AIM:To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride(CCl4).METHODS:Using cre-loxp technique,we created an Ad-myr-HA-Akt virus,in which ...AIM:To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride(CCl4).METHODS:Using cre-loxp technique,we created an Ad-myr-HA-Akt virus,in which Akt is labeled by a HA tag and its expression is driven by myr promoter.Further,through measuring enzyme levels and histological structure,we determined the efficacy of this Ad-myrHA-Akt virus in inhibiting the development of cirrhosis induced by CCl4in rats.Lastly,using western blotting,we examined the expression levels and/or phosphorylation status of Akt,apoptotic mediators,endothelial nitric oxide synthase(eNOS),and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus.RESULTS:The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment,which was consistent with the sequence reported in the GenBank.The concentrations of Admyr-HA-Akt and adenoviral enhanced green fluorescent protein(Ad-EGFP)virus used in the current study were5.5×1011vp/mL.The portal vein diameter,peak velocity of blood flow,portal blood flow and congestion index were significantly increased in untreated,saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection.In contrast,these parameters in the Akt cirrhosis group were comparable to normal control group.Compared to the normal control,the liver function(Alanine aminotransferase,Aspartate aminotransferase and Albumin)was significantly impaired in the untreated,saline and Ad-EGFP cirrhosis groups.The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated,saline and Ad-EGFP cirrhosis groups.The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups.The results of HE and Van Gieson staining indicated that Akt group has better preservation of histological structure and less fibrosis than other cirrhosis groups.The percentage of apoptotic cell was greatly less in Akt cirrhosis group than in other cirrhosis groups.Akt group showed positive HA tag and an increased level of phosphorylated Akt as well as decreased levels of Fas.In contrast,Caspase-3 and Caspase-9 levels in Akt group were significantly lower than other cirrhosis groups.Noticeable decrease of DR5 andα-SMA and increase of phosphorylated eNOS were observed in the Akt group when compared to other cirrhosis groups.The NO level in liver was significantly higher in Akt group than other cirrhosis groups,which was consistent with the level of phosphorylated eNOS in these groups.CONCLUSION:This study suggest that Ad-myr-HA-Akt virus is a useful tool to prevent CCl4-induced cirrhosis in rat model and Akt pathway may be a therapeutic target for human cirrhosis.展开更多
文摘AIM:To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride(CCl4).METHODS:Using cre-loxp technique,we created an Ad-myr-HA-Akt virus,in which Akt is labeled by a HA tag and its expression is driven by myr promoter.Further,through measuring enzyme levels and histological structure,we determined the efficacy of this Ad-myrHA-Akt virus in inhibiting the development of cirrhosis induced by CCl4in rats.Lastly,using western blotting,we examined the expression levels and/or phosphorylation status of Akt,apoptotic mediators,endothelial nitric oxide synthase(eNOS),and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus.RESULTS:The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment,which was consistent with the sequence reported in the GenBank.The concentrations of Admyr-HA-Akt and adenoviral enhanced green fluorescent protein(Ad-EGFP)virus used in the current study were5.5×1011vp/mL.The portal vein diameter,peak velocity of blood flow,portal blood flow and congestion index were significantly increased in untreated,saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection.In contrast,these parameters in the Akt cirrhosis group were comparable to normal control group.Compared to the normal control,the liver function(Alanine aminotransferase,Aspartate aminotransferase and Albumin)was significantly impaired in the untreated,saline and Ad-EGFP cirrhosis groups.The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated,saline and Ad-EGFP cirrhosis groups.The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups.The results of HE and Van Gieson staining indicated that Akt group has better preservation of histological structure and less fibrosis than other cirrhosis groups.The percentage of apoptotic cell was greatly less in Akt cirrhosis group than in other cirrhosis groups.Akt group showed positive HA tag and an increased level of phosphorylated Akt as well as decreased levels of Fas.In contrast,Caspase-3 and Caspase-9 levels in Akt group were significantly lower than other cirrhosis groups.Noticeable decrease of DR5 andα-SMA and increase of phosphorylated eNOS were observed in the Akt group when compared to other cirrhosis groups.The NO level in liver was significantly higher in Akt group than other cirrhosis groups,which was consistent with the level of phosphorylated eNOS in these groups.CONCLUSION:This study suggest that Ad-myr-HA-Akt virus is a useful tool to prevent CCl4-induced cirrhosis in rat model and Akt pathway may be a therapeutic target for human cirrhosis.