| Liver fibrosis is a common condition that is associated with chronic liver injury due to viral, metabolic, genetic and cholestatic liver disease. Liver fibrosis ultimately leads to cirrhosis, which is a major public health problem worldwide. Hepatic stellate cells (HSCs) are recognized as the primary cellular source of extracellular matrix (ECM) in chronic liver disease, and they play a critical role in the development and maintenance of liver fibrosis.In normal liver, HSCs are nonparenchymal, quiescent cells whose main functions are to store vitamin A and probably to maintain the normal basement membrane-type matrix. However, numerous in vivo and in vitro studies indicate that in response to liver injury, HSCs undergo an"activation"process in which they loss vitamin A stores and appearance of the cytoskeleton proteinα-smooth muscle actin (α-SMA), become highly proliferative, and synthesize"fibrotic"matrix rich in type I collage. Several cytokines, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor beta 1 (TGF-β1) stimulate HSC proliferation and collagen synthesis. Therefore, this understanding has helped to identify underlying mechanisms, are will likely lead to new therapies for liver fibrosis.Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the ErbB family that binds to and activates two ErbB receptors, epidermal growth factor receptor (EGFR/ErbB1) and ErbB4. The activation of ErbB receptors provokes mitogen-activated protein kinase (MAPK). Recent study indicated that HB-EGF induces proliferation responses in a variety of cell types. As a nontoxic mutant of diphtheria toxin, cross-reacting material 197 (CRM197) binds the soluble form and the membrane-anchored form of HB-EGF, thus blocking its binds to ErbB receptors and its subsequent downstream mitogenic activity. Extracellular signal-regulated kinase (ERK) is an important member of the MAPK family. PDGF and EGF promote HSC proliferation by activated the Ras/Raf/ERK signaling pathway. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is activated by mitogens and growth factors. Both Ras/Raf/ERK and PI3K/Akt signal pathways play crucial roles in the proliferation, apoptosis and collagen synthesis in HSCs.The purpose of the present study is to investigate the effects of HB-EGF on the proliferation and apoptosis of HSC cell lines LX2, T6 and primary rat HSCs, as well as the possibly associated molecular mechanisms. The experiments contain three parts as below:Part 1: The expression of HB-EGF in primary rat HSCsObjective: To explore the role of HB-EGF on HSCs activation.Methods: (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) The expression of HB-EGF, phosphorylated EGFR and ErbB4 receptors were low in quiescent primary HSCs, but there were significantly increased in activated primary HSCs. Total EGFR was unchanged in quiescent and activated HSCs. (4) The expression of phosphorylated EGFR and ErbB4 receptors were unchanged in with or without CRM197-treated quiescent HSCs.Results: (1) The expression of HB-EGF, phosphorylated EGFR and ErbB4 receptors were low in quiescent primary HSCs, but there were significantly increased in activated primary HSCs. Total EGFR was unchanged in quiescent and activated HSCs. (2) The expression of phosphorylated EGFR and ErbB4 receptors were unchanged in with or without CRM197-treated quiescent HSCs.Conclusions: The expression of HB-EGF, phosphorylated EGFR and ErbB4 receptors were significantly increased in activated primary HSCs. The changes of HB-EGF in quiescent and activated HSC maybe indicated that HB-EGF and its downstream signaling pathways are closely related with liver fibrosis.Part 2: HB-EGF promotes proliferation and inhibits apoptosis in HSCsObjective: To explore the effects of HB-EGF on HSCs proliferation and apoptosis.Methods: LX2, T6 and primary HSCs were preincubated with or without CRM197 (20μg/ml) for 24 h, and then stimulated with or without HB-EGF (10, 20 and 40 ng/ml) for 12 h, 24 h and 48 h. The proliferation of HSCs was detected by 3-(4, 5-dimethylthiazol-2-yl)-3, 5- diphenyltetrazolium bromide (MTT) assay. DNA synthesis was explored by bromodeoxyuridine (BrdU) incorporation assay. T6, LX2 and primary HSCs were preincubated with CRM197 (20μg/ml), AG1478 (5μmol/L), PD98059 (50μmol/L) and LY294002 (25μmol/L) for 24 h, and then stimulated with HB-EGF (40 ng/ml) for 24 h. The proliferation of HSCs was detected by MTT assay. DNA synthesis was explored by BrdU incorporation assay. LX2, T6 and primary HSCs were incubated with CRM197 (20μg/ml) for 24 h. Morphological examination via transmission electron microscopy (TEM) evaluation and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique were performed. HSCs were preincubated with CRM197 (20μg/ml) for 24 h and stimulated by HB-EGF for 24 h. Apoptosis rate assay by the Annexin-V/Propidium iodide (PI) double-labeled flow cytometry and Caspase-3 activity were detected. CRM197 (20μg/ml), AG1478 (5μmol/L), PD98059 (50μmol/L) and LY294002 (25μmol/L) for 24 h, and then stimulated with HB-EGF (40 ng/ml) for 24 h. Apoptosis