| Ghrelin is a 28-amino acid peptide recently identified as endogenous ligand for the growth hormone secretagogue receptor(GHS-R).It is secreted mainly by gastric tissue. Circulating levels of total ghrelin include two forms,acylation of ghrelin is required for GHSR-1a activation,whereas des-acyl ghrelin,does not bind GHSR-1a.Ghrelin and its receptor are widely distributed in cardiovascular tissues,there is no doubt that ghrelin has an important role in the regulation of cardiovascular function.There are many studies on cardiovascular effects of ghrelin,including metabolic effects,cardiovascular effects,endocrine effects and cellular effects.Along with the people living condition's improvement and life style's change,the prevalence rate of diabetes increases year by year,already became one kind of worldwide epidemic disease.The Diabetes Control and Complications Trial(DCCT) and the U.K.Prospective Diabetes Study(UKPDS)indicate that it is the persistent hyperglycemia of uncontrolled diabetes that causes vascular complications.Vascular disease is the principal cause of morbidity and mortality in patients with diabetes.A wide variety of studies suggest that early site at which these vascular complications develop is the endothelium.When the endothelial cells under the influence of some cardiovascular high-risk factor like hypertention,dyslipidemia,hyperglycemia and increased oxidative stress,may occur the endothelial cells apoptosis and the endothelium dysfunction.Therefore,deeply study the function of ghrelin on endothelial cells will be helpful clear the inner link between ghrelin and diabetes atherosclerosis.Oxidative stress participate many kinds of disease's pathophysiology processes, and cause the endothelial dysfunction.Some reports that the oxidative stress is "the common soil" of the insulin resistance,diabetes and cardiovascular disease.Oxidative stress produces excessively free radical in the organization or in the cell,the free radical close related to oxidative stress to be reactive oxygen species(ROS).Some scholars discovered that ROS through the activation of poly ADP-ribose polymerase (PARP)suppresses GAPDH,then activates PKC,causes NF-κB expression upward, promotes the vascular complication occurrence development.Many evidences indicated that the hyperglycemia cause the massive ROS production,cause the vascular endothelial cells dysfunction directly,as well as in cell nucleic acid,protein expression mistake,these molecular level harm in cell and organization level displays for various systems' complication.Ghrelin caused a dose-dependent increase in SOD activity in 3T3-L1 preadipocytes cell culture.This result suggested that ghrelin has the resistance function of oxidation stress.In vitro studies showed that ghrelin inhibited ROS generation by human polymorphoneuclear cells in a dose-dependent manner.This experiment through culture endothelial cells in vitro under hyperglycemia environment, studied the effect of ghrelin on ROS production,thus further clear about ghrelin's function.The cell signal transduction is an important front topic on the biological information and the cell communication,there has a very important significance on deeply study the process of cell signal transduction.The PI3-Kinase/AKT pathway participated in diabetes,the tumor and many kinds of disease's pathology processes,it is also a cell survival most important pathway.In many growth factor induced cell growth and the survival response,there has the AKT/PKB high expression and the activation,it induces many kinds of code anti-apoptosis protein genes expression. Ghrelin as a growth hormone's endocardial ligand,it still lacked the explicit report under the hyperglycemia environment with the relationship of PI3-Kinase/AKT signal transduction pathway.NF-κB has the widespread biology activity,it participates in many genes's regulation,it play a role in the infection,the inflammation responded,the oxidized stress,the cell proliferation,the cell apoptosis and so on.Recent studies indicated that NF-κB close to the cell apoptosis,it participate many kinds of apoptosis related gene regulation and play an important role in disease's occurrence and the developing process.NF-κB and the cell apoptosis have bidirectional adjustment relations: Suppresses the cell apoptosis,also promotes the cell apoptosis,it relies on the cell type and the stimulating factor difference.Some reports that ghrelin inhibit the activation of NF-κB induced by TNFα,but still hasn't research under the hyperglycemia environment.High glucose concentration level has been shown to induce cell apoptosis in kidney,neuron and vascular endothelial cells,which are supposed to be related to the acceleration of diabetic complications and atherosclerosis.Therefore,in the present study,we evaluated that the effects of ghrelin on high glucose-induced apoptosis in human umbilical vein endothelial cells(ECV304)and then explored the mechanisms for its anti-apoptotic effect.Methods1.Cell cultureECV304 were cultured in low glucose concentration(5.5mmol/L)Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%fetal bovine serum(FBS), 100units/ml of penicillin and 100mg/ml streptomycin.Cells in passage 4-5 were used. For all cell culture experiments,cells were permitted to adhere for 24 hours following plating before the experiment reagent were administered.2.Acridine Orange(AO)and Hoechst33258 stainingCells were incubated with each experimental treatment,dyed with 0.01%AO and 100μL Hoechst33258,respectively.The plates were sealed and observed under a fluorescence microscope.3.Flow cytometry assay apoptosisCell apoptosis was detected by flow cytometry with PI staining.Cells were incubated with each experimental treatment.Cells were collected and centrifuged at 1000rpm/min for 10min,the cells were washed twice with pre-cold PBS,and fixed with 