| Background Liver fibrosis is the common scarring reaction associated with chronic liver injury that results from prolonged parenchymal cell injury and inflammation. The fibrogenic response is characterized by progressive accumulation of extracellular matrix components enriched in fibrillar collagens and a failure of matrix turnover. This process is driven by a heterogeneous population of hepatic myofibroblasts, which mainly derive from hepatic stellate cells and portal fibroblasts. Hepatic fibrosis is the common result of the development of a variety of chronic liver disease, the inevitable stage of cirrhosis, especially hepatocellular carcinoma precursor. Causes of liver fibrosis, including hepatitis, chronic alcoholism, nonalcoholic fatty hepatitis, drug, industrial chemicals, hepatic vein is blocked, schistosomiasis, autoimmune hepatitis, cholestasis, genetic metabolic diseases and hidden source sex hepatitis, etc. Our country is a high incidence of viral hepatitis, global has chronic hepatitis b virus infection(HBV) is about350million; hepatitis c virus infection (HCV) is about170million and about120million4million HBV and HCV infection in our country.Chronic inflammation of the liver caused by chronic hepatitis b virus, hepatitis c virus (HCV) infection, promote the liver fibrosis, further develop liver cirrhosis, which greatly increased the risk of hepatocellular carcinoma (HCC), related studies have shown that more than85%of hepatocellular carcinoma (HCC) associated with chronic HCV, HBV infection. At present there is no effective method to cure the disease case, in-depth study of the molecular mechanism of formation and development of liver fibrosis, find the corresponding specific therapeutic targets, to prevent or reverse fibrosis progression, thereby preventing the further development of liver cirrhosis, liver cancer, even has become a very important goal of treatment. Related to the pathogenesis of liver fibrosis study showed that hepatic stellate cells (HSC) activation and transformation is the key to the formation of liver fibrosis. While Transforming growth factor-beta1(TGF-β1) plays a very important role in the process of liver fibrosis formation. TGF-β1is promoting the HSC activation, and promote the HSC express the key factor of the ECM. TGF-β1can promote the synthesis, adhesive proteins, collagen and proteoglycan of extracellular matrix of HSC elastin, reducing the degradation of protease synthesis, to prevent decomposition of the new synthesis of ECM, break the balance of the ECM synthesis and degradation of the ECM deposition increases, accelerate the development of liver fibrosis.TGF-β1effect on promoting fibrosis mainly through a signal transmitted downstream Smads family, related studies have shown that TGF-β1/Smads signaling pathways in the pathogenesis of hepatic fibrosis has played a key role. In addition to Smad signaling, TGF-β1can also activate the mitogen activated protein kinase (MAPK) signal transduction pathway, mainly including the c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase(ERK) and p38lightning kinase three subtribe. Our preliminary results indicate that TGF-β1in HepG2cells may promoted Smad2/3, Smad3L phosphorylation through the JNK signaling pathway and p38lightning. On this basis, this topic will continue to study the MAPK pathway of TGF-beta/Smad other important regulating role in signal transduction mechanisms.Objectives1. To clarify TGF-β/Smad signal transduction was regulated by MAPK pathway in HSC cells;2. To clarify TGF-β/Smad signal transduction was regulated by MAPK pathway in HepG2cells.Methods1. Cell culture and treatment HepG2cells or HSC cells were starved overnight in serum-free medium, in the absence or presence of ERK inhibitor (PD98059), JNK inhibitor (SP600125) or p38inhibitor for5h, respectively; subsequently the cells were treated with TGF-β1for the determined concentrations and time depending on the different purposes. The cells of the control group were added to an equal volume of vehicle. Each experiment was replicated at least three times in this study.2. Smad2/3phosphorylation and Smad2/3/4complex formation HSC cells or HepG2cells were detected by immunoprecipitation and western blot methodAfter incubation, HSC cells or HepG2cells were harvested and lysed for total protein extraction. The supernatants of pretreated cell lysates were subjected to immunoprecipitation with anti-Smad2/3antibody. The