The Role Of Multikinase Inhibitor Sorafenib In Collagen Metabolism Of Rat Fibrosis And Hepatic Stellate Cells

Liver fibrosis, a wound-healing response to a variety of chronic stimuli, is characterized by excessive deposition of extracellular matrix (ECM), of which type I collagen predominates. It is in the early stage of cirrhosis and may be reversed under some circumstances. If the liver injury is persistent, the excess deposition of ECM disrupts the normal architecture of the liver resulting in the formation of nodules, and fibrosis will develop to liver cirrhosis. It is well established that the activated hepatic stellate cells (HSCs) play a key role in the excess synthesis and deposition of ECM following a fibrotic stimulus. HSCs are not only the major source of ECM, but also the major cells releasing matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), which responsible for regulating matrix degradation. The activated HSC express tyrosine kinase receptors, including platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) and nerve growth factor receptor (NGFR). With these receptors binding to specific ligands, some intracellular signaling pathways were trigged. It has been shown that the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K) play important roles in proliferation, apoptosis, angiopoiesis and collagen metabolism in HSCs stimulated by different growth factors.Multikinase inhibitor is a kind of novel multi-targeted agents studied in recent years. It has been shown to inhibit tumor cell growth and angiogenesis by blocking the receptor tyrosine kinases, such as PDGFR, VEGFR, FGFR, and NGFR. This is attributed to block signaling pathways including Ras/Raf/mitogen-activated protein kinase (MAPK) and PI3K/Akt/70-kDa ribosomal S6 kinase (p70S6K) and provide theoretical basis for antifibrotic therapy. Yoshiji H et al found imatinib, a multikinase inhibitor, markedly attenuated liver fibrosis, and decreased hepatic hydroxyproline and serum fibrosis markers. Imatinib markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner in vitro. Sunitinib, another multikinase inhibitor, resulted in significant decreases in hepatic vascular density, inflammatory infiltrate, alpha-SMA abundance, collagen expression and portal pressure. Sorafenib, a novel oral multikinase inhibitor, is more effective in inhibiting ERK1/2 and PI3K signaling pathways. Mejias M et al found sorafenib treatment resulted in a reduction in portal pressure, as well as a remarkable improvement in liver damage and intra- hepatic fibrosis, inflammation, and angiogenesis. Although sorafenib exerts in reducing portal pressure, the role of sorafenib in the reversal of fibrosis, the activation of HSCs, collagen synthesis and degradation are unclear. The effects of sorafenib on collagen metabolism in vivo and in vitro are investigated in this study, and the mechanism is also explored. The experiments are composed of four parts as below.Part 1: The therapic effect of sorafenib on intrahepatic fibrosis of experimental rats.Objective : To explore the therapic effect of sorafenib on liver pathological changes, hydroxyproline content and expression of type I collagen in experimental fibrotic rats.Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL) and in Wistar rats by intraperitoneal injections of dimethylnitrosamine (DMN). Sorafenib was administered orally by gavage once a day during the third and the fourth week for two weeks. Histopathological changes were evaluated by hematoxylin and eosin staining and by Masson's trichrome method. Hepatic hydroxyproline was measured using a hydroxyproline detection kit. Type I collagen protein expression in the livers were determined by Western blot, while the expression of collagenα1(I) mRNA in the livers was assessed by Real-time PCR analysis . Results:①Hepatic fibrosis was successfully induced by BDL and by intraperitoneal injections of dimethylnitrosamine (DMN) by hematoxylin and eosin staining and Masson's trichrome method.