In this study, our results showed (1) 18α-GA reduced the mRNA expression of Smad3, COL1A2 and COL3A1, and increased that of Smad7 in both LX-2 and CFSC, and similar trend was also found in protein expression of these factors; (2) 18α-GA reduced the phosphorylation level of Smad3 in LX-2 and CFSC; (3) 18α-GA inhibited the activities of SP-1, AP-1 and NF-κB in both LX-2 and CFSC.
TGF-β signals are transmitted via serine / threonine kinase transmembrane receptors that phosphorylate cytoplasmic mediators of Smad family. Phospho-Smads then translocate into the nucleus, where they function as transcription factors and bind to DNA either directly or with the help of other proteins, regulating collagen I and III expression.
Over-expression of Smad3 can increase the deposition of fibronectin and collagen I and accelerate α-SMA production in HSCs 10
, and HSCs from mice lacking Smad3 had decreased ability to respond to stimulation of fibers immunogenicity, indicating that Smad3 plays a promotive role in the TGF-β1-mediated fibrogenesis. Wang et al have shown that glycyrrhizin can significantly reduce the levels of Smad2 and Smad3 in primary HSCs isolated from animals with carbon tetrachloride-induced hepatic fibrosis 11
. Other studies also reveal that glycyrrhizin or glycyrrhizic acid can inhibit COL1A2 promoter activity in HSCs of mice with carbon tetrachloride-induced hepatic fibrosis, in which the decrease of Smad3 in the nucleus of HSCs plays an important role 12
. Our findings also demonstrated that the level of Smad3 was also decreased after 18α-GA treatment in a time-dependent manner in both LX-2 and CFSC, but that of Smad2 remained unchanged, which may be related to the time point and the cell type.
Ubiquitin- proteasome plays a regulatory role in the TGF-β1/Smad signaling, in which E3 ubiquitin ligase, such as Smurfs, is an important one participant. Smad7 can bind to TGF-β1 receptor complex and promote the degradation of TGF-β1 through recruiting ubiquitin ligase Smurfs. Wang et al found that the level of Smad7 was increased after administration of glycyrrhizin for 2 weeks in rats with carbon tetrachloride induced hepatic fibrosis, and the hepatic pathology presented significant improvement after 4-week treatment, suggesting that this may be due to increase of Smad7. Our results also indicated that 18α-GA increased Smad7 expression significantly in a time-dependent manner. The phosphorylation of Smad2 and Smad3 are their active form, TGF-β type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create phosphoisoforms. After acute liver injury, TGF-β and pro-inflammatory cytokines synergistically enhance collagen synthesis by activated hepatic stellate cells via phosphorylation of Smad2 and Smad3 pathways 13,14
. And then the phosphorylation level of Smad2 and Smad3 was determined by immunocytochemistry in both LX-2 and CFSC. Results showed that 18α-GA reduced the phosphorylation level of Smad3, while that of phosphorylation of Smad2 remained unchanged. Previous studies have shown that Smad2 and Smad3 play different roles in the activation of HSCs: over-expression of Smad3 can significantly increase the collagen I and α-SMA expression, which is not observed after Smad2 over-expression, suggesting that Smad3 plays a more important role in the activation of HSCs 10
. Smad2 phosphorylation and nuclear translocation as a response to TGF-β1 are mainly found in resting HSCs, while Smad3 phosphorylation observed in activated HSCs. The cells in the present study were in the activated form, which may explain why no change in the phosphorylation level of Smad2 was observed.
Hepatic fibrosis is a dynamic wound healing process, during which both of the quantity and quality of ECM in the liver are significantly changed. Previous studies have revealed that the mRNA and protein expression of collagen I and III was significantly reduced in HSCs collected from rats with carbon tetrachloride and ethanol-induced hepatic fibrosis 11
. The -376~-108 bp in COL1A2 have the most potent promoter activity and contain a C/EBP binding site, three SP-1 binding sites and one AP-1 binding site. A large number of studies have shown that SP-1 and AP-1 are the transcription factors of COL1A2 as a response to TGF -β1 15-17
. In addition, SP-1 and AP-1 can also promote the synthesis of ECM and inhibit ECM degradation through regulating TIMP, MMP and IL-6 expressions in the liver fibrogenesis 18-21
It has been noted that NF-κB involves in the activation of HSCs 22
. Upon stimulation by cytokines, mitogen and CD40 ligand, the NF-κB activity increased rapidly, which promotes the production of intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase 2 (COX2), IL-6 and IL-8 in HSCs, and triggers or exacerbates liver inflammation 23, 24
Our findings revealed that 18α-GA could inhibit the activities of SP-1, AP-1 and NF-κB, which may also involve in the down-regulation of collagen expression after 18α-GA treatment.
However, this study was an in vitro one, and more in vivo studies are required to confirm our findings. Our results showed the expression of Smad3, Smad7, AP-1, SP-1, NF-κB, type I and III collagen was affected by 18α-GA, but we could not confirm that whether this effect was directly or indirectly related to 18α-GA. That is, the target of 18α-GA in HSCs was not determined.
In summary, our findings revealed that 18α-GA significantly reduced the expression of collagen I and III, which may be attributed to the regulation of expression of Smad3, Smad7, AP-1, SP-1 and NF-κB by 18α-GA. Furthermore, the activities of collagen-related transcription factors are also detected, and results also demonstrate that 18α-GA down-regulates the expression of type I and III collagen.