The Effects Of Ghrelin On Palmitate Induced Apoptosis In Rat Aortic Endothelial Cells And Its Mechanism

IntroductionGhrelin is a novel 28-amino-acid peptide that is secreted mainly by gastic tissue. Ghrelin exists in two major molecular forms, acylated ghrelin (AG) and unacylated ghrelin (UAG). Ghrelin has been identified as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Ghrelin stimulates growth hormone (GH) release and induces a positive energy balance by stimulating food intake while decreasing fat use through GH-independent mechanisms. Ghrelin also exerts numerous peripheral effects including direct effects on exocrine and endocrine pancreatic functions, carbohydrate metabolism, the cardiovascular system, gastric secretion, stomach motility and sleep. Ghrelin protects several cell types such as adipocytes, cortical neuron, pancreaticβ-cells, cardiomyocytes and endothelial cells by inhibiting apoptotic stimuli. Ghrelin has been shown to bind to the vascular tissures of normal human and rats. Recently, there are many studies about the cardiovascular effects of ghrelin, but the cellular and molecular mechanisms are unknown. Low ghrelin plasma levels have been reported in pathological conditions characterized by high cardiovascular risk, such as uncomplicated obesity, insulin resistant, hypertension and type 2 diabetes mellitus.Endothelial cell injury is a critical event in the pathogenesis of diabetes-associated atherosclerosis and vascular complications. Increased apoptosis may contribute to the loss of endothelial integrity and leads to cardiovascular diseases. Free fatty acid have been implicated in causing endothelial dysfunction, atherosclerosis and inflammation. Palmitate is the most abundant fatty acid in vivo. Palmitate was shown to promote apoptosis and increase reactive oxygen species (ROS) generation in cultured human endothelial cells in vitro. Ghrelin has been reported to have protective effects against many types of extracellular stimuli in vascular endothelial cells. But there was no report on the environment of palmitate in vascular endothelial cells. Oxidative stress is implicated in most cardiovascular diseases. The endothelial dysfunction may result from an imbalance between the oxidant stress and a depletion of the antioxidant reserve. Survival factors inhibit cell death by activating specific signaling pathways, which then lead to the inhibition of the apoptotic signaling cascade. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway have been implicated in the regulation of cell survival. Previous studies suggest the pathway play an important role in the mechanisms of ghrelin-mediated protection. However, the intracellular signaling mechanisms underlying this ghrelin-induced protection remain elusive. Based on these observations, we hypothesized that ghrelin may protect vascular endothelial cells from palmitate-induced injury. In this study we have investigated the effect of ghrelin on cell proliferation and palmitate-induced rat aortic endothelial cells apoptosis. Because mitochondria are involved in a variety of key events in apoptosis. we investigated the effect of ghrelin on palmitate-induced ROS production. Because Bcl-2 inhibits apoptosis by binding to the proapoptotic Bax protein, it is believed that the Bcl-2/Bax ratio is a determining factor for the cells'fate. Finally, we investigated the effects of ghrelin on the proteins of the Bcl-2 and Bax.Materials and methodsMaterials Wistar rats, Ghrelin, Fatty acid-free bovine serum albumin(BSA), Palmitate, Hoechst33258, MTT, LY294002, PD98059 Antibodies used were anti-phospho-ser473-AKT, anti-total AKT, Caspase-3 activity assay kit; AnnexinV-PI apoptosis detection kit.Methods1. Primary culture and differentiation of rat aortic endothelial cellsA male wistar rat was anesthetized with an intraperitoneal injection of ethylcarbamate. The thoracic aorta was dissected out and immersed in PBS. The fat or connecting tissue was rapidly removed with forceps. The aorta was cut lengthwise. Drop 0.1% typeⅠcollagenase, then incubated 30min at 37℃. With a scalpel, the aorta was cut into pieces that are approximately square and 2mm on each side. Put the pieces onto a 6-well plate with the endomembrane adown. Droped a little DMEM to moisten the pieces. Put the palte upside down at 37℃incubator for 1h, then added culture medium. Replaced the medium every 3 days. Cells were digestived by 0.25% trypsin digestion juice when grown to 80-90% confluency around the pieces. After centrifugation, cells were inoculated in 6-well plates rinse, replaced the medium every 2-3 days. Passaged cells with 0.25% trypsin digestion digestive fluid when cells were grown to 90% confluency. The pieces or the cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco,Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS),1% penicillin-streptomycin, and 0.15mg/ml endothelial cells growth supplement (Cambrex, Walkersville, MD, USA) at 37℃in a 95% air/5% CO2 incubator. Experiments were performed on cells at passage 3-5 from primary culture. Cells were confirmed to be endothelial cells by their cobblestone-like shape and positive staining for von Willebrand factor.2. Assessment of cell viability by MTT Cells were seeded on a 96-well plate containing 