②Sorafenib suppresses hepatic fibrosis by inhibiting the collagen deposition and inflammation in BDL and DMN rats. In BDL rats after sorafenib treatment at dose of 20 mg/kg and 40 mg/kg, hepatic fibrosis score (11.92±1.35 and 8.64±2.13, respective- ly) were both lower than that in vehicle-treated group(16.97±2.82), P<0.01; In DMN rats after sorafenib treatment at dose of 1 mg/kg and 5 mg/kg, hepatic fibrosis score(12.41±3.34 and 9.65±3.02, respectively) were both lower than that in vehicle-treated group(17.17±3.65), P<0.01.③Treatment with sorafenib reduced hydroxyproline content in BDL and DMN rats. In BDL rats, hydroxyproline content (247.00±16.05μg/g and 200.00±50.81μg/g, respect- tively) at dose of 20 mg/kg and 40 mg/kg were both lower than that in vehicle- treated group(442.50±139.14μg/g), P<0.01. In DMN rats, hydroxyproline content (214.40±35.71μg/g ) at doses of 5 mg/kg was lower than that in vehicle-treated group(355.80±33.05μg/g), P<0.05.④Sorafenib down- regulated the expression of type I collagen and collagenα1 (I) mRNA in a dose-dependent manner in BDL rats. The expression of type I collagen protein (0.54±0.02 and 0.51±0.01, respectively) at doses of 20 mg/kg and 40 mg/kg by Western blot were both lower than that in vehicle-treated group, reduced by 14.86% and 17.89% , respectively, P<0.05. The expression of collagenα1 (I) mRNA (1.77±0.33 and 1.56±0.18, respectively ) at doses of 20 mg/kg and 40mg/kg by real-time PCR were both lower than that in vehicle-treated group(3.32±0.67), reduced by 46.69% and 53.01% , respectively, P<0.01.⑤Sorafenib inhibited the expression of type I collagen and collagenα1 (I) mRNA in a dose-dependent manner in DMN rats. The expression of type I collagen protein at doses of 1 mg/kg and 5 mg/kg reduced by 14.92% and 32.18%, respectively, P<0.05. The expression of collagenα1 (I) mRNA reduced by 43.17% and 49.78%, respectively, P<0.01. Furthermore,sorafenib 5 mg/kg was more significantly than that at dose of 1 mg/kg in inhibiting type collagen I protein and mRNA. Conclusions: During the fibrogenesis, hydroxyproline content, type I collagen and collagenα1 (I) mRNA are upregulated in BDL and DMN rats. Sorafenib suppresses hepatic fibrosis by inhibiting the collagen deposition and inflammation, reduces hepatic fibrosis score and hydroxyproline content, as well as downregulats the expression of type I collagen and collagenα1 (I) mRNA. All data suggest that sorafenib may be a potential therapeutic agent in the treatment of liver fibrosis.Part 2: The effect of sorafenib on the expression ofα-SMA,MMP-13 and TIMP-1 in experimental fibrotic rats.Objective:To explore the effect of sorafenib on the expression ofα-SMA, MMP-13 and TIMP-1 in experimental fibrotic rats. Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL) and in Wistar rats by intraperitoneal injections of dimethylnitrosamine (DMN). Sorafenib was administered orally by gavage once a day during the third and the fourth week for two weeks (BDL rats: 20, 40 mg/kg; DMN rats: 1, 5 mg/kg). The expression ofα-SMA were evaluated by immunohistochemistry. MMP-13 and TIMP-1 protein expression in the livers were determined by Western blot and immunohistochemistry. While the expression of MMP-13 and TIMP-1 mRNA in the livers was assessed by Real-time PCR analysis.Results:①During fibrogenesis, interstitial cells strongly positive forα-SMA appeared along the portal area, fibrous septum, the hepatic sinusoids and the periportal area. Sorafenib reduced the number of the cells positive forα-SMA. The mean density forα-SMA were 30.72±1.87% and 18.05±4.02% in BDL rats after administration of sorafenib (20 mg/kg and 40 mg/kg), respectively, and lower than that in vehicle-treated rats (38.19±2.10%), P<0.01. In DMN rats, after sorafenib (1 mg/kg and 5 mg/kg) treatment, the mean density forα-SMA were significantly lower than that in vehicle-treated rats(23.06±2.20%, 20.63±2.50% vs 32.32±4.40%), P<0.01.②During the progress of liver fibrosis, the expression of TIMP-1 was up- regulated in BDL and DMN rats. The expression of MMP-13 was up-regulated at the early stage, wheras downregulated at end-stage of liver fibrosis. The balance between MMP-13 and TIMP-1 was disturbed.