Search

Objective: To explore the protective functions of recombinant protein RANK-Fc against hepatic ischemia/reperfusion injury and clarify its possible mechanism., Methods: Sixty male Balb/c mice were randomly divided into 3 groups according to different treatments: serum-free medium control (Sham) group, target gene retrovirus (RANK-Fc) group and empty vector retrovirus (eGFP) group. All mice were injected with 2.5 ml solution (with or without retrovirus) within 6 seconds via tail vein. After 3 days, the model of 70% hepatic ischemia/reperfusion was induced under warm conditions for 90 minutes after different periods of reperfusion in RANK-Fc and eGFP groups; Sham group underwent the same procedure without the occlusion of blood supply. Blood and liver samples were obtained at different time points (1, 3, 6 and 24 h; n = 5 in each). Reverse transcription-polymerase chain reaction (RT-PCR) was used for the evaluation of eGFP mRNA expression. RANK-Fc was assessed by Western blot. Liver transaminases and histopathological changes were used for the evaluation of hepatic injury. The activity of NF-κB in liver nucleus was analyzed by Western blot and immunohistochemistry. The activation level of JNK was also assessed by Western blot. Liver homogenate levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were detected by enzyme-linked immunosorbent assay (ELISA). Apoptosis was identified by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. The differences between three treatment groups at each time point were detected by the one-way ANOVA. Statistical analysis for inter-comparison was performed by Student's t test., Results: RANK-Fc and eGFP were successfully expressed in liver after hydrodynamics-based transfection. Compared with eGFP group, RANK-Fc significantly improved liver functions at the same time point (P < 0.01), decreased NF-κB nuclear translocation (t = 6.726, P < 0.01)and JNK phosphorylation (t = 6.713, P < 0.01)and obviously suppressed the release of pro-inflammatory cytokine TNF-α (t = 4.779, P < 0.01) and IL-6 (t = 5.482, P < 0.01). Morphological injuries were markedly alleviated while the expressions of immunohistochemical positive cells and TUNEL staining positive cells decreased in RANF-Fc group., Conclusion: RANK-Fc has protective functions against hepatic ischemia/reperfusion injury in mice. Its mechanism is at least partially related with the suppressions of proinflammatory NF-κB and proapoptotic JNK signaling pathways.

Aim: To explore a simple method to create intestinal autotransplantation in rats and growing pigs and to investigate the effect of L-arginine supplementation on serum nitric oxide (NO), nitric oxide synthase (NOS) and intestinal mucosal NOS and Na+-K+-ATPase activity during cold ischemia-reperfusion (IR) in growing pigs., Methods: In adult Wistar rat models of small bowel autotransplantation, a fine tube was inserted into mesenteric artery via the abdominal aorta. The superior mesenteric artery and vein were occluded. Isolated terminal ileum segment was irrigated with Ringer's solution at 4 degrees and preserved in the same solution at 0-4 degrees for 60 min. Then, the tube was removed and reperfusion was established. In growing pig models, a terminal ileum segment, 50 cm in length, was isolated and its mesenteric artery was irrigated via a needle with lactated Ringer's solution at 4 degrees. The method and period of cold preservation and reperfusion were described above. Ten white outbred pigs were randomly divided into control group and experimental group. L-arginine (150 mg/kg) was continuously infused for 15 min before reperfusion and for 30 min after reperfusion in the experimental group. One, 24, 48, and 72 h after reperfusion, peripheral vein blood was respectively collected for NO and NOS determination. At the same time point, intestinal mucosae were also obtained for NOS and Na+-K+-ATPase activity measurement., Results: In adult rat models, 16 of 20 rats sustained the procedure, three died of hemorrhage shock and one of deep anesthesia. In growing pig models, the viability of small bowel graft remained for 72 h after cold IR in eight of 10 pigs. In experimental group, serum NO level at 1 and 24 h after reperfusion increased significantly when compared with control group at the same time point (152.2+/-61.4 micromol/L vs 60.8+/-31.6 micromol/L, t=2.802, P=0.02<0.05; 82.2+/-24.0 micromol/L vs 54.0+/-24.3 micromol/L, t=2.490, P=0.04<0.05). Serum NO level increased significantly at 1 h post-reperfusion when compared with the same group before cold IR, 24 and 48 h post-reperfusion (152.2+/-61.4 micromol/L vs 75.6+/-16.2 micromol/L, t=2.820, P=0.02<0.05, 82.2+/-24.0 micromol/L, t=2.760, P=0.03<0.05, 74.2+/-21.9 micromol/L, t=2.822, P=0.02<0.05). Serum NOS activity at each time point had no significant difference between two groups. In experimental group, intestinal mucosal NOS activity at 1 h post-reperfusion reduced significantly when compared with pre-cold IR (0.79+/-0.04 U/mg vs 0.46+/-0.12 U/mg, t=3.460, P=0.009<0.01). Mucosal NOS activity at 24, 48, and 72 h post-reperfusion also reduced significantly when compared with pre-cold IR (0.79+/-0.04 U/mg vs 0.57+/-0.14 U/mg, t=2.380, P=0.04<0.05, 0.61+/-0.11 U/mg, t=2.309, P=0.04<0.05, 0.63+/-0.12 U/mg, t=2.307, P=0.04<0.05). In control group, mucosal NOS activity at 1 and 24 h post-reperfusion was significantly lower than that in pre-cold IR (0.72+/-0.12 U/mg vs 0.60+/-0.07 U/mg, t=2.320, P=0.04<0.05, 0.58+/-0.18 U/mg, t=2.310, P=0.04<0.05). When compared to the normal value, Na+-K+-ATPase activity increased significantly at 48 and 72 h post-reperfusion in experimental group (2.48+/-0.59 micromol/mg vs 3.89+/-1.43 micromol/mg, t=3.202, P=0.04<0.05, 3.96+/-0.86 micromol/mg, t=3.401, P=0.009<0.01) and control group (2.48+/-0.59 micromol/mg vs 3.58+/-0.76 micromol/mg, t=2.489, P=0.04<0.05, 3.67+/-0.81 micromol/mg, t=2.542, P=0.03<0.05)., Conclusion: This novel technique for intestinal autotransplantation provides a potentially consistent and practical model for experimental studies of graft cold preservation. L-arginine supplementation during cold IR may act as a useful adjunct to preserve the grafted intestine.