Effects Of Recombinant Adenovirus Containing Akt On Carbon Tetrachloride-induced Liver Fiberosis In Rats

As an important stage that influences the prognostic treatment' for chronic liver diseases,hepatic fibrosis is a histopathological change that is commonly seen in the process of many chronic liver diseases developing into hepatic cirrhosis.Hepatic fibrosis is a dynamic process that is a reversible pathological change.However,it becomes irreversible if hepatic fibrosis develops into the structural change of hepatic lobule and the formation of pseudo-lobule(hepatic cirrhosis stage).Up to now, anti-hepatic fibrosis drug with widely approved effect has not been available for clinic treatment.Therefore,the preventive and therapeutic research for hepatic fibrosis will still remain a hot but difficult topic for home and abroad researchers in the future.Apoptosis represents the physiological way to eliminate excessive cells during embryogenesis and tissue remodeling.Under these conditions,apoptosis occurs in a controlled environment where dying cells are promptly removed by phagocytosis and replaced by new cells generated by mitosis.Apoptosis,however,is also an essential feature of a wide variety of acute and chronic diseases,including liver diseases. Imbalance between cell proliferation and death always leads to loss of tissue homeostasis and onset of various diseases.Excessive apoptosis has, indeed,been identified in acute and chronic viral hepatitis.Sustained apoptosis has also been linked with the development of hepatic fibrosis. In contrast,insufficient apoptosis has been associated with development and profession of tumours of the liver and the biliary tree.Thus identification of target molecules involved in apoptosis may offer new options for pharmacological and/or gene mediated therapies for patients with liver diseases.Many studies have shown that apoptosis play an important role in hepatic ischemia/reperfusion(I/R)injury,heart failure,parkinsonism and Alzheimer's disease.Akt,a serine-threonine protein kinase,is frequently referred to as protein kinase B(PKB),and it is the major anti-apoptosis in cells.Nowadays,researchers have shown that it is useful inhibit diseases above in rats by inhibiting cell apoptosis via Akt.So,we produced recombinant adenovirus Ad-myr-HA-Akt by homologoas recombination in 293 cells and transducted into rats via the tail vein.Methods1.50 male SD rats were randomly divided into two groups,The experiment group(40)were treated with 40%CCL₄(0.3ml/100g)by subcutaneous injection twice a week.Since the second week,The fodder feed was 80%corn flour,20%animal oil,and 0.15%cholesterol,the only beverage was 5%alcohol.The control group(10)were fed adlibitum with standard chow and distilled water as drinking fluid and injected with normal saline,the frequency,dose and period as same as the experiment group.Liver biopsy after 8 weeks,HE,VG staining were performed with liver tissue.Expression of Akt and p-Akt were evaluated by Western blotting.2.Myr-HA-Akt cDNA obtained from the plasmid pcDNA3.1-myr-HA-Akt was cloned into the plasmid pDC316.Then, pDC316-myr-HA-Akt was cotransferred with adenoviral backbone vector into 293 cells.The recombinant adenovirus was reproduced and purified. The recombinant adenovirus was identified by the cytopathic effect of 293 cells,polymerase chain reaction(PCR),and gene sequencing for myr-HA-Akt cDNA.TCID50 assay was performed to determine the titer of virus.After the adenovirus infected the hepatic fibrosis rat models via the tail vein,protein abundance and phosphorylation status of Akt were examined by Western blotting.3.10 normal rats were divided into the group normalI.The methods of compound factor,carbon tetrachloride(CCl4),corn flour and cholesterol plus alcohol were used to construct the hepatic fibrosis rat models.The other 60 rats were divided into four groups randomly: negative control group;saline group;Ad-EGFP group and Ad-myr-HA-Akt group.Rat hepatic fibrosis was induced by CCl4 for 8 weeks.Rats in the saline group,Ad-EGFP group and Ad-myr-HA-Akt group simultaneously received saline,Ad-EGFP and Ad-myr-HA-Akt via the tail vein at the second and the sixth week respectively.The pathological changes in liver tissues were observed after Van Gieson(VG) staining.Fas antigen in rat livers were determined with immunohistochemical method.Apoptosis of hypeatocytes of every group were evaluated by FACS with Annexin V FITC/P1.The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),albumin(ALB) and hydroxyproline(Hyp)were measured.Expression of Akt,p-Akt,Fas,caspase 3,caspase 9,DR5 andα-SMA were evaluated by Western blotting.Frozen sections of the liver,heart,lung,kidney,brain,spleen and testis were made to examine the expression of EGFP by fluorescence microscopy in EGFP group.Result1.After being induced with above means,the rat mortality of the experiment group was 12.5%,and the formation rate was 80%.Compared with the control group,the expression of p-Akt in livers was significantly decreased in the experiment group.The fiver of fibrosis animals showed a significant impaired in Akt phosphorylation.2.Infected 293 cells showed significant cytopathic effect.Products of PCR confirmed the presence of recombinant adenovirus.The identification result by DNA sequence analysis showed that myr-HA-Akt cDNA was cloned to pDC316 correctly and homologous recombinate with pBHGloxâ–³E1,3Cre and Ad-myr-HA-Akts were packaged successfully.The titer of virus was 5.5×10¹¹vp/ml.The expression of Akt in the livers of cirrhotic rats was verified by Western blotting.3.In comparison with other cirrhosis rats,the pathological changes in the Ad-myr-HA-Akt-treated group was markedly attenuated.The ratios of apoptosis of hepatocytes and the levels of ALT,AST and Hyp were significantly lowered in Ad-myr-HA-Akt-treated group.Western blot showed that the protein expression of p-Akt in the Ad-myr-HA-Akt-treated group was higher significantly as compared with those in the negative control group,saline group and Ad-EGFP group. Western blot also showed that the protein expression of Fas.Caspase 3,caspase 9and DR5,α-SMA in the Ad-myr-HA-Akt-treated group was lower significantly.EGFP expression was mainly demonstrated by fluorescence microscopy on the frozen section of liver,very little fluorescene were detected in lung and kidney and there was no detectable EGFP in other organs.Conclusions1.The methods of compound factor can established reliable rat model of fiver fibrosis,Akt may be a new target of pharmacological and/or gene mediated therapies for liver fibrosis.2.The recombinant adenoviral vector containing myr-HA-Akt was constructed and the transgene hepatic fibrosis rats expressed Akt gene in vivo successfully.It provided foundation for the further study of treatment for hepatic fibrosis by Akt gene.3.Ad-myr-HA-Ak inhibits CCl4-induced liver fibrosis and is a potential pharmacological target for gene therapy in liver fibrosis.