Sirolimus Induces Rat Na(?)ve CD4~+ T Cells To Regulatory T Cells And Its Application

Part 1: Sirolimus Induces Na?ve CD4⁺ T Cells to Regulatory T Cells In VitroObjective: Sirolimus Induces Naive CD4⁺ T cells to regulatory T cells In Vitro and insight its possible mechanism.Methods: Na?ve CD4⁺T cells were separated from rat PBMC. These cells were stimulated with anti-CD3/CD28-beads. IL-2, TGF-β, and SRL were added to some culture. The cells were harvested and stained the phenotypes of nature regulatory cells. Membran bound TGF-βwas stained after restimulation for 72 hours. The suppress function of different condition cells was analyzed by the dilution of CFSE of T cells. Results: Combine SRL with TGF-βnot only enhanced FoxP3 expression by CD4⁺cells, but also appeared more resistant to apoptosis compared to others. The combination of SRL and TGF-βenabled CD4⁺cells to express a phenotype and trafficking receptors similar to natural Tregs, and no significant cytotoxicity was observed. CD4_SRL were anergic and had potent in vitro suppressive activity.Conclusion: Stimulation of rat CD4⁺T cells in the presence of SRL results in a highly increased suppressor function is TGF-βdependent. Part 2: Sirolimus Induce CD4⁺CD25⁺ FoxP3⁺ T Regulatory Cells Proliferations in RatsObjective: to investigate sirolimus (SRL) induce CD4⁺CD25⁺FoxP3⁺ T regulatory cells proliferations in rats.Methods: 20 male SD rats weight 200²20g were randomized into experiment group and control group (10/group), experimental rats were pretreated with sirolimus (2 mg/kg/day) by oral gavage for two weeks, the rats were given with saline as the control group. All rats were housed in controlled environmental conditions with a 12-h light–dark cycle, and freely access to standard rodent diet and water. Peripheral blood was collected, spleen cells and thymus cells were prepared, CD4⁺CD25⁺T cells were detected with flow cytometer. The expression of FoxP3 mRNA was detected in spleen by real-time PCR detection. The serum TGF-β, IL-10 levels were measured by enzyme-linked immunosorbent assay (ELISA).Results: The present of CD4⁺CD25⁺T cells among mononuclear cells was significantly higher in peripheral blood, spleen and thymus compared with control group (P<0.05). Furthermore, real-time PCR showed that the expression of the FoxP3 mRNA of experimental rat spleen was 4.1 folds as that of control group (P<0.05). The ELISA showed that sirolimus could significantly increased the content of TGF-βand IL-10 (P<0.05).Conclusion: RPM can effectively induce CD4⁺CD25⁺ FoxP3⁺ T regulatory cells proliferations in rat, and increased TGF -β, IL-10 secretion.? Part 3: Protection of Sirolimus Pretreatment Against Liver Ischemian Reperfusion Injury in RatsObjective: to investigate the protective effect of sirolimus pretreatment against liver ischemic reperfusion (I/R) injury in rat model and find its possible mechanism.Methods: 48 male SD rats were randomized into four groups (12/group), A: sham group with saline, B: sham group with sirolimus, C: saline-operated group, D: sirolimus-operated group. The rats were pretreated with either saline or sirolimus (2 mg/kg/day) by oral gavage for two weeks. The establishment of rat partial liver model of I/R injury, the samples were collected at 24h after the I/R, the serum ALT and AST levels were measured, HE staining to obverse the histologic changes by light microscopy, used flow cytometer to analyze the frequency of CD4⁺CD25⁺T cells among mononuclear cells of liver tissue, the expression of FoxP3 mRNA was detected in liver by real-time PCR detection, the serum TGF-β, IL-10 levels were measuered by enzyme-linked immunosorbent assay (ELISA).Results: Serum ALT and AST were significantly decreased in the sirolimus-operated group compare with saline-operated group (P<0.05), histological damage was also significantly lighter. The present of CD4⁺CD25⁺T cells among mononuclear cells were (6.12±1.87)%, (22.36±6.75)%, (4.53±1.02)% and (13.29±3.16)% by respectively in liver tissue, furthermore, the expression of the FoxP3 mRNA were significantly higher in sirolimus group than saline group (P<0.05). The ELISA showed that sirolimus could significantly increased the content of TGF-βand IL-10 (P<0.05).Conclusion: Pretreatment of sirolimus can effectively protect against liver ischemia-reperfusion injury in rat, which may be related to induce CD4⁺CD25⁺FoxP3⁺ T regulator cells by sirolimus, the cells increased TGF -β, IL-10 secretion, and inhibited the inflammatory response.