O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is an important form of post-translational protein modification, mainly intracellular. It is closely related to cellular signaling pathways, and is involved in signal transduction, gene transcription and other important biological processes. Studies have found that O-GlcNAc glycosylation is directly related with diabetic retinopathy (DR), further studies may help us to uncover the DR mechanism, and develop new strategies for the diagnosis and treatment of this disease.
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.
Objective To study the effects of advanced glycation end (AGEs) products induced by bovine serum albumin (BSA) on the survival and the morphology of bovine retinal endothelial cells (BREC) and pericytes (BRP). Methods BSA with the final concentration of 50 mg/ml was incubated in PBS, containing 500 mmol/L D-glucose, for 12 weeks under 37℃. AGEs-BSA was purified by Sephacryl S-300 column chromatography and was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of AGEs-BSA was determined by the method of commassie protein assay. In order to detect the toxic effects of AGEs-BSA on cultured BREC and BRP, groups of AGEs-BSA and BSA with different concentration and untreated control were set up. Phase contrast microscope was used to observe the effect of AGEs-BSA and BSA (with the concentration of 500mu;g/ml and actuation duration of 48 hours) on morphology of BREC and BRP. Results As the dosage of AGEs-BSA increased, the number of inhibited cells increased. When the concentration of AGEs-BSA was 500mu;g/ml, the inhibited BREC in AGEs-BSA group was (72.8plusmn;15.9)% of which in untreated control group, and the inhibited BRP was (64.8plusmn;9) % of which in untreated control group. AGEs-BSA with low concentration promoted the proliferation of endothelial cells, but there was no significant difference between AGEs-BSA and the control group (P=0.231). Inhibited proliferation and abnormal morphology were seen under the phase contrast microscope while the normal morphology of cells was found in BSA and control group. Conclusion AGEs-BSA with the high concentration may inhibit the growth of both BREC and BRP, which leads the loss of BRP and damage of vascular function. These results suggest that nonenzymatic glycosylation plays a major role in diabetic complications. (Chin J Ocul Fundus Dis, 2006, 22: 11-15)
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 observe the protective effect of tanshinone Ⅱ A on the mouse liver ischemia-reperfusion injury (IRI) model and preliminarily explore its mechanism of alleviating liver injury.MethodsThe IRI mouse model was established after the pre-treating with tanshinone Ⅱ A. Then, the serum and liver tissue of mice were collected to detect the changes of liver function, histopathology, liver cell apoptosis, and inflammatory factors. In addition, the protein expression levels of high mobility group box 1 (HMGB1), advanced glycosylation end-product specific receptor (RAGE), and Toll like receptor 4 (TLR4) in the liver tissues were detected by the Western blot method.ResultsAll data were analyzed by the homogeneity of variance test. The results of factorial design showed that the levels of ALT and AST in the serum, the pathological score and apoptosis index, the inflammatory response, as well as the expressions of HMGB1, TLR4 and RAGE proteins in the liver tissues were decreased significantly (P<0.05) in the sham operatation plus tanshinone Ⅱ A mice, which were increased significantly (P<0.05) in the IRI mice, which were antagonized synergistically by the tanshinone ⅡA and IRI (P<0.05).ConclusionsTanshinone ⅡA could reduce the liver IRI and inflammatory response in mouse. These effects might be related to the down-regulations of TLR4, HMGB1, and RAGE expressions.