③Sorafenib inhibited the expression of TIMP-1 protein and mRNA. Sorafenib down-regulated TIMP-1 protein by 31.69% and 47.87% and mRNA by 44.63% and 43.65% in BDL rats at the dose of 20 mg/kg and 40 mg/kg, respectively. Sorafenib (1 mg/kg and 5 mg/kg) decreased the expression of TIMP-1 protein by 44.14% and 55.18% and mRNA by 72.03% and 67.56% in DMN rats, respectively.④Sorafenib upregulated the expression of MMP-13 protein and mRNA in experimental fibrotic rats. Sorafenib (20 mg/kgand 40 mg/kg) up-regulated MMP-13 protein by 164.35% and 175.52% and mRNA by 78.50% and 56.07% in BDL rats, respectively. Sorafenib increased the expression of MMP-13 protein by 119.74% and 152.7% and mRNA by 33.87% and 57.26% in DMN rats at the dose of 1 mg/kg and 5 mg/kg, respectively. Hepatocyte and interstitial cells strongly positive for MMP-13 appeared along the portal area and fibrous septum.⑤Sorafenib increased the ratio of MMP-13 to TIMP-1 and improved the disbalance between MMP-13 and TIMP-1.With the progress of liver fibrosis, the ratio of MMP-13 to TIMP-1 decreased and was the lowest in model 4wk, the value was 0.32±0.06 in BDL rats and 0.51±0.07 in DMN rats, respectively. The ratio of MMP-13 to TIMP-1 was increased after treatment with sorafenib in BDL and DMN rats and improved the disbalance between MMP-13 and TIMP-1.Conclusions: Sorafenib has potent to reduceα-SMA expression and inhibit HSC activation. For another, sorafenib down-regulates TIMP-1 protein and mRNA and up-regulates MMP-13 protein and mRNA. It improves the disbalance between MMP-13 and TIMP-1. As a result, sorafenib promotes degradation of ECM and plays a key role in the reversal of fibrosis.Part 3: Effects of sorafenib on the Collagen Metabolism of Hepatic Stellate CellsObjective:To investigate the effects of sorafenib on collagen synthesis and MMP-13 (or MMP-1), TIMP-1 expressions in PDGF-stimulated HSC,so as to study the mechanism of regulating collagen metabolism of HSC with sorafenib.Methods: Three types of cell lines, freshly isolated rat HSC, HSC-T6 and LX-2 cell lines, were used in this study. Collagen synthesis was determined by 3H-pro incorporation. Expressions of type I collagen, MMP-13 and TIMP-1 were evaluated by western blot and quantitative real-time RT-PCR. Immuocytochemistry was used to detect type I collagen andα-SMA.Results:①Stimulation with PDGF-BB induced increase of collagen Iα1 mRNA by 5.45-, 2.49- and 2.20-fold in primary HSC , HSC-T6 and LX-2. The expression of type I collagen protein was increased by 1.26- and 1.10- fold in HSC-T6 and LX-2. Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated collagen Iα1 mRNA by 54.90%, 64.88% and 64.51% in primary HSC, by 62.64%, 67.94% and 82.52% in HSC-T6 and by 58.66%, 67.06% and 81.64% in LX-2, respectively. Furthermore, sorafenib decreased type I collagen protein by 21.28%, 28.72% and 38.30% in HSC-T6 and by 17.40%, 18.84% and 18.84% in LX-2, respectively (P<0.05).The same results were obtained by immuocytochemistry in HSC-T6 and LX-2 cells.②In the range of sorafenib (2.5～10.0μM), 3H-pro incorporation could be inhibited in dose- dependent manner with or without stimulation with PDGF-BB, P<0.01. The inhibition rates with sorafenib(10.0μM) were 14.51%, 36.67% , 63.44% (HSC-T6)and 22.69%, 37.52% and 71.74% (LX-2) at 12h,24h and 48h, respectively. So sorafenib could also dramatically inhibit the collagen synthesis of HSC in dose- and time-dependent manner.③Stimulation with PDGF-BB induced increase of TIMP-1 protein by 1.29-, 2.31-fold and mRNA by 2.57-, 2.11-fold in HSC-T6 and LX-2, respectively(P<0.01). Increased TIMP-1 expression was inhibited after treatment with sorafenib for 24 h, with a significant reduction at 10.0μM in T6 cells and at 5.0μM in LX-2 cells. Sorafenib dose-dependently downregulated the expression of TIMP-1 mRNA in the three HSC.