| Liver fibrosis (LF) represents the consequences of injury-recovery response to various chronic liver diseases, which leads to the over-produced extracellular matrix (ECM) accumulate in the liver. Though the origin and mechanisms of each liver fibrogenesis are difference, without effective therapy, LF can progress into cirrhosis even liver cancer. Because the mechanisms of LF are too complicated, the knowledge of LF is poor by now which limit the progress of its therapy. As one of the most important producer of ECM, hepatic stellate cell (HSC) became the focus of research on IF. Such as how to suppress the activation of HSC or enhance its apoptosis, how to suppress the production of ECM by HSC or enhance its degradation have attracted the interest of numerous researchers in this field. The mutual contact, interaction, mutual effect between HSC and the cells around or cytokines secreted by HSC or other cells around regulate the behavior and function of HSC. Integrin, one the membrane receptor of ECM, play an important role in regulating HSC functions as a signal transporter between HSC and ECM. The identification of Integrin-linked kinase in 1996 described a novel signaling pathway as well as the reaction platform for the interaction and transduction between ECM and cytoskeleton. It is known that ILK is a serine/threonine protein kinase which can bind to the ?1 or ?3 Integrin cytoplasmic domain. ILK can regulate multifunction of cells, such as proliferation, differentiation, apoptosis and migration, mediated by ECM or growth factor (GF) signaling pathway. There are evidences supporting the involvement of ILK in fibrogenesis of lung as well as kidney. Our research tried to investigate the possibility of involvement of ILK in fibrogenesis of liver fibrosis via regulatory of HSC. Therefore, we try to answer the following questions by experiments in vitro or in vivo: ①whether ILK is involved in the liver fibrogenesis happened in SD rat? ②If ILK really participate in the liver fibrogenesis, whether the involvement is realized via or partially via regulation of HSC? What is the possible way to regulate the behavior of HSC by ILK? ④How to control the HSC by regulation of ILK expression to achieve the anti-fibrosis effect? To demonstrate the hypothesis above, (1) A SD rat LF model was established induced by CCl4. The immunohistochemical stain (IH stain) and immunofluorescence stain (IF stain) were used to determine the changes of ILK expression in liver tissues along with the progress of fibrosis. Meanwhile, the α-smooth actin (α-SMA) was also detected by stain as the activity marker of HSC; (2) Liver tissues'ILK kinase activities were also determined in different periods (from 1 to 5 weeks) of fibrosis on SD LF model respectively; (3) The protein expression of ILK by quiescent or different stages of culture-activated HSC, isolated from normal SD rat were investigated by Western Blot. (4) Dual immunofluorescence stain were performed to identify the co-localization of ILK and α-SMA produced by culture-activated HSC isolated from normal SD rat; (5) The subcellular location of ILK was determined by detecting the expression of ILK on Cytosol and membrane by Western Blot respectively; (6) The expression of ILK by quiescent HSC as well as activated HSC, which were activated in vivo by the injection of CCl4 from 1 to 5 weeks, were determined; (7) The expression of total PKB/Akt and phosphor-PKB/Akt ser473 expressed by quiescent HSC were compared with those expressed by culture-activated HSC to investigated phosphorylation kinase functional pathway of ILK; oh the other hand, theexpressions were also determined in both transfected and without transfected CFSC-2G cell to elucidate the regulatory effect of ILK on the phosphorylation of PKB/Akt; (8) Culture-activated HSC, isolated from normal SD rat were incubated with PI3K inhibitor-Ly294002 (10nM) for 6, 12 or 24h respectively and the expression of ILK were determined by Western Blot; (9) ILK SiRNA were transfected into CFSC-2G cell line by Lipofectamine 2000 and the expression of ILK was detected by Quantitative real-time PCR; (10) BrdU ELISA kit was used to certify the proliferation of CFSC-2G cells which have been transfected by ILK SiRNA for 48h; (11) the apoptosis was detected by Hoechst 33342 immunofluorescence stain in the CFSC-2G before and after transfection by ILK SiRNA for 48h; (12) The migration ability was presented by wound healing assay; (13) The morphological changes were monitored by immunofluorescence stain; (14) WB was used to demonstrate the involvement of MAPK families. (15)Real-time PCR was used to detect the changes of mRNA synthesis of procollagen type I,TGF?1and MMP9 before and after ILK SiRNA transfection. The result showed: (1) The IH stain showed that with the injection of CCl4 and the progress of fibrosis, the ILK expression in liver tissue was increased, especially in Disse's space. The increase intend similar to that of α-SMA. The IF stain also suggested that ILK expression by liver was up-regulated along with the progress of LF; (2) With the progress of LF, Not only the ILK protein synthesis, but also the ILK activity was increased in liver; (3) The expression of ILK by Culture-activated HSC isolated from normal SD rat increased dramatically compared with that expressed by freshly isolated quiescent HSC (P<0.05) and keep on increasing with the prolongation of culture, paralleling to the changes of α-SMA; (4) Compared with quiescent HSC freshly isolated from normal rat , the ILK expression by activated HSC which was activated in vivo by CCl4 injection was elevated significantly and went on increasing with the culture after isolation(P<0.05); (5) The co-localization of ILK and α-SMA at cell-cell junction or margin of the cells was identified by dual IF stain. Instead of Cytosol, ILK was mainly located at the membrane part; (6) Compared with the freshly isolated quiescent HSC, the expression of phosphor-PKB/Aktser473 by culture-activated HSC was increased; (7) The expression of ILK by culture-activated HSC incubated with PI3K inhibitor (Ly294002) for 12 or 24h were suppressed dramatically (P<0.05), no significant difference between control group and incubation for 6h group. The difference between 12h and 24h incubation was also denied by Western Blot detection; (8) Both of WB and Real-time PCR proved that the expression of ILK by CFSC-2G cells was suppressed significantly by ILK SiRNA transfection; (9) BrdU incorporation assay implied that the proliferation of CFSC-2G was decreased by 52.67±12.22%; (10) Compared with un-transfected cells, ILK SiRNA transfection for 48h induced the apoptosis of transfected cells dramatically; (11) Under the same injury, time required for wound healing was prolonged for transfected cells compared with those without transfection; (12) After transfection, the morphology of CFSC-2G was altered and cells turned into smaller, spindle like cells with denser actin fiber; (13) phosphor-JNK as well as phosphor-ERK were down-regulated as the result of the ILK SiRNA transfection which implied the potential involvement of MARPK family in regulatory effect of ILK; (14) accompanied with the suppression of ILK via ILK SiRNA transfection, the mRNA synthesis of procollagen type I,TGF?1and MMP9 were dramatically suppressed too. According to all the results showed above, it is evidently that activated HSC can synthesize ILK. ILK plays a pivotal role in liver fibrogenesis via regulating the morphology as well as function (such as proliferation, activation, migration and apoptosis) of HSC. Furthermore, as the central factor of PI3K signaling pathway, ILK exerts regulatory function on HSC depending or partially depending...
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