Role of integrin- linked kinase in phenotypic transformation of rat hepatic stellate cells induced by recombinant connective tissue growth factor

Objective To investigate the role of integrin- linked kinase ( ILK) in the phenotypic transformation of rat's primary hepatic stellate cells ( HSCs) induced by recombinant connective tissue growth factor ( rCTGF) . Methods Primary HSCs were isolated from normal Sprague Dawley rats by in situ perfusion of collagenase and density gradient centrifugation. After 24 h of adherent cell culture, HSCs were treated with rCTGF for another 24 h before they were transfected with ILK small interfering RNA ( siRNA) or control siRNA. An rCTGF control group ( HSCs treated with only rCTGF) and a blank control group ( HSCs treated with no rCTGF) were also set up. The gene expression levels of α- smooth muscle actin ( αSMA) , ILK, and type I collagen in HSCs were measured by RT- PCR and Western blot at 24 h, 48 h, and 72 h after transfection with siRNA. The t- test was used for comparisons of measurement data. Results rCTGF significantly upregulated αSMA protein, ILK protein, and type I collagen mRNA in rat HSCs compared with the blank control group. Transfection of rat HSCs with ILK siRNA specifically inhibited the rCTGF- induced ILK upregulation. Compared with those in the rCTGF control group, the expression level of ILK protein in the ILK siRNA- transfected HSCs decreased by 72% ± 6% ( t = 21. 39, P < 0. 01) at 24 h, 87% ± 9% ( t = 68. 25, P < 0. 01) at 48 h, and 47% ± 3% ( t = 18. 25, P = 0. 003) at 72 h; the expression levels of αSMA protein and type I collagen mRNA decreased by 5% ± 1% ( t =2.52, P >0.05) and 6% ±3% ( t =1.63, P >0.05) at 24 h, 31% ±7% ( t =34.77, P <0.01) and 20% ±5% ( t =6.71, P <0.05) at 48 h, and 67% ± 8% ( t = 58. 82, P < 0. 01) and 43% ± 6% ( t = 15. 21, P < 0. 01) at 72 h. No changes were observed in the expression levels of ILK protein, αSMA protein, and type I collagen mRNA in HSCs transfected with control siRNA at the same time points. Conclusion ILK may partly mediate the phenotypic transformation of rat HSCs induced by rCTGF.