Expression Of C-flip_l In The Process Of Formation And Recovery Of Hepatic Fibrosis And Effects Of Regulation On Trail-induced Hepatic Stellate Cells Apoptosis

Hepatic fibrosis is a pathological process that a large number of collagen-based extracellular matrix (ECM) deposited in the liver when the liver did the repairment to a variety of chronic stimulus. Hepatic stellate cells (HSC) occupies the core position in hepatic fibrosis. It has been widely reported that hepatic fibrosis can be reversed to recover spontaneously, and the apoptosis of active HSC is the key to its reversal. In the process of hepatic fibrosis, the apoptosis of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays an important role. Preliminary study found that in the processes of formation and resolution of hepatic fibrosis, TRAIL secreted by Kupffer cells (KC) was increased; the expressions of DR4 and DR5 by HSC were also significantly increased. This raises a question: in the process of resolution of hepatic fibrosis, the expression of TRAIL and its death receptor was increased, HSC showed apoptosis; while in the process of the formation of hepatic fibrosis the expression of TRAIL and its death receptors were also increased, why HSC performed proliferation? It is speculated that in the procession of the formation and the resolution of hepatic fibrosis the expression of TRAIL apoptosis pathway changes, affecting the TRAIL apoptosis pathway, and resulted in different sensitivity of apoptosis induced by TRAIL in different stages of hepatic fibrosis.According to the important effects of cell type Fas associated death domain-like interleukin-1βconverting enzyme inhibitory protein (c-FLIP) in the regulation of TRAIL apoptosis, this study chosed hepatic fibrosis formation and the resolution model induced by CCl4 to investigate the expression of c-FLIP at different stage of hepatic fibrosis and its effects on the TRAIL-induced apoptosis of HSC, and further to discuss the regulation of c-FLIP protein signaling pathway and the mechanism of leflunomide (Lef) which promote HSC apoptosis . This research included three parts as follows:1. The model preparation of spontaneous resolution of rat hepatic fibrosisIn this section, we used the model induced by CCl4 for 12 weeks and spoatntaneous resolution for 4 weeks to investigate the mechsim of the sensitivity of HSC at different stage of hepatic fibrosis to the apopsis of TRAIL. The results showed that: compared with the hepatic fibrosis model group, resolution rat's ALT, AST were significantly lower, the serum index of hepatic fibrosis (HA, HA, LN, PCⅢ, CⅣ) and histological index of hydroxyproline content (Hydroxyproline, Hyp) also significantly reduced; histopathological analysis showed that the content of collagen in liver tissue decreased in the process of resolution, meanwhile the extent that collagen fibers extending to tissue were also significantly reduced. In the immunohistochemical detection ofα-SMA which is the marker of the active HSC, the quantity of active HSC was significantly decreased in the liver tissue of resolution comparing to model group. These results showed that the hepatic fibrosis model was successfully established.2. Expression of c-FLIPL in the process of formation and recovery of hepatic fibrosis and Effects of regulation on TRAIL-induced hepatic stellate cells apoptosisHSCs of different periods were isolated from normal male SD rats by sequential in situ perfusion with collagenase and protease as described previously, and used exogenous TRAIL to induce apoptosis.The tests by DNA ladder and flow cytometry showed that hepatic fibrosis apoptosis rate of HSC was higher than the model group. Further testing showed that the activity of caspase-3 in the resolution group was significantly higher comparing with the model group; It was also found that the expressions of c-FLIPL in liver tissue and HSC were significantly reduced comparing with model group. Interference by siRNA to test the expression of c-FLIPL model group, the apoptosis rate of HSC induced by TRAIL was significantly increased. It was showed that the expressions of p-Akt, mTOR, and p-p70S6K were significantly increased in the resolution period comparing with the model group. Using of phosphatidylinositol 3-kinases (PI3K) inhibitor LY294002 and the mammalian target of rapamycin, (mTOR) inhibitor rapamycin can inhibit the expression of c-FLIPL and increase the sensitivity of HSC to TRAIL; Using siRNA to inhibit the expression of 40S small subunit ribosomal S6K protein kinase (p70S6K) in model group also significantly increased the apoptosis rate of HSC induced by TRAIL. These results suggested that the expression change of c-FLIPL at different stages of hepatic fibrosis influnced the apoptosis sensitivity of HSC induced by TRAIL. PI3K/Akt/mTOR/ p70S6K signaling pathways inhibited the TRAIL-induced apoptosis of HSC by increasing the expression of c-FLIPL in the process of hepatic fibrosis.3. The mechanism of leflunomide promoting TRAIL-induced apoptosis of HSC-T6Lef is an immunomodulatory agent which can promote TRAILL-induced apoptosis of HSC, however the mechanism was not clear. The results showed that: Lef inhibited Leptin-induced c-FLIPL expression in HSC-T6. The activity of caspase-3 and the apoptosis of active HSC-T6 were significantly increased. It showed that Lef promoted TRAIL-induced apoptosis by inhibiting the expression of c-FLIPL in active HSC-T6. Further study demonstrated Lef pretreated inhibited the expressions of p-Akt, mTOR, and p-p70S6K and induced HSC-T6 apoptosis.These results demonstrated that Lef inhibited the expression of c-FLIPL by inhibiting PI3K/Akt/mTOR pathway which made active HSC-T6 more sensitivity to TRAIL-induced apoptosis.