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 RT-PCR 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.