Study On The Mechanism Of Human Augmenter Of Liver Regeneration-Mediated Immunosuppression

Objective: Human augmenter of liver regeneration (hALR) is a non-specific, with thermal stability, and promoting cell regeneration factor, which inhibits the proliferation of mononuclear cells and production of cytokines INF-γ, IL-2. However, the mechanism is unclear. The signaling pathways of MAPK / ERK, PKC-NF-KB and calcium are involved in activation of peripheral blood mononuclear cells and also targets for many immunosuppressive agents. Whether ALR as an immunosuppressive agent inhibits these three main pathways or not is needed to be clarified. Therefore, this study was to observe the effect of ALR on these three signaling pathways.Apoptosis is another important way of immune suppression.we have noticed that rat ALR can promote monocyte apoptosis. It is unclear whether ALR induces apoptosis by inhibiting IL-2 production or stimulating the apoptosis signaling pathway? Therefore, in this study we also observe the effects of ALR on the signaling pathway of apoptosis-related Caspse-3 and cytokines production such as IL-2.Methods1 Role of ALR on MAPK / ERK1) Determination of the optimcal time and dose for ALR the optimal time was selected in the series of time 16h,40h and 60h at which PBMCs stimulated with ConA (5ug/ml) proliferated significantly (p<0.01) by the MTT method.The optimcal dose of ALR was determined among a series concentration of 0.5ug/ml, 1ug / ml, 2ug/ml, 7.5ug/ml, 10 ug / ml, 15 ug / ml and 30 ug / ml , at whith ALR inhibited the proliferation stimulated by ConA significantly at 60h.2) Alteration of MAPK/ERK according with the established dose and time to observe whether ALR 30ug/ml had effect on the MAPK / ERK at 60h. Cells were divided into N, ALR, ConA and ALR +ConA group . Western blot was intaked to detect expression of MAPK / ERK.3) Dynamic alteration of MAPK / ERK. Based on the changes of ERK at 60h, courses of ERK altreration were observed at a series time of 10 min, 30min,1h,2h,4h,8h,16h,32h and40h.2 Role of ALR on Ras The expression of Ras were observed at the same time point of ERK using Western Blot to further determine whether ALR suppression was through the pathway of Ras-MAPK/ERK.3 Role of ALR on PKC-NF-KB The PKC-NF-KB pathway function was measured by the expression in PBMC followed the observison that the ras didn't manipulate the activation of ERK in the metaphase. 4 Role of ALR on calcium In the above two signaling pathway, no one was observed to work in the period of 4-8h. Therefore the intracellular calcium and supernatant calcium were studied by calcium-sensitive probe loading and methyl thymol blue colorimetric assay to assert the role of ALR.5 The role of ALR on apoptosis Flow cytometry was intake to appraise ALR effect on apoptosis in PBMC. Gel electrophoresis was used to observe whether there was DNA degradation. Western Blot used to dectect the activation of Caspase-3.6 Affect of ALR on IL-2,IL-4 and IL-10 The levels of IL-2, IL-4 and IL-10 were detected by ELISA in cell supernatants at the same time of ERK.Results1 Action for ALR on MAPK / ERK1) The proliferation of monocytes stimulated with ConA enhanced in time course-dependent manner, with the statistical significance at 16h ,40h (p = 0.0413,0.0479) and 60h (p <0.01). ALR inhibited cell proliferation in a dose-dependent maner and it attained the significant inhibitory effect (p <0.01) while the concentration reached to 30 ug / ml.2) Alteration of ERK at 60h compared with the normal group, the content of phosphorylated and non-phosphorylated ERK in ConA group were increased; with ConA group, ALR+ConA group non-phosphorylated ERK phosphorylation levels were reduced especially ERK2 phosphorylation; with the normal group, ALR group ERK2 phosphorylation decreased. In each group, there was no difference between the ratios of phosphorylated ERK to non-phosphorylated ERK.3) The role of ALR for ERK alteration The content of phosphorylated ERK in ALR group at 10 min and ConA group at 30min were significantly increased than that in normal group. The content of phosphorylated ERK in normal group peaked at 2h. The content of phosphorylated ERK,especially ERK2, in ALR+ConA group were significantly reduced at 10min,30min,1h and 2h.There was no difference of phosphorylated ERK among the groups at 4h.The total ERK content in each group Cells gradually increased, at 4-16h achieved a higher level, followed by decline, 40h reached the lowest level.The content of ERK in each group at the same time point showd no difference before 60h, while there were significant alteration between ERK contents at 60h.2 The role of ALR for Ras pathway In the whole culture process, Ras showed multiple fluctuations in each group. Ras in N and ConA group cells showed fluctuations 3 times (10min-1h,1-8h,8-40h) and (10min-1h,1-4h,4-40h) respectively , ALR and ALR+ConA group ras appeared 2 times (1-8h,8-40h). The content of Ras in ConA in the period of 10min-1h was significantly increased rather than that in normal group. Compared with the normal group the situation reversed in ALR+ConA and ConA groups at 16h. ALR+ConA cells'Ras in 10min-1h was significantly reduceed than ConA group especially at 30min.3 The role of ALR for PKC-NF-KB pathway In each group the expression of PKC and NF-KB shew the same trendency line.PKC-NF-KB system in the cell culture course altered after 8h and ALR made ConA-induced peak delay.4 The role of ALR for calcium alteration The level of calcium shew increasing and declining in turn besides of the group of ALR+ ConA. In ALR group, the concentration of calcium ion peaked at 30min, ConA group at 1h, and N group at 2h. Calcium concentration in each group at 4h was no significant difference, then rapid decline. After 8h calcium ion in ALR group fluctuate again. Compared with the ConA group, intracellular calcium ion concentration in ALR+ConA group was significant differences at 1h.5 Apoptosis of PBMC caused by ALR compared with the normal group, apoptosis in ALR and ConA group significantly increased at 60h, while apoptosis in ALR+ConA group significantly reduced than that in ConA group. Caspase-3 expression in ALR+ ConA group significantly decreased than ConA group at 60h time point. 6 Affect of ALR on IL-2,IL-4 and IL-10 IL -2 in ALR group's supernantant peaked at 16h and IL-10 at 32h as well as in ConA group. Wihle the peaks of IL-2 was delayed and IL-10 was in advance in ALR+ConA group supernantant.Conclusion1 ALR inhibits PBMC proliferation by suppressing Ras-MAPK/ERK2 pathway.2 ALR has a role in two-way on MAPK / ERK in human PBMC without ConA stimulation, not only promote the phosphorylation of ERK, but also inhibite the phosphorylation of ERK2.3 ALR inhibits cell proliferation by inhibiting intracellular calcium signals.4 The role of ALR to modulate calcium is also two-way trip, activating the calcium signal and suppressing calcium signaling pathway.5 ALR inhibits the PKC-NF-KB pathway to affect cell proliferation and cytokine secretion.6 ALR affects PBMC apoptosis to regulate immune response.7 ALR inhibits IL-2 produce by IL-10 secreting.