Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats.

BACKGROUND: No efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen. METHODS: Real-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type I collagen. In addition to hepatic hydroxyproline content, hepatic collagen types I and III were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining. RESULTS: Exogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P < 0.01), hepatic hydroxyproline content and the accumulation of collagen types I and III were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P < 0.01) as compared with the model group. CONCLUSION: The results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis.

Blockage of transforming growth factor beta 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 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.

[Effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis].

OBJECTIVE: To study the effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis. METHODS: RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TGFbetaRII recombinant plasmid which can be expressed in eukaryotic cells. Thirty-six male SD rats were randomly distributed into five groups: 10 in experimental liver fibrosis model induced by pig-serum as disease control group; 10 in antisense TGFbetaRII transfection as treatment group; 10 in pCDNA3 transfection as treatment control group and 6 in normal control group. The recombinant plasmid and empty vector (pCDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model respectively. Expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR and Western blot. We also tested ELISA of serum TGF-beta1, the contents of hepatic hydroxyproline, immunohistochemistry of type I and III collagen, and VG staining for pathological study. RESULTS: The antisense TGFbetaRII expressing plasmid could be well expressed in vivo, and could block the mRNA and protein expression of TGFbetaRII in the fibrotic liver induced by pig serum. Its expression also reduced the level of TGF-beta1 [antisense treatment group (23.16+/-3.13) ng/ml, disease control group (32.96+/-3.79) ng/ml; F=36.73, 0.01]. Compared with the disease control group, the contents of hepatic hydroxyproline [antisense treatment group (0.17+/-0.01) mg/g liver, disease control group (0.30+/-0.03) mg/g liver; F=15.48, 0.01] and the deposition of collagens type I and type III decreased in the antisense group (antisense treatment group collagen type I 650.26+/-51.51, collagen type III 661.58+/-55.28; disease control group type I 1209.44+/-116.60, collagen type III 1175.14+/-121.44; F values are 69.87, 70.46, 0.01). And its expression also improved the pathologic classification of liver fibrosis models (0.01). CONCLUSION: The results demonstrate that TGF-beta plays a key role in liver fibrogenesis and the prevention of liver fibrosis by antisense TGFbetaRII recombinant plasmid intervention may be therapeutically useful.

[Effects of cisapride on intestinal bacterial and endotoxin translocation in cirrhotic rats].

OBJECTIVES: To further investigate the effects of cisapride on intestinal bacterial overgrowth (IBO), bacterial and endotoxin translocation, intestinal transit and permeability in cirrhotic rats. METHODS: 25 normal control rats, 25 cirrhotic rats, 20 cirrhotic rats received saline, and 20 cirrhotic rats treated with cisapride were included in the study. All animals were assessed with many variables including bacterial and endotoxin translocation, IBO, intestinal transit and permeability. RESULTS: Bacterial translocation was found in 48%(12/25) cirrhotic rats and none of control rats. Among the 20 rats with IBO, there were 11 rats with bacterial translocation (BT) while only one rats occurred BT out of the 5 rats without IBO. Cirrhotic rats with IBO had a significantly higher rate of endotoxin translocation, higher intestinal permeability and longer intestinal transit than those without IBO. BT of a specific organism was always associated with IBO of that organism. Compared with the placebo group, cisapride-treated rats had lower rates of bacterial and endotoxin translocation and IBO, which had close relationship with shorter intestinal transit and lower permeability. CONCLUSION: Endotoxin and bacterial translocation in cirrhotic rats may be the result of IBO and higher permeability. IBO may be the result of longer transit. Cisapride which can accelerate intestinal transit and improve intestinal permeability is helpful in preventing and treating intestinal bacterial and endotoxin translocation.

Effect of cisapride on intestinal bacterial and endotoxin translocation in cirrhosis.

