| ObjectThis study aims to investigate the protective effects of the novel helix B surface peptide (HBSP) through mice renal ischemia reperfusion injury model, investigate the expression and distribution of the tissue protective receptor subunits and the effect of ischemia reperfusion injury on them, including beta common receptor (βcR), EPOR monomer (EPOR) and their heterodimer form (βcR/EPOR) and to investigate the impact of HBSP on the activation of caspase-9and caspase-3in apoptosis and further to have a try to reveal the role of PI3K/Akt pathway during the renal protection process.The results of this research may provide basic evidence for its further translation.Methods Male BALB/c mice (weighted20-25g) of SPF grade were randomly divided into:1) sham group (Sham; n=6);2) Vehicle group, I/R injury with phosphate-buffered saline vehicle intraperitoneally injected (I/R; n=6);3) HBSP group, IR injury with HBSP of8nmol/kg intraperitoneally injected at1min,6h,12h after the operation (HBSP; n=6);4) HBSP plus Wortmannin group, treated with HBSP plus one dose of Wortmannin,1mg/kg, i.p.,30min before operation (H+W; n=6). The IR operation was performed as clamping the bilateral renal pedicle for30min and samples were obtained after the48-h reperfusion. The renal function was evaluated basing on the level serum creatinine and blood urea nitrogen in each group. HE staining and TUNEL were performed in renal sections to assess the tissue damage and apoptosis. To detect the expression and distribution of βcR and EPOR in the kidney, immunohistochemistry was performed in sections obtained in the part Ⅰ study. Co-Immunoprecipitation was used to quantitatively detect the expression of βcR in combination with EPOR. Immunohistochemistry was used to detect the activated cleaved caspase-3positive cells in kidney. Further investigation through western blot was performed to detect the expression of inactivated full-length caspase-9and activated37and39kD fragments of caspase-9and cleaved17kD caspase-3; The expression of PI3K p85and Akt, as well as the phosphorylated Akt was also investigated by western blot.Results The level of serum creatinine and blood urea nitrogen in HBSP group was much lower than IR group (23.83±1.32vs.119.80±6.41μmol/L, p<0.01;10.62±0.79vs.50.33±5.09mmol/L, p<0.01). The HE staining section also showed a significantly lower score of renal tubule damage in HBSP group than IR group (1.17±0.31vs.4.50±0.43,p<0.01), including remarkably ameliorated renal structure, clearer glomeruli and tubule, milder tubular epithelia oedema, more intact basal membrane and less cas. While the TUNEL results showed much less amount of apoptosis positive in HBSP treated mice than that in IR ones (3.83±1.40vs.24.17±1.45,p<0.05). Both the βcR and EPOR existed constructively on the surface of renal tubular epithelia and could be up-regulated in the ischemia reperfusion injury (94.83±4.19vs.130.70±13.80,p<0.05;184.3±6.965vs.246.50±15.43,p<0.01). The heterodimeric PcR with EPOR was also constitutively expressed in kidney and ischemia reperfusion injury played a stimulating role to it (EPO co-precipitated βcR/GAPDH,0.47±0.12vs.0.95±0.12,p<0.05). There were no statistical differences in the expression of PI3K p85among groups. The expression of Akt and its phosphorylation at both Ser473and Th308sites were stimulated significantly by HBSP (/β-actin,0.44±0.04vs.0.21±0.04,p<0.05;2.09±0.55vs.0.13±0.01,p<0.05;0.77±0.12vs.0.12±0.06, p<0.01. However, the inhibition of PI3K/Akt pathway by wortmannin could obviously revers the effects of HBSP on Akt and its phosphorylation (/β-actin,0.07±0.04vs.0.44±0.04,p<0.05;0.05±0.01vs.2.09±0.55, p<0.05;0.01±0.00vs.0.77±0.12,p<0.01). The number of caspase-3positive cells in kidneys in HBSP appeared much fewer than IR (1.33±0.42vs.8.67±0.67, p<0.01), which was also reversed by wortmannin. Western blot investigation showed no significant differences in the expression of the49kD full-length caspase-9. However, its activated fragments, including both37kD and39kD were remarkably reduced by HBSP (/β-actin,0.60±0.07vs.1.55±0.32, p<0.05;0.05±0.01vs.0.89±0.31, p<0.05). The impact of HBSP on the17kD caspase-3activated cleavage was similar (/β-actin,0.08±0.03vs.0.26±0.04, p<0.05). In the same way, wortmannin increased these HBSP reduced caspases by inhibiting the PI3K/Akt pathway (/β-actin,2.95±0.52vs.0.60±0.07, p<0.05;2.01±0.17vs.0.05±0.01, p<0.01;1.85±0.62vs.0.08±0.03, p<0.05).Conclusion The novel erythropoietin derived peptide, HBSP, could efficiently protect the kidney from ischemia and reperfusion injury. It significantly ameliorated the renal dysfunction and reduced both the tissue damage tubule apoptosis. The tissue protective receptor PcR/EPOR exists in kidney and can even be up-regulated in ischemia reperfusion injury, which well indicates the capacity of HBSP in renal protection. HBSP can stimulate the PI3K/Akt pathway by increasing the expression and phosphorylation of Akt, in order to reduce the activation of capase-9and caspase-3and achieve its anti-apoptosis role. Therefore, HBSP should be a potential candidate of reno-protective agent. |
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