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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (5): 561-568.doi: 10.3736/jcim20120512

• Original Experimental Research • Previous Articles     Next Articles

Chinese herbal medicine Fuzheng Huayu recipe inhibits liver fibrosis by mediating the transforming growth factor-β1/Smads signaling pathway

Qing-lan Wang1,2, Yan-yan Tao1, Li Shen1, Hong-yan Cui1, Cheng-hai Liu1,2,3()   

  1. 1. Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    2. Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China
    3. Key Laboratory of Liver and Kidney Diseases (Shanghai University of Traditional Chinese Medicine), Ministry of Education, Shanghai 201203, China;
  • Received:2011-12-15 Accepted:2011-12-27 Online:2012-05-20 Published:2018-06-15
  • Contact: Liu Cheng-hai

Objective: To investigate the mechanism of Fuzheng Huayu recipe (FZHY), a compound traditional Chinese herbal medicine, against liver fibrosis related to transforming growth factor-β1 (TGF-β1)/Smads signaling transduction.

Methods: The research consisted of in vitro and in vivo experiments. In the in vivo experiment, 37 male Wistar rats were divided into 3 groups: 5 rats in normal group, 18 and 14 rats respectively in model and FZHY groups. Liver fibrosis was induced in rats of the model group and the FZHY group by intraperitoneal injection of dimethylnitrosamine with a dose of 10 μg/kg body weight for 4 weeks. Rats in the FZHY group were administered with FZHY for 4 weeks after liver fibrosis was induced. After the treatment of FZHY, hydroxyproline (Hyp) content in rat liver tissue was assayed by Jamall’s method and protein expressions of TGF-β1, TGF-β1 receptor Ⅰ (TβR- Ⅰ), Smad2, Smad3 and phosphorylated-Smad2/3 were analyzed by Western blotting. In the in vitro experiment, hepatic stellate cells (HSCs) were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. On the 4th day of cell culture, HSCs were stimulated by 2.5 ng/mL TGF-β1 for 24 h, then incubated with the medium containing 10% FZHY-medicated serum or 10 μmol/L SB-431542 (a potent and specific inhibitor of TGF-β1 receptor Ⅰ kinase) for 24 h. And the HSCs without TGF-β1 stimulating were used as control group. Protein expressions and location of α-smooth muscle actin (α-SMA) and Smad3 in HSCs were assayed by immunofluorescent staining, and the image was analyzed by Image-Pro Plus 6.1 System.

Results: In the in vivo experiment, liver Hyp content in the FZHY group was reduced significantly compared with the model group. FZHY also down-regulated the protein expressions of TGF-β1, TβR-Ⅰ and p-Smad2/3 in fibrotic liver tissue. In the in vitro experiment, FZHY-medicated serum incubated with TGF-β1-stimulated HSCs significantly down-regulated the protein expression of α-SMA. It also inhibited Smad3 nuclear translocation in TGF-β1-stimulated HSCs.

Conclusion: The mechanism of FZHY against liver fibrosis is related to the regulation of TGF-β1 signaling transduction pathway by inhibition of TGF-β1 and TβR-Ⅰ expressions and Smads activation in fibrotic liver tissue and HSCs.

Key words: drugs, Chinese herbal, liver fibrosis, hepatic stellate cell, transforming growth factor-beta1, Smad proteins, receptor-regulated, rats

Table 1

Quality control standard for FZHY"

Compound (Marker) Quality criterion
Salvianolic acid B Referred to Radix Salviae Miltiorrhizae, not less than 15.6 mg in 24 g of FZHY crude drug
Sodium Danshensu Referred to Radix Salviae Miltiorrhizae, not less than 13.2 mg in 24 g of FZHY crude drug
Adenosine Referred to Cordyceps hyphae, not less than 4.8 mg in 24 g of FZHY crude drug
Schisandrin B Referred to Fructus Schisandrae Chinensis, not less than 2.28 mg in 24 g of FZHY crude drug

Table 2

Antibodies used in this study"

Antibody Isotype Supplier Cat. No. Dilution
α-SMA Mouse IgG2a Sigma A2547 1∶200
TGF-β1 Mouse IgG1 R&D Systems MAB240 5 μg/mL
TβR-Ⅰ Rabbit IgG Cell Signaling Technology #3712 1∶1 000
Smad2 Mouse IgG1 Cell Signaling Technology #3103 1∶800
Smad3 Rabbit IgG Zymed Laboratories 51-1500 1∶200
p-Smad2/3 Rabbit IgG Santa Cruz sc-11769-R 1∶100

Table 3

Effect of FZHY on Hyp content in fibrotic liver of rats (x±s)"

