The Effect Of Eukaryotic Peptide Chain Release Factor ERF3a/gspt1on Rat Liver Fibrosis Model

Objective： The chronic hepatitis B is the majority of chronic liverdisease in our country, the hepatitis due to hepatitis virus replication andstimulate the immune system, causing immune lymphocytes during clearingthe virus,"friendly fire" liver cells to produce liver inflammation inducedproliferation of fibrous tissue, resulted in liver fibrosis. The pathologicalchanges in liver fibrosis is the excessive deposition of extracellular matrix inthe liver, and the majority of chronic liver disease with varying degrees ofliver fibrosis is a common pathological basis and features a variety of chronicliver disease, liver cells based on collagen the components of the extracellularmatrix synthes is increased, the relative lack of degradation, excessivedeposition in the liver. Further development will cause the hepatic lobulealterations and pseudolobules nodule formation, of which25%to40%eventually develop into cirrhosis and liver cancer. Occlusion of hepaticfibrosis becomes a key issue in the treatment of chronic liver disease. Theliver fibrosis is a common pathological basis in the progression of a variety ofchronic liver disease developed into cirrhosis, then routine liver function testscan only reflect liver function, without detecting the degree of liver fibrosis. Itis difficult to do the dynamic observation and efficacy assessment of liverfibrosis based on liver puncture biopsy.Eukaryotic peptide chain release factor involved in intracellular proteinsynthesis termination process nascent polypeptide chain releasing a group ofimportant proteins in eukaryotes, include two categories, whose name areeRF1and of eRF3. As many studies showed, eRF3is the major factor in theprocess of mammalian translation termination, and its expression level affectsthe eRF1stability of the protein, and also determines the termination of theformation of complexes. Therefore, in this test the expression level of eRF3a was altered to observe whether different expression level of eRF3a couldimpact the liver fibrosis.Methods:1The clone of the gspt1gene molecular. The whole-genome DNAextraction kit is used to extract DNA, and then amplify gspt1gene. Cloningthe gspt1gene into the eGFP-C2plasmid, then extrac the recombinant plasmidusing alkaline lysis method, and proceed the double digestion and sequencinganalysis.2The modeling of Liver fibrosis. CCL₄and phenobarbital were used toinduce liver fibrosis model in SD rats, containing intraperitoneal injection of5%carbon tetrachloride with a mixture of olive oil, a dose of5ml/kg twice aweek for8weeks injection. During themodeling process,animals were addedthe water with25mg/L of phenobarbital. Normal control group contained14animals, according to the model for the establishment of the group of the sameprogram intraperitoneal injection of olive oil for8weeks. The remaining ratswere divided into the plasmid processing, negative group and colchicinepositive control treatment after CCL₄processing four weeks.The plasmidprocessing group was divided into five subgroups: CCL₄+eGFP-C2-gspt1plasmid0.125mg/kg group once a week （treated1）, CCL₄+eGFP-C2-gspt1plasmid0.125mg/kg twice a week group （treated2）, CCL₄+eGFP-C2-gspt1plasmid0.25mg/kg once a week group （treated3）, CCL₄+eGFP-C2-gspt1plasmid0.25mg/kg twice a week group （treated4）, CCL₄+eGFP-C2-gspt1plasmid0mg/kg group （model group） once a week, there were12rats in eachgroup, by the rapid tail vein injection of plasmid; Positive control group forcolchicine, a total of14mg/kg lavage, once per day; Negative control groupfor injecting CCL₄+eGFP-C2plasmid0.125mg/kg group once a week, atotal of12rats..3The test of liver function. Microplates were used to assay liver functionmarkers changes such as AST, ALT in specimens from rats serum.4Liver histopathology. After the experiment, rat liver tissue wereextracted, then embedded in paraffin, and line thickness of5μm serial sections, respectively, and then used HE staining and immunohistochemistry.5The detection of liver fibrosis factor gene expression. Fluorescencequantitative polymerase chain reaction （Real-Time RT-PCR）2-ΔΔCtrelativequantification method was used to detect fiber relevant factor α-SMA, TGF-β1,CTGF, Mmp13, Col