Objective To investigate the effect of CD44 fucosylation on fluid adhesion force of rabbit bone marrow mesenchymal stem cells (BMSCs). Methods The rabbit BMSCs were isolated and purified by density gradient centrifugation combined with adherent culture method. The morphology of cells were observed by inverted microscope, and the cell surface markers of CD44, CD34, CD29, and CD105 were assessed by flow cytometry. BMSCs fucosylated by alpha-(1, 3)-fucosyltransferase Ⅵ (FTⅥ) were as the experimental group, and the non-fucosylated BMSCs were as the control group, and then the positive rate of sialyl-LewisX (sLeX) and the binding rate of E-selectin were detected by flow cytometry. The fucosylated BMSCs resuspended in Hank balanced salt solution (HBSS) were assigned as the experimental group (group A), at same time, the non-fucosylated BMSCs resuspended in HBSS solution as the study control group (group B), and the fucosylated BMSCs resuspended in HBSS solution which was added EDTA as negative control group (group C). The fluid adhesion force of rabbit BMSCs were detected by the parallel flow chamber adhesion test. Results Primary BMSCs mainly shaped as spindle and kept strong growth. The third generation BMSCs were negative for CD34, but positive for CD44, CD29, and CD105. After fucosylation, the positive rate of sLeX in the experimental group was 32.52%±1.76%, which was significantly higher than that in the control group (1.48%±0.51%) (t=29.277, P= 0.000). The binding rate of E-selectin in the experimental group was 41.05%±1.84%, which was also significantly higher than that in the control group (4.33%±0.92%) (t=35.674, P=0.000). With the increase of fluid shear force, the number of BMSCs adhering to the surface of human umbilical vascular endothelial cells (HUVEC) in group A was increased at first and then decreased, while there was few BMSCs adhering to the surface of HUVEC in groups B and C. Under the different fluid shear stress, the number of BMSCs adhered to the surface of HUVEC in group A was significantly higher than that in groups B and C (P<0.05), and there was no significant difference between groups B and C (P>0.05). Conclusion CD44 fucosylation on BMSCs can enhance the fluid adhesion force of rabbit BMSCs.
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.
Objective To explore the influence and mechanism of mechanistic target of rapamycin kinase (mTOR)/ receptor of advanced glycation end products (RAGE) pathway mediated-ferritinophagy on high glucose consumption promoting invasion and migration of colorectal cancer (CRC). Methods① Patients and tissue samples. Clinical data and tissues were collected from CRC patients underwent surgery and completed the dietary questionnaire in the Second Affiliated Hospital of Harbin Medical University from October 2022 to October 2023. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to analyzed the expression of nuclear receptor coactivator 4 (NCOA4) and ferritin in CRC and para-carcinoma tissues. ② Cell culture and treatment. The HT29 and HCT116 cells were treated by RPMI1640 medium containing 0, 35, 70, 105, 140 mmol/L glucose, and cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) activity analysis were performed to confirm 105 mmol/L glucose was the optimal concentration in the current study. Then the HT29 and HCT116 cells were randomly divided into: control group, glucose group; control group, glucose group, si-RAGE group, and glucose+si-RAGE group; control group, glucose group, rapamycin group, and glucose+rapamycin group. Untreated HT29 and HCT116 cells were considered as control group. The cells in glucose group were treated with 105 mmol/L glucose for 48 h. The CRC cells in the si-RAGE group were transfected with si-RAGE for 6 h. The CRC cells in the rapamycin group were treated with 10 nmol/L rapamycin for 48 h. The CRC cells in the glucose+si-RAGE group were treated with 105 mmol/L glucose for 48 h combination transfected with si-RAGE for 6 h. The CRC cells in the glucose+rapamycin group were treated with 105 mmol/L glucose for 48 h combination treated with 10 nmol/L rapamycin for 48 h. Then electron microscopy and Western blot, wound healing assay and transwell assay were exhibited, respectively. ③ Azoxymethane (AOM)-induced CRC rat model. The effects of glucose consumption on malignant behavior and ferritinophagy mediated by mTOR/RAGE pathway were evaluated in AOM-induced CRC rat models. A total of 16 rats were randomly divided into control group and glucose group, the CRC number was recorded and HE staining of CRC tissues was further performed. The expression of RAGE, mTOR, NCOA4, and ferritin in colorectal tissues of rats from each group was detected by RT-qPCR. Results① More lymphatic node metastasis and TNM Ⅲ/Ⅳ stages were observed in CRC patients from high glucose consumption group (P=0.004, P=0.004). Moreover, we confirmed that NCOA4 expression was significantly decreased (P<0.001) while ferritin was significantly increased (P<0.001) in CRC tissues especially in the CRC tissues from patients with positive lymph nodes metastasis. ② High glucose treatment significantly decreased