Effects Of Glycyrrhizin On TGF-β/smad Signal Transduction In Hepatic Stellate Cells In Rats

Background: Liver fibrosis is the common pathological basis for diverse liver injuries. During the formation of liver fibrosis or cirrhosis, the activation of hepatic stellate cells (HSC) and the imbalance between synthesis and degradation of extracellular matrix (ECM) are important in the pathologenesis. Molecular mechanisms involved in fibrogenesis reveal that transforming growth factor β (TGF-β ), especially TGF- β 1, plays a pivotal role. The signal transduction of TGF-β through a family of transmemberane ser/thr kinase receptors to the nucleus is predominantly mediated by phosphorylation of evolutionarily conserved cytoplasmic mediators of the smad family. The smad family is divided into three functional groups: R-smads (receptor-associated smads) can directly interact with activated type 1 receptor and then be phosphorylated; Co-smads(Common mediated smads) is a common necessary mediator, upon R-smads phospharylation and oligomerization with Co-smads, they move into nucleus to regulate transcription of target genes; and I-smads (inhibitory smads) may function as a negative feedback loop in TGF- β signaling pathway. Smad2, 3, 4 and 7 are involved in TGF-β signaling. Glycyrrhizin is a widely used and effective agent in current clinic treatment for chronic hepatitis. Its effect might be associated with inducing α -Interferon, reducing the expression levels of interlukin 6 and tumor necrosis factor, producing prostaglandin E, and inhibiting the activities of Phospholipase A2. Previous research showed glycyrrhizin may inhibit fibrosis by intervention of TGF β /smad signal. However the accurate mechanism of glycyrrhizin still needs to be investigated. Objective: To investigation the effects of glycyrrhizin on TGF- β /smad pathway in cultured HSC stimulated by TGF- β₁ and then to reveal the possible molecular mechanism of glycyrrhizin on anti-fibrosis..Methods: Primary HSCs were isolated from SD rats and cultured in vitro. The inhibitory rate of proliferation was measured by MTT method, the viability of cells was tested by evaluation of supernant LDH and Trypan blue dye exclusion within control and of glycyrrhizin 4 groups (in different concentration of 1 μ mol/L-1000 μ mol/L). Protein expression of α -SMA was detected by western blot. TGF- β₁ concentration was also detected by ELISA. The gene expression profiles were compared within control group, TGF- β₁ group or TGF- β₁ with glycyrrhizin (100 μmol/L) group. Using a cDNA microarray GEArray^?Q to screen changed target genes with significant change in TGF-β /smad pathway. Then HSCs were divided into 6 groups, as control group, TGF β₁ only group and TGF β₁ with different concentration glycyrrhizin(1 μ mol/L-1000 μ mol/L) groups respectively. The mRNA levels of smad2, smad3, smad7, procollagen I α 2, procollagen III α1, PAI-1, TGF β receptor 2 were assessed by semi-quantitative RT-PCR method , The protein level of smad2, p-smad2, smad3, N-smad3, smad7, type I Collagen, type III Collagen, PAI-1 and TGF β receptor 2 were assessed by Western blot analysis. Results: MTT method showed an obvious inhibition of HSC proliferation after 100 μ mol/L -1000 μ mol/L glycyrrhizin treatment. Different concentrations of glycyrrhizin do not affect the vitality on HSC by LDH measurement and Trypan Blue dye exclusion method. Glycyrrhizin of different concentration could gradually decrease protein expression of α-SMA. Glycyrrhizin of 1000 μ mol/L could inhibit supernant TGF- β₁ secreted by HSC. The cDNA microarray identified that 16 genes were down-regulated by glycyrrhizin after being up-regulated by TGF β₁; 5 genes were up-regulated by glycyrrhizin after being down-regulated by TGF β₁; and 2 genes were up-regulated predominantly by glycyrrhizin after being up-regulated by TGF β₁ in HSC. Among these 7 genes were associated with TGF- β /smad signaling. The results of RT-PCR and Western blot identified the coincidence with those of cDNA microarray. It also demonstrated that TGF β₁ could increase mRNA level of Smad2, 3, 7, procollagen I α 2, procollagen III α 1, PAI-1 in HSC, and TGF-β₁ could also increase protein expression of Smad2, 3, 7, p-smad2, N-smad3, type I collagen, type III collagen PAI-1 and TGF-13 receptor 2. Glycyrrhizin of 1 μ mol/1-1000 μ mol/1 could gradually decrease the mRNA level and protein expression of Smad2, 3, 7, type I collagen, type III collagen and PAI-1, but increase those of TGF-β receptor 2.Conclusion: Glycyrrhizin with high concentration could inhibit HSC proliferation and TGF β₁ secretion. Glycyrrhizin in different concentrations could also decrease the mRNA level and protein expression of smd2, smad3, smad7, type I , type III collagen and PAI-1 gradually. It could also up-graduate the mRNA level and protein expression of TGF- β receptor 2.