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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 E-mail:chenghai_liu@yahoo.com.cn

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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."

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