The Role Of CT-1 In Cirrosis And Hepatocellular Carcinoma And The Application Of Micro-CT In The Assessment Of Liver Injury

Objective: The role of cardiotrophin-1(CT-1)has been widely studied in various liver diseases such as hepatic steatosis and liver ischemia/reperfusion injury,but its role in cirrhosis and hepatocellular carcinoma remains unclear.Our study aimed to investigate the expression and the role of CT-1 in pediatric cholestatic liver disease(PCLD)and hepatocellular carcinoma(HCC)and explore the potential of contrast-enhanced computed tomography(CECT)using ExiTron nano6000 for assessment of liver lesions in mouse models.Methods: qRT-PCR,Western blot,immunochemistry and confocal were utilized to detect the expression and distribution of CT-1 in PCLD and HCC liver tissues.We used Elisa to detect the plasma CT-1 levels in PCLD.In the in vitro analysis,the effect of CT-1 on Lx-2 and HCC cells proliferation was investigated by CCK-8.Transwell was utilized to detect the effects of CT-1 on the invasiveness of liver cancer cells.qRT-PCR and Western blot were utilized to detect the activation of STAT3 and p38 MAPK pathways in CT-1 treated Lx-2.Vascular endothelial cell line-HUVEC cells were treated by H2O2 and AP-1 siRNA.The luciferase reporter gene assay and chromatin immunoprecipitation assay were conducted to provide direct evidence for the AP-1-mediated regulatory effect on the CT-1 promoter.Three mouse models of liver lesions were used: bile duct ligation(BDL),lipopolysaccharide(LPS)/D-galactosamine(D-GalN),and alcohol.After injection with the contrast agent ExiTron nano6000,the mice were scanned with micro-CT.Liver lesions were evaluated using CECT images,hematoxylin and eosin staining,and serum aminotransferase levels.qRT-PCR was used to detect the mRNA level of Cardiotrophin1 in injuried livers treated with BDL.Macrophage distribution in the injury models was shown by immunohistochemical staining of CD68.The in vitro studies measured the densities of RAW264.7 under different conditions by CECT.Results: CT-1 mRNA and protein expression levels were upregulated in PCLD liver biopsy tissues compared with controls.Immunohistochemistry and confocal microscopy of liver sections showed that hepatic vascular endothelial cells,proliferative biliary tracts and inflammatory cells were the main cell types in PCLD and cancer liver tissues.Serum CT-1 was elevated significantly in the children with PCLD compared with controls.Serum CT-1 levels exhibited a moderate positive correlation with the Scheuer stage of hepatic fibrosis.In vitro analysis indicated that LX-2 cells preconditioned with CT-1 exhibited significant increments in proliferation and accumulation of extracellular matrix components,while also positively regulating the STAT3 and p38 MAPK pathways.Administration of CT-1 significantly decreased the cell proliferation of liver cancer cells,while increased the invasiveness of HCC cells.H2O2 could elevate the expression of CT-1 in HUVEC,which could be eliminated by AP-1 siRNA.At last,AP-1 could directly bound to the CT-1 promoter and trans-activated its expression.In the in vivo studies,CECT provided specific and strong contrast enhancement of liver in mice.After BDL treatment,the texture of the liver CT image became more heterogeneous and black regions more extensive and the liver density was significantly greater compared to that of the sham controls.At the same time,BDL-treated liver tissues showed a significantly elevated CT-1 mRNA level.In the CECT imaging of LPS/D-GalN-induced liver lesions,the liver density showed an up-down trend in that it increased in the early period and decreased in the advanced stages of liver lesions.For acute alcohol-induced liver lesions,the predominant changes were the increased density of the injured liver.The liver histology and immunochemistry of CD68 demonstrated that both dilated biliary tracts and necrosis in the injured livers could lead to the heterogeneous distribution of macrophages.The in vitro study showed that the RAW264.7 cell masses had higher densities after LPS activation.Conclusions: Our data suggests that biliary epithelium-derived CT-1 may exert a profibrogenic potential in PCLD.AP-1-regulated paracrine CT-1 has a negative effect on proliferation of HCC cells while increasing their invasiveness.Micro-CT with the contrast agent ExiTron nano6000 is feasible for detecting various liver lesions by emphasizing the heterogeneous textures and densities of CECT images,which may be due to the different number,distribution and function of macrophages in the injured livers.