The Role Of Smad2 In The Sensitivity Of TRAIL-induced Hepatic Stellate Cell Apoptosis

Hepatic fibrosis (HF) is considered to be a wound-healing process in response to chronic liver injury and is characterized by the excessive deposition of extracellular matrix (ECM). The activation of hepatic stellate cells (HSC) plays a vital role in the development and maintenance of HF. In the recovery of liver fibrosis, tumor necrosis factor related apoptosis inducing ligand (TRAIL) could promote the apoptosis of HSC, resulted in the degradation of ECM. Inducing activated hepatic stellate cell apoptosis may be one way of liver fibrosis reversal.As is known to all, ECM could be generated through activating HSC by TGF-β1, resulted in the formation of liver fibrosis. Smad2 is downstream molecules of TGF-β1 pathway. Our earlier studies have shown that Smad2 on liver fibrosis in rats induced by CCl4 has a protective effect. Activation and proliferation HSC are key of liver fibrosis, which promote the activation of HSC apoptosis that avoid or reduce ECM accumulate. It could also be one of the important measures of anti fibrosis treatment. However, the effect of Smad2 on HSC function has not been reported.This experiment adopts the CCl4 subcutaneous injection for liver fibrosis model; TGF-β1 stimulates the activation of hepatic stellate cell LX-2 as the cell model, discusses the effect of Smad2 on the apoptosis of activated HSCs. The study includes the following three parts:(1) The effect of Smad2 HSC apoptosis during the different stages of HF CCl4 subcutaneous injection is performed for liver fibrosis model, stop injection 4 weeks as the recovery model. Observing the pathological and histological changes of liver fibrosis in different periods of HF, Q-PCR and Western blot are used to test the changes of a-SMA and Col. I mRNA and protein expression. Smad2 and HSC apoptosis changes are observed during different periods of liver fibrosis. The results showed that:P-Smad2 was increased during rat liver fibrosis compared to normal liver tissues, while P-Smad2 expression levels during the spontaneous recovery from fibrosis were not obviously decreased compared with those during the progressive stage of HF, TRAIL expression was significantly increased during the recovery phase. TUNEL and SMA double staining showed that HSC apoptosis during the spontaneous recovery from fibrosis is closely related to TRAIL expression. The above results suggest that the HSC apoptosis induced by TRAIL in the recovery of hepatic fibrosis may be related to Smad2.(2) Smad2 affects LX-2 apoptosis induced by TRAIL In the LX-2 cells, using plasmid transfection and small interfering RN A technology to establish overexpression and silence Smad2 cell model. Flow cytometry instrument detecting LX-2 apoptosis, Caspase-3 enzyme activity kit and Western blot detecting Caspase-3 enzyme activity and protein expression, Q-PCR and Western blot were used to detect a-SMA and Col. I mRNA and protein expression to study the effect of Smad2 on TRAIL-induced apoptosis of LX-2. It is found that TRAIL-induced LX-2 apoptosis was increased, the protein expression and activity of Caspase-3 were increased when overexpression of Smad2, a-SMA and Col. I expression also were decreased. TRAIL induced activation LX-2 apoptosis was decreased, the protein expression and activity of Caspase-3 were decreased when silence of Smad2, a-SMA and Col. I expression also were increased. The above results suggest that Smad2 might increase the sensitivity of LX-2 apoptosis induced by TRAIL.(3) The mechanism research of Smad2 affecting LX-2 apoptosis induced by TRAIL Q-PCR and Western blot were used to detect the mRNA and protein expression of XIAP. The results showed that:the mRNA and protein expression of XIAP were decreased resulted from overexpression of Smad2. The mRNA and protein expression of XIAP were increased when silence of Smad2. These results indicate that Smad2 might increase TRAIL-induced apoptosis sensitivity of LX-2 by inhibiting the expression of XIAP.