Blockage of transforming growth factor beta receptors prevents progression of pig serum-induced rat liver fibrosis.
OBJECTIVE
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).


CONCLUSIONS
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.
Jiang
Wei
W
Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Yang
Chang-Qing
CQ
Liu
Wen-Bin
WB
Wang
Yi-Qing
YQ
He
Bo-Ming
BM
Wang
Ji-Yao
JY
D030201:Activin Receptors, Type I / Q000037:antagonists & inhibitors* / Q000235:genetics*; D000818:Animals; D001798:Blood Proteins / Q000276:immunology* / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D016373:DNA, Antisense; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D015316:Genetic Therapy; D006909:Hydroxyproline / Q000378:metabolism; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008103:Liver Cirrhosis / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D008297:Male; D010957:Plasmids / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000235:genetics*; D013552:Swine; D016212:Transforming Growth Factor beta / Q000097:blood; D053773:Transforming Growth Factor beta1
D001798:Blood Proteins; D024042:Collagen Type I; D024043:Collagen Type II; D016373:DNA, Antisense; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; C000625843:Tgfbr1 protein, rat; D006909:Hydroxyproline