Effects And Molecular Mechanism Of Sorafenib On Rat Liver Fibrosis And Hepatic Stellate Cell Proliferation And Apoptosis

Liver fibrosis characterized by the excessive accumulation of extracellular matrix represents the pathologic response of a sustained wound healing response to chronic liver disease induced by a variety of causes, including viral, autoimmune, drug-related, ethanol, cholestatic and metabolic damage. A key role in hepatic fibrogenesis is attributed to activated hepatic stellate cells (HSCs), which have been identified as major collagen-producing cells in an injured liver and are responsible for excessive deposition of extracellular matrix (ECM), of which type I collagen predominates.Following liver injury of any etiology, HSCs undergo a response known as"activation", which is the transition of quiescent cells into proliferative, fibrogenic and contractile myofibroblasts. Morphological changes associated with HSC activation include a loss of vitamin A stores and appearance of the cytoskeleton proteinα-smooth muscle actin (α-SMA). Numerous studies, performed in animal models of acute or chronic liver injury, have shown a potential reversibility of liver fibrosis associated with inhibition of HSC proliferation and induction of HSC apoptosis. Consequently,inhibition of HSC proliferation and induction of HSC apoptosis become major antifibrotic therapeutic strategies.The most potent mitogenic factor for HSCs is platelet-derived growth factor (PDGF), which combines with PDGF receptor (PDGFR) on the surface of HSCs and activates intracellular signal transduction as autocrine factors, stimulate cell proliferation, synthesis of ECM including collagens and activate quiescent HSCs to matrix-secreting myofibroblasts. Multiple signaling pathways are implicated in HSC proliferation activated by PDGF.Extracellular signal-regulated kinase (ERK) is an important member of the mitogen-activated protein kinase (MAPK) family, which is composed of three core units: mitogen-activated protein kinase kinase kinase (MAPKKK, Raf), mitogen-activated protein kinase kinase (MAPKK, MEK) and MAPK (ERK). It has been found that MAPK/ERK signal pathway is involved in hepatic fibrosis and activation of Ras due to PDGF is followed by sequential activation of Raf, MEK, and ERK. The phosphatidylinositol 3-kinase (PI3K)/Akt/70-kDa ribosomal S6 kinase (p70S6K) signaling pathway is activated by mitogens and growth factors including PDGF, and is required for cell cycle progression, cell differentiation, and cell growth. Both MAPK/ERK and PI3K/Akt/p70S6K signal pathways play crucial roles in the DNA synthesis, collagen synthesis and cell survival in HSCs. Inhibiting either of the pathways blocks HSC proliferation and type I collagen synthesis and induces HSC apoptosis.Sorafenib is farthest along in clinical development and has been approved in several countries worldwide for treatment of a wide variety of tumors including renal cell carcinoma, hepatocellular carcinoma, et al. As a multikinase inhibitor, sorafenib potently blocks the tyrosine kinases of vascular endothelial growth factor receptor-2 (VEGFR-2) and PDGFR-β, as well as the Raf serine/threonine kinases along the Raf/MEK/ERK pathway, which is known to be important in tumor cell signaling and tumor cell proliferation. Simultaneouly, sorafenib decreased the phosphorylations of Raf/MEK/ERK and PI3K/Akt/p70S6K signal pathways. It has also been reported that sorafenib inhibits proliferation accompanied by the inhibition of Cyclins and Cyclin-dependent kinases (Cdks) and induces apoptosis accompanied by the inhibition of the down-regulation of myeloid cell leukemia-1 (Mcl-1), Bcl-2 and up-regulation of Fas/Fas ligand (Fas-L) in multiple human tumor cell lines. Furthermore, Sorafenib could substantially