Oleanolic acid synergizes with cyclosporine A to prolong renal allograft survival in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the synergistic effect of oleanolic acid (OA) and cyclosporine A (CsA) on the survival of renal allografts in rats.</p><p><b>METHODS</b>Renal allograft transplantation was performed using BN rats as donors and LEW rats as recipients. Forty male LEW rats were randomized into 4 equal groups for interventions with DMSO-PBS (control), OA, CsA, or CsA+OA, starting from 1 day before transplantation. Serum creatinine levels were regularly examined, and the survival of rats were recorded. On day 5 after transplantation, CD4(+) and CD8(+) T-cell infiltration in the renal grafts was analyzed by immunohistochemistry; the concentrations of the proinflammatory cytokines (IL-1β, IFN-γ, IL-2, IL-4, and IL-17), anti-inflammatory cytokine IL-10 and chemokines (IP-10, MCP-1, MIP, and Mig) were analyzed with Luminex; the T-cell phenotypes (IFN-γ, IL-10, IL-4, and IL-17) were analyzed using ELISpot.</p><p><b>RESULTS</b>In OA+CsA group, renal allograft survival was markedly prolonged and CD4(+) and CD8(+) T cell infiltration in the graft significantly decreased as compared to other groups. A significant decrease in IL-2 was observed in OA group and OA+CsA group, especially the latter. Compared with the control group, all the 3 treated groups showed significantly decreased IL-1β, IP-10 and MCP-1, increased IL-10 levels, decreased percentages of T cells secreting IFN-γ, IL-4 and IL-17, and increased percentage of T cells secreting IL-10. The increments of serum IL-10 level and T cell percentage were more prominent in OA+CsA group than in the other two intervention groups.</p><p><b>CONCLUSIONS</b>OA and CsA synergistically ameliorate renal graft rejection and inflammation and promote allograft survival and function in rats.</p>

Effects of GW5074 in the process of imDCs inducing differentiation of na(i)ve CD4+T cells into Treg cells in vitro / 中华器官移植杂志

Objective To establish a stable and efficient method of culturing imDCs in vitro,and to explore the effect of GW5074, which blocks ERK1/2 signal pathway in the process of imnature dentritic cells (imDCs) on inducing differentiation of the na(i)ve allogeneic CD4+ T cells into Treg cells in vitro. Methods The imDCs and mature DCs (mDCs) were isolated and cultured from the peripheral blood mononuclear cells (PBMC) derived from a healthy adult male volunteer, and they were identified by cell morphology, cell surface marker and cell functions respectively. Na(i)ve CD4+ T cells were isolated from newborn umbilical vein blood and were divided into 5 groups to be cultured: (1) Blank control group: Na(i)ve CD4+ T cells were cultured alone;(2) Positive control group: The irrDCs were Middle-concentration GW5074 group;(5) High-concentration GW5074 group. In the last three groups, imDCs and na(i)ve CD4+ T cells were co-cultured, the same as the positive control group, but these groups were added by GW5074 dilution at the concentrations of 8, 24, and 40μmol/Lrespectively. After co-culture for 5 days, the transformation ratio from naive CD4+T cells to Treg T cells was detected by flow cytometry. Results On the surface of imDCs, there was stronger pression of CD1a, but weaker expression of CD80 and CD83. On the contrary, on the surface of mDCs, there was weaker expression of CD1a, but stronger expression of CD80 and CD83. The stimulation index in imDCs group and mDCs group was 1.12±0.03 and 2.85±0. 07 respectively. The transformation ratio of Treg T cells in blank control group, positive control group, low-concentration GW5074 group, middle-concentration GW5074 group and high-concentration GW5074 group was (5. 81±1.36)%, (35.73±2.07)%, (22.53±2.11)%, (11.55±1.73)%, and (4.97±1.83)%respectively. One-way ANOVA analysis revealed that there was no significant difference between high-concentration GW5074 group and blank control group, P＞0. 05, but significant difference between the remaining groups, P＜0.01. Conclusion High purity of imDCs can be obtained from PBMC by induction with rhGM-CSF and rhIL-4. ERK1/2 signal pathway plays a role in inducing the immune tolerance. GW5074 can inhibit differentiation of na(i)ve CD4+ T cells into Treg T cells.

Effects of alkaloid sinomenine on expression levels of nuclear factor of activated T cells and interferon-gamma in CD4+ T lymphocytes of human periphery blood in vitro / 中华器官移植杂志

Objective To study the immunosuppressive mechanism of alkaloid sinomenine (SIN) by observing the effects of SIN on the proliferation and intracellular protein expression levels of nuclear factor of activated T cells (NF-AT) and interferon-gamma (IFN-γ) in CD4+ T lymphocytes of human periphery blood. Methods CD4+ T lymphocytes were isolated from PBMC suspensions with immunomagnetic beads and divided into five groups to culture. (1) Negative control group: no medicine was added to cell culture medium; (2) Positive control group: CsA solution (final concentration: 50ng/ml) was added to cell culture media; (3) Low-concentration SIN group (L-SIN): low-concentration SIN solution (final concentration: 10 μmol/L) was added to cell culture media; (4) Middle-concentration SIN group (M-SIN): middle-concentration SIN solution (final concentration: 200 μmol/L) was added to cell culture media; (5) High-concentration SIN group (H-SIN): high-concentration SIN solution (final concentration: 1000 tmol/L) was added to cell culture media. The proliferations of CD4+ T lymphocytes were observed. Western blotting was performed to detect the protein expression levels of NF-AT. FCM was used to determine the levels of IFN-γ. Results Compared with negative control group, the cell proliferation was significantly inhibited in H- and M-SIN groups (P＜0. 01 ). SIN concentration-dependently inhibited the protein expression levels of NF-AT and IFN-γ in CD4+ T lymphocytes of human periphery blood (P＜0.01). The protein expression levels of NF-AT and IFN-γ were lowest in positive control group. There was a close negative correlation between intracellular levels of NF-AT and cell proliferation inhibition ratio in CD4+ T lymphocytes of human periphery blood (rs = - 0. 969, P = 0. 000). Conclusion SIN can inhibit the protein expression of NF-AT and IFN-γ in CD4+ T lymphocytes of human periphery blood probably by decreasing protein levels of NF-AT to inhibit the activity and proliferation of CD4+ T lymphocytes.