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1 Lab. Anim. Res. 2010: 26(1), 1-6 Anti-hepatofibrogenic Effect of Turnip Water Extract on Thioacetamide-induced Liver Fibrosis Lan Li 1#, Dae-Hun Park 2#, Yong-Chun Li 1, Seung-Kee Park 3, Yun-Lyul Lee 4, Hyon-Min Choi 1, Dae-Sup Han 1, Hyuck-Joo Yang 1, Eun-Hye Lee 1, Hyung-Kwan Jang 4, Young-Jin Kim 5, Ja-June Jang 6 and Min-Jae Lee 1 * 1 Department of Laboratory Animal Medicine, Kangwon National University, Chuncheon, Korea 2 College of Pharmacy, Kangwon National University, Chuncheon, Korea 3 Department of Microbiology, College of Medicine, Hallym University, Chuncheon, Korea 4 Department of Physiology, College of Medicine, Hallym University, Chuncheon, Korea 5 Department of Veterinary Infectious Diseases and Avian Disease, College of Veterinary Medicine, Chonbuk National University, Cheongju, Korea 6 Traditional Food Research Group, Korea Food Research Institute, Seoul, Korea 7 Department of Pathology, College of Medicine, Seoul National University, Seoul, Korea Liver fibrosis is a chronic liver disease and lots of people in Korea are suffered. There are many efforts to find candidates to suppress liver fibrogenesis and several chemical-induced model or bile duct ligation model have been used to research and develop hepatic fibrogenic suppressor. From the previous study about functional effects of turnip which cultivated in Kangha Island, we got the feasibility which turnip might be able to inhibit heptatic fibrogenesis. TAA is a representative hepatic fibrosis inducer, repeated 7- weeks i.p. injection of it results in hepatic fibrosis. We compared the level of hepatic fibrosis in TAAturnip group, TAA group, and vehicle control group. Nodules-formed by TAA were observed; they were rarely shown in vehicle control group, observed in most area in TAA group, but only shown in periportal regions in TAA-turnip group. These results were confirmed through Masson's trichrom stain; fibrous structures increased in TAA group (fibrosis score: 4) but significantly decreased in TAA-turnip group (fibrosis score: 2-3). In conclusion, we got the result that turnip water extract has a potency to protect TAA-induced hepatic fibrogenesis but it is necessary further study to find its mechanism. Key words: Turnip water extract, thioacetamide (TAA), hepatofibrogenic animal model, nodular formation (Received 16 September 2009; Revised version received 2 February 2010; Accepted 10 March 2010) y y,, yƒ š w, (Zhou and Lu, 2009). y k w {lx (scarring) x mw, ƒ 16 wš (Hillebrandt et al., 2005). x y w ƒ š š, s w ƒ me», s» # These authors equally contributed to this study *Corresponding author: Min-Jae Lee, School of Veterinary Medicine, Kangwon University, Chuncheon, Kangwon-do , Korea Tel: Fax: mjlee@kangwon.ac.kr (extracellular matrix) wš s» y w ƒ (Friedman et al., 2008). ù y w» yw š y w» w» w yw, dimethylnitrosamine (DEN) (Chang et al., 2008), carbon tetrachloride (CCl 4 ) (Young et al., 2007), ethanol (Hu et al. 2009) thioacetamide (TAA) (An et al., 2006) w» (Krahenbuhl et al., 2000) w ƒ w š. p TAA w y xkw, yw p w» TAA w w. ¾ y w curcumin (Shapiro et al., 2006), Inchin-ko-to (TJ-135) (Inao et al., 2004), polyenylphosphatidylcholine (PPC) (Ma et 1

2 2 Lan Li et al. al., 1996) y p ½e y š, s w 2x wš(jung et al., 2008), g wwš (Kim et al., 2006), k y w (Shin et al., 2006), z w (Shin et al., ) z ƒ, w k x transaminase (AST & ALT) lactic dehydrogenase z superoxide dismutase catalase y z (Choi et al., 2006)ƒ š. w ww m w TAA y w w w, y w z ³ w y ƒ w ³ wš w. w t w,. w w z,» 3 ƒ w š, q- ƒƒ 1% ƒwš 65 o C 22 wš w ww 60 C w w o. sww AIN76 Table 1 ƒ 1% yww w x w. Table 1. Composition of turnip feed Feed Ingredients Content (%) Turnip water extracts 1.0 Corn Starch 49.0 Casein 20.0 Sucrose 15.0 Cellulose 5.0 Mineral Mixture 3.5 Vitamin Mixture 1.0 DL-methionine 0.3 Choline Chloride 0.2 Corn Oil 5.0 Total Ò l x 120 g 6 f F344 w Figure 1 TAA n TAA n w. w» w thioacetamide (TAA, Sigma, USA) saline kg 200 mg 3z 7 w. TAA n w w. x» 1z d w š x óù z d w x ƒ e w w. x w x z» w w ( x x ƒ y: KIACUC ). l j x l (Junsei Chemical Co., Led., Japan) Figure 1. The protocol of TAA-induced hepatic fibrogenetic animal model. Arrows mean administrated a intraperitoneal injection of TAA at 200 mg/kg a week.

