RESUMEN
OBJECTIVE: To investigate the changes in Smad 2, 3, 4 and 7 of the transforming growth factor-beta 1 (TGF-b1)/Smad signaling pathways in carbon tetrachloride (CCL4)-induced hepatic fibrosis rats treated with TGF-b1 small interfering (si)RNA. METHODS: Rats were randomly divided among five groups: non-fibrotic (normal); fibrosis-induced (model); fibrotic treated with 0.125 mg/kg TGF-b1 siRNA; fibrotic treated with 0.250 mg/kg TGF-b1 siRNA; and fibrotic treated with negative control TGF-b1 siRNA. The expression of Smad 2, 3, 4 and 7 was detected by real-time polymerase chain reaction (for mRNA), immunohistochemistry and Western blotting (for protein). RESULTS: The mRNA and protein levels of Smad 2, 3 and 4 were significantly lower in the the fibrotic rats treated with either 0.250 mg/kg or 0.125 mg/kg TGF-b1 siRNA than in the fibrotic model or the negative control TGF-b1 siRNA rats (P less than 0.01). Moreover, the mRNA and protein expression levels of Smad 2, 3 and 4 were significantly lower in the 0.250 mg/kg TGF-b1 siRNA group than in the 0.125 mg/kg group (P less than 0.05). Comparing the 0.250 mg/kg and 0.125 mg/kg TGF-b1 siRNA groups to the model group and the TGF-b1 siRNA negative control group showed significantly increased levels of mRNA and protein expression of Smad 7 (P less than 0.01). In addition, the expression levels of Smad 7 were significantly higher in the 0.250 mg/kg TGF-b1 siRNA group than in the 0.125 mg/kg group (P less than 0.05). CONCLUSION: siRNA-mediated silencing of TGF-b1 in rats led to significantly reduced expression of Smad 2, 3 and 4, but significantly increased expression of Smad 7. TGF-b1 regulation of Smad signaling molecules may contribute to hepatic fibrosis in rats and represent a target of future therapeutic intervention.
Asunto(s)
Silenciador del Gen , Cirrosis Hepática/metabolismo , ARN Interferente Pequeño , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/genética , Animales , RatasRESUMEN
BACKGROUND/AIMS: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-ß1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-ß1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). METHODS: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-ß1 siRNA 0.125mg/kg treatment group, TGF-ß1 siRNA 0.25mg/kg treatment group and TGF-ß1 siRNA negative control group (0.25mg/kg). CCL4 and a high-fat diet were used for 8weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-ß1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-ß1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-ß1 by quantitative real-time polymerase chain reaction. RESULTS: Comparing the TGF-ß1 siRNA 0.25mg/kg treatment group to the model group, the TGF-ß1 siRNA negative control group and the TGF-ß1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-ß1, type I collagen and type III collagen (P<0.01). CONCLUSIONS: Using siRNA to target TGF-ß1 can inhibit the expression of TGF-ß1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-ß1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.
Asunto(s)
Cirrosis Hepática/terapia , Interferencia de ARN , ARN Interferente Pequeño/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Cirrosis Hepática/patología , Masculino , Plásmidos/genética , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: To construct the siRNA eukaryotic expression vectors targeting on TGFß1, TIMP-1 and TIMP-2 and to investigate the inhibitory efficiency of target genes expression on rat hepatic stellate cell in vitro. METHODS: The siRNA cDNA sequences of TGFß1, TIMP-1 and TIMP-2 were designed, synthesized and inserted into plasmid pGenesil-1 respectively to generate eukaryotic expression plasmids. The plasmids were transfected into HSC T6 cells in vitro and the inhibitory efficiency of target genes expression was observed with real-time PCR and Western blot. RESULTS: The eukaryotic expression vectors were constructed successfully. The expressions of TGFß1 mRNA, TIMP-1 mRNA and TIMP-2mRNA in siRNA-transfected groups were decreased by 63.4% ± 8.0%, 64.5% ± 9.0% and 55.0% ± 17.0% respectively and the expressions of TGFß1 protein, TIMP-1 protein and TIMP-2 protein were decreased by 57.8% ± 3.0%, 55.1% ± 5.0%, 49.3% ± 1.0% respectively as compared to the control groups. CONCLUSIONS: The siRNA eukaryotic expression vectors constructed targeting on TGFß1, TIMP-1 and TIMP-2 could reduce the expressions of target genes and they might be able to used for the exploration of new anti-fibrosis drugs genetically.