Effect of tissue inhibitor of metalloproteinase-1 and 2 siRNA on the expression of smad2/3/4 protein in hepatic stellate cells

Abstract

Objective: To study the effect of tissue inhibitor of metalloproteinases (TIMP)-1 siRNA and TIMP-2 siRNA on the expression of smad2/3/4 protein in CCl4-induced liver fibrosis rat hepatic stellate cells (HSC). Methods: Rat's liver tissues with liver fibrosis after treatment with pre-built TIMP-1siRNA and TIMP-2 siRNA were used as the research subjects. Immunohistochemistry, Western blotting and real-time PCR were used to detect the protein and corresponding mRNA expression levels on smad2/3/4. TUNEL and α-smooth muscle actin (α-SMA) positive cells were quantified by double-labeled immunofluorescence. Analysis of variance (ANOVA) was used to compare the means between multiple groups, and the SNK test was used for the pairwise comparison of means. Results: The results of immunohistochemistry showed that the protein expressions of smad2, smad3, and smad4 in the TIMP-1 siRNA group and TIMP-2 siRNA group were significantly reduced than those of the model and the negative control group (P < 0.05). In addition, Western blotting results had also shown the same trend. The protein expression of smad2, smad3, and smad4 in the TIMP-1siRNA group and TIMP-2siRNA group were significantly reduced than those of the model and the negative control group (P < 0.01). The mRNA expression of smad2, smad3, and smad4 in TIMP-1siRNA group and TIMP-2siRNA group was significantly reduced than those of the model and negative control group (P < 0.05). Immunofluorescence showed that the apoptosis of activated HSC in the TIMP-1 siRNA group(0.014 3 ± 0.002 4) and TIMP-2 siRNA group(0.010 7 ± 0.004 4) was increased than those of the model(0) and the negative control group (0.002 4 ± 0.002 4, P < 0.05). Conclusion: TIMP-1 siRNA and TIMP-2 siRNA promote the apoptosis of activated HSCs. In addition, it also has a significant inhibitory effect on the expression of smad protein.