Aim: To investigate the inhibition effects on the production of collagen type I, III secreted by activated rat hepatic stellate cells (rHSCs) by antisense tissue inhibitors of metalloproteinase 1 (TIMP-1) recombinant plasmid through elevating interstitial collagenase activity.
Methods: rHSCs were extracted from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation, and were cultured on plastic dishes until they were activated to a myofibroblastic phenotype after 7-10 days. RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryotic cells. The recombinant plasmid and the pcDNA3 empty plasmid were transfected in rHSCs by Effectene (QIAGEN) separately. Cells were selected after growing in DMEM containing 400 microg/ml G418 for 2-3 weeks. Expression of exogenous gene was assessed by Northern blot, and expression of TIMP-1 in rHSCs was determined by Northern blot and Western blot. We tested the interstitial collagenase activity with FITC-labled type I collagen as substrate. Ultimately, we quantified the type I, III collagen by Western blot.
Results: The exogenous antisense TIMP-1 recombinant plasmid could be expressed in rHSCs well, which could block the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNA level (P<0.05); the ratio of TIMP-1/beta-actin was 0.31, 0.98 and 1.32 separately at protein level (P<0.05); It might elevate active and latent interstitial collagenase activity, the collagenase activity was 0.3049, 0.1411 and 0.1196 respectively. (P<0.05), which led to promotion the degradation of type I, III collagen, the ratio of collagen I/beta-actin was 0.63, 1.78 and 1.92 separately (P<0.05); and the ratio of collagen III/beta-actin was 0.59, 1.81 and 1.98 separately (P<0.05).
Conclusion: These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on the production of type I, III collagens secreted by activated rHSCs in vitro. It could be a novel method to reverse hepatic fibrosis in the future.