Objective To study the expression of receptor of advanced glycation end products (RAGE) in autogenous vein graft of streptozotocin induced diabetic rats and the inhibitory effects of aminoguanidine on intimal hyperplasia. Methods Sixty male Sprague-Dawley rats were randomly divided into three groups: aminoguanidine group, distilled water group and control group. Autogenous vein graft models were established in all groups. Streptozotocin was injected into abdominal cavity to induce diabetes in both aminoguanidine group and distilled water group, and they were intragastric administrated with aminoguanidine or distilled water, respectively before and after transplantation. Specimens were collected from autogenous vein graft 7 days and 14 days after surgery to undergo histological examination. At the same time, the level of serum advanced glycation end products (AGE) was tested. Western blotting and immunohistochemistry were used to detect the protein expression of RAGE and NF-κB p65. RAGE and NF-κB p65 mRNA were measured by reverse transcription-PCR. Results The mRNA and protein expressions of RAGE, NF-κB p65, the level of serum AGE and the intimal thickness of vein graft in distilled water group increased in comparison with those in control group 7 days and 14 days after surgery （P＜0.05). The level of serum AGE, mRNA and protein expressions of NF-κB p65 and the intimal thickness of vein graft in aminoguanidine group were lower than those in distilled water group （P＜0.05), and showed no significant difference compared with control group （P＞0.05). Conclusion The over-expression of RAGE in vein graft activats NF-κB in streptozotocin-induced diabetic rat, which has a close relation with intimal hyperplasia. Aminoguanidine can block the binding of AGE and RAGE by inhibiting the production of AGE, which will prevent intimal hyperplasia of vein graft.
Obiective lt;brgt;To investigate the change of the activity of proliferation in cultivated Muuml;ller cells treated by advanced glycation endoproducts (AGEs), and the effect of these changes on expression of occludin in bovine retinal vascular endothelial cells (BREC). lt;brgt;Methods lt;brgt;The cultivated Muuml;ller cells were devided into normal growth group and cultured with AGEs group. The cultured BREC were devided into 4 groups:group 1, without any medium; group 2, with normal growth Muuml;ller cell medium (MCM); group 3,MCM treated by AGEs; group 4, without cell as the control. Enzyme-linked immuno sorbent assay was used to detect the content of occludin in the medium in the 4 groups. lt;brgt;Results lt;brgt;The content of expression of occludin was the most in group 2, less in group 1, and the least in group 3. lt;brgt;Conclusion lt;brgt;AGEs may promote the abnormal proliferation of Muuml;ller cells and inhibit the expression of occludin in BREC. lt;brgt;(Chin J Ocul Fundus Dis, 2006, 22: 28-30)
ObjectiveTo observe the protective effect of polypyrimidine bundle-binding protein-related splicing factor (PSF) over-expression on RPE cell injury induced by advanced glycation end products (AGEs).MethodsThe human RPE cells cultured in vitro were divided into three groups: normal control group (N group), blank control group (N + AGEs group), empty vector control group (Vec + AGEs group), and PSF high expression group (PSF + AGEs). group). RPE cells in N group were routinely cultured; RPE cells in N + AGEs group were only transfected but did not introduce any exogenous genes combined with AGEs induction; Vec +AGEs group and PSF + AGEs group were transfected with pcDNA The empty vector or pcDNA-PSF eukaryotic expression plasmid was introduced into RPE cells and induced by AGEs. Except the N group, the other 3 groups of cells were transfected accordingly, and were stimulated with 150 μg/ml AGEs for 72 h after 24 h. HE staining and Hoechst 33258 staining were used to observe the effect of high PSF expression on the morphological changes of RPE cells; ROS level detection was used to analyze the effect of PSF high expression on the ROS expression of RPE cells induced by AGEs; MTT colorimetric method was used to detect the high PSF expression Effects on the viability of RPE cells; Western blot was used to detect the effects of different time and dose of PSF on the expression of heme oxygenase 1 (HO-1).ResultsHE staining and Hoechst 33258 staining observation showed that the cells in group N were full in shape, the nucleus was round, the cytoplasm was rich, and the staining was uniform; the cells in N + AGEs group and Vec + AGEs group were reduced in size, the eosinophilic staining was enhanced, and the nucleus was densely densely stained. Pyrolysis and even fragmentation; the morphology of cells in the PSF + AGEs group was still full, the cytoplasm staining was more uniform, and the nucleus staining was uniform. The results of MTT colorimetry showed that high expression of PSF can effectively improve the viability of RPE cells, but this effect can be effectively antagonized by ZnPP, and the difference is statistically significant (F=33.26, P＜0.05). DCFH-DA test results showed that compared with the N + AGEs group and Vec + AGEs group, the ROS production in PSF + AGEs group decreased, the difference was statistically significant (F=11.94, P＜0.05). Western blot analysis showed that PSF protein up-regulated HO-1 expression in a time- and dose-dependent manner. The relative expression level of HO-1 at 24, 48, and 72 h after PSF protein was significantly higher than that at 0 h, and the difference was statistically significant (F=164.91, P＜0.05). The relative expression level of HO-1 under the action of 0.1, 0.5, 1.0, 1.5, and 2.0 μg PSF protein was significantly higher than 0.0 μg, and the difference was statistically significant (F=104.82, P＜0.05).ConclusionPSF may inhibit the production of ROS by up-regulating the expression of HO-1, thus protecting the RPE cells induced by AGEs.
