Ghrelin Attenuates PAI-1 Secretion Induced By TNF-α In HepG2 Cells

Introduction and objectiveThere is increasing evidence of the coupling of immune status to diabetes and insulin resistant.The communication between the state of systemic and cellular energy balance to immune compartment is mediated via a complex array of cytokines, hormones and neuropeptides.Inflammatory markers like cross-reacting protein(CRP),Plasminogen activator inhibitor type-â… (PAI-1) are present in higher concentrations in insulin resistant people than in normal people.The focus of current diabetes research is the clarification of the pathogenetic relationships between subclinical inflammation,diabetes and arteriosclerosis.Although circulating TNF-αlevels are relatively low and have no clear correlation with obesity or insulin resistance,tissue expression levels of TNF-αcorrelate positively with both conditions.TNF-αand other proinflammatory cytokines have also been shown to participate in the pathogenesis of atherosclerosis.PAI-1,a glycoprotein,is the major physiologic inhibitor of tissue-type plasminogen activators(tPA).PAI-1 is an important mediator of atherosclerosis and liver fibrosis in insulin resistance.Circulating levels of PAI-1 are elevated in obese individuals.Insulin resistance prior to the development of frank type 2 diabetes and type 2 diabetes itself is associated with a significant increase in the risk of atherothrombotic disease,which is due in part to a disruption in the balance of factors regulating coagulation and fibrinolysis.Emerging evidences implicate PAI-1 as a significant risk factor for macrovascular and microvascular complications of diabetes. Indeed,circulating PAI-1 levels are elevated at an early stage of impaired glucose tolerance and continue to be elevated as diabetes and the metabolic syndrome develop. In addition,PAI-1 was shown to be important in the development of atherosclerosis. Thus,PAI-1 may constitute a link between insulin resistance and its related disorders.In addition to regulation of energy metabolism,liver has been reported to be a source of PAI-1 in circulatory system.There is no sufficiens in vivo or in vitro evidence of regulated PAI-1 expression in human hepatocytes.In steatotic liver,TNF pathway dysregulation could be involved in increased plasma PAI-1 in obesity with IR.Nuclear factor-kappa B(NFκB) is a critical signaling molecule in TNF-α-induced inflammation.It was also seen that NFκB is active in the cells which were induced by TNF-α.The p38,ERK,JNK pathways are also involved in TNF-α-induced PAI-1 production in vitror.PI3K/Akt pathway activation could inhibit PAI-1 production.Ghrelin is a 28-amino-acid acylated polypeptide secreted predominantly from X/A-like enteroendocrine cells of the stomach.Several lines of evidence implicate ghrelin in growth hormone(GH) release,energy balance in rodents and humans.In addition,the wide tissue distribution of growth hormone secretagogue receptor(GHS-R) suggests that ghrelin may function as signal modulators among the endocrine,nervous, and immune systems.Fasting plasma ghrelin levels were negatively correlated with BMI.Ghrelin was also negatively correlated with the PAI-1 concentrations.Because TNF-α-induced PAI-1 is critical in these pathological states and because GHS-R has been identified in liver,we hypothesized that ghrelin may have anti-inflammatory effects in the liver.On the basis of the aforementioned observations,we examined whether ghrelin inhibits PAI-1 in HepG2 cells and the mechanism involve in the faction.In the present report,we describe a novel function of ghrelin on PAI-1 secretion and PAI-1 mRNA expression by HepG2 cells upon TNF-αexposure.The mechanism whether NFκB,MAPK and PI3K/Akt pathways invoved in the regulation were also investergated.These results have implications in the potential use of ghrelin as a therapeutic target associated with pro-thrombotic changes that occur in association with type 2 diabetes and insulin resistance.HepG2 cells have been studied in detail with respect to their potency to syntheisze acute phases proteins and are generally accepted to be a good in vitro model to study acute phase proteins.As the HepG2 cell line possesses most of the characteristics of mature human hepatocytes,and the GHS-R is found in hepatoma cells,we used this cell culture model system to determine the effect of ghrelin on PAI-1 expression.Methods1.Cell culture.HepG2 cells were cultured in T-75 tissue culture flasks with 15 ml of Dulbecco's modified Eagle's medium(DMEM) containing 10%NBCS,100 U/ml penicillin and100 ug/ml streptomycin.Cells were grown in a humidified incubator at 37℃in an atmosphere of 5%CO₂ and 95%air.Subcultures were made from confluent stock cultures by trypsinization with 0.25%Trypsin and 0.02%EDTA solution. Cultures were allowed to reach 80%to 