is a kind of important negative regulatory cytokines. In this section, we analyzed the effects of Lariciresinol on IL-10 secretion by DCs with different concentrations of Lariciresinol and LPS. The results showed that Lariciresinol cooperated with LPS could promote IL-10 secretion in a dose-dependent manner, and 8nmol/ml of Lariciresinol is the best concentration. We then investigated the signal pathways of Lariciresinol on IL-10 secretion by DCs. DCs treated with Lariciresinol (8nmol/ml) for 24 hours and then stimulated with (100ng/ml) on 0,15,30 and 60 minutes were collected for protein extraction. Then phosphorylation of ERK1/2, p38 and JNK was detected by Western Blot. The results showed that Lariciresinol could significantly enhance the phosphorylation of ERKl/2 and PD98059, an inhibitor of ERKl/2 signaling pathway, could inhibit the phosphorylation. The data indicated that ERK1/2 signaling pathway plays an important role in up-regulation IL-10 by Lariciresinol. Interestingly, SB203580, an inhibitor of P38 MAPK, can also inhibit the production of IL-10 augmented by Lariciresinol,though we did not find the enhancement of the P38 MAPK phosphorylation.PartⅢThe inhibitory effects of Lariciresinol and Lariciresinol treated DCs on the experimental autoimmune encephalomyelitis(EAE)In order to observe the inhibitory effects of Lariciresinol in vivo, we established the experimental autoimmune encephalomyelitis (EAE) animal model in C57BL/6 mice, and then the protective effect of Lariciresinol treated DCs on EAE were observed. The immature DCs were treated with Lariciresinol(8nmol/ml) to treat bone marrow-derived for 24 hours and then stimulated with LPS for 18 hours before the load of myelin oligodendrocyte glycoprotein peptide (MOG35-55) for another 6 hours. Then the DCs were collected and injected into normal C57BL/6 mice intraperitoneally. EAE were induced 24 hours later. The results showed that Lariciresinol treated DCs could delay the onset and decrease the incidence rates significantly compared with the control mice. The clinical neurological function scores was also improved. The Lariciresinol was also injected into normal C57BL/6 mice intraperitoneally and the similar inhibitory effects were obtained. Our results indicated that Lariciresinol might be a novel immunosuppressive drug with important clinical value.In conclusion, Lariciresinol might be a novel pharmacological monomer which could up-regulate the secretion of IL-10 by DCs through the ERK1/2 signaling pathway. It may show clinical potential in treatment of immune related diseases.