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 phosphorylation 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 and plays a key role in LDP.