X Cheng1, M Zhang1, Y Xue1, H Sun1, Q Liu1, X F Shi2. 1. Key Laboratory of Molecular Biology for Infectious Diseases, Institute for Viral Hepatitis, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China. 2. Department of Infectious Diseases, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
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.
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 fibrosisrat 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.