rate assay by the Annexin-V/PI double-labeled flow cytometry.Results: (1) HB-EGF stimulated the proliferation of LX2, T6 and activated primary HSCs in time- and dose-dependently manner. HB-EGF (40μg/ml) increased the proliferation of LX2 (137.11±8.44%, P<0.05), T6 (138.74±6.21%, P<0.05) and activated HSCs (129.92±4.68%, P<0.05) compared with those of the control group (100%). (2) The proliferation effect of HB-EGF is equivalent to that of PDGF at the same concentration. The effect of CRM197 inhibited the proliferation is similar to AG1478. (3) HB-EGF stimulated the DNA synthesis of LX2, T6 and activated HSCs in dose- and time-dependently. HB-EGF (40μg/ml) significantly increased the DNA synthesis of LX2 (160.77±13.62%, P<0.05), T6 (154.33±5.55%, P<0.05) and activated HSCs (130.92±1.74%, P<0.05) compared with those of the control group (100%). (4) CRM197 inhibited the DNA synthesis of HB-EGF-stimulated LX2, T6 and activated HSCs. CRM197 (20μg/ml) significantly decreased the DNA synthesis of LX2 (68.87±5.57%) compared with that of HB-EGF group (160.90±13.57%) (P<0.05), decreased the DNA synthesis of T6 (74.67±5.55%) compared with that of HB-EGF group (154.33±5.55%) (P<0.05) and decreased the DNA synthesis of activated HSCs (74.67±5.55%) compared with that of HB-EGF group (130.92±1.74%) (P<0.05). (5) Morphological changes of LX2, T6 and activated HSCs after treatment with CRM197 (20μg/ml) for 24 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. (6) CRM197 (20μg/ml) up-regulated the positive-LX2, T6 and activated HSCs of TUNEL staining by 8.87 times, 7.54 times and 6.54 times respectively compared with the control groups (P<0.05). (7) CRM197 induced apoptosis of HSCs. HB-EGF decreased HSC apoptotic rates of CRM197-pretreated LX2, T6 and activated HSCs in dose- and time-dependent manner. (8) HB-EGF inhibited the activity of Caspase-3, whereas CRM197 increased the activity of Caspase-3 in LX2, T6 and activated HSCs by 1.56 times (P<0.05), 1.65 times (P<0.05) and 1.70 times (P<0.05) respectively compared with the control groups.Conclusions: HB-EGF stimulated the proliferation and DNA synthesis in HSCs. However, CRM197 induced HSC apoptosis, which was related to the up-regulation of Caspase-3.Part 3: The mechanism of intracellular signal transduction that contribute to the impacts of HB-EGF on HSC proliferation and apoptosisObjective: To explore the regulation of HB-EGF on ErbB-mediated MAPK/ERK and PI3K/Akt signalling pathways in HSCs.Methods: The expression of ErbB4 and total or phospho-EGFR were analysis by immunocytochemistry and Western blot. LX2, T6 and activated HSCs were preincubated with or without CRM197 (20μg/ml) for 24 h, and then stimulated with or without HB-EGF (10, 20 and 40 ng/ml) for 24 h. Another set of HSCs were preincubated with or without CRM197 (20μg/ml) for 24 h, and then stimulated with or without HB-EGF (40 ng/ml) for 12, 24 and 48 h. The protein expressions of EGFR, ErbB4, ERK, Akt, p-EGFR, p-ERK and p-Akt were determined by Western blot analysis. The mRNA levels of EGFR and ErbB4 were measured by RT real-time PCR. Simultaneously, LX2, T6 and activated HSCs were treated with CRM197 (20μg/ml), AG1478 (5μmol/L), PD98059 (50μmol/L) or LY294002 (25μmol/L), stimulated with or without HB-EGF (40 ng/ml). The protein expressions of EGFR, ErbB4, ERK, Akt, p-EGFR, p-ERK and p-Akt were determined by Western blot analysis.Results: (1) The protein expression of EGFR, phospho-EGFR and ErbB4 in both the nucleus and cytoplasm. The expression of phospho-EGFR and ErbB4 were increased in HB-EGF-stimulated nucleus and cytoplasm of HSCs. However, CRM197 inhibited the expression of phospho-EGFR and ErbB4 in HSCs. (2) HB-EGF increased the mRNA expressions of ErbB4 as well as the protein levels of ErbB4 in LX2, T6 and activated HSC. However, the EGFR mRNA levels were not significantly changed in each group of activated HSCs. (3) HB-EGF significantly increased EGFR phosphorylation and ErbB4 in dose- and time-dependent manner. These further increased ERK and Akt phosphorylations. In contrast, CRM197 markedly decreased the ErbB receptors and reduced the activities downstream signaling molecules. (4) CRM197 showed the inhibiting effects on phosphorylation of EGFR similar to the specific blocking agent of the EGFR. AG1478 was effective in inhibition of phosphorylation of EGFR, whereas no significant different impact in ErbB4. CRM197 inhibited both the ErbB4 and phosphorylation of EGFR. (5) CRM197 showed the inhibiting effects on ERK and Akt signals similar to the specific blocking agent of the two pathways. PD98059 and LY294002 were effective in inhibition of phosphorylation of ERK and Akt, respectively. CRM197 inhibited both the phosphorylated ERK and Akt. However, compared with the specific blocking agent groups, the phosphorylated ERK and Akt were significantly increased in CRM197 group.Conclusions: HB-EGF promoted HSC proliferation and inhibited apoptosis probably via ErbB receptors activation and up-regulation of phosphorylations of ERK and Akt signaling pathways.
|
PDF Full Text Request