Group n Hyp content (μg/g liver tissue)
Normal 5 130.55±18.84
Model 12 163.59±31.47*
FZHY 12 137.78±29.11

Figure 1

Effects of FZHY on TGF-β1, TβR-Ⅰ, Smad2 and Smad3 proteins and p-Smad2/3 expressions in fibrotic liver Results of Western blot analysis. The values are represented as the density of TGF-β1, TβR-Ⅰ or p-Smad 2/3 vs GAPDH from three different samples. *P<0.05, vs normal group; △P<0.05, vs model group. FZHY: Fuzheng Huayu recipe; α-SMA: α-smooth muscle actin; TGF-β1: transforming growth factor-β1; TβR-Ⅰ: TGF-β1 type Ⅰ receptor; p-Smad2/3: phosphorylated Smad2/3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Figure 2

Effect of FZHY on α-SMA expression in HSCs (a) Western blot analysis. The values are represented as the density of α-SMA vs GAPDH. ▲▲P<0.01, vs control; □P<0.05, vs TGF-β1. (b) Immunofluorescent staining of α-SMA (×200) acquired by an Olympus IX70 fluorescence microscope. The blue-colored stain is nuclear stained with Hoechst 33258; the green-colored stain is for α-SMA. FZHY: Fuzheng Huayu recipe; SB-431542: a potent and specific inhibitor of TGF-β1 receptor Ⅰ kinase; α-SMA: α-smooth muscle actin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Figure 3

Effect of FZHY on Smad3 nuclear translocation in HSCs (a) Immunofluorescent staining (×200) acquired by an Olympus IX70 fluorescence microscope. The blue-colored stain is nuclear stained with Hoechst 33258 and the red-colored stain is for Smad3. (b) Smad3 nuclear translocation was analyzed by Image-Pro Plus 6.1 System. ▲▲P<0.01, vs control; □□P<0.01, vs TGF-β1. FZHY: Fuzheng Huayu recipe; TGF-β1: transforming growth factor-β1; SB-431542: a potent and specific inhibitor of TGF-β1 receptor Ⅰ kinase."