70%ethanol overnight at 4℃.Cell pellets were resuspended in PBS after being washed twice with pre-cold PBS.Then 500μL of propidium iodide(1000μg/L)were added to the cells for 30min at room temperature in the dark,and then flow cytometry analysis was performed.Data were analyzed using CellQuest 3.0 Software.4.TUNEL detect apoptosisCells were incubated with each experimental treatment.We detected TUNEL-positive cells according to the manufacturer's instructions,cells were fixed with 4%paraformaldehyde and stained using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay.The number of stained cells was assessed using the computer software program MetaMorph/DP10/BX41 image,to avoid potential subjective errors.5.Spectrofluorometer assay caspase-3 activityCaspase-3 activity was tested in the presence or absence of pharmacological inhibitors of PI3-Kinase(LY294002)and with or without ghrelin pretreated for 24h, then exposed to test media containing 1%FBS and normol(5.5mmol/L)or high glucose(33.3mmol/L)for 24h to 72h in accordance with TUNEL assay.Caspase-3 activity assay according to the manufacturer's instructions.Briefly,after being washed twice with phosphate buffered saline(PBS),cells centrifuged at 10000rpm/min for 1min,followed by the addition of 50μL 2×Reaction Buffer/DTTMix.Cell lysates were incubated at 37℃with 5μL caspase-3 substrate(Ac-DEVD-pNA)for 4h,the caspase-3 activity was measured by a spectrofluorometer with a wavelength at 400nm.6.Flow cytometry assay ROSThe level of intracellular ROS was monitored by flow cytometry using peroxide-sensitive fluorescent probe 2'7'-dichlorlfluorescin diacetate(DCFH-DA). DCFH-DA is converted by intracellular esterases to DCFH,which is oxidized into the highly fluorescent dichlorofluorescin(DCF)in the presence of a proper oxidant. Samples were loaded with 2μmol/L DCFH-DA in Hank's Balanced Salt Solution (HBSS)for 20min at 37℃,after incubation,the cells were resuspended in ice-cold PBS,and used immediately for flow cytometry.Mean fluorescence was calculated by using the program CellQuest.7.Western-Blot assay P-Akt(Ser473)and NF-κB(p65)The cells were lysed using RIPA lysis buffer and Nuclear-Cytosol Extraction Kit. Proteins were separated by 10%SDS-PAGE and transferred to polyvinylidine fluoride membranes.After being washed three times with 1×TBS(PH:7.6)buffer,the membranes were soaked in 5%nonfat dry milk for 2h and incubated overnight at 4℃with the anti-phospho-S473 Akt polyclonal antibody(diluted 1:200)and anti-NF-κB p65 monoclonal antibkdy(diluted 1:1000).After incubation with HRP-conjugated secondary antibody(diluted 1:5,000)for 2h at room temperature,the immune complexes were visualized by enhanced chemiluminescence methods,the band intensity was measured and quantitated.The resulting images were analyzed with Scion Image software.8.Statistical analysisResults are expressed as means±SE for three or more independent experiments. Statistical significance was estimated by oneway ANOVA followed by Student Newman-Keuls test for comparison of several groups,p＜0.05 was considered statistically significant.Results1.We measured apoptosis by TUNEL assay and flow cytometry.The apoptosis cells was significantly greater in the ones incubated during 72h at the high glucose concentration(33.3mmol/L)than normal glucose concentration(5.5mmol/L)(p＜0.05). However,pretreatment with ghrelin(10-7mol/L)for 24h prevented the increase in apoptosis caused by hyperglycemia at 72h(p＜0.05).2.An increase in caspase-3 activity,which is thought to be a key enzyme in the process of apoptosis,was evident in cells incubated with 33.3 mmol/L glucose for 24h to 72h(p＜0.01),and it was prevented by pretreatment of ghrelin(10-7mol/L)for 24h (p＜0.01).However,a significant decrease in caspase-3 activity was observed when addition of PI3-Kinase inhibitor(20μmol/L LY294002)before ghrelin(10-7mol/L) pretreated at 72h(p＜0.05).3.Cells were pre-treated with or without ghrelin(10-7mol/L)for 24h,then incubated with normal(5.5mmol/L)and high(33.3mmol/L)glucose concentrations for 24h and 48h,levels of intracellular ROS under high glucose conditions increased by 27%and 35%,respectively,however,levels of intracellular ROS with pretreatment of ghrelin under high glucose conditions increased by 11%and 14%,respectively, significant lower than that of high glucose group.4.To explore the possible mechanism for anti-apoptotic effect of ghrelin,we investigated the effect of ghrelin on AKT phosphorylation and activation of NF-κB in ECV304.Exposure of cells to ghrelin(10-7mol/L)caused rapid activation of AKT, reached the peak at 30min,in addition,ghrelin concentrations ranging from 10-6mol/L to 10-9mol/L resulted in also rapid activation of AKT at 30min.Exposure of cells to high glucose(33.3mmol/L)from 6h to 24h caused a time-dependent activation of NF-κB(p＜0.05),treatment with ghrelin(10-7mol/L)inhibited high glucose induced NF-κB activity at 24h(p＜0.05).Conclusion1.Exposure to high glucose concentration for 72h caused a significant increase in apoptosis,but pretreatment of ghrelin eliminated high glucose-induced apoptosis in ECV304.2.Exposure of cells to high glucose concentration caused a time-dependent activation of caspase-3,treatment with ghrelin inhibited high glucose concentration induced caspase-3 activity.3.Ghrelin protected endothelial cells from high glucose concentration by inhibiting ROS generation.4.Ghrelin induced endothelial cells AKT phosphorylation,together with the result that PI3-Kinase inhibitor attenuated ghrelin's inhibitory effect on caspase-3 activity suggests that the anti-apoptotic effect of ghrelin may be partly via PI3-Kinase/AKT pathway.5.High glucose concentration increase activation of NF-κB in vascular endothelial cells,treatment with ghrelin inhibited high glucose concentration induced NF-κB activity.
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