Liver And Serum Comparative Proteomics Study Of The Anti-hepatic Fibrosis Effect Of RhCygb In Rats

Chronic liver disease has become a worldwide problem that seriously endanger to human health,and liver fibrosis is a common pathological basis of all chronic liver disease.Liver fibrosis is a healing response to chronic liver injury and is a total pathological changes induced by sustained liver injury,liver cells necrosis or inflammation.A large number of fiber proliferation accompanied by the relative or absolute lack of fiber degradation and great numbers of extracellular matrix(ECM)deposition,leading the blood micro-circulation passage between normal liver cells circulation disorders and the blood supply of liver cells inefficiency,so that damage of the hepatocytes due to inflammation is not easy to repair or even heavier damage because of fewer functioning liver cells,leading to further cirrhosis and even liver cancer development.Cytoglobin is a hexacoordinated heme-containing globin found in recent years,it located in the fibroblast cytoplasm and nucleus of most visceral tissue.Like other globin family members(myoglobin,hemoglobin,neuroglobin),Cygb has the same effect of binding and transport oxygen.More and more evidence suggests that Cygb also has the anti-fibrosis function that up-regulated expression under the fibrogenic stimulation due to a variety of stress conditions,including hypoxia and oxidative stress.Its molecular mechanism is still not clear,but its nitrogen oxide dioxygenase activity and lipid peroxidase activity has been confirmed.From more and more evidences,we can conclude that Cygb is a protective protein at the physiological function.With the implementation and promotion of the human genome project,life science research has entered the post-genomic era,which is mainly epitomized by the rise of proteomics.Proteomics is the study of the entire mass of intracellular levels of the protein or tissue characteristics,including the level of protein expression,post-translational modifications,protein-protein interactions,whereby we can have a overall and comprehensive understanding on the occurrence of the disease or the mechanism of cell metabolism on the protein level.This paper is based on previous studies in our laboratory,aimed at establishing a disease rat model of liver fibrosis and recombinant human cytoglobin(rhCygb)treatment model,and then find out the differentially expressed protein between the fibrosis model group and the treatment group in liver and serum 2D-gels through optimizing the condition of two-dimensional electrophoresis,and then through the comprehensive and overall proteomic analysis of the anti-fibrosis effect of rhCygb,we conduct a theory of the antifibrotic mechanism of Cygb.The topic is mainly divided into four parts,the first part is to establish hepatic fibrosis rat models with CC14 through subcutaneous method,as a basis,the rats were divided into three groups including control group,model group and treatment group.Through the detection of alanine aminotransferase(ALT),aspartate aminotransferase(AST),Liver fibrosis indexes(LN,HA,P III NP,IV-C)and biopsy,we can determine the successful construct of the models.Then the liver and serum were collected for further analysis;The second part is to establish and optimize rat liver and serum two-dimensional electrophoresis conditions,and then analysis differential expression proteins of rat liver and serum between different groups in the electrophoresis;the third part is to identify the differential expression protein spots from two-dimensional electrophoresis gel,purified and MALDI-TOF/MS determination the peptide mass fingerprinting(PMF)and then uploaded to the corresponding protein database to determine the protein;the final part is to discuss the protein function of the differential expression proteins,and adopted GO(Gene Ontology)analyze to enrich the different biological function terms of the proteins,and to make forecasts for rhCygb's anti-fibrotic mechanism.From the results,the indicators(ALT,AST,and Liver fibrosis indexes)of model group were significantly different(P<0.01)with the control group and the treatment group,and liver biopsy prompted that rhCygb have significant effect on the reversal effect ofliver fibrosis.By two-dimensional electrophoresis of serum we have found 43 differentially expressed protein spots and successfully identified 30 spotsby MALDI-TOF/MS,while the liver have found 50 differentially expressed protein spots and finally successfully identified 35 spots.Over these differentially expressed protein spots,some molecular positive correlation with liver fibrosis were specifically up-regulated in model group,such as Nrap,Cathepsin D,FGFR1 oncogene partner 2 homolog,Clusterin,Haptoglobin,Hsp60,Asl,BACAT,etc.While after rhCygb treatment,these proteins in the treatment group decreased to the normal level of the control group.Some molecules negative correlation with liver fibrosis,including proteins participated in redox reactions and liver biotransformation were significant up-regulated expressed after treatment with rhCygb,such as Peroxiredoxin-2,Nmrall,Glutathione S-transferase,Glutathione peroxidase 1 and Cytochrome P450.Conclusions can be drawn from the above statement that we have successfully constructed the animal model of liver fibrosis,and rhCygb obtained a good therapeutic effect of anti-hepatic fibrosis in rats.We speculate that,through its antioxidant and lipid peroxidase activity,Cygb may effectively lower the oxidative stress of liver and prevent the activation of hepatic stellate cells(HSC)and exhibits a powerful potential on the prevention of the occurrence and development of hepatic fibrosis.