【摘要】 目的 观察晚期糖基化终产物（advanced glycosylation end prodrcts，AGE）对人结肠癌细胞株SW-480增殖的影响，并探讨其可能机制。 方法 不同浓度AGE干预SW-480细胞，噻唑蓝（MTT）法比较各组细胞活力，流式细胞术观察AGE对SW-480细胞周期的影响，蛋白质印迹法观察AGE对SW-480细胞CyclinD1表达的影响，端粒重复序列扩增法（telomeric repeat amplification protocol，TRAP）银染法观察AGE对SW-480细胞端粒酶活性的影响。MTT测细胞活力的检测设置空白对照组、100 μg/mL小牛血清白蛋白（bovine serum albumin，BSA）组及50、100、500 μg/mL AGE组，其余检测只设置100 μg/mL BSA组和100 μg/mL AGE组。 结果 MTT结果示AGE促进SW-480细胞的增殖，且呈浓度依赖性。100 μg/mL BSA组与100 μg/mL AGE组72 h后的细胞G0/G1期所占百分比分别为56.02%±0.58%、51.93%±1.01%，差异有统计学意义（Plt;0.05）。蛋白质印迹法示100 μg/mL AGE组72 h后CyclinD1的表达较100 μg/mL BSA组增加，差异有统计学意义（Plt;0.05）。TRAP银染法检测示100 μg/mL AGE干预SW-480细胞72 h后可以增加端粒酶活性（Plt;0.05）。 结论 AGE可促进人结肠癌细胞SW-480生长，呈剂量依赖性。其作用机制可能与AGE上调CyclinD1的表达加速G1/S期转换及增加端粒酶活性有关。【Abstract】 Objective To observe the effects of advanced glycosylation end products (AGE) on proliferation of SW-480 cells and study the possible mechanism. Methods Various concentrations of AGE were designed to have impact on SW-480 cells. Proliferation of SW-480 cells was assessed by thiazolyl blue tetrazolium bromide (MTT) assay; The impact of AGE on the cell cycle of SW-480 cells was analyzed by flow cytometry (FCM); the influence of AGE on expression of CyclinD1 was checked by Western blotting; and the impact of AGE on telomerase activity was examined by telomeric repeat amplification proctol (TRAP) sliver staining. For the MTT assay, blank control group, 100 μg/mL bovine serum albumin (BSA) group, 50, 100 and 500 μg/mL AGE groups were designed, while for other examinations, there were only 100 μg/mL BSA group and 100 μg/mL AGE group. Results MTT result showed that AGE increased the proliferation of SW-480 cells in a dose-dependent mode. The proportion of the cells at G0/G1 stage of the 100 μg/mL BSA group and the 100 μg/mL AGE experimental group were (56.02±0.58)% and (51.93±1.01)% respectively after 72 hours, with a significant difference (Plt;0.05); western blotting showed that the expression of CyclinD1 in the 100 μg/mL AGE group was significantly higher than that in the 100 μg/mL BSA group after 72 hours; TRAP silver staining demonstrated that telomerase activity increased significantly after treated with 100 μg/mL AGE for 72 hours. Conclusions AGE can promote the growth of SW-480 cells in a dose-dependent mode. Its mechanism is mainly by up-regulating the expression of CyclinD1 to shorten G0/G1 and increasing the telomerase activity significantly.
