The Protective Effects Of OPG On Apoptosis And Its Mechanisms In Vascular Endothelial Cells Induced By Palmitic Acid

IntroductionWith the developing of social economy and the changing of life styles, diabetes and obesity have been increased dramatically all over the world. Metabolic diseases, such as obesity and diabetes, have been closely associated with human health problems. Diabetic vascular complications are the main reasons to result in disability and death in diabetic patients. Vascular endothelial dysfunction is the early pathological change of diabetic vascular complications. Some studies showed that apoptosis of endothelial cells can be induced by high glucose in vitro. The results suggested apoptosis of vascular endothelial cells is closely related to vascular complication in diabetes mellituss. In addition to high glucose, many researches have showed that Free fatty acid (FFA) is markedly increased in obesity and diabetes. Palmitic Acid is one of the main constituent of FFA, which not only can cause insulin resistance, but also lead to vascular endothelium dysfunction, atherosclerosis and inflammatory response. Many signal pathways, such as Extracellular signal-regulated kinasel/2(ERK1/2)-tuberous sclerosis complex-mammals rapamycin target protein compound1(ERK1/2-TSC-mTOR) may be involved in the occurrence of apoptosis and play a very important role in the development of vascular complications in diabetes mellitus.Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor superfamily, which can compete with receptor activator of nuclear factor-κB (RANK) for the binding of RANK ligand (RANKL) to regulate osteoclast differentiation and bone resorption. OPG can compete either with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), preventing binding to membrance-associated death receptors (DR4and DR5) to trigger apoptosis of multiple cell type. Therefor, some studies have suggested that OPG also acts as an important regulatory molecule in vasculature. Clinical studies suggested that plasma concentration of OPG were higer in diabetic than in nondiabetic subjects, and it is strong associated with microangiopathy and macroangiopathy. However, to date, the exact effects of OPG and its mechanisms on vascular endothelial cells are still unclear.However, we hypothesized that plasma OPG has a protect effect on vascular endothelial cells induced by PA. For this assumption, following aspects will be discussed in this study,(1) we will build a damage model of human umbilical vein endothelial cells induced by palmitic acid in vitro, by which to explore the protective effect of OPG on VEC induced by palmitic acid.(2) Protein expression levels of ERK1/2-TSC-mTOR signaling pathway will be tested to explore the possible mechanisms by which OPG protect endothelial cells. Our objectives are to explore the mechanism of development of vascular complications anf to provide the evidence that OPG has protective effects in preventing and controlling diabetes complications.Materials and MethodsMeterialsHUVECs lines (ATCC CRL-2873), palmitic acid, bovine serum albumin with non free fatty acid, dimethyl sulfoxide (DMSO), antibodies (ERK, P-ERK, tuberin, P-tuberin, S6K, P-S6K, Bcl-2, Bax), high concentration of glucose DMEM, recombination-Osteoprotegerin, PD98059, fetal calf serumMethods1. The damage model of HUVECs induced by PA HUVECs were divided into two groups:①Normal control group (0.4%Bovine Serum Albumin),②palmitic acid group (0.2mmol/L,0.4mmol/L,0.8mmol/L PA). All the cells were cultured for24hours and48hours, morphological changes of HUVECs were observed by inverted phase contrast microscope, cell viability was measured by MTT, flow cytometry was used to assessed cell apoptosis and Hoechst33258staining is for observing changes of nucleus.2. The protective effects of OPG on HUVECs cultured with PA.HUVECs were divided into three groups:①Normal control group (0.4%Bovine Serum Albumin),②palmitic acid group (0.4mmol/L PA),③osteoprotegerin group (Cells were pretreated by different concentrations of OPG for24h, then be treated with0.4mmol/L PA). Afetr the cells were cultured for24hours, cell viability was measured by MTT, cell apoptosis was assessed by flow cytometry and apoptotic nuclei were detected using Hoechst33258.3. The mechanism of protective effects of OPG on HUVECs induced by PAHUVECs were divided into five groups:①Control group,②palmitic acid group (0.4mmol/L),③osteoprotegerin group(400μg/L),④osteoprotegerin+PD98059group (Pretreated with20μmol/L PD98059for12hours, then is400μg/L OPG, cells be treated with0.4mmol/L palmitic acid at last),⑤palimitic acid+PD98059group (Pretreated with20μmol/L PD98059for12hours, then cells be treated with0.4mmol/L palmitic acid), all the cells were cultured for24hours. The expression levels of extracellular signal-regulated kinase1/2(ERK1/2), tuberin, ribosomal protein S6kinase (S6K) protein and their phosphorylated forms, and also the levels of Bcl-2and Bax were measured by western blot analysis using specific antibodies.Statistical analysisData were presented as mean±standard deviation (SD) and analysed with SPSS13.0software. ANOVE was used for comparing among groups, and LSD was used for between groups. Factorial analysis was used in multifactor comparison. Differences were considered significant if p was<0.05. Results1. The damage model of HUVECs induced by palmitic acid.1) Morphological change of HUVECs (inverted phase contrast microscope).When the cells were cultured for4hours, HUVECs partly