phosphorylation of Smad2/3at the C-terminal region (pSmad2C, pSmad3C) and at the linker region (pSmad2L, pSmad3L) was then monitored by western blot analysis using each domain-specific antibody against the phosphorylation site:anti-pSmad2C, anti-pSmad3C, anti-pSmad2L or anti-pSmad3L. In addition, after immunoprecipitation, the Smad2/3/4complex was monitored using anti-Smad4antibody to detect Smad4that bound to Smad2/3. The endogenous Smad2/3expression and Smad4in cell extracts were monitored as the reference.3. The intracellular localization of phosphorylated Smad2/3and MAPK proteins in HSC cells or HepG2cells were examined using immunofluorescence assayHSC cells or HepG2cells were seeded on sterile slides in24-well plates. After incubation, the pretreated cells were fixed and blocked. The cells were then incubated with each primary antibody overnight followed by incubation with fluorescein isothiocyanate-conjugated IgG. After that, the slides were viewed and photographed under a fluorescence microscope to observe the intracellular localization of phosphorylated Smad2/3and MAPK proteins in keloid fibroblasts.4. The protein levels of Imp7/Imp8and MAPKs in HSC cells or HepG2cells were measured by western blot method After incubation, HSC cells or HepG2cells were harvested and lysed for total protein extraction. The expression of Imp7/Imp8protein as well as pJNK1/2, pERK1/2, or pp38protein in HSC cells or HepG2cells was measured by western blot method; the expression of GAPDH and non-phosphorylated pJNKl/2, pERK1/2, or pp38in in HSC cells or HepG2cells was measured as the reference, respectively.5. The PAI-1mRNA level in HSC cells or HepG2cells was detected by real-time quantitative RT-PCRTotal RNA was extracted from HSC cells or HepG2cells by using RNAiso Reagentand was reverse-transcribed to cDNA followed by amplification using the SYBR(?)rimeScriptTM RT-PCR kit. Threshold cycles (Ct) at which emission rises above baseline were automatically calculated by the real-time RT-PCR System. Each Ct value was normalized to the housekeeping gene β-actin Ct value and a control sample. Relative quantization was expressed as fold-induction compared to control conditions. Melting curves were generated after each run to confirm amplification of specific transcripts. The experiments were repeated for three times.Results1. Effects of three MAPK inhibitors on the formation and nuclear translocation of phosphorylated Smad3L induced by TGF-β1in HSC cellsUnder the condition of no TGF-β1stimulation, phosphorylation of Smad3C, Smad3L level is very low and expressed is very weak in the nucleus. TGF-β1induce phosphorylation of Smad3L, and promote pSmad3L into the nucleus; Three kinds of MAPK inhibitors (10microns) can inhibit pSmad3L phosphorylation of Smad3L. JNK inhibitors can inhibit pSmad3L into the nucleus.2. Effects of three MAPK inhibitors inhibited Smad2/3/4complex formation and nuclear translocation induced by TGF-β1in HSC cells TGFG-β1to induce Smad4expression increases, and promote its into the nucleus; Three kinds of MAPK inhibitors can inhibit Smad4into the nucleus, but had no significant effect on its expression.3. Effect of three mitogen-activated protein kinase (MAPK)-specific inhibitors on the level of Imp7/imp8protein induced by TGF-β1in HSC cells3.1Effect of three mitogen-activated protein kinase (MAPK)-specific inhibitors on the level of Imp7protein induced by TGF-β1in HSC cellsUnder the condition of no TGF-β1stimulation, Imp7degree is very low and within the nucleus of protein expression is very weak. TGF-β1can induce Imp7expression and into the nucleus; Three kinds of MAPK inhibitors can inhibit Imp7expression and into the nucleus.3.2Effect of three mitogen-activated protein kinase (MAPK)-specific inhibitors on the level of Imp8protein induced by TGF-β1in HSC cellsUnder the condition of no TGF stimulation, Imp8degree is very low and within the nucleus of protein expression is very weak. TGF-β1can induce Imp8expression and into the nucleus; Three kinds of inhibitors can inhibit Imp8expression, p38lightning inhibitory factor can reduce Imp8into the nucleus.4. Effect of three mitogen-activated protein kinase (MAPK)-specific inhibitors on the expression of plasminogen activator inhibitor(PAI)-1mRNA induced by transforming growth factor (TGF)-β1in HSC cellsTGF-β1can obviously induced PAI-1mRNA expression, ERK, JNK and p38lightning inhibitory factor can suppress