Volume 41, Issue 2, Pages 242-250 (August 2004) Herb medicine Inchin-ko-to (TJ-135) regulates PDGF-BB-dependent signaling pathways of hepatic stellate cells in primary culture and attenuates development of liver fibrosis induced by thioacetamide administration in rats  Yukihiro Imanishi, Naoto Maeda, Kohji Otogawa, Shuichi Seki, Hiroko Matsui, Norifumi Kawada, Tetsuo Arakawa  Journal of Hepatology  Volume 41, Issue 2, Pages 242-250 (August 2004) DOI: 10.1016/j.jhep.2004.04.005

Fig. 1 Effect of TJ-135 on TAA-induced liver fibrosis in rats. (A) Histology. a, c, and e: TAA-treated group. b, d, and f: TAA+TJ-135-treated group. a and b: H.E. staining. c and d: Sirius red staining. e and f: SM α-actin immuno-staining. (B) Expression of PDGFRβ, SM α-actin and STAP in rat liver tissues. Expression of PDGFRβ, SM α-actin and STAP was determined by western blotting. Densitometric analysis of each band. The arbitrary unit is the % volume measured by using a BIO RAD GS-700 densitometer. *P<0.01. (C) RT-PCR analysis of TGFβ1 and GAPDH mRNA expression in liver tissues of four individuals in each treated group. The arbitrary unit is the % volume of TGFβ1/% volume of GAPDH measured by using a BIO RAD GS-700 densitometer. *P<0.01. (D) Serum level of AST, ALT and hyaluronic acid (H.A.) of rats treated with TAA and TJ-135 for 6 weeks (n=5). *P<0.05. Journal of Hepatology 2004 41, 242-250DOI: (10.1016/j.jhep.2004.04.005)

Fig. 2 Effects of TJ-135 on morphology, expression of PDGFRβ and SM α-actin and DNA synthesis of primary-cultured HSCs. (A) Morphology. Adherent HSCs were cultured in serum-free DMEM containing TJ-135 until day 4. The morphology of HSCs was observed under a phase-contrast microscope and photographed. (a) Non-treated control culture. Addition of 1 (b), 10 (c), and 100 (d) μg/ml of TJ-135. (B) Western blot analysis of the expression of PDGFRβ and SM α-actin in HSCs. Adherent HSCs were cultured in DMEM±FBS supplemented with TJ-135 at the indicated doses until day 3 or 4. PDGFRβ and SM α-actin was detected by western blotting. Representative data from three individual experiments are depicted here. (C) BrdU incorporation assay. a through d; HSCs cultured in the presence of serum. e through h; HSCs cultured in serum-free DMEM. a and e: non-treated control culture. b and f: addition of 1 μg/ml TJ-135. c and g: addition of 10 μg/ml TJ-135. d and h: addition of 100 μg/ml TJ-135. (D) BrdU labeling index (BrdU L.I.). Data are expressed as the means±SD of three different experiments. *P<0.01. Journal of Hepatology 2004 41, 242-250DOI: (10.1016/j.jhep.2004.04.005)

Fig. 3 Effects of TJ-135 on DNA synthesis, migration and signaling pathways of HSCs stimulated by PDGF-BB. (A) BrdU incorporation assay. a: 20 ng/ml PDGF-BB. b: 20 ng/ml PDGF-BB+1 μg/ml TJ-135. c: 20 ng/ml PDGF-BB+10 μg/ml TJ-135. d: 20 ng/ml PDGF-BB+100 μg/ml TJ-135. e: BrdU L.I. Data are expressed as the means±SD of three different experiments. *P<0.01. (B) Effect of TJ-135 on the migratory activity of HSCs. Data are expressed as the means±SD of three different experiments. *P<0.01. (C) Effect of TJ-135 on the phosphorylation of PDGFRβ, c-Raf, MEK1/2, ERK1/2 and Akt upon stimulation by PDGF-BB. Representative data from three individual experiments are depicted here. Journal of Hepatology 2004 41, 242-250DOI: (10.1016/j.jhep.2004.04.005)

Fig. 4 Analysis of active components included in TJ-135 that inhibit HSC proliferation. (A) BrdU incorporation assay. A. C. Spica, Artemisiae capillari spica; G. Fructus, Gardeniae fructus; R. Rhizoma, Rhei rhizoma. Data are expressed as the means±SD of three different experiments. *P<0.01. (B) BrdU incorporation assay. HSCs were treated with a component of R. rhizoma, i.e. garlic acid, cinnamic acid or emodin, at 5 μg/ml for 24 h and successively incubated in the identical medium including 100 μM BrdU for the next 24 h. *P<0.01. (C) BrdU incorporation assay. HSCs were treated with emodin at the indicated doses for 24 h and successively cultured in the identical medium including 100 μM BrdU for the next 24 h. *P<0.01. (b) Western blot. Representative data from three individual experiments are depicted here. Journal of Hepatology 2004 41, 242-250DOI: (10.1016/j.jhep.2004.04.005)

Fig. 5 Effect of emodin on PDGF-BB-stimulated DNA synthesis, migration and activation of signaling pathways of HSCs. (A) BrdU incorporation assay. a: 20 ng/ml PDGF-BB. b: 20 ng/ml PDGF-BB+1 μg/ml emodin. c: 20 ng/ml PDGF-BB+2 μg/ml emodin. d: 20 ng/ml PDGF-BB+5 μg/ml emodin. e: calculated BrdU L.I. Data are expressed as the means±SD of three different experiments. *P<0.01. (B) (a) Effects of emodin on the migratory activity of PDGF-BB-stimulated HSCs. *P<0.01. (C) Effect of emodin on the phosphorylation of PDGFRβ, c-Raf, MEK1/2, ERK1/2 and Akt under stimulation with PDGF-BB. Representative data from three individual experiments are depicted here. Journal of Hepatology 2004 41, 242-250DOI: (10.1016/j.jhep.2004.04.005)

Fig. 6 Effect of TJ-135 and emodin on ECM-related gene expression. RT-PCR was performed to detect mRNAs for collagen α1(I), collagen III, fibronectin and GAPDH. (A) a: TJ-135-treated HSCs. Representative data from three individual experiments are depicted here. b: Densitometric analysis of bands. The arbitrary unit is the % volume of collagen α1(I), collagen III and fibronectin/% volume of GAPDH. Data are expressed as the means±SD of three different experiments. *P<0.01. **P<0.05. (B) a: emodin-treated HSCs. b: densitometric analysis of bands. Data are expressed as the means±SD of three different experiments. *P<0.01. Journal of Hepatology 2004 41, 242-250DOI: (10.1016/j.jhep.2004.04.005)

Fig. 7 Effect of emodin on TAA-induced liver fibrosis in rats. (A) Histology. a, c, and e: TAA-treated rat liver tissues. b, d, and f: TAA+emodin (60 μg/g body weight)-treated rat liver tissues. a and b: H.E. staining. c and d: Sirius red staining. e and f: SM α-actin immuno staining. (B) Expression of TGFβ1 in rat liver tissues. The arbitrary unit is the % volume of TGFβ1/% volume of GAPDH measured by using a BIO RAD GS-700 densitometer. *P<0.01. (C) Serum levels of AST, ALT and hyaluronic acid (H.A.) in rats administered TAA (40 mg/body, twice a week, intraperitoneally) for 6 weeks. n=each 5. *P<0.01, **P<0.05. Journal of Hepatology 2004 41, 242-250DOI: (10.1016/j.jhep.2004.04.005)