The Mechanism Of Effect Of Augmenter Of Liver Regeneration On Immunosuppression And The Relationship To Regulatory T Cells

Part Ⅰ The Effect of hALR on Proliferation of PBMC and the Relationship to TregObjective:Augmenter of liver regeneration (ALR) has effect of immunoloregulation, but the underlying mechanism is not completely understood. The objective of this study is to observe the effect of hALR on proliferation of ConA-stimulated PBMC and on differentiation and proliferation of CD4+CD25+FoxP3+Tregs, and to investigate the underlying mechanism of immunosuppressive role of hALR.Methods:The PBMC from healthy donors was isolated and stimulated with ConA, then cultured in three groups(Control、ConA and ConA+hALR) in vitro for60h. MTS assay was used to determine the cellular viability. Flow cytometry (FCM) and western blot assay were used to detect the early apoptotic ratios and the main proteins of apoptosis. FoxP3mRNA was detected by Real-time fluorescent quantitation PCR, and the ratios of CD4+CD25+FoxP3+Treg and the mitotic cycle of Treg were observed by FCM.Results:The proliferation of ConA-stimulated PBMC was obviously inhibited by hALR (ConA group vs ConA+hALR group, P<0.01), and the apoptosis of ConA-stimulated PBMC was decreased by hALR (ConA group vs ConA+hALR group, P<0.01). Meanwhile, hALR increased the expression of FoxP3mRNA and up-regulated the percentage of Tregs (ConA group vs ConA+hALR group, P<0.01). In addition, hALR could relieve the G2/M cell cycle arrest of Treg induced by ConA (ConA group vs ConA+hALR group,P<0.01)Conclusions:The data demonstrated that hALR inhibited the proliferation of ConA-stimulated PBMC and up-regulated the percentages of Treg. It indicated us that hALR inhibited the proliferation of ConA-stimulated PBMC through the pathway of induction of Tregs possibly. Part Ⅱ The Mechanism of Induction of Treg and the Pathways of Immunosuppression of hALRObjective:Cytokines played important roles in the differentiation and proliferation of Treg. In this study, we detected the levels of the cytokines (TGF-β1、IL-10、IL-2) in the supernatant and observed the main signaling pathway related molecular (ERK1/2、NF-κB), then discussed the possible mechanism of hALR on induction of Treg and the underlying signaling pathway of immunosuppressive role.Methods:The PBMC from healthy donors was isolated and stimulated with ConA, then cultured in three groups(Control、ConA and ConA+hALR) in vitro. The levels of TGF-β1, IL-10and IL-2in the supernatant were determined by ELISA, and the expressions of the main signaling pathway related molecular were detected by western blot assay.Results:The levels of TGF-β1and IL-10in the supernatant were significantly increased by hALR. On the contrary, the level of IL-2was decreased notably (ConA group vs ConA+hALR group, P<0.01) Meanwhile, hALR inhibited the activation of ERK1/2and the expression of NF-κB (p65) obviously (ConA group vs ConA+hALR group, P<0.01).Conclusions:The levels of the cytokines in the supernatant were regulated by hALR, increasing the production of TGF-β1and IL-10, but decreasing IL-2. Notably, hALR inhibited the activation or expression of the signaling pathway relative molecular. On one hand, it was indicated that cytokines regulated by hALR might be the mechanism of induction of Treg and the approach of immunosuppressive role. On the other hand, the molecular mechanism of hALR on immunosuppressive role was the regulation of the signaling pathway molecular. Part Ⅲ The Effect of hALR on Immunological Rejection of HCT in ALF RatsObjective:As hALR up-regulated Treg and inhibited proliferation of immunocytes in vitro, in this part of study, we investigated the potential effect and mechanism of hALR on ALF rats treated with intraperitoneal HCT in vivo.Methods:ALF rats induced by D-GalN were studied, and were intraperitoneal injected with or without hepatocytes and hALR24h after the induction. The hepatocytes were isolated from healthy S-D rats with an improved two-step semi-situ recirculating collagenase perfusion method. The ALF rats were divided in four groups (PS group, HCT group、 HCT+hALR group and hALR group). Animal survival was assessed, and the levels of TNF-α and IL-1β were detected by ELISA. Histological examination was performed and immunological responses were identified by immunohistochemistry assay. FCM was used to determinate the percentages of Treg in immunocytes in the greater omentum. The expressions of TGF-β1and IL-10mRNA were detected by Real-time fluorescent quantitation PCR.Results:It showed that hALR improved the survival of ALF rats treated with HCT (P<0.05). The inflammatory damage was reduced significantly in the transplanted islets by hALR after being strained with PAS. Markedly lower percentages of CD68, CD4and CD8positive immunocytes and a higher percentage of Foxp3positive immunocytes in the greater omentum were observed in HCT+hALR group. Additionally, hALR up-regulated the percentage of Treg of the immunocytes in the greater omentum, and increased the expression of TGF-β1and IL-10mRNA, decreased pro-inflammation cytokines (HCT group vs HCT+hALR group, P<0.05)。Conclusions:In the treatment of ALF rats, hALR up-regulated Treg, inhibited the inflammation, attenuated the damage of immunological rejection, and improved the overall survival. It indicated that hALR inhibited the immunological rejection, promoted the transplantation tolerance of ALF rats, and the underlying mechanism was the up-regulation of Treg by hALR.