| Objective (1) To investigate the acute allograft rejection occurred after orthotopic liver transplantation (OLT) in Lewis-BN rats and the effect of augmenter of liver regeneration (ALR) on acute allograft rejection (AR) after OLT. (2) To study the effect of ALR on the activation, apoptosis of intragraft lymphocytes and the gene transcripts and expressions of IFN-γand IL-2 intragraft lymphocytes after OLT (3) To observe the effect of ALR on the gene transcripts and expressions of TNF-αand IL-10 in Kupffer after OLT, explore the possible mechanisms to affect AR. (4) To investigate the effect of ALR on the proliferation, activation, apoptosis and the gene transcript and expression of cytokines of lymphocytes in spleen, clarity its possible mechanisms, find out the exact roles of Kupffer cells in these effect.Methods (1) Allogenetic orthotopic liver transplantation model was performed from Lewis to BN rat and isogenetic liver transplantation model was performed from BN to BN rat. All recipients were randomly divided into three groups: ALR group (with ALR 100μg/kg/d intraperitoneal injections postoperatively), Allograft group (with the same volume of saline intraperitoneal injections) and Isograft group. 3, 5 and 7 days posttransplantation, Recipient survival, histopathological characteristics of hepatic allografts were investigated, the AR was scored by baff code with rejection activity index (RAI). Plasma levels of alanine aminotransferase (ALT), aspartase mainotransferase (AST) and total bilirubin (TBIL) were measured with an automatic biochemical analyser. (2) On 7 days posttransplantation, the expressions of IL-2 and INF-γwere performed by Immunohistochemistry. Lymphocytes in liver grafts were isolated by Lydroxypropylmethyl Cellulose, the IL-2R positive and apoptosis ratios were investigated with flow cytometer (FCM) in isolated lymphocytes, the IL-2 and INF-γgene transcripts and expressions in isolated lymphocytes were performed by realtime-PCR and western-blotting. (3) Kupffer cells in liver grafts were isolated 7 days after OTL using the technique of isopyknic gradient centrifugation with Percoll, IL-10 and TNF-αgene transcripts and expressions in the isolated kupffer cells were performed by realtime-PCR and western-blotting. (4) The spleen lymphocytes in Lewis rat and the kupffer cells in BN rat were isolated, cultured and divided into four groups: group A: LCs (5×106) + ConA (50ug/ml), group B: LCs (5×106) + ALR (30ug/ml) + ConA (50ug/ml), group C: LCs (5×106) + ConA (50ug/ml)+KCs (1×106) , group D: LCs (5×106) +ALR (30ug/ml) + ConA (50ug/ml) + KCs (1×106). IL-2, IL-10, INF-γand TNF-αlevels in supernatant were measured with enzyme linked immunosorbent assay (ELISA), respectively. The activation and apoptosis of lymphocytes were detected by FCM, and the proliferations of lymphocytes were detected with 3H-TdR method, respectively.Results (1) Duration of operation was not significantly different among Allograft, Isograft and ALR groups. Without using any immunosuppressive therapy, allograft group showed typical acute rejection with severely impaired liver function after 5 and 7 days, but there were no difference in Isograft and ALR groups. Compared the RAI scores with in allograft group (2.17±0.75) vs ALR (2.67±1.03) and isograft (1.67±0.75) groups after 3 days, there were no significantly different (P> 0.05). But 5 and 7 days after OLT, compared them with in allograft group (6.33±1.03 and 8.17±0.75) vs ALR group (2.83±0.75 and 2.83±0.75) and isograft group (1.50±0.55 and 1.33±0.52), there were different significantly (P<0.05). Comparing the mean survival times showed that allograft group was only 11.17±1.47d, it was fewer significantly than ALR (46.17±10.22d) and isograft (49.67±17.31d) groups (P<0.05).(2) In ALR group, the IL-2R positive ratio of infiltrated lymphocytes (0.08±0.03) was decreased accompaning with higher apoptotic rate (0.45±0.06), compared those with in Allograft group (0.25±0.07 and 0.16±0.04) after OLT 7 days (P<0.05). But compared those with in Isograft group, there was no significant difference (P> 0.05). In gene and protein levels, IL-2 and IFN-γin infiltrated lymphocytes were notably upregulated in Allograft group, but in ALR and Isograft groups, they were all kept in normal levels (P<0.05). The expressions of them investigated by immunohistochemisty in the three groups showed the same trend.(3) At 7 days post transplantation, the expression in graft of TNF-αinvestigated by immunohistochemisty in Allograft group enhanced higher than in Isograft and ALR groups. However, the expression of IL-10 in Allograft group descended significantly compared with in ALR and Isograft groups (P<0.05). Meanwhile, the levels of TNF-αand IL-10 investigated with western blot reveal the same trend. The levels of TNF-αand IL-10 mRNA in KCs showed that: the level of TNF-αin ALR group (3.86±0.42) was lower than Allograft group (8.69±0.63) significantly (P<0.05) and mild higher than in isograft group (3.43±0.34), but this step up was not signicant (P>0.05), The levels of IL-10 in Allograft, ALR and isograft groups are 1.24±0.18, 5.76±0.32 and 4.58±0.57 respectively, showed a clashing trend.(4) The proliferation and the activation rates in group B (2.0083±0.245×10~6cpm and 0.2817±0.0392) were markedly lower than those in group A (3.9817±0.2101×10~6cpm and 0.465±0.0568) (P<0.05), but they were higher than that in group C (1.545±0.2239×10~6cpm, 0.2983±0.0319) and group D (0.64±0.1401×10~6cpm, 0.0783±0.0271) (P<0.05). Compared those with in group D, the proliferation and activation rates in group C were enhancer significantly (P<0.05). There were no difference in apoptosis rates between group A (0.0457±0.008) and B (0.0622±0.0148) (P>0.05). However, they were all lower than that in group C (0.1617±0.0263) (P<0.05). Furthermore, the apoptosis rate in group D (0.2775±0.0199) was higher than group C (P<0.05). The levels of cytokines in supernatant showed that the levels of IL-2, IFN-γand TNF-αin group B descended significantly compared with in group A, but the IL-10 step up markedly (P<0.05). The levels of IL-2, IFN-γand TNF-αin group C were descended compared with in group A (P<0.05), but the IL-10 step up significantly (P<0.05). Comparing them in group C with group D, the levels of IL-2, IFN-γand TNF-αin group D are lower than group C, but the IL-10 is higher than group C (P <0.05).Conclusions (1) ALR could elevate the survival and attenuate acute rejection of rats which experience acute rejection after allograft liver transplantation. (2) ALR could inhibit the function and activation of infiltrated lymphocytes in liver allograft. Furthermore, ALR promotes the apoptosis of lymphocytes, thus ameliorates the situation of liver allograft. (3) ALR could accommodate KCs function of TNF-αand IL-10 expression in liver allograft, thus inhibits the function and activation, promotes the apoptosis of infiltrated lymphocytes. (4) ALR could inhibit the proliferation and activation of lymphocytes in spleen, but can promote the apoptosis of lymphocytes in spleen directly. (5) ALR can promote the KCs function of killing to lymphocytes in spleen by reducing TNF-αexpression and augmenting IL-10 expression.
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