90%confluence before experiments initiated. Cells were seeded at 1.0×10⁶ per well in 6-well dishes and grown to confluence.2.After serum starvation,HepG2 cells were treated with or without TNF-αin different Concentration or time,Cells lysate and conditioned media were collected for measurement.The induction of PAI-1 was determined by enzyme linked immunosorbent assay.The effect on mRNA expression of PAI-1 induced by TNF-αwas studied using semiquantitative reverse transcription-polymerase chain reaction.3.After serum starvation,cells were exposed to DMEM with selected concentrations of ghrelin(0,1.0,10,100ng/ml).To elicit the effect of ghrelin on expression of PAI-1,the cells were pretreated with ghrelin 1h in different concentrations,then,were stimulated by addition of TNF-α1.0ng/ml for 24 h.At the end of the experiment,media was then collected and assayed for PAI-1 by ELISA.The effect of ghrelin on mRNA expression of PAI-1 induced by TNF-αwas studied using semiquantitative RT-PCR.4.HepG2 cells were preincubated with PDTC in the presence or absence of ghrelin for 1 hour,and PAI-1 release was determined 24 hours later in both unstimulated cells and cells treated with 1 ng/ml TNF-α.PAI-1 contents in the supernatant were measured by ELISA and mRNA levels of PAI-1 were analyzed using semiquantitative RT-PCR.5.HepG2 were pretreated with or without 100 ng/ml ghrelin for 1 hour and exposed to TNF-αfor 30 minutes.Cytosolic or nuclear proteins were extracted,and Western blotting was performed to detect NFκBp65,IκBα,p38MAPK,ERK1/2,p-JNK, p-Akt andβ-actin.6.HepG2 were pretreated with or without PDTC for 1 hour and exposed to TNF-αfor 30 minutes.Cytosolic or nuclear proteins were extracted,and Western blotting was performed to detect NFκBp65,IκBα,andβ-actin.7.HepG2 were pretreated with or without ghrelin for 1 hour and exposed to TNF-αfor 30 minutes.Immunocytochemical method was performed to identify the localization of NFκBResults1.Unstimulated HepG2 cells secreted PAI-1 into the culture medium.After incubation with different concentrations of TNF-α,from 0.1 to 10.0 ng/mL,PAI-1 concentrations were elevated in a dose-dependent manner.The effect of TNF-αon PAI-1 secretion was also time-dependen manner.After incubation with 1.0 ng/mL TNF-α,PAI-1 concentrations were elevated in a time-dependent manner,peaking at 24 hours.TNF-αinduced a dose-dependent increase in PAI-1 mRNA,with a maximum at 100ngl/mL;and 1.0 ng/mL TNF-αinduced a time-dependent increase in PAI-1 mRNA, which peaked at 6h.2.Ghrelin inhibited both basal and TNF-α-induced PAI-1 release in a dose and time dependent manner.Semiquantitative RT-PCR analysis showed that ghrelin reduced TNF-α-induced PAI-1 mRNA expression significantly.3.PDTC could markedly inhibit the increase of PAI-1 secretion and mRNA expression induced by TNF-α.PDTC did not have additive inhibitory effects of ghrelin (P＞0.05). 4.NFκBp65 translocation from cytosol to nucleus were increased in cultures treated with TNF-α,Ghrelin or PDTC pretreate for 1 h repressed nuclear accumulation of NFκBp65 induced by TNF-α..NFκBp65 were increased in cytosolic protein pretreated with ghrelin than in cultures treated with TNF-α.IκBαwas also lower in cultures treated for 30 min with TNF-αthan in the controls,IκBαwas increased in cultures preincubated with ghrelin or PDTC than in the cultures treated for 30 min with TNF-α.5.Phospho-p38MAPK,phospho-JNK and phospho-ERK1/2 were increased in cultures treated for 30 min with TNF-αthan in the controls;phospho-p38MAPK, phospho-JNK were decreased in cultures preincubated with ghrelin than in the cultures treated for 30 min with TNF-α,nevertheless,pretreatment with ghrelin augmented ERK1/2 activation induced by TNF-α.6.In control cells,phospho-Akt was expressed slightly,treatment with TNF-αdid not influenced Akt expression obviously,Nevertheless,pretreatment with ghrelin augmented phospho-Akt expression induced by TNF-α.7.Immunocytochemistry results manifested that NFκBp65 was distributed uniformly in cytosol in control.After cells induced by TNF-α,NFκBp65 translocation from cytosol to nucleus were increased.NFκBp65 was enriched in nucleus.Ghrelin pretreat could decrease the nuclear transfer induced by TNF-α.Conclusions1.The level of PAI-1 concentration and mRNA in HepG2 cells were increased after TNF-αexposure.2.Ghrelin could markedly inhibit the increase of PAI-1 mRNA and secretion both basal and induced by TNF-α.3.Ghrelin could inhibit PAI-1 production and PAI-1 mRNA expression via attenuating NFκB activation inducd by TNF-α.4.Ghrelin maybe inhibit PAI-1 production and mRNA expression via attenuating p38MAPK and JNK signal pathway in HepG2 cells. 5.The PI3K/Akt signaling pathways were significant activated in response to pretreatment with ghrelin.Ghrelin maybe inhibit PAI-1 secretion via this signaling pathways.