Overexpression of thioredoxin prevents thioacetamide-induced hepatic fibrosis in mice

Upregulation of endogenous Trx in TAA-induced murine and rat hepatic fibrosis. Immunohistochemical staining for Trx in normal murine liver (A), TAA-induced murine fibrotic liver (B), normal rat liver (C) and TAA-induced rat fibrotic liver (D). Endogenous Trx expression was increased in hepatocytes of TAA-treated murine and rat fibrotic liver (B and D). Magnification, ×200.

Effect of Trx in TNF-α-induced apoptosis of HepG2 cells. (A) Western blotting of stable Trx transfectants of HepG2 cells. EV indicates empty vector. Trx wt indicates wild type Trx. Trx dm indicates double mutant Trx. Trx is overexpressed in Trx wt HepG2 cells. This antibody recognizes double mutant Trx as well. (B) Determination of viable number of HepG2 cells treated with TNF-α plus CHX by MTS assay. Cell death was significantly inhibited in Trx wt HepG2 cells (solid line) compared with control (dotted line) (^*P<0.05). Cell death was significantly enhanced in Trx dm HepG2 cells (grey line) compared with control (^*P<0.05). (C) Flow cytometry analysis of DNA fragmentation in stable Trx transfectants of HepG2 cells treated with TNF-α plus CHX. Apoptosis was significantly inhibited in Trx wt HepG2 cells (black bar) compared with control (white bar) (^*P<0.05). There was no difference in apoptosis between Trx dm HepG2 cells (hatched bar) and control (white bar).

Trx inhibited TAA-induced hepatic fibrosis. (A–D). Azan staining. (A) Non-treated WT mouse liver. (B) Non-treated Tg mouse liver. (C) TAA-treated WT mouse liver. (D) TAA-treated Tg mouse liver. Fibrosis is apparent in TAA-treated WT mouse liver (C). Fibrosis is hardly detected in TAA-treated Tg mouse liver (D). Magnification, ×100. (E) Evaluation of fibrotic area observed by Azan staining. We used image analysis soft Mac Scope. ^*P<0.05. (F) MDA content in the liver. MDA was measured at the absorbance of 532nm. ^*P<0.05 (G and H). Serum level of AST (G) and ALT (H) after TAA administration for 3 months.

Effect of Trx on serum- and PDGF-stimulated proliferation of primary-cultured HSC. (A) Western blot. Samples were prepared from HSC that were isolated from WT and Tg mice and cultured for the indicated days. hTrx, human Trx. mTrx, mouse Trx. HSC isolated from Tg mice express hTrx. (B) The morphology of HSC isolated from WT and Tg mice. Upper left: quiescent HSC isolated from WT mice and cultured for 1 day. Upper right: quiescent HSC isolated from Tg mice and cultured for 1 day. Lower left: activated HSC isolated from WT mice and cultured for 7 days. Lower right: activated HSC isolated from Tg mice and cultured for 7 days. (C) The number of cultured HSC isolated from WT and Tg mice. Cell number was determined at the indicated days by Trypan blue staining. (D) Effect of human recombinant Trx on serum-stimulated DNA synthesis of activated rat HSC. ^*P<0.01. (E) Effect of recombinant human Trx on PDGF-dependent DNA synthesis of activated rat HSC. ^*P<0.05. (F) Expression of phospho-specific ERK, total ERK, phospho-specific Akt, and total Akt in activated rat HSC treated with recombinant Trx (10μM) and PDGF-BB (20μg/ml). After culture for 5 days, rat HSC were preincubated with 10μM of recombinant Trx suspended in sterile phosphate buffer saline (PBS) for 24h at 37°C and then incubated with 20μg/ml of PDGF-BB for 10min at 37°C. (G) Expression of collagen type I alpha 1 mRNA in HSC that were isolated from WT and Tg mice and treated with 10μM of TGF-β for 24h after culture for 5 days.