the PAI1mRNA expression induced by TGF-β1.5. Inhibitory effects of three mitogen-activated protein kinase (MAPK)-specific inhibitors on transforming growth factor (TGF)-β1-induced activation of mitogen-activated protein kinase (MAPK)5.1Inhibitory effects of PD98059on transforming growth factor (TGF)-β1-induced activation of extracellular signal-regulated protein kinase (ERK) In the absence of TGF-β1stimulus, ERK pathway has a certain degree of phosphorylation in HepG2cells; TGF-β1further enhance pERK expression.And ERK inhibitors (PD98059) can inhibit the expression of pERK.5.2Inhibitory effects of SP600125on transforming growth factor (TGF)-β1-induced activation of c-Jun N-terminal kinase (JNK)In the absence of TGF-β1stimulus, JNK pathway has obvious phosphorylation in HepG2cells; TGF-β1further enhance pJNK expression. And JNK inhibitors (SP600125) can obviously inhibit the expression of pJNK.5.3Inhibitory effects of SB203580on transforming growth factor (TGF)-β1-induced activation of c-Jun N-terminal kinase (p38)In the absence of TGF-β1stimulus, JNK pathway has obvious phosphorylation in HepG2cells; TGF-β1further enhance pp38expression. And p38lightning inhibitory factor (SB203580) can obviously inhibit the expression of pp38.6. Effects of three MAPK inhibitors on TGF-β1-induced translocation into nuclei of phosphorylated Smad3C and Smad3LUnder the condition of no TGF-β1stimulation, Smad3C and Smad3L phosphorylation level is very low and expressed in the nucleus is very weak. TGF-β1can induce phosphorylation of Smad3C and Smad3L, and promote pSmad3C, pSmad3L into the nucleus. ERK inhibition factor, JNK inhibition factor significantly inhibits phosphorylation of Smad3L and into the nucleus, p38lightning inhibitory factor inhibiting phosphorylation of Smad3L, but has no obvious influence on its nuclear plasma distribution. TGF-β1and three kinds of MAPK inhibitors for Smad3C phosphorylation were not seen significant influence, but increase pSmad3C into nuclear TGF-β1, three kinds of MAPK inhibitors can inhibit pSmad3C into the nucleus.7. Effects of three MAPK inhibitors on TGF-β1-mediated Smad2/3/4complex formation in HepG2cellsUnder the condition of no TGF-β1stimulation, There have not Smad2/3/4complexe formed in HepG2cells, TGF-β1can significantly induce Smad2/3/4complex formation; P38lightning inhibitory factor (SB203580) almost completely blocked Smad2/3/4complex formation; JNK inhibitors (SP600125) is concentration dependent inhibition Smad2/3/4complex formation induced by TGF-β1; ERK inhibitors (PD98059) has no apparent influence on the formation complexes. Smad2/3groups of HepG2cell protein expression quantity did not change, at the same time, the Smad4protein expression in cell extracts there was no difference between groups.8.Effect of three mitogen-activated protein kinase (MAPK)-specific inhibitors on the level of Imp7/imp8protein induced by TGF-β1in HepG2cells8.1Effect of three mitogen-activated protein kinase (MAPK)-specific inhibitors on the level of Imp7protein induced by TGF-β1in HepG2cellsUnder the condition of no TGF-β1stimulation, Imp7degree is very low and within the nucleus of protein expression is very weak. TGF-β1can induce Imp7expression and into the nucleus; Three kinds of MAPK inhibitors can inhibit Imp7into the nucleus, especially p38lightning inhibiting factor.8.2Effect of three mitogen-activated protein kinase (MAPK)-specific inhibitors on the level of Imp8protein induced by TGF-β1in HepG2cells Under the condition of no TGF stimulation, Imp8degree is very low and within the nucleus of protein expression is very weak. TGF-β1can induce Imp8expression and into the nucleus; ERK inhibition factor, JNK inhibitors can inhibit Imp8into nuclear, p38lightning inhibitory factor had no obvious effect on Imp8nuclear plasma distribution.9. Effect of three mitogen-activated protein kinase (MAPK)-specific inhibitors on the expression of plasminogen activator inhibitor(PAI)-1mRNA induced by transforming growth factor (TGF)-β1in HepG2cellsTGF-β1can obviously induced PAI-1mRNA expression, ERK, JNK and p38lightning inhibitory factor can suppress the PAI1mRNA expression induced by TGF-β1.Conclusions1. TGF-β1induced phosphorylation of Smad3C and Smad3L in HSC cells and HepG2cells,and thus promote Smad2/3/4complex formation and its transfer into the nucleus and target genes PAI-1mRNA expression may be the important mechanism of liver fibrosis-liver cancer