The Study Of Natural Immune Cytokine HMGB1 In Renal Ischemia Reperfusion Injury And Xenotransplant Rejection

PartⅠUpregulation of HO-1 to protect renal ischemia reperfusion injury via inhibiting release of HMGB1.[Objective] Based on the results of the previous experiments, the mechanism of protection of the protective gene HO-1 on the rat renal ischemia reperfusion injury is studied more thoroughly.[Method] A dose of 2.5mg/kg of CoPP was administered intraperitoneally into rats 48h and 24h before 47min of renal ischemia. Blood and kidney samples were collected 24h after reperfusion. Serum Cr and BUN were determined to evaluate kidney function. HO-1 expression in kidney was detected by immunohistochemistry and western-blot. Prolongation of renal ischemia time to 80min was undertaken to study the protective effects of CoPP on rat mortality. The expression of HMGB1, TRL4, MPO, and TNF-αwas detected by immunohistochemistry, and the cell apoptosis was detected by TUNEL assay with cell counted.[Result] The mean serum Cr and BUN levels of the IRI control rats were 134.37±24.26μmol/L and 30.10±3.09mmol/L, CoPP treatment significantly improved renal function after I/R, the mean serum Cr and BUN levels were 48.92±12.92μmol/L and 13.99±5.00mmol/L respectively(p<0.05 vs.IRI control group). CoPP therapy significantly protected rats from lethal kidney ischemia. The expression of HMGB1 and TLR4 in the tissues treated with CoPP was significantly weaker than the control group and the marker of monocyte MPO and cytokine TNF-αin the protected group is also much weaker than the control. The apoptotic cells counted in the HO-1 protected group were statistically less than in the control group.[Conclusion] Inhibition the release of HMGB1 maybe one of the mechanisms that protective gene HO-1 prevents rat renal ischemia reperfusion injury; furthermore it inhibits the inflammation response and reduces injury. PartⅡNeutralizing extracellular HMGB1 by using antibody prevents mice from renal ischemia-reperfusion injury[Objective] To investigate the pattern that High mobility group box 1 (HMGB1) releases during renal ischemia/reperfusion injury (IRI) and relative mechanism that neutralizing the extracellular HMGB1 prevents mice from renal IRI.[Methods] Renal IRI was induced by clamping the left renal pedicle for 60 min, and some of the kidneys were removed immediately as others collected 24 h later for detection of HMGB1 expression with Immunohistocheistry and Western blot. Rabbit anti-mouse HMGB1 antibody was administered 24 h and 30 min before ischemia as control mice with normal sodium. The therapeutic effects were evaluated in renal function after 24 h of reperfusion, also with histologic examination. Deposits of anti-HMGB1 antibody were detected by Immunohistochemistry and MPO expression was measured by Immunofluorescence. Additionally, TUNEL assays were used to evaluate the apoptosis of renal cells.[Results] HMGB1 expressed in cell nucleus was passively released by ischemic renal cells as to cytoplasm or even out of cells early as the time prior to reperfusion. When mice were treated, the therapic anti-HMGB1 antibody was detected to deposit at local renal tubules, peritubular capillaries and glomcruli, but no deposits were found in control mice. Consequently, levels of serum creatinine and blood urea nitrogen were significantly decreased in mice treated with anti-HMGB1 antibody ((32.24±22.51)μmol/L, (25.81±11.41)μmol/L, respectively) compared with control mice ((82.46±30.13)μmol/L, (46.46±9.61)μmol/L, respectively,p<0.05). Correspondently, Tissue damage caused by IRI was markedly reduced with less necrosis and hemorrhage as a result of neutralizing antibody treatment. In addition, MPO expression was extremely diminished in treated mice compared with control kidneys, and apoptosis was also significantly decreased (p<0.05).[Conclusion] HMGB1 may act as a crucial mediator during IRI, and blockage of HMGB1 before ischemia can protect mice against renal IRI, and the mechanism of protection might come from targeting HMGB1 to interfering process of inflammation induce by IRI. PartⅢAnti-HMGB1 antibody prolongs rat to mouse cardiac xenograft survival xia inhibiting products of anti-donor antibody.[Objective] To investigate the role of High mobility group box 1 (HMGB1) in the rejection of rat to mouse cardiac xenotransplantation, and relative mechanism that neutralizing the extracellular HMGB1 prolongs xenograft survival.[Methods] Heterotopic cardiac xenotransplantation was established from One-week-old SD rat to BALB/c mouse. Rabbit anti-mouse HMGB1 antibody was administered by intraperitoneal injection on day-1,0 and every other day until the study end point or graft rejection, while control mice was received control IgG or PBS instead of neutralizing antibody. The therapeutic effects were evaluated by the survival of xenograft, and some of hearts were obtained from recipients which were therapied at day 6 as symotanians control. MPO, CD3, HMGB1, TLR4 expression were measured by Immunohistochemical staining and cytockine TNF, IL2, IL10 were evaluated by RT-PCR. TUNEL assays were used to evaluate the apoptosis of renal cells. Additionally, serum or intra graft IgG and IgM were measured and CRPs alse were avaluated to analyse the mechanism.[Results] The xenograft survival was significantly prolonged by neutrolizing antibody from 5 days to 9 days. In concurrent control groups, the neutralizing group exibited clear vessels, fewer interstitial hemorrhages, and integrated endothelial cell with mass JG12-positive cells and less apotosis cells. Furthermore, less MPO and CD3 positive cells were observed and HMGB1 with its receptor TLR4 were much weaker than the control group. More interestingly, the FCM and immunohistochemistry showed the less IgG and IgM in the circle and xenograft, and much more expression of CRPs in the treated gourp.[Conclusion] we have shown that anti-HMGBl antibody efficiently delayed AVR by markedly inhibiting anti-donor xAb production and up-regulating CRPs in the xenograft in rat to mouse cardiac transplantation.