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Volume 39, Issue 5, Pages 742-748 (November 2003) A blocking peptide for transforming growth factor-β1 activation prevents hepatic fibrosis in vivo Hiroki Kondou, Sotaro Mushiake, Yuri Etani, Yoko Miyoshi, Toshimi Michigami, Keiichi Ozono Journal of Hepatology Volume 39, Issue 5, Pages 742-748 (November 2003) DOI: 10.1016/S0168-8278(03)00377-5

Fig. 1 A timeline of the experimental protocol. Rats received an intraperitoneal injection of 1% DMN at a dose of 10 mg DMN/kg for three consecutive days a week for 4 weeks. The animals were treated simultaneously with one of the following regimens: (i) DMN-treatment and 100 μg LSKL in 0.5 ml saline i.p. daily (DMN+LSKL group); (ii) DMN-treatment and 100 μg SLLK in 0.5 ml saline i.p. daily (DMN+SLLK group); (iii) DMN-treatment and 0.5 ml saline i.p. daily (DMN+saline group); (vi) 100 μg LSKL in 0.5 ml saline i.p. daily (LSKL group); (v) 100 μg SLLK in 0.5 ml saline i.p. daily (SLLK group); and (vi) 0.5 ml saline only i.p. daily (saline group). After 4 weeks of treatment, the gain of body and liver weights were evaluated. In addition, a histological analysis of damage and fibrosis of the liver in each group was performed. Journal of Hepatology 2003 39, 742-748DOI: (10.1016/S0168-8278(03)00377-5)

Fig. 2 Macroscopic appearance of the liver. Livers obtained from rats treated with DMN+LSKL i.p. (A); and DMN+SLLK i.p. (B). Bar=1 cm. The livers of the DMN+SLLK group were atrophic. Journal of Hepatology 2003 39, 742-748DOI: (10.1016/S0168-8278(03)00377-5)

Fig. 3 HE staining (A, B); and Mallory azan staining (C, D) of the liver sections in the DMN+LSKL (B, D); and DMN+SLLK (A, C) groups. In HE staining, scattered foci of hepatocellular necrosis, ballooning degeneration, and enlargement of the nuclei of the hepatocytes were marked in the DMN+SLLK group (A). However, these findings were markedly less in the DMN+LSKL group (B). In Mallory azan stained liver, severe bridging fibrosis was found in the DMN+SLLK group (C), and those findings were ameliorated in the DMN+LSKL group (D). Bar=100 μm. Journal of Hepatology 2003 39, 742-748DOI: (10.1016/S0168-8278(03)00377-5)

Fig. 4 Semiquantitative analysis for necrosis/degeneration scoring (A); and fibrosis scoring (B). Rats were treated with DMN+LSKL (n=15) DMN+SLLK (n=8), and DMN+saline (n=11). Scoring (Table 1) was performed by an independent pathologist blinded to the treatment protocol. The necrosis/degeneration score was evaluated in HE staining, and significantly lower in the DMN+LSKL group than in the DMN+SLLK and DMN+saline groups. The degree of fibrosis score was significantly lower in the DMN+LSKL group than in the DMN+SLLK and DMN+saline groups. aP<0.001, bP<0.01, cP<0.05. Journal of Hepatology 2003 39, 742-748DOI: (10.1016/S0168-8278(03)00377-5)

Fig. 5 Hydroxyproline content of the liver (A); and the results of real-time quantitative PCR (B). Hydroxyproline contents and mRNA levels of collagen α2 (I) were measured in the liver of the DMN+LSKL group (n=15), DMN+SLLK group (n=8), DMN+saline group (n=11), LSKL group (n=6), SLLK group (n=5), and saline group (n=6). Results are expressed as mean±SD. aP<0.001 versus DMN+LSKL group, bP<0.01 versus DMN+LSKL group, cP<0.05 versus DMN+LSKL group, dP<0.001 versus DMN+SLLK group, eP<0.01 versus DMN+SLLK group, fP<0.001 versus DMN+saline group, gP<0.01 versus DMN+saline group. Journal of Hepatology 2003 39, 742-748DOI: (10.1016/S0168-8278(03)00377-5)

Fig. 6 Active/total TGF-β1 ratio. Active and total TGF-β1 were measured using an ELISA kit for TGF-β1 in the liver of the DMN+LSKL group (n=15), DMN+SLLK group (n=8), DMN+saline group (n=11), LSKL group (n=6), SLLK group (n=5), and saline group (n=6). Results are expressed as mean±SD. aP<0.001 versus DMN+LSKL group, bP<0.05 versus DMN+LSKL group, cP<0.01 versus DMN+SLLK group, dP<0.05 versus DMN+SLLK group. Journal of Hepatology 2003 39, 742-748DOI: (10.1016/S0168-8278(03)00377-5)

Fig. 7 Immunoprecipitation/Western blotting of Smad 2 and phospho-Smad 2. Tissue extracts were immunoprecipitated with anti-Smad 2 antiserum, and SDS-PAGE and Western blotting were performed using the Smad 2 or the phospho-Smad 2 antibodies. The bands were quantitated by densitometry, and these data were evaluated as a percent of control (saline group). The percent of controls are shown as mean±SD. Results are expressed as mean±SD. aP<0.001 versus DMN+saline group, bP<0.01 versus DMN+saline group, cP<0.05 versus DMN+saline group, dP<0.05 versus DMN+LSKL group. Journal of Hepatology 2003 39, 742-748DOI: (10.1016/S0168-8278(03)00377-5)