ABSTRACT
Objective:To investigate the mechanism of sinomenine (SIN) in inducing the immunosuppressive effects of rat-derived dendritic cells (DCs).Methods:The bone marrow-derived precursor cells from Wistar rats were cultured in vitro. The morphological differences between sinomenine treated DCs (sinomenine modified group, SIN group) and conventional induced DCs (conventional induced group, control group) were observed under microscope. The CD phenotype of DCs was detected by flow cytometry. DCs were induced maturation by lipopolysaccharides (LPS) stimulation. The impact of SIN on the expressions of Toll-like receptor (TLR)2, TLR3 and TLR4 on the DCs surface were detected by flow cytometry. Results:In the conventional induction group, the cells showed clusters or suspension growth, with obvious granular sense on the cell surface; while in the sinomenine induction group, the cells were clustered together, with no significant change in cell volume and morphology. The relative expressions of CD80 and CD86 were 70.7% and 71.3% in the conventional induction group, while 51.7% and 49.4% in the SIN group. The relative expression of TLRs on DCs in SIN + LPS group was TLR2 (51.2±0.34)%, TLR3 (50.3±0.14)%, TLR4 (52.1±0.16)%, which were significantly lower than those in LPS group [(94.35±0.16)%, (97.55±0.16)%, (94.6±0.12)%].Conclusions:SIN may induce immune tolerance by inhibiting the maturation of DCs via inhibiting the TLRs signaling pathways.
ABSTRACT
BACKGROUND:It may be an important approach to avoiding organ transplant rejection by utilizing immature dendritic cells to induce donor-specific immunologic tolerance. OBJECTIVE:To study the effect of sinomenine on the differentiation and maturation of rat bone marrow-derived dendritic celsin vitro. METHODS:Bone marrow-derived dendritic cells were isolated from the rat femur and tibia, and immature dendritic cells were induced by granulocyte-macrophage colony stimulating factor and interleukin-4. On day 7, lipopolysaccharide was added and the cells were cultured to generate mature dendritic cells. Cells were divided into control group and low-, middle- and high-dose sinomenine treatment groups (SNL, SNM, SNH groups). Forty hours later, dendritic cels were harvested, and cell morphology was observed by inverted phase contrast microscope. The expression of CD80 and RT1B was detected by flow cytometry. ELISA was used to detect the expression of interleukin-12. The mixed lymphocyte reaction was used to detect the ability of dendritic cells to stimulate the activation of allogeneic T lymphocytes. RESULTS AND CONCLUSION: (1) Under the inverted microscope, the morphology of mature dendritic cells was observed in the control group; in the SNL group most dendritic cells were visible; in the SNM group, there were partially suspended cells with poor maturation; and in the SNH group, most of the cells were not mature. (2) The expression of CD80 in the control group was significantly lower than that in the SNL, SNM and SNH groups (P < 0.05), and the expression of RT1B was significantly reduced in the SNM and SNH groups than the control group. (3) Compared with the control group, the level of IL-12p70 in the cell supernatant was significantly decreased in the SNM and SNH groups (P < 0.01). (4) The ability of dendritic cells to stimulate T lymphocyte proliferation in the SNM and SNH groups was significantly decreased compared with the control group (P < 0.05). To conclude, sinomenine can inhibit the maturation of dendritic cells.
ABSTRACT
Objective To investigate the effect of immature dendritic cell (imDC) derived from donor-derived bone marrow induced by alkaloid sinomenine (SN) on the Th1/Th2 cytokines in venous blood of receptors,and to probe into the mechanism which imDC induced by SN can lead transplantation immune tolerance.Methods Inbred strain Wistar and Sprague Dawley (SD) rats were selected as kidney transplant donor and recipient,respectively.Vessel sutures of the microsurgery technique were used to build the bilaterally renal transplantation model of rats.By the injection of imDC to the recipient rats preoperatively,enzyme-linked immunosorbent assay (ELISA) was used to determine the level of the interleukin (IL)-2,IL-4,IL-10 and interferon-γ (INF-γ).Results (1) Successful rate of transplantation was 89.5％.Arterial anastomosis time was (12.5 ±5.7)min,and venous anastomosis time was (17.3 ± 3.4)min.(2) The content of the IL-2,INF-γ,IL-4 and IL-10 in SN-imDC 106 group was (17.25 ± 3.41) pg/ml,(239.80 ± 9.06) pg/ml,(337.60 ± 25.07) pg/ml,and (1 432.00 ± 106.39) pg/ml,respectively.Among the same concentration,the level of the IL-4 and IL-10 that stood for the Th2 cytokines was significantly higher in SN-imDC group than imDC group and control group (P ＜ 0.05),and was significantly higher in SN-imDC 106 group than SN-imDC 105 group (P ＜ 0.05).Among the same concentration,the lever of IL-2 and INF-γ that stood for the Th1 cytokines was significantly lower in SN-imDC group than imDC group and control group (P ＜ 0.05),and was significantly lower in SN-imDC 106 group than SN-imDC 105 group (P ＜ 0.05).Conclusions (1) The use of microsurgery for anastomosis could make the model of singel kidney transplantation in rats.(2) Specific imDC induced by SN could induce the migration to Th2 immune response,which proved imDC induced by SN could mediate immune tolerance to the recipient.