decrease the extensive deposition of fibrillar collagen in common bile duct ligation (BDL) rats. Another multitargeted receptor tyrosine kinase inhibitor sunitinib induce significant decreases ofα-SMA expression and ECM accumulation of rat liver fibrosis and down-regulates HSC viability and collagen expression. We speculate the anti-fibrotic effects of sorafenib on liver fibrosis in vivo and inhibition of proliferation as well as promotion of apoptosis of HSCs in vitro.The purpose of the present study is to investigate the impact of sorafenib on liver fibrosis in two animal models: BDL and dimethylnitrosamine (DMN) induced liver fibrosis models and the effects of sorafenib on the proliferation and apoptosis of HSC cell lines T6, LX2 and primary rat HSCs, as well as the possibly associated molecular mechanisms. The experiments contain four parts as blow:Part 1: The effects of oral sorafenib treatment on liver fibrosis and the potential mechanismObjective: To explore the impacts of sorafenib on liver fibrosis induced by common bile duct ligated (BDL) and dimethylnitrosamine (DMN). Methods: Hepatic fibrosis was induced by BDL and intraperitoneal injections of DMN. Sorafenib 20 mg/kg and 40 mg/kg in BDL rats, 1 mg/kg and 5 mg/kg in DMN rats or vehicle were administered orally by gavage once a day during the third and the fourth week. Livers in model group were harvested at fixed time points: 1 wk, 2 wk, 3 wk and 4 wk after operation. Livers in sham operation group were harvested at 4 wk after operation. Histopathological changes were evaluated by hematoxylin and eosin (HE) staining and by Masson's trichrome method. The latter was quantified for collagen by analyzing Masson-stained area as a percentage of total area. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (BIL) and albumin (ALB) were evaluated in samples of serum. The protein expressions of ERK, phospho-ERK, Akt, phospho-Akt, p70S6K and phospho-p70S6K were determined by Western blot, while the distribution of phospho-ERK phospho-Akt, phospho-p70S6K andα-SMA in the livers was assessed by immunohistochemistry.Results: (1) HE and Masson's trichrome staining of liver confirmed the establishment of hepatic fibrosis models via BDL and DMN. The liver tissues of BDL rats appeared to exhibit a marked increase in the number of bile ductules and an extensive deposition of collagen, which contributed to the formation of pseudolobuli. DMN-induced liver injury and fibrosis exhibited extensive hemorrhagic necrosis and lobular architecture with thin bands of reticulin joining central areas. Sorafenib, especially the higher concentration group, attenuated the histopathologic changes of both fibrotic models. (2) Both fibrotic models developed hepatic injury as evidenced by significantly higher plasma concentrations of AST, ALT, BIL and lower concentration of ALB. Sorafenib treatment did not change the injury of liver function in BDL rats. In DMN rats, 1 mg/kg sorafenib ameliorated the increase of ALT and BIL, and the decrease of ALB. 5 mg/kg sorafenib increased AST and ALT levels significantly with a 101.10% (P<0.001) and a 20.08% (P<0.001) increase as compared to vehicle-treated group respectively. (3) Sorafenib attenuated the collagen deposition in a dose-dependent fashion in both fibrotic models by analyzing Masson-stained area (P<0.001). (4) The expression ofα-SMA of both models at week 1 to 4 in liver during the process of liver fibrogenesis increased by immunohistochemistry analysis. Sorafenib treatment decreased the positive cells ofα-SMA in a dose-dependent manner (P<0.001). (5) The relative protein expressions of phospho-ERK/ERK, phospho-Akt/Akt, phospho-p70S6K/p70S6K by Western blot and the positive areas of phospho-ERK, phospho-Akt and phospho-p70S6K by immunohistochemistry were increased