The Molecular Mechanism Of The Apoptosis Of Rat Hepatic Stellate Cells During Recovery From Hepatic Fibrosis And The Effect Of Leflunomide

Hepatic fibrosis is a common consequence of chronic liver injury of any etiology, and hepatic stellate cells (HSCs) are the major source of increased extracellular matrix (ECM) proteins in chronic liver diseases. If hepatic injury persists, HSCs proliferate and undergo dramatic transdifferentiation from quiescent vitamin A-storing cells to activated myofibroblast-like cells. These activated HSCs secrete large amounts of ECM proteins, which is a seminal event in hepatic fibrogenesis.The factors that modulate HSC activation influence the progression of liver fibrosis. Kupffer cells, the resident macrophages in the liver, act as a double-edged sword in the progression of and recovery from hepatic fibrosis. It has been widely reported that Kupffer cells can promote HSC activation during the progression of fibrosis. On the other hand, Kupffer cells can negatively regulate activated HSCs during recovery from liver fibrosis associated with enhancing matrix degradation. The mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis, however, is still far from being fully understood. Leflunomide, an isoxazole derivative, is a unique immunomodulatory agent, capable of treating rheumatoid arthritis (RA), allograft and xenograft rejection,and systemic lupus erythematosus. After administration, it is metabolized to its active form, A771726, which leads to the immunosuppressive activity. It has also been reported to block cell cycle progression in the G0/G1 phase. Recent evidence has suggested that A771726 can significantly inhibit the proliferation and activation of HSCs by inhibiting the Janus kinase (JAK) and protein kinase B (Akt) pathways. In vivo, A771726 showed an inhibitory effect on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats. Based on these results, the aim of this study was to elucidate the mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis and the effect of leflunomide on this process.Following stimulation with recovery KCCM, HSCs showed a decrease in proliferation and an increase in apoptosis by a caspase-dependent mechanism. Furthermore, pretreatment with A771726 markedly enhanced these effects. Real-time quantitative PCR (Q-PCR) analysis showed increased expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Kupffer cells during the spontaneous recovery phase. The pro-apoptotic function of KCCM prepared from TRAIL siRNA-treated Kupffer cells was obviously decreased, suggesting that TRAIL played an important role in recovery from hepatic fibrosis. Moreover, A771726 enhanced recovery KCCM-induced apoptosis of HSCs by a mechanism involving the inhibition of nuclear factor-kappa B (NF-κB) activation. Our results showed the role of TRAIL in recovery Kupffer cell-induced apoptosis of activated HSCs and provided insights into the resolution of fibrosis and the mechanisms by which leflunomide might act upon liver fibrosis.During the resolution phase of hepatic fibrosis, a crucial mechanism is the apoptosis of activated hepatic stellate cells (HSCs). It is necessary to find more anti-fibrosis drugs that would modulate HSCs to be more susceptible to apoptotic stimuli. Here we showed that A771726, the active metabolite of leflunomide, markedly enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in the human hepatic stellate cell line LX-2. A771726 could increase caspase activity in LX-2 cells in a dose-dependent manner. A771726 did not increase the expression of TRAIL receptors in LX-2 cells but could inhibit activation of the c-Jun NH2-terminal kinase (JNK) pathway through decreasing TRAIL-induced JNK and c-Jun phosphorylation. Moreover, A771726 could accelerate TRAIL-induced apoptosis via inhibiting nuclear factor-kappaB (NF-κB) activation in LX-2 cells. In conclusion, our results indicated leflunomide could enhance the sensitivity of LX-2 cells to TRAIL-induced apoptosis via inhibiting the survival pathways and provided a promising approach to anti-fibrotic therapy with leflunomide.