④Sorafenib (5.0μM) induced the increase of MMP-1 protein by 167.39% in LX-2. The expression of MMP-13 mRNA were upregulated at the dose of sorafenib 2.5μM and 5.0μM in primary HSC. Sorafenib had no effect on the expression of MMP-13 in HSC-T6.Conclusions: Sorafenib inhibited collagen synthesis in primary HSCs, HSC-T6 and LX-2 cells dose-dependently. As a result, the production of ECM was decreased. For another, the expressions of TIMP-1 protein and mRNA were downregulated by treatment with sorafenib. The dissolution of ECM was increased because of abolishing the inhibition of activity of MMPs. These data demonstrate that sorafenib is a potential therapeutic agent in the treatment of liver fibrosis.Part 4: Mechanism of sorafenib on the collagen metabolism in experimental fibrotic rats and in hepatic stellate cellsObjective:To investigate the effects of sorafenib on Akt , phosphorylated Akt,ERK and phosphorylated ERK in PDGF-stimulated HSC and in experimental fibrotic rats and further to explore the mechanism of signal transduction in regulating collagen synthesis.Methods:Two types of HSC cell lines (HSC-T6 and LX-2) and two different fibrotic models were used in this study. Fibrosis was induced by bile duct-ligation (BDL) or intraperitoneal injections of dimethylnitrosamine. HSC were preincubated with or without sorafenib and then stimulated with PDGF-BB. The ERK inhibitor PD98059 and Akt inhibitor LY294002 were used to treat HSC. The expression of ERK, phosphorylated ERK, Akt and phosphorylated Akt in HSC and rat livers were evaluated by western blot. The phosphorylated ERK and phosphorylated Akt in liver were detected by immunohistochemistry.Results:①Sorafenib had no effect on total ERK and total Akt in HSC-T6 and LX-2 cells. The difference among groups was no significant in HSC-T6 and LX-2 cells. P value was 0.16, 0.24, 0.26and 0.17, respectively.②Sorafenib inhibited the activation of ERK and Akt in HSC-T6 cells. Stimulation with PDGF-BB induced increase of phosphorylated ERK and phosphorylated Akt by 1.27-fold (P=0.071) and 1.48-fold (P<0.001) in HSC-T6 cells. Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated phosphorylated- ERK by 24.24%, 34.85% , 50% and inhibited phosphorylated Akt by 25.15%,40% and 50.29%,respectively. The expression were lower than that in PGDF- stimulated group, P<0.01. Furthermore, sorafenib inhibited the activation of ERK and Akt in dose-dependent manner from 2.5μM to 10.0μM.③Sorafenib decreased the expression of phosphorylated ERK and phosphorylated Akt in LX-2 cells. Stimulation with PDGF-BB induced increase of phosphorylated ERK and phosphorylated Akt by 1.61- and 1.23-fold in LX-2, the difference was significant in statistic (P<0.01). Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated phosphorylated ERK by 60.38%, 69.81%, 73.58% and inhibited the activation of Akt by 32.12%,36.50% and 45.26%,respectively. The expression were less than that in PGDF-stimulated group, P<0.05.④Sorafenib played similar roles in inhibiting the activation of signal transduction molecule in HSC-T6 cells, compared with special inhibitor. Both phosphorylated ERK and phosphorylated Akt were significantly blocked by sorafenib. The inhibition of phosphorylated ERK was similar after treatment with sorafenib and PD98059, and so was the phospho-Akt inhibition by sorafenib and LY294002. P value was 0.24 and 0.11, respectively. The same results were obtained in LX-2 cells.⑤Sorafenib decreased the expression of phospho-ERK and Akt in in experimental fibrotic rats. During fibrogenesis, the expression of phospho-ERK and phospho-Akt gradually increased and decreased after administration of sorafenib (20 mg/kg and 40 mg/kg) for two weeks. The expression of phospho-ERK was downregulated by 39.82% and 46.02%, respectively, P<0.01;and the expression of phospho-Akt was decreased by 38.75% and 40%, respectively, P<0.05 ; As the evidence showed by immunochemistry, the phosphorylation of ERK and Akt were very low in normal rats. With the development of hepatic fibrosis, the positive staining cells of phospho-ERK and Akt increased obviously, and mainly resided in the interstitial cells in portal ducts, fiber septa and perisinusoidal space. Part hepatocytes were accompanied. The expressions of phospho-ERK and phospho-Akt were both decreased by administration of sorafenib. The same results were obtained in DMN rats. Conclusions: Sorafenib blocks ERK/MAPK and PI3K/Akt ERK signal transduction pathways by inhibiting the phosphorylations of ERK and Akt in HSCs. Sorafenib ameliorates fibrosis by downregulating the expressions of phosphorylated ERK and phosphorylated Akt in liver fibrosis.