during the process of liver fibrogenesis in both models. Sorafenib treatment diminished those increased the phosphorylations of ERK, Akt and p70S6K in a dose-dependent manner. Furthermore, the phosphorylations of higher concentration groups (BDL: 40 mg/kg; DMN: 5 mg/kg) decreased to the most degree (P<0.001).Conclusions: Sorafenib inhibited the phosphorylation of ERK and Akt/p70S6K signaling pathways, consequently suppressed the activation of HSCs, by which sorafenib attenuated liver fibrosis.Part 2: Sorafenib inhibits HSC proliferation and regulates HSC cell cycleObjective: To explore the effects of sorafenib on HSC proliferation and cell cycle.Methods: Primary rat HSCs were isolated by circulating perfusion of the liver and density gradient centrifugation. The quiescent HSCs immediately after plating were tested by fluorescence microscope to evaluate the purity of the cultures andα-SMA monoclonal antibody was used to identify the activated primary HSCs by immunocytochemistry. T6, LX2 and primary HSCs were preincubated with or without sorafenib (2.5, 5.0, and 10.0μmol/L) for 2 h, and then stimulated with or without PDGF-BB for 12 h, 24 h, 48 h and 72 h. The viability of HSCs was detected by 3-(4, 5-dimethylthiazol-2-yl)-3, 5- diphenyltetrazolium bromide (MTT) assay. DNA synthesis was explored by [3H]-Thymidine ([3H]-TdR) incorporation assay. Cell cycles of HSCs were analyzed by flow cytometry. The protein expressions of Cyclin-D1 and Cdk-4 were determined by Western blot analysis.Results: (1) Primary rat HSCs were isolated successfully by sequential digestion of the liver with pronase and collagenase, followed by single step density gradient centrifugation with Nycodenz. Cell viability and cell purity were greater than 90%, with a yield ranging from 1.2×107 to 2.0×107 HSCs/rat. The primary HSC immediately after plating is round and in rich of lipid droplet under the inverted microscope, while HSCs show blue under the ultraviolet light (λ=328 nm). After 10 days culture, activated HSCs lose retinoid and become fusiform myofibroblast-like cell. (2) To indentify the primary HSCs identificationα-SMA staining was performed at day 1 (quiescent,α-SMA negative cells) and day 10 (activated,α-SMA positive cells) by immunocytochemistry. (3) Sorafenib inhibited the viabilities of T6, LX2 and primary HSCs with or without the stimulation of PDGF-BB time- and dose-dependently. Sorafenib (10μmol/L) decreased the viabilities of T6 (16.37±3.85%, P<0.001), LX2 (22.49±1.86%, P<0.001) and primary HSCs (15.49±2.16%, P<0.001) compared with those of the control group (100%). (4) Sorafenib inhibited the DNA synthesis of T6 and LX2 activated by PDGF-BB dose-dependently. Sorafenib (10μmol/L) significantly decreased the cpm of T6 (891.50±160.33) compared with that of PDGF group (10023.00±2442.25) (P<0.001), and decreased the cpm of LX2 (1411.17±908.39) compared with that of PDGF group (4416.00±667.63) (P<0.001). (5) Cell cycle analysis of T6 and LX2 cells showed an increase in S phase cells and a decrease in G1 and M phase. (6) Western blot analysis indicated that sorafenib reduced the increased Cyclin-D1 and Cdk-4 protein levels by PDGF-BB in both T6 and LX2 cells. The inhibition of Cyclin-D1 of sorafenib is in a dose-dependent manner.Conclusions: Sorafenib diminished the expressions of Cyclin-D1 and Cdk-4, which contributed to the S-phase arrest of cell cycle and the inhibition of HSC proliferation.Part 3: Sorafenib induces HSC apoptosisObjective: To investigate the effects of sorafenib on HSC apoptosis and apoptosis regulatory proteins.Methods: T6 and LX2 cells were incubated with sorafenib (2.5, 5.0, and 10.0μmol/L) for 12 h or 24 h. Morphological examination via transmission electron microscopy (TEM) evaluation and apoptosis rate assay by the