autophagosomes in HT29 and HCT116 cells while si-RAGE transfection increased autophagic vacuoles compared to the control group. When compared with the glucose group, autophagosomes were increased in the glucose+si-RAGE group. Moreover, compared to the control group, the expressions of RAGE, p-mTOR, and ferritin were increased (P<0.001) while the expression of NCOA4 was decreased (P<0.001) in glucose group, but the expressions of RAGE, p-mTOR, and ferritin were decreased (P<0.001) while the expression of NCOA4 was increased (P<0.001) in the si-RAGE group; when compared with the glucose group, the expressions of RAGE, p-mTOR, and ferritin were downregulated (P<0.001) while the expression of NCOA4 was upregulated (P<0.001) in HT29 and HCT116 cells from the glucose+si-RAGE group. Compared to the control group, the HT29 and HCT116 cells in the glucose group performed enhanced wound scratch healing , migration and invasion viabilities (P<0.05); but the HT29 and HCT116 cells in the si-RAGE group performed impaired wound scratch healing, migration and invasion viabilities (P<0.05). When compared with the glucose group, the HT29 and HCT116 cells in the glucose+si-RAGE group performed impaired wound scratch healing, migration and invasion viabilities (P<0.05). ③ Rapamycin treatment significantly inhibited the expression of ferritin (P<0.05) but induced the expression of NCOA4 (P<0.05) compared to the control group. When compared with the glucose group, the expression of ferritin was downregulated (P<0.05) while the expression of NCOA4 was upregulated (P<0.05) in HT29 and HCT116 cells from the glucose+rapamycin group. Additionally, compared to the control group, rapamycin treatment performed inhibited effect on wound scratch healing, migration and invasion viabilities in the HT29 and HCT116 cells (P<0.05); while the HT29 and HCT116 cells in the glucose+rapamycin group performed impaired wound scratch healing, migration and invasion viabilities (P<0.05) when compared with the glucose group. ④ In the AOM induced CRC rat models, we found the more CRCs, aggravated cellular pleomorphism and upregulated expressions of RAGE, mTOR, ferritin (P<0.05) while downregulated expression of NCOA4 (P<0.05) in the glucose group than those of the control group. ConclusionHigh glucose consumption promote invasion and migration in CRC through suppressing ferritinophagy via activating the mTOR/RAGE pathway.
Objective To observe the correlation between the level of advanced glycosylation end products (AGE) in skin and diabetic retinopathy (DR), and establish and preliminatively verify the nomogramolumbaric model for predicting the risk of DR. MethodsA clinical case-control study. A total of 346 patients with type 2 diabetes mellitus (T2DM) who were admitted to the Department of Endocrinology and Ophthalmology of the First Affiliated Hospital of Zhengzhou University from January 2023 to June 2024 were included in the study. Among them, 198 were males and 148 were females. The mean age was (54.77±10.92). According to whether the patients were accompanied by DR, the patients were divided into the non-DR group (NDR group) and the DR group (DR group), 174 and 172 cases, respectively. All patients underwent skin AGE detection using a noninvasive diabetes detector. Diabetes duration, hemoglobin A1c (HbA1c), fasting plasma glucose, Urea, creatinine (Crea), uric acid, total cholesterol, triglyceride, estimated glomerular filtration rate (eGFR), urinary albumin concentration (UALB), and body mass index (BMI) were collected in detail. Univariate analysis and multivariate logistic regression analysis were used to determine the independent risk factors for T2DM concurrent DR, and to construct a nomogram prediction model for DR risk. Receiver operating characteristic curve (ROC curve), calibration curve and decision curve (DCA) were used to evaluate the model. ResultsHypertension prevalence rate (χ2=3.892), Diabetes duration (Z=−7.708), BMI (Z=−2.627), HbA1c (Z=−4.484), Urea (Z=−4.620), Crea (Z=−3.526), UALB (Z=−6.999), AGE (Z=−8.097) in DR group were significantly higher than those in NDR group, with statistical significance (P＜0.05); eGFR was lower than that in NDR group, the difference was statistically significant (Z=−6.061, P＜0.05). Logistic regression analysis showed that AGE, diabetes duration, HbA1c, UALB and eGFR were independent risk factors for DR (P＜0.05). Based on the results of multi-factor regression analysis, a nomogram prediction model was constructed. The area under ROC curve of the model was 0.843, 95% confidence interval was 0.802-0.884, sensitivity and specificity were 79.1% and 75.9%, respectively. The calibration curve was basically consistent with the ideal curve. The results of DCA analysis showed that when the model predicted the risk threshold of patients with DR between 0.17 and 0.99, the clinical net benefit provided by the nomogram model was＞0. ConclusionsSkin AGE level is an independent risk factor for DR. The nomogram prediction model based on AGE, diabetes duration, HbA1c, eGFR and UALB can accurately predict the risk of DR, and has good clinical practicability.