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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (7): 783-788.doi: 10.3736/jcim20110713

• Original Experimental Research • Previous Articles     Next Articles

Effects of Chinese herbal medicine Liuwei Dihuang Pill and its main monomer catalpol on transforming growth factor-β1/Smad pathway in HK-2 cells

Yu-min Liu1, Yue Zhang2(), Yan-peng Hao1, Zhi-jie Li1   

  1. 1. Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    2. Basic Medical College, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
  • Received:2010-11-04 Accepted:2011-04-12 Online:2011-07-20 Published:2018-10-14

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Objective: To observe the effects of sera containing Liuwei Dihuang Pill (LDP), a compound traditional Chinese herbal medicine, and its monomer catalpol on transforming growth factor-β1 (TGF-β1)/Smad pathway in HK-2 cells.
Methods:Wistar rats were intragastrically administered with LDP twice daily for 3 days and then sera were obtained. HK-2 cells were cultured at different concentrations of serum containing LDP. Lactate dehydrogenase (LDH) activity in the medium and cell activity, tested by methyl thiazolyl tetrazolium (MTT) method, were combined to determine the best concentration of the pill. Hepatocyte growth factor (HGF) was used as a positive control, while Western blotting was applied to observe the effects of sera containing LDP and catalpol on Smad2 phosphorylation and protein expressions of Smad2, Smad7 and SnoN.
Results: The 10% concentration of serum containing LDP was selected to carry out this study, as it showed no change of LDH activity in the medium and vitality of cells cultured with serum from normal rats. Similar to the mechanisms of HGF, the 10% concentration of serum containing LDP significantly inhibited the phosphorylation of Smad2 protein and up-regulated the expression of the Smad transcriptional co-repressor SnoN in TGF-β1-stimulated HK-2 cells. Catalpol inhibited phoshporylation of Smad2 without affecting SnoN protein expression. Neither serum containing LDP nor catalpol showed typical regulation effects on Smad7 expression in TGF-β1-stimulated HK-2 cells.
Conclusion: Serum containing LDP at 10% concentration has a potent antagonistic action on TGF-β1/Smad pathway in TGF-β1-stimulated HK-2 cells, which is similar to HGF. Catalpol is one of the most important monomers that plays a key role in LDP.

Key words: drugs,Chinese herbal, Liuwei Dihuang pill, Chinese herb main monomers, hepatocyte growth factor, HK-2 cells, Smad proteins

Table 1

Effects of different concentrations of sera on HK-2 cell proliferation at 48 h ($\bar{x}$±s)"

Group n Optical density
Normal serum
5% 3 0.68±0.16
10% 3 0.76±0.12
20% 3 0.88±0.19
Liuwei Dihuan Pill-containing serum
5% 3 0.82±0.12*
10% 3 0.83±0.15
20% 3 0.83±0.14

Table 2

Effects of different concentrations of sera on HK-2 cell leaking LDH at 48 h ($\bar{x}$±s, IU/L)"

Group n LDH
5% Liuwei Dihuan Pill-containing serum 5 213.40±10.26
10% Liuwei Dihuan Pill-containing serum 5 220.40±22.90
20% Liuwei Dihuan Pill-containing serum 5 376.00±26.60*

Figure 1

Effects of medicated serum with LDP on phosphorylation of Smad2 observed by Western blotting A: Control group; B: Normal serum group; C: Liuwei Dihuang Pill-containing serum group; D: TGF-β1 group; E: Normal serum plus TGF-β1 group; F: Liuwei Dihuang Pill-containing serum plus TGF-β1 group; G: HGF plus TGF-β1 group; H: HGF group. *P<0.05, vs control group; △P<0.05, vs TGF-β1 group. TGF-β1: transforming growth factor-β1; Smad: Sma- and Mad-related protein; HGF: hepatocyte growth factor; LDP: Liuwei Dihuang Pill."

Figure 2

Effects of medicated serum with LDP on Smad7 and SnoN protein expressions observed by Western blotting A: Control group; B: Normal serum group; C: Liuwei Dihuang Pill-containing serum group; D: TGF-β1 group; E: Normal serum plus TGF-β1 group; F: Liuwei Dihuang Pill-containing serum plus TGF-β1 group; G: HGF plus TGF-β1 group; H: HGF group. △P<0.05, vs TGF-β1 group. TGF-β1: transforming growth factor-β1; Smad: Sma- and Mad-related protein; HGF: hepatocyte growth factor; LDP: Liuwei Dihuang Pill."

Figure 3

Effects of catalpol at different concentrations on phosphorylation of Smad2 observed by Western blotting A: Control group; B: Low-dose catalpol group; C: High-dose catalpol group; D: TGF-β1 group; E: Low-dose catalpol plus TGF-β1 group; F: High-dose catapol plus TGF-β1 group; G: HGF plus TGF-β1 group; H: HGF group. *P<0.05, vscontrol group; △P<0.05, vs TGF-β1 group. TGF-β1: transforming growth factor-β1; Smad: Sma- and Mad-related protein; HGF: hepatocyte growth factor."

Figure 4

Effects of catalpol at different concentrations on Smad7 and SnoN protein expressions observed by Western blotting A: Control group; B: Low-dose catalpol group; C: High-dose catalpol group; D: TGF-β1 group; E: Low-dose catalpol plus TGF-β1 group; F: High-dose catalpol plus TGF-β1 group; G: HGF plus TGF-β1 group; H: HGF group. △P<0.05, vsTGF-β1 group. TGF-β1: transforming growth factor-β1; Smad: Sma- and Mad-related protein; HGF: hepatocyte growth factor."

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