Annexin-V/Propidium iodide (PI) double-labeled flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique were performed. HSCs were preincubated with sorafenib (2.5, 5.0, and 10.0μmol/L) for 2 h and stimulated by PDGF-BB for another 22 h. Caspase-3 activity was detected, and the protein expressions of Bcl-2, Bax, Fas, Fas-L, and Caspase-3 were analyzed by Western blot. The mRNA levels of Bcl-2, Bax, and Caspase-3 were measured by RT real-time PCR.Results: (1) Morphological changes of T6 and LX2 cells after treatment with sorafenib (10μmol/L) for 12 h under the TEM showed that cells became smaller, the chromatins condensed along inside the nuclear membrane, the crescent cell nuclear formed, the nuclear-cytoplasmic ratio decreased, caryotheca was damaged, and the endoplasmic reticulum dilated. (2) Sorafenib induced the apoptosis of HSCs. Sorafenib (2.5, 5.0, and 10.0μmol/L) increased HSC apoptotic rates of T6 and LX2 cells in dose- and time-dependent manner. The apoptotic rate of T6 cells treated with 10.0μmol/L sorafenib for 12 h (72.62±4.05%) was 2.74 times higher than the control group (P<0.001), and that of LX2 cells (92.87±2.81%) was 2.15 times higher than the control group (P<0.001). (3) Sorafenib (2.5, 5.0, and 10.0μmol/L) up-regulated the positive-T6 and LX2 cells of TUNEL staining by 5.28 and 3.24 times respectively compared with the control groups (P<0.001). (4) Sorafenib reduced Bcl-2 protein and mRNA levels and increased the mRNA expressions of Bax and Caspase-3 as well as the protein levels of Bax, Fas, Fas-L and Caspase-3 in both T6 and LX2 cells. The ratio of Bcl-2 to Bax decreased. (5) PDGF inhibited the activity of Caspase-3, whereas 10μmol/L sorafenib increased the activity of Caspase-3 in both T6 and LX2 cells by 2.83 times (P<0.001) and 1.58 times (P=0.021) respectively compared with the control groups.Conclusions: Sorafenib induced HSC apoptosis, which was related to the down-regulation of Bcl-2 and up-regulations of Bax, Fas, Fas-L and Caspase-3.Part 4: The mechanism of intracellular signal transduction that contribute to the impacts of sorafenib on HSC proliferation and apoptosisObjective: To explore the regulation of sorafenib on MAPK/ERK and Akt/p70S6K signalling pathways in HSCs.Methods: T6 and LX2 cells were preincubated with or without sorafenib (2.5, 5.0, and 10.0μmol/L) for 2 h, and then stimulated with or without PDGF-BB (20 ng/ml) for 22 h. Simultaneously, T6 and LX2 cells were treated with sorafenib, LY294002 (25μmol/L), or PD98059 (50μmol/L), stimulated with or without PDGF-BB. The protein expressions of ERK, Akt, p70S6K, p-ERK, p-Akt and p-p70S6K were determined by Western blot analysis.Results: (1) Sorafenib inhibited the phosphorylations of ERK and Akt/p70S6K signals increased by PDGF-BB. PDGF-BB up regulated the ratios of p-ERK/ERK, p-Akt/Akt and p-p70S6K/p70S6K, which were diminished by sorafenib dose-dependently. Sorafenib (10μmol/L) in T6 cells decreased the ratios of p-ERK/ERK, p-Akt/Akt and p-p70S6K/p70S6K by 50.00% (P=0.001), 46.71% (P<0.001), and 24.73% (P<0.001) compared with PDGF-BB group. Those of LX2 cells were decreased by 61.11% (P<0.001), 37.50% (P=0.001), and 19.70% (P<0.001) compared with PDGF-BB group. (2) Sorafenib showed the inhibiting effects on ERK and Akt/p70S6K signals similar to the specific blocking agent of the two pathways. PD98059 and LY294002 were effective in inhibition of phosphorylation of ERK and Akt/p70S6K, respectively. Sorafenib inhibited both the phophorylated ERK and Akt/p70S6K, and no significant different impact was found between sorafenib vs PD98059 and sorafenib vs LY294002 on p-ERK and p-Akt respectively.Conclusions: Sorafenib inhibited HSC proliferation and induced apoptosis probably via down-regulation of phosphorylations of ERK and Akt/p70S6K signaling pathways.