100μl of culture media, and 20μl of MTT (Amresco, Dallas, USA) stock solution (5 mg/ml) was added to the medium. Cells were incubated at 37℃for 4h in the dark and then lysed with 150μl of dimethyl sulphoxide (DMSO). The absorbance was read on a micro plate reader at a wavelength of 570nm.3. Assessment of apoptosis by hoechst33258 stainingCells were incubated with each experimental treatment, then were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 10 min at 4 C. Cells were then washed twice with PBS and stained in 100μl Hoechst 33258. After 10 min incubation at room temperature, washed twice with PBS and observed under a fluorescence microscope.4. Assessment of apoptosis by Annexin V-FITC/PI double stainingCells were trypsinized and washed in medium, centrifuged and then resuspended. Cells were counted and a volume of media containing 1×106 cells was centrifuged to obtain a pellet. After dislodging the pellet,160μl assay buffer and annexin V-FITC were added. Following an incubation for 10 min at room temperature in the dark, cells were centrifuged and resuspended. Then 190μl assay buffer and annexin V-FITC were added. PI were added and the samples were analysed immediately using flow cytometer.5. Spectrofluorometer assay caspase-3 activityAfter being washed twice with phosphate-buffered saline (PBS), cells centrifuged at 2000 rpm/min for 10 min. Removed PBS, followed by the addition of 50μL icecold lysis buffer and 50μL DTT. Lysis the cells on ice for 60min. Cells were centrifuged at 10,000 rpm/min for 1 min, followed by the addition of 50μL 2×Reaction Buffer/DTTMix. Cells were incubated at 37℃with 5μL caspase-3 substrate (Ac-DEVD-pNA) for 4 h. The caspase-3 activity was measured by a spectrofluorometer with a wavelength at 400 nm.6. Measurement of intracellular ROS formationThe level of intracellular ROS was monitored by flow cytometry using peroxide-sensitive fluorescent probe 2'7'-dichlorofluorescein diacetate (DCFH-DA). Samples were loaded with 2μM DCFH-DA in Hank's Balanced Salt Solution (HBSS) for 20 min at 37℃. After incubation, the cells were resuspended in ice-cold PBS, and used immediately for flow cytometry.7. Western blot assay P-Akt, total Akt, Bcl-2 and BaxCells were lysed using RIP A lysis buffer. Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After being washed three times with 1×TBS (pH 7.6), the membranes were soaked in 5% nonfat dry milk for 2 h and incubated overnight at 4℃with primary antibody. After incubation with HRP-conjugated secondary antibodyfor 2 h at room temperature, the immune complexes were visualized by enhanced chemiluminescence methods, the band intensity was measured and quantitated.8. Statistical analysisSPSS 13.0 softwarewas used to analysis the data. Data are expressed as the mean±standard error of mean (SE) from at least three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnett's test. P<0.05 was considered statically significant.Results1. Rat aortic endothelial cells were were cobblestone-like shape. Von Willebrand factor staining is positive. 2. Cells were incubated in ghrelin or ghrelin complex palmitate for 24h. Ghrelin promoted cell proliferation in both experimental condition as deteated by MTT assay (P<0.05).3. Following a 24h incubation, palmitate significant induced cell apoptosis as assessed by Hoechst33258 and Annexin V-FITC/PI double staining (P<0.01). In the ghrelin and palmitate treated groups, apoptotic cells was significantly lower than the groups treated with palmitate alone (P<0.01). An increase in caspase-3 activity was evident in cells incubated with 0.3mM palmitate for 24h (P<0.01). In the palmitate and ghrelin treated groups, the activation of caspase-3 was singificantly lower than that in the groups treated with palmitate alone (P<0.05).4. Ghrelin (100nM) caused a rapid phosphorylation of Akt, reached the peak at 30 min and last at least 60 min. Ghrelin caused activation of Akt in a dose dependent manner. Ghrelin-induced Akt phosphorylation was inhibited by LY294002, the specific inhibitors of PI3K (P<0.01). Moreover, LY294002 decreased the protective effect of ghrelin against palmitate-induced apoptosis as evaluated by flow cytometry with Annexin V-FITC/PI double staining (P<0.01).5. Incubated with palmitate (0.3 mM) for 24h increased ROS generation than the control group (P<0.01), while in the ghrelin and palmitate treated groups, the level of intracellular ROS was significantly decreased than the groups treated with palmitate alone (P<0.05).6. Incubated with palmitate (0.3 mM) for 24h decreased Bcl-2/Bax ratio (P<0.01), while cotreatment with ghrelin increased Bcl-2/Bax ratio (P<0.01).Conclusion1. Exposure to palmitate (0.3mM) caused a significant increase in apoptosis.2. Ghrelin eliminate palmitate-induced apoptosis in rat aortic endothelial cells. 3. Ghrelin protected endothelial cells from palmitate-induced endothelial injury by inhibiting ROS generation.4. Ghrelin induced endothelial cells Akt phosphorylation. PI3K inhibitor attenuated the anti-apoptotic effect of ghrelin on palmitate-induced apoptosis. It is suggested that the protective effect of ghrelin was at least partly mediated by PI3K/Akt pathway.5. Palmitate (0.3 mM) decrease Bcl-2/Bax ratio, while treament with ghrelin increase Bcl-2/Bax ratio.