began to adhere to the flask and stretch out pseudopodia under the normal condition. About24hours after incubated, all the cells adhered to the culture bottle, presented a monolayer arrangement and a variety of shapes, such as rounded, short spindle or flat polygonal shape. During3～4days after incubation, HUVECs coalesced together and aligned like cobble-stone shape. During5～6days after inoculation, HUVECs presented a dense monolayer and then grew slowly. However, the cells began to turn bigger and turn to round cultivated with palmitic acid. The cells gradually appeared apoptosis and necrosis over time.2) Proliferation of HUVECs induced by palmitic acid (MTT assay)Compared with control group, the proliferation ratio in palmitic acid group was significantly decreased with the time extending, which was in a dose-time dependent manner.3) Apoptosis of HUVECs induced by palmitic acid (flow cytometry (Annexin V-FITC/PI double staining)).Compared with control group, the apoptosis ratio in palmitic acid group was significantly increased (P<0.05), and which is in a palmitic acid-dose dependent manner.4) Morphological changes of HUVECs induced by palmitic acid (Hoechst33258staining).Morphological changes of endothelium were analyzed by fluorescence microscopy. Compared with the diffuse fluorescence of nuclei in control group, pyknotic and hyperchromatic nuclei appeared in palmitic acid group.2. The protective effects of OPG on damage of HUVECs induced by palmitic acid.1) Proliferation of OPG on HUVECs induced by palmitic acid (MTT assay). Compared with control group, the viability of HUVECs was significantly decreased in palmitic acid group (P<0.05). Compared with palmitic acid group, proliferation ratios were obviously increase in an OPG-dose-dependent manner (P<0.05).2) Apoptosis of OPG on HUVECs induced by palmitic acid (flow cytometry, Annexin V-FITC/PI double staining).Apoptosis ratios in all the groups were assessed by flow cytometry. Compared with control group, the apoptosis ratios were significantly increased in palmitic acid group (P<0.05). Compared with palmitic acid group, the apoptosis ratios were dramatictly lower than that in palmitic acid group (P<0.05), which was in an OPG-dose-dependent manner.3) Morphological change of OPG on HUVECs induced by palmitic acid (Hoechst33258staining).After cells were dyed by DNA fluorescent dyes, the cellular morphological changes were observed through fluorescence microscope. We chose three visions of each group to calculate fifty nuclei of each field, and to calculate the average apoptosis ratio. Our results showed that DNA distribution within apoptosis nuclei were relatively uniform, and no pyknotic nuclei in control group. Cells in control group presented a larger and light-staining nuclear morphology. However, it presented a DNA densitied style within apoptosis nuclei. Apoptosis nuclei were found in different degrees of shrinkage, and they presented densely and condensedly in the field of view or showed in a fragment, dense and condensed form. Compared with apoptosis ratios in control group, the proportions in palmitic acid group is significantly increased (P<0.05). However, the apoptosis ratios in OPG group was dramatically lower than that in palmitic acid group (P<0.05), and which was in an OPG dose-dependent manner.3. The mechanisms of protection of OPG on HUVECs induced by PA.To explore the action of ERK-TSC-mTOR signal pathway on the protective effects of OPG on vascular endothelial cells induced by Palmitic Acid, western blot was used to determine the expression of ERK-TSC-mTOR.1) The expression of upstream of mTOR.Compared with control group, the level of P-ERK1/2and P-tuberin increased significantlyinpalmitic acid group(P<0.05), the level of P-ERK1/2and P-tuberin in osteoprotegerin group were evidently lower (P<0.05); pretreated with the ERK inhibitor PD98059, the expression levels of P-ERK1/2and P-tuberin both in Osteoprotegerin+PD98059group and Palmitic Acid+PD98059group significantly decreased compared with those similar group without PD98059(P<0.05)2) The expression of downstream of mTOR.Compared with control group, the level of P-S6K were dramaticlly increased (P<0.05), however, which were significantly decreased in OPG group (P<0.05). After blocked ERK1/2by PD98059, the expression of P-S6K in Osteoprotegerin+PD98059group and Palmitic Acid+PD98059group were lower than that those similar group without PD980593) The expression of apoptosis-related protein.To clarify whether TSC/mTOR signal pathway was associated with apoptotic signals, the expression levels of Bcl-2, Bax and Caspase3were determined by western blot. Compared with normal control group, the expression levels of Bax, and caspase3were increased markedly (P<0.05), and the expression of Bcl-2was decreased significantly in PA group (P<0.05). The levels of Bax, and caspase3were lower in OPG group than that in PA group, and the level of Bcl-2was higer in OPG group compared with PA group (P<0.05)Conclusion1. The apoptosis of HUVECs can be induced by palmitic acid, which has a palmitic acid dose-dependent manner.2. Palmitic acid is via activating ERK-TSC-mTOR signal pathway, downregulating Bcl-2and upregulating Bax to result in apoptosis.3. OPG protects HUVECs induced by palmitic acid far from apoptosis, which is in an OPG dose-dependent manner.4. The protective mechanisms of OPG on HUVECs induced by palmitic acid associated with inactivating ERK-TSC-mTOR signal pathway and increasing the expression level of Bcl-2.