[1] Liu P, Liu C, Xu LM, Hu YY, Xue HM, Liu CH, Zhang ZQ . Effects of Fuzheng Huayu 319 recipe on liver fibrosis in chronic hepatitis B[J]. World J Gastroenterol, 1998,4(4):348-353
doi: 10.3748/wjg.v4.i4.348
[2] Liu C, Hu Y, Xu L , Liu P . Effect of Fuzheng Huayu formula and its actions against liver fibrosis[J].Chin Med, 2009, 4: 12
doi: 10.1186/1749-8546-4-12 pmid: 19558726
[3] Zhou T, Yan XC, Chen Q, Tao YY, Hu YY, Liu P, Liu CH . Effects of Chinese herbal medicine Fuzheng Huayu recipe and its components against hepatocyte apoptosis in mice with hepatic injury[J]. J Chin Integr Med, 2011,9(1):57-63.
周滔, 闫秀川, 陈倩, 陶艳艳, 胡义扬, 刘平, 刘成海 . 扶正化瘀方及其治法拆方对肝损伤小鼠肝细胞凋亡的干预作用[J]. 中西医结合学报, 2011,9(1):57-63
[4] Ji G, Liu P, Liu C, Xu LM, Hong JH . Effect of sera from the rats treated with Fuzheng Huayu(FZHY) decoction on rat hepatocyte proliferation and collagen synjournal in vitro[J]. Zhongguo Shi Yan Fang Ji Xue Za Zhi, 1997,3(3):20-23.
季光, 刘平, 刘成, 徐列明, 洪嘉禾 . 扶正化瘀方药物血清对原代培养大鼠肝细胞增殖及胶原生成率的影响[J]. 中国实验方剂学杂志, 1997,3(3):20-23
[5] Moreira RK . Hepatic stellate cells and liver fibrosis[J]. Arch Pathol Lab Med, 2007,131(11):1728-1734
doi: 10.1002/cphy.c120035
[6] Gressner AM, Weiskirchen R, Breitkopf K , Dooley S.Roles of TGF-β in hepatic fibrosis[J].Front Biosci, 2002, 7: d793-d807
doi: 10.2741/gressner pmid: 11897555
[7] Liu CH, Wang XL, Liu P, Liu C, Tan YZ, Gu HT . Serapharmacological effect of Fuzheng Huayu 319 Decoction on expression of type Ⅰ collagen and transforming growth factor β1 in hepatic stellate cells[J]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 1999,19(7):412-414.
doi: 10.7661/CJIM.1999.7.412
刘成海, 王晓玲, 刘平, 刘成, 谭英姿, 顾宏图 . 扶正化瘀319方药物血清对肝星状细胞Ⅰ型胶原及转化生长因子β1表达的影响[J]. 中国中西医结合杂志, 1999,19(7):412-414
doi: 10.7661/CJIM.1999.7.412
[8] Inman GJ, Nicolás FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS . SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type Ⅰ activin receptor-like kinase(ALK) receptors ALK4, ALK5, and ALK7[J]. Mol Pharmacol, 2002,62(1):65-74
doi: 10.1124/mol.62.1.65
[9] Tao YY, Cui HY, Liu CH . Dynamic characteristics of SARA during liver fibrogenesis in rats[J]. Zhonghua Gan Zang Bing Za Zhi, 2006,14(12):909-913.
doi: 10.3760/j.issn:1007-3418.2006.12.009
陶艳艳, 崔红燕, 刘成海 . 大鼠肝纤维化病理过程中Smads锚着蛋白表达变化[J]. 中华肝脏病杂志, 2006,14(12):909-913
doi: 10.3760/j.issn:1007-3418.2006.12.009
[10] Jamall IS, Finelli VN, Que Hee SS . A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues[J]. Anal Biochem, 1981,112(1):70-75
doi: 10.1016/0003-2697(81)90261-X pmid: 7258630
[11] Liu CH, Liu P, Liu C, Ji G, Xu LM . Study on a seropharmacological method for an effective antifibrotic formula[J]. Zhongguo Shi Yan Fang Ji Xue Za Zhi, 1998,4(2):16-19.
刘成海, 刘平, 刘成, 季光, 徐列明 . 抗肝纤维化有效中药复方血清药理学方法探讨[J]. 中国实验方剂学杂志, 1998,4(2):16-19
[12] Zhao JF, Liu CH, Hu YY, Xu LM, Liu P, Liu C . Effect of salvianolic acid B on Smad3 expression in hepatic stellate cells[J]. Hepatobiliary Pancreat Dis Int, 2004,3(1):102-105
pmid: 14969849
[13] Hernandez-Gea V , Friedman SL.Pathogenesis of liver fibrosis[J].Annu Rev Pathol, 2011, 6: 425-456
doi: 10.1146/annurev-pathol-011110-130246
[14] Kawada N . Evolution of hepatic fibrosis research[J]. Hepatol Res, 2011,41(3):199-208
doi: 10.1111/j.1872-034X.2011.00776.x pmid: 21338451
[15] Galicia-Moreno M, Rodríguez-Rivera A, Reyes-Gordillo K, Segovia J, Shibayama M, Tsutsumi V, Vergara P, Moreno MG, Muriel P . N-acetylcysteine prevents carbon tetrachloride-induced liver cirrhosis: role of liver transforming growth factor-β and oxidative stress[J]. Eur J Gastroenterol Hepatol, 2009,21(8):908-914
doi: 10.1097/MEG.0b013e32831f1f3a
[16] Ikushima H, Miyazono K . Biology of transforming growth factor-β signalin. Curr Pharm Biotechnol. 2011 May 27. [Epub ahead of print]
[17] Zhao CQ, Wu YQ, Xu LM . Curative effects of Fuzheng Huayu capsules on hepatic fibrosis and the functional mechanisms: a review[J]. J Chin Integr Med, 2006,4(5):467-472.
赵长青, 吴艺青, 徐列明 . 扶正化瘀胶囊抗肝纤维化的临床疗效和作用机制[J]. 中西医结合学报, 2006,4(5):467-472
[18] Liu X, Hu H, Yin JQ . Therapeutic strategies against TGF-β signaling pathway in hepatic fibrosis[J]. Liver Int, 2006,26(1):8-22
doi: 10.1111/j.1478-3231.2005.01192.x pmid: 16420505
[19] Lan HY, Chung AC.Transforming growth factor-β and Smads[J].Contrib Nephrol , 2011, 170(): 75-82
[20] Liu C, Gaca MD, Swenson ES, Vellucci VF, Reiss M, Wells RG . Smads 2 and 3 are differentially activated by transforming growth factor-β(TGF-β) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-β-independent[J]. J Biol Chem, 2003,278(13):11721-11728
doi: 10.1074/jbc.M207728200
[21] Hofman K, Hall B, Cleaver H, Marshall S . High-throughput quantification of hydroxyproline for determination of collagen[J]. Anal Biochem, 2011,417(2):289-291
doi: 10.1016/j.ab.2011.06.019 pmid: 2021741948
[22] Friedman SL . Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver[J]. Physiol Rev, 2008,88(1):125-172
doi: 10.1152/physrev.00013.2007 pmid: 2888531
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