Functions Of Cytoglobin In The Oxidative Stress Of Liver Fibrosis And The Mechanism Of Its Upregulation

Cytoglobin (Cygb) is a member of the newly discovered class of the hexacoordinate hemoglobin superfamily. Preliminary research of our group has demonstrated that Cygb over-expression could protect against damage-induced oxidative stress, thus inhibiting free radical-mediated activation of fibroblasts and induction of tissue fibrosis. In this thesis, the specific functions of Cygb towards oxidative stress was systematically investigated, using two representative oxidative models in the liver, the H2O2 model and the iron overload model, and the possible mechamism of the upregulation of Cygb in activated hepatic stellate cells was preliminarily investigated via the microRNA technique. First of all, the oxidative stress related in vitro model of liver fibrosis was constructed and the recombinant Cygb protein was successfully purified. Both the recombinant Cygb protein and the Cygb gene transfection were indicated to exert significant protective effect on LX-2 and Chang liver cells treated with H2O2 and Fe-NTA/AA of different doses. The cytoplasmic ROS scanvaging capacity of the recombinant Cygb protein was not as good as its capacity in scanvaging ROS outside the cells, perhaps owing to the lacking of active transporting mechanisms. Moreover, interferring Cygb mRNA with specific siRNA could sensitize LX-2 cells to the injury of H2O2 or Fe-NTA/AA, which further confirmed that upregulation of Cygb was the protective response of activated hepatic stellate cells in face of oxidative stress. Further study on primary rat hepatic stellate cells (rHSC) also validated that recombinant Cygb protein showed a significant protective effect on rHSC from the injury elicited by H2O2, and could protect rHSC from excessive proliferation and injury when Fe-NTA/AA was used.In order to find out the possible mechanism of the upregulation of Cygb in activated HSC, bioinformatics measures, gene arrays and miRNA arrays were combined in this thesis to predict the most possible miRNAs targeting Cygb mRNA. It was proved that the upregulation of Cygb in activated hepatic stellate cells was closely related to the downregulation of corresponding miRNAs. Also, the potential miRNAs targeting HSC activation markers and Col1a1 mRNA were investigated with the combination of the above three methods. MiR-29 family was finally tested to be capable of downregulating Col1a1 mRNA via the experiment. Thus, an effective method to predict the possible function of a specific miRNA in the HSC was established.All the above results provided comprehensive explanation to the anti-oxidative fuction of Cygb and its mechanism, which would accelerate the exploitation of new anti-fibrotic target.