developing, and phosphorylation of Smad3L induced by TGF-β1may be the most key link of liver fibrosis-liver cancer developing.2. ERK, JNK, P38lightning inhibitory factor can significantly inhibit the expression of pSmad3L induced by TGF-β1in HSC cells, JNK inhibitors can inhibit pSmad3L into nuclear; Prompt, Smad3protein phosphorylation connection area depends on the activation of ERK, JNK, p38lightning channel, and pSmad3L transposition into the nucleus are mainly related to the activation of JNK pathway. ERK inhibition factor, JNK inhibition factor significantly inhibits Smad3L phosphorylation and its transfer into the nucleus in HepG2cells, p38lightning inhibitory factor inhibiting Smad3L phosphorylation, but has no obvious influence on its nuclear plasma distribution. Three kinds of MAPK inhibitors can inhibit pSmad3C transposition into the nucleus. Prompt, Smad3L phosphorylation in HepG2cells depends on the activation of ERK, JNK, p38lightning pathways, while pSmad3L transposition into nuclear mainly related to the activation of ERK, JNK pathway. PSmad3C transposition into the nucleus is related to the activation of ERK, JNK, p38lightning channel. 3. Three MAPK inhibitors can inhibit Smad2/3/4complex into the nucleus, but had no significant effect on its expression in HSC cells. Prompt, Smad2/3/4complex formation mainly is regulated by p38lightning, ERK pathway in HSC cells, and Smad2/3/4complex transposition into the nucleus is mainly affected by the JNK pathway. P38lightning inhibitory factor (SB203580) almost completely blocked Smad2/3/4complex formation; JNK inhibitors (SP600125) is concentration dependent inhibition Smad2/3/4complex formation induced by TGF-β1; ERK inhibitors (PD98059) has no apparent influence on the formation complexes. Prompt, Smad2/3/4complex formation mainly is regulated by p38lightning,JNK pathway in HepG2cells, and Smad2/3/4complex transposition into the nucleus is mainly affected by the ERK%JNK pathway.4. In HSC cells, the three kinds of MAPK inhibitors can inhibit Imp7expression and into the nucleus. Three kinds of inhibitors can inhibit Imp8expression, p38lightning inhibitory factor can reduce Imp8into the nucleus. Prompt, Smad2/3/4complex transposition into nuclear regulated by ERK, JNK pathway may be related to Imp7/Imp8expression and Imp7into the nucleus; Smad2/3/4complex transposition into nuclear regulated by p38pathway may be related to Imp7/Imp8expression and into the nucleus. The three kinds of MAPK inhibitors can inhibit Imp7into the nucleus, especially p38lightning inhibiting factor in HepG2cells. ERK inhibition factor, JNK inhibitors can inhibit Imp8into nuclear, p38lightning inhibitory factor had no obvious effect on Imp8nuclear plasma distribution. Prompt, Smad2/3/4complex transposition into nuclear regulated by ERK, JNK pathway may be related to Imp7/Imp8into the nucleus; Smad2/3/4complex transposition into nuclear regulated by p38pathway may be related to Imp7into the nucleus.5. ERK, JNK, P38lightning inhibitory factor can significantly inhibited the expression of target genes of PAI-1mRNA induced by TGF-β1. Prompt, ERK, JNK, p38lightning pathways are involved in TGF-β1signaling pathways regulating gene transcription activity of PAI-1target in HSC cells. ERK, JNK, P38lightning inhibitory factor can significantly inhibited the expression of target genes of PAI-1mRNA induced by TGF-β1. Prompt, ERK, JNK, p38lightning pathways are involved in TGF-β1signaling pathways regulating gene transcription activity of PAI-1target in HepG2cells.In summary, P38lightning, ERK pathway may mainly through regulating the protein phosphorylation of Smad3connection area and Smad2/3/4complex formation and influence of target gene transcription of PAI-1, JNK pathway mainly through regulating Smad2/3/4of transposition into nuclear complex influence target genes PAI-1transcription in HSC cells. P38lightning channel may mainly by regulating the Smad3protein phosphorylation and Smad2/connection area3/4complex formation and influence target gene transcription of PAI-1in HepG2cells; ERK pathway is mainly through regulating Smad2/3/4of transposition into nuclear complex influence target genes PAI-1transcription; JNK pathways may through regulating Smad3protein connection area phosphorylation and Smad2/3/4Smad2/3/4complex compounds formation and regulation of transposition into nuclear influence target genes PAI-1transcription. |
PDF Full Text Request