ObjectiveTo observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.MethodsExperimental study. Müller cells were cultured and divided into groups according to the project design, plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro, then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay. The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit. Meanwhile, 2′,7′-dichlorofluorescin diacetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.ResultsThe morphology of cells in normal group was full and the cytoplasm staining was uniform. In N+AGEs group and Vec+AGEs group, cell volume decreased, cytoplasm was dense and concentrated, and eosinophilic staining was enhanced. The cell morphology of PSF+AGEs group was still full, with uniform cytoplasm staining and uniform nucleus staining. The viability of N+AGEs group, Vec+AGEs group and PSF+AGEs group were 0.42±0.11, 0.35±0.12 and 0.68±0.12. The apoptosis values were 1.08±0.16, 0.96±0.20 and 0.44±0.08. The intracellular ROS levels were 28 833.67±3 550.06, 28 356.67±4 854.81, 186 163.00±382.54. Compared with N+AGEs group and Vec+AGEs group, the cell viability of PSF+AGEs group was significantly improved (F=20.65, P=0.000), cell apoptosis value (F=43.43, P=0.000) and intracellular ROS level (F=18.86, P=0.000).ConclusionPSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müller cells.
Objective To investigate the effects of advanced glycation endproducts (AGEs) on proliferation of pericytes of bovine retinal capillary vessels and expression of transforming growth factor beta;(TGF-beta;). Methods The proliferation of pericytes detected by methyl thiazolyl tetrazolium (MTT) colorimetric assay, cellular cycle of pericytes was analyzed by flow cytometry was used to analyze cell, and TGF-beta; protein expression of pericytes was observed by immunofluorescent staining. Results AGEs inhibited the proliferation of pericytes of bovine retinal capillary vessels, stopped the cellular cycle of pericytes in synthesis phase (S phase), increased the number of apoptotic cells obviously (Plt;0.01), and promoted the expression of TGF-beta; proteinof perycytes. Conclusions AGEs may promote the apoptosis of pericytes by inhibiting the proliferation of pericytes to lead the decrease of pericytes number, and may accelerate diabetic retinopathy by promoting the expression of TGF-beta; protein of pericytes. (Chin J Ocul Fundus Dis, 2006, 22: 20-23)
ObjectiveTo investigate the protection and the corresponding molecular mechanisms of polypyramidine tract binding protein-associated splicing factor (PSF) overexpression on human retinal microvascular endothelial cells (hRMECs) induced by advanced glycation end-products (AGEs).MethodsThe hRMECs were divided into the normal group, the vector group, PSF group, zinc protoporphyrin (ZnPP) group and PSF+ZnPP group for experiment. Cells in the normal group were cultured in a DMEM medium containing 10% fetal calf serum, penicillin/streptomycin, and placed in a closed constant temperature incubator at 37 °C, 95% air, and 5% CO2. Cells in the vector group were infected with empty lentivirus. The cells in the PSF group were infected with overexpressing PSF lentivirus. Cells in the ZnPP group were treated with ZnPP (10 mol/L) for 2 h. The PSF+ZnPP group cells were infected with overexpressing PSF lentivirus, and then pretreated with ZnPP (10 mol/L) for 2 h. With the last four groups of cells stimulated with AGEs, HE, Hoechst33258 staining and flow cytometry were used to observe the protective effect of high expression of PSF on cell damage and the antagonistic effect of ZnPP on PSF. Western blot was used to detect the protein expression of heme oxygenase-1 (HO-1), phosphorylated (p) extracellular regulatory protein kinase (ERK), and Nrf2 in the cells. U0126, a specific antagonist of ERK pathway, was introduced, and Western blot verified the reversal effect of U0126 on the expression of HO-1 induced by PSF protein.ResultsHE staining and Hoechst33258 staining showed that the number of nuclei of damaged cells of PSF group were significantly increased compared with control group, while decreased compared with PSF+ZnPP group (F=27.5, 38.7; P＜0.05). The results of flow cytometry showed that the ROS produced by cells in the PSF group was significantly increased compared to the normal group, and significantly decreased compared to the PSF+ZnPP group, the difference was statistically significant (F=126.4, P＜0.05). Western blot results showed that HO-1 expression of PSF group was significantly increased compared with control and the vector group (F=70.1, P＜0.05). AGEs inducement of 30, 60, 120 and 240 min could significantly improve pERK expression compared with 15 min (F=474.0, P＜0.05). The expression of HO-1 and Nrf2 proteins in the PSF+/U0126－ group was significantly more than those in the PSF－/U0126－ group, the expression of HO-1 and Nrf2 proteins in the PSF+/U0126+ group was significantly lower than that in the PSF+/U0126－ group, and the differences were statistically significant (F=30.2, 489.4; P＜0.05).ConclusionOver expression of PSF can promote the HO-1 expression by activating ERK pathway and promoting the Nrf2 to the nucleus, thus protect hRMECs against AGEs-induced oxidative damage.