Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice.
BACKGROUND
Previous studies have shown that transforming growth factor-beta 1 (TGF-beta1) is the most potent means of stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells. Thus, TGF-beta1 could be a target for treating hepatic fibrosis. This study aimed to investigate the inhibitory effects of specific TGF-beta1 small interference RNA (siRNA) on immune hepatic fibrosis induced by Concanavalin A (Con A) in mice.


METHODS
Three short hairpin RNAs targeting different positions of TGF-beta1 were designed and cloned to the plasmid pGenesil-1 to obtain three recombinant expression vectors (pGenesil-TGF-beta1-m1, pGenesil-TGF-beta1-m2 and pGenesil-TGF-beta1-m3). Thirty male Kunming mice were randomly divided into 6 groups: normal, model, control, and three treatment groups. The immune hepatic fibrosis models were constructed by injecting Con A via the tail vein at 8 mg/kg per week for 6 weeks. At weeks 2, 4 and 6, pGenesil-TGF-beta1-m1, pGenesil-TGF-beta1-m2 or pGenesil-TGF-beta1-m3 was injected by a hydrodynamics-based transfection method via the tail vein at 0.8 ml/10 g within 24 hours after injection of Con A in each of the three treatment groups. The mice in the control group were injected with control plasmid pGenesil-HK at the same dose. All mice were sacrificed at week 7. The levels of hydroxyproline in liver tissue were determined by biochemistry. Liver histopathology was assessed by Van Gieson staining. The expression levels and localization of TGF-beta1, Smad3, and Smad7 in liver tissue were detected by immunohistochemistry. The expression of TGF-beta1, Smad3, Smad7 and alpha-smooth muscle actin (alpha-SMA) mRNAs in the liver were assessed by semi-quantitative RT-PCR.


RESULTS
The levels of hydroxyproline in the liver tissue of the treatment groups were lower than those of the model group (P<0.01). Histopathologic assay showed that liver fibrogenesis was clearly improved in the treatment groups compared with the model group. The expression levels of TGF-beta1 and Smad3 of liver tissue were also markedly lower in the treatment groups than in the model group (P<0.01), while the levels of Smad7 were higher in the treatment groups than in the model group (P<0.01). RT-PCR further showed that the expression of TGF-beta1, Smad3 and alpha-SMA mRNA was significantly inhibited in the treatment groups compared with the model group, while the levels of Smad7 were increased. There was no difference in the above parameters among the three treatment groups or between the control and model groups (P>0.05), but the inhibitory effect of pGenesil-TGF-beta1-m1 was the highest among the treatment groups.


CONCLUSIONS
Specific siRNA targeting of TGF-beta1 markedly inhibited the fibrogenesis of immune hepatic fibrosis induced by Con A in mice. The anti-fibrosis mechanisms of siRNAs may be associated with the down-regulation of TGF-beta1, Smad3 and alpha-SMA expression and up-regulation of Smad7 expression in liver tissue, which resulted in suppressing the activation of hepatic stellate cells.
Xu
Wei
W
Department of Infectious Diseases, State Key Laboratory of Virology, Renmin Hospital of Wuhan University, Wuhan, China.
Wang
Lu-Wen
LW
Shi
Jin-Zhi
JZ
Gong
Zuo-Jiong
ZJ
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003208:Concanavalin A / Q000276:immunology*; D015536:Down-Regulation; D006909:Hydroxyproline / Q000032:analysis; D007150:Immunohistochemistry; D008099:Liver / Q000737:chemistry / Q000473:pathology; D008103:Liver Cirrhosis / Q000276:immunology* / Q000517:prevention & control*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics* / Q000378:metabolism
D000199:Actins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D053773:Transforming Growth Factor beta1; C544013:alpha-smooth muscle actin, mouse; D003208:Concanavalin A; D006909:Hydroxyproline