Blockage of transforming growth factor β receptors prevents progression of pig serum-induced rat liver fibrosis

AIM: To test the hypothesis that introduction of antisense T beta R I and T beta R II eukaryotic expressing plasmids into a rat model of immunologically induced liver fibrosis might block the action of TGF-beta(1) and halt the progression of liver fibrosis. METHODS: RT-Nest-PCR and gene recombination techniques were used to construct rat antisense T beta R I and T beta R II recombinant plasmids which could be expressed in eukaryotic cells. The recombinant plasmids and empty vector (pcDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model. Expression of exogenously transfected gene was assessed by Northern blot, and hepatic expressions of T beta R I and T beta R II were evaluated by RTPCR and Western blot. We also performed ELISA for serum TGF-beta(1), hydroxyproline of hepatic tissues, immunohistochemistry for collagen types I and III, and VG staining for pathological study of the liver tissues. RESULTS: The exogenous antisense T beta R I and T beta R II plasmids could be well expressed in vivo, and block mRNA and protein expression of T beta R I and T beta R II in the fibrotic liver at the level of mRNA respectively. These exogenous plasmid expressions reduced the level of TGF-beta(1) (antisense T beta R I group 23.998 +/- 3.045 ng/mL, antisense T beta R II group 23.156 +/- 3.131 ng/mL, disease control group 32.960 +/- 3.789 ng/mL; F=38.19, 36.73, P<0.01). Compared with disease control group, the contents of hepatic hydroxyproline (antisense T beta R I group 0.169 +/- 0.015 mg/g liver, antisense T beta R II group 0.167 +/- 0.009 mg/g liver, disease control group 0.296 +/- 0.026 mg/g liver; F=14.39, 15.48, P<0.01) and the deposition of collagen types I and III decreased in the two antisense treatment groups (antisense T beta R I group, collagen type I 669.90 +/- 50.67, collagen type III 657.29 +/- 49.48; antisense T beta R II group, collagen type I 650.26 +/- 51.51, collagen type III 661.58 +/- 55.28; disease control group, collagen type I 1209.44 +/- 116.60, collagen type III 1175.14 +/- 121.44; F=15.48 to 74.89, P<0.01). Their expression also improved the pathologic classification of liver fibrosis models (compared with disease control group, chi(2)=17.14, 17.24, P<0.01). No difference was found in the level of TGF-beta(1), the contents of hepatic hydroxyproline and collagen types I and III and pathologic grade between pcDNA3 control group and disease control group or between the two antisense treatment groups (F =0.11 to 1.06, chi(2)=0.13 to 0.16, P>0.05). CONCLUSION: Antisense T beta R I and T beta R II recombinant plasmids have certain reverse effects on liver fibrosis and can be used as possible candidates for gene therapy.