Study Of Fusion Protein TAT-Cytoglobin On Acceleratigg Skin Woound Heealing Annd Liver Fibrosis Treatment

Cytoglobin (Cygb), a21.4kDa plasmosin originally identified in cultured rathepatic stellate cells, is a member of hexacoordinate globin superfamily. Itsperoxidase activity and effect of oxygen free redical scavenger have beendemonstrated. Cygb has also been found to suppress hepatic fibrosis throughup-regulation of Cygb gene expression. However, as a cytoplasmic protein lacking ofactive transporting mechanisms, exogenous Cygb could not enter cells to exert itsbiological functions. In this study, a novel fusion protein was made by thetransmenbrane cell-penetrating peptide TAT with the anti-oxidant, anti-inflammatoryand anti-fibrotic protein, and then was subjected for further analysis, in order todetermine whether the fusion protein TAT-Cygb could accelerate skin wound healingand reverse liver fibrosis.Firstly, the fusion protein TAT-Cygb, whose expression was induced by lactosefrom E. coli BL21/pET22b-TAT-Cygb, was purified by CM Sepharose Fast FlowProtocol, Sephadex S-100molecular sieving, Sephadex G-25molecular sieving, andverified by SDS-PAGE, LC/MS, Western blotting. The final TAT-Cygb product hada concentration of3.273mg/mL with95%purity, exhibiting a high specificperoxidase activity up to (422.63±3.17) U/mg and its penetrating ability wasidentified by immunocytochemistry. Secondly, TAT-Cygb could reduce the level ofsuperoxide free radical in cells, indicating protective effect and treatment against thetoxic effect of the H2O2, while Cygb did not remedy cells injuried by H2O2, this mightdue to lack of penetration ability. The anti-inflammatory effect of TAT-Cygb wastested and verified by xylene-induced mouse ear swelling. Besides, TAT-Cygbshowed beneficial effects in skin wound healing owing to its anti-inflammatory effect.By means of inhibiting oxidative stress to prevent excessive fibroblast proliferation ormigration, TAT-Cygb could reduce the size of scar tissue effectively. In addition,TAT-Cygb exhibited a significant therapeutic effect on CCl4induced hepatic fibrosismice when compared with the model group, the content of liver function indexs and serum fibrosis markers of the TAT-Cygb treatment group were all close to the nomal,with obvious structure of liver lobules and few collagen fibers.This work first reported that the fusion protein TAT-Cygb could be useful foraccelerating skin wound healing and liver fibrosis treatment. It provides useful andbetter information for further study on anti-inflammatory, anti-aging and anti-fibroticmechanism of Cygb.