3 Turnip water extract inhibits TAA-induced hepatofibrogenesis in rat model 3 Figure 2. TAA treated groups showed lower body weight gain compared to TAA non-treated group during the experiment. All values are expressed as mean±sd. *P<0.05, TAA untreated groups vs. TAA treated groups. z w xw. yw»(fuji DRI-CHEM 3500i, Japan) w, x x 3000rpm 15 w x mw aspartic acid transaminase (AST), alanine transaminase (ALT), total bilirubin (T-Bil) d w. j mw ƒƒ 2 r üš 10% s š w z w -k q v w 4µm Ì r g. hematoxylin and eosin (H&E) Masson's trichrome (MT) ww Ÿwx w. y ùkü» w w score w score 0: x (no), score 1: y(portal fibrosis), score 2: y(periportal fibrosis), score 3: y(septal fibrosis), score 4: (cirrhosis) q w. SAS v (ANOVA) w m (P<0.05) q w. m TAA n ƒ e w y w TAA n ƒ w k y w (P<0.05). wr w w z j y w (Figure 2). e í m l j w ùkü TAA n TAA n ùkû ù TAA n ƒ w e. w ƒw ùkù (Cha et al., 2009),» (Figure 3A). x ü AST ALT s w z, sƒ q x ù x eƒ s d w y (Yang and Leow, 2006). Total bilirubin x ƒ ü s eƒ w. Figure 3B TAA n w TAA n AST, ALT, Total bilirubin eƒ w ƒw w. w ƒ, ƒ w e q. ù TAA n w x z y j z ƒ, y q w w ƒ wš x z y w x z yƒ y w š q w». ô e l j m e m w w TAA n t yƒ ù TAA n x y (data not shown). H&E g e y w

4 4 Lan Li et al. Figure 3. Physiological changes by TAA treatment or untreatment (A) Relative liver weight of experimental animals. TAA treated groups is larger than that in TAA untreated groups. All values are expressed as mean±sd. P<0.05, TAA untreated groups vs. TAA treated groups. (B) Serological analysis of liver enzymes in TAA treated rat liver with turnip water extract. It is not different from activities of serum enzymes of control and those of turnip water extract treatment group. All values are expressed as mean±sd. P<0.05, TAA untreated groups vs. TAA treated groups.» w MT w (Figure 4A). TAA n v e wš, y y. TAA n y ƒ š MT g e w w x (nodular formation)w x x. TAA n w w xk š, y v e q. w y w, TAA n fibrosis scoreƒ 0 y, TAA n fibrosis scoreƒ 4 y š TAA n w fibrosis scoreƒ 2-3 q (Figure 4B). mw w y, y w y w. q 2010 w w ww.