Ι relative expression level in rat liver. GAPDH gene as aninternal reference, specimens of RNA quality and reverse transcriptionefficiency differences standardized. The expression levels of each factor innormal liver tissue as a calibration sample, compared with expression levels invarious tissues.6The detection of liver fibrosis factor protein expression. The Westernblot was used to detect the eRF3a and α-SMA expression level while β-actinas an internal differences, and all plasmid treatment groups compared withmodel group.Results:1gspt1gene molecular clone. The subject successfully cloned eukaryoticpeptide chain releasing factor gspt1gene. The rat gspt1gene coding regionhas1911bp, encoding637amino acids, suggesting that the relative molecularmass of approximately80kD.2Liver function tests. Compared with the model group, the level of ASTand ALT of plasmid treated group dcreasesd with the dose increasing.3Liver histopathology. HE staining showed that the normal rats are withclear lobular architecture and the mononuclear liver cells, and the liver plateswere streak arranged radially around the central vein, the interlobular and aportal area basic distribution of collagen fibers, only in minor collagen fiberdistribution of the central vein and hepatic sinus. In the liver tissue lesion area,there were damaged normal lobular structure,liver cell cords arranged indisorder, enlarged portal area,and there were a large number of inflammatorycell infiltration, hepatocyte edema, with extensive fatty degeneration ofcollagen fibers visible in the model rats HE staining hyperplasia. The collagenfibers of the plasmid treated group decreased significantly, which was widelyin cords separated hepatic lobule, diffuse distribution form pseudolobules. 4Liver fibrosis factor gene expression detection. Real-time quantitativePCR results showed that compared with the model group fiber-related factorα-SMA, CTGF, TGF-β1, Col Ι in the plasmid treated group has declined,MMP13elevated. CTGF expression level in treated3and treated4was lowerthan model group （P=0.01）, and treated4CTGF expression level was lowerthan the other group （P=0.04）; TGF-β1expression level in treated3, treated4was significantly lower than model group （P=0.01）, and TGF-β1expressionlevel no difference in treated3, treated4; Col Ι expression level in treated3,treated4was significantly lower than model group （P=0.02）, and Col Ιexpression level no difference in treated3, treated4;α-SMA expression levelin treated3, treated4was lower than that in model group （P=0.01）, andα-SMA expression level no difference between them; MMP13expression levelsin treated3, treated4were significantly higher than those in model group （P=0.01）, and MMP13expressed level was no difference between them.5Liver fibrosis factor protein expression detection. Using Western Blotto detect eRF3a protein and α-SMA expression, visiblely specific bandseRF3a protein, located between72KD and95KD, about80KD, calculatedratio of eRF3a/β-actin, normal group, model group, Negative group, positivegroup and four treatment groups relative expression levels were0.98,0.77,0.72,1.44,0.80,0.92,1.40,1.67respectively, eRF3a protein expression levelin treated3and4were higher than the model group（P=0.03）, and the eRF3aprotein expression level in treated4was higher than that in treated3（P=0.02）;while α-SMA protein specific bands about44KD, calculated ratio ofα-SMA/β-actin, The ratio of the normal group, the model group, Negativegroup, positive group and four treatment groups relative expression were0.87,1.17,1.21,1.54,1.32,1.28,1.05, respectively, α-SMA protein expressionlevel in treated3and4were higher than the model group（P=0.03）, and theeRF3a protein expression level in treated4was higher than that in treated3（P=0.02）.Conclusions:1After eight weeks, the liver fibrosis biochemical makers such as AST, ALT in rats decreased, suggesting the liver function is improving.2The liver histopathology showed the lessening of plasmid treated ratsinjury. And with the dose increasing, the degree of injury reduced.3In rat liver, the level of promoting liver fibrosis factor expressiondeclined, while the inhibiting fibrosis factor expression levels increased.Suggesting that eRF3a could inhibit hepatic fibrosis in rats.