AIM: To investigate the effects of cisapride on intestinal bacterial overgrowth (IBO), bacterial and endotoxin translocation, intestinal transit and permeability in cirrhotic rats. METHODS: All animals were assessed with variables including bacterial and endotoxin translocation, intestinal bacterial overgrowth, intestinal transit and permeability. Bacterial translocation (BT) was assessed by bacterial culture of MLN, liver and spleen, IBO by a jejunal bacterial count of the specific organism, intestinal permeability by determination of the 24-hour urinary (99m)Tc-DTPA excretion and intestinal transit by measurement of the distribution of (51)Cr in the intestine. RESULTS: Bacterial translocation (BT) and IBO was found in 48 % and 80 % cirrhotic rats respectively and none in control rats. Urinary excretion of (99m)Tc-DTPA in cirrhotic rats with BT (22.2+/-7.8) was greater than these without BT (10.5+/-2.9). Intestinal transit (geometric center ratio) was significantly delayed in cirrhotic rats (0.31+/-0.06) and further more delayed in cirrhotic rats with BT (0.24+/-0.06) than these without BT (0.38+/-0.11). Cirrhotic rats with IBO had significantly higher rates of intestinal bacterial and endotoxin translocation, slower intestinal transit time and higher intestinal permeability than those without IBO. It was also found that BT was closely associated with IBO and the injury of intestinal barrier. Compared with the placebo group, cisapride-treated rats had lower rates of bacterial/endotoxin translocation and IBO, which was closely associated with increased intestinal transit and improved intestinal permeability by cisapride. CONCLUSION: These results indicate that endotoxin and bacterial translocation in cirrhotic rats may be attributed to IBO and increased intestinal permeability. Cisapride that accelerates intestinal transit and improve intestinal permeability might be helpful in preventing intestinal bacterial and endotoxin translocation.

Inhibition on the production of collagen type I, III of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid.

AIM: To investigate the inhibition effects on the production of collagen type I, III secreted by activated rat hepatic stellate cells (rHSCs) by antisense tissue inhibitors of metalloproteinase 1 (TIMP-1) recombinant plasmid through elevating interstitial collagenase activity. METHODS: rHSCs were extracted from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation, and were cultured on plastic dishes until they were activated to a myofibroblastic phenotype after 7-10 days. RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryotic cells. The recombinant plasmid and the pcDNA3 empty plasmid were transfected in rHSCs by Effectene (QIAGEN) separately. Cells were selected after growing in DMEM containing 400 microg/ml G418 for 2-3 weeks. Expression of exogenous gene was assessed by Northern blot, and expression of TIMP-1 in rHSCs was determined by Northern blot and Western blot. We tested the interstitial collagenase activity with FITC-labled type I collagen as substrate. Ultimately, we quantified the type I, III collagen by Western blot. RESULTS: The exogenous antisense TIMP-1 recombinant plasmid could be expressed in rHSCs well, which could block the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNA level (P<0.05); the ratio of TIMP-1/beta-actin was 0.31, 0.98 and 1.32 separately at protein level (P<0.05); It might elevate active and latent interstitial collagenase activity, the collagenase activity was 0.3049, 0.1411 and 0.1196 respectively. (P<0.05), which led to promotion the degradation of type I, III collagen, the ratio of collagen I/beta-actin was 0.63, 1.78 and 1.92 separately (P<0.05); and the ratio of collagen III/beta-actin was 0.59, 1.81 and 1.98 separately (P<0.05). CONCLUSION: These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on the production of type I, III collagens secreted by activated rHSCs in vitro. It could be a novel method to reverse hepatic fibrosis in the future.

Glyco-poly-l-lysine is better than liposomal delivery of exogenous genes to rat of liver.

AIM:To compare the effects of liposomes and glyco-poly-L-lysine on liver targeted uptake and expression of plasmid in rat liver.METHODS:After binding with lipofectamine or galactose-terminal glyco-poly-l-lysine, the plasmid could be expressed in eukaryotic cells when injected in to Wistar rats by intravenous route. At different time intervals after the injection, the distribution and expression of the plasmid in liver of rats were observed and compared using in situ hybridization and immunohistochemistry.RESULTS:The expression of the plasmidbinding to liposomes or G-PLL cou ld be markedly observed 24 h later, and began to decrease one week later,but it still could be observed up to three weeks.Both liposomes and G-PLL coul d deliver the plasmid to the liver effectively, but the effect of the latter was better than the former concerning the distribution and expression of the plasmid targeted uptake in the liver.