5 Turnip water extract inhibits TAA-induced hepatofibrogenesis in rat model 5 Figure 4. Anti-hepatofibrogenic effects of turnip water extract (A) Photos of livers in control and turnip water extract treatment group are normal but that in TAA treatment is shown typical hepatic fibrosis. Turnip water extract almost inhibits TAA-induced nodular formation and fibrosis. (B) Fibrosis scores in each group. Semi-quantitative scoring system is adopted to evaluate the degree of liver fibrosis (0: no, score 1: portal fibrosis, score 2: periportal fibrosis, score 3: septal fibrosis, score 4: cirrhosis). Mean of fibrosis scores in TAA treatment group is 4 but turnip water extract decreases mean of fibrosis scores by 2-3. All values are expressed as mean±sd. P<0.05, TAA untreated groups vs. TAA treated groups. k An, J.H., Seong, J, Oh, H., Kim, W., Han, K.H. and Paik, Y.H. (2006) Protein expression profiles in a rat cirrhotic model induced by thioacetamide. Kor. J. Hepatol. 12(1), Cha, J.Y., Park, J.C., Ahn, H.Y., Eom, K.E., Park, B.K., Jun, B.S., Lee, C.H. and Cho, Y.S. (2009) Effect of Monascus purpureus -fermented Korean red ginseng powder on the serum lipid levels and antioxidative activity in rats. J. Kor Soc Food Sic Nutr. 38(9), Chang, J.J., Jeon, S.Y., Song, J.Y., Kim, J.H., Li, L., Park, D.H., Lee, Y.L., Park, J.J., Woo, D.W., Kim, G.J. and Lee, M.J. (2008) Alteration of X-linked inhibitors of apoptosis (XIAP) expression in rat model with DEN-induced hepatocellular carcinogenesis. Mol. Cell. Toxicol. 4(4), Choi, H.J., Han, M.J., Baek, N.I., Kim, D.H., Jung, H.G. and Kim, N.J. (2006) Hepatoprotective Effects of Brassica rapa (Turnip) on d-galactosamine Induced Liver Injured Rats. Kor. J. Phamacogn. 37(4), Friedman, S.L. (2008) Mechanisms of Hepatic Fibrogenesis. Gastroenterology 314, Hillebrandt, S. (2005) Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans. Nature Genetics 37, Hu, S.L., Yin, S., Jiang, X.D., Huang, D.B. and Shen, G. (2009) Melatonin protects against alcoholic liver injury by attenuating oxidative stress, inflammatory response, and apoptosis. Europ. J. Pharm. 616, Inao, M., Mochida, S., Matsui, A., Eguchi, Y., Yulutuz, Y., Wang, Y., Naiki, K., Kakinuma, T., Fujimori, K., Nagoshi, S. and fujiwara, K. (2004) Japanese herbal medicine inchin-ko-to as a therapeutic drug for liver fibrosis. J. Hepatol. 41, Jung, U.J., Baek, N.I., Chung, H.G., Bang, M.H., Jeong, T.S., Lee, K.T., Kang, Y.J., Lee, M.K., Kim, H.J., Yeo, J. and Choi, M.S. (2008) Effects of the ethanol extract of the roots of Brassica rapa on glucose and lipid metabolism in C57BL/KsJ - db/db mice. Clinical. Nutrition 27, Kim, D.H., Kim, J.H., Kim, C.H., Kwon, M.C., Kim, H.S., Chung, H.G., Kang, H.Y., Lee, H.J. and Lee, H.Y. (2008) Effects of alcohol oxidation of Brassicarapa L. extraction process in Kang-Hwa. Kor. J. Medicinal Crop Sci. 14(1), Krahenbuhl, S., Brass, EP. and Hoppel, C.L. (2000) Decreased carnitine biosynthesis in rats with secondary biliary cirrhosis. Hepatology 3(1), Ma, X., Zhao, J. and Lieber, C.S. (1996) Polyenylphosphatidylcholine Attenuates non-alcoholic hepatic fibrosis and

6 6 Lan Li et al. accelerates its regression. J. Hepatol. 24, Shapiro, H., Ashkenazi, M., Weizman, N., Shahmurov, M., Aeed, H. and Bruck, R. (2006) Curcumin ameliorates acute thioacetamide-induced hepatotoxicity. J. Gastroenterology & Hepatology 21(2), Shin, H.W., Park, S.Y., Lee, K.B., Shin, E., Lee, M.J., Kim, Y.J. and Jang, J.J. (2006) Inhibitory effect of turnip extract on thioacetamide-induced rat hepatic fibrogenesis. Cancer Prevention Research 11, Shin, H.W., Park, S.Y., Lee, K.B., Shin, E., Ryu, H.S., Kim, M.S., Ahn, J.B., Kim, Y.J. and Jang, J.J. (2006-1) Inhibitory Effect of Turnip extract fractions on Hepatic Fibrogenesis. Lab. Anim. Res. 22(3), Yang, S. and Leow, C.K.. (2006) Expression patterns of cytokine, growth factor and cell cycle-related genes after partial hepatectomy in rats with thioacetamide-induced cirrhosis. World J. Gastroenterol. 12, Young, S.C., Wang, C.J., Lin, J.J., Peng, P.L., Hsu, J.L. and Chou, F.P. (2007) Protection effect of piper betel leaf extract against carbon tetrachloride-induced liver fibrosis in rats. Arch Toxicol. 81, Zhou, K. and Lu, G. (2009) Assessment of fibrosis in chronic liver diseases. J. Digestive Diseases 10, 7-14.

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