Experimental animals. Sixty cleaning grade healthy adult male Sprague-Dawley (SD) rats weighing 180±20g were obtained from the Experimental Animal Center of Soochow University. Animals were housed under standard diurnal conditions according to the guidelines established by the Animal Research: Reporting of In Vivo Experiments (ARRIVE). The room temperature was maintained at 20-23℃ with a relative humidity of 50-70% and the noise was kept＜50dB. Before experiments, all rats were fed a basal diet for one week. For the use of experimental animals, guidelines were followed from the "Regulations on the Management of Laboratory Animals" promulgated by the National Science and Technology Commission and the Declaration of Helsinki.
Experimental materials. The following materials were obtained for this study: Curcumin [Taishi (Shanghai) Chemical Industry Development Co.,Ltd., China], trypsin (Invitrogen, USA), type II collagenase, thiazole blue (MTT), 0.04% trypan blue (Sigma, USA), rabbit monoclonal antibody to von Willebrand factor (Abcam, Cambrige, UK), rabbit anti-CD31 antibody (Abcam, Cambrige, UK), rabbit anti-NF-κB p65 antibody (Abcam, Cambrige, UK), rabbit phosphor-NF-κB p65 antibody (Cell signaling Technology, USA), rabbit anti-IKBα antibody (Abcam, Cambrige, UK), rabbit anti-Bax antibody ((Wuhan Sanying Biotechnology Co., Ltd., Hubei, China), rabbit anti-Bcl2 antibody ((Wuhan Sanying Biotechnology Co., Ltd., Hubei, China), rabbit anti-IL-1β antibody (Cell signaling Technology, USA), endothelial cell culture medium, fluorescent (Cy3) tagged goat anti-rabbit IgG (Wuhan Biosciences Co., Ltd., Hubei, China), fluorescent (FITC) tagged goat anti-rabbit IgG (Wuhan Biosciences Co., Ltd., Hubei, China), rabbit anti-phosphoglyceraldehyde Dehydrogenase (GAPDH, Hangzhou Xianzhi Biotechnology Co., Ltd., Zhejiang, China), Light shift chemiluminescent EMSA kit (Thermo Scientific, USA), bicinchoninic acid (BCA) protein concentration determination kit, cell reactive oxygen species (ROS) detection kit (Biyuntian Biotechnology Institute, Hubei, China), diamino Diphenylamine (DAB) Chromogenic Kit (Beijing Solvay Technology Co., Ltd., China), apoptosis detection kit (Jiangsu Keyi Biotechnology Co., Ltd.,Jiangsu, China), Immunohistochemistry Kit (Beijing ZhongshanJinqiao Biotechnology Co., Ltd., China). Model IX51 inverted microscope, Eppendorf, BX53 fluorescence microscope (Olympus, Japan), Nikon E100 biomicroscope (Nikon, Japan), microplate reader (American Thermo Electron Corporation, USA), and flow cytometry (BD Biosciences, USA).
Rat retinal vascular endothelial cells (RRVECs) culture, identification and activity detection.
The RRVECs were isolated and cultured as previous described.The rats were anesthetized by intraperitoneally injection of 10% chloral hydrate with 3.3% ketamine.The eyes were placed in a petri dish containing iced PBS gauze, and the retinal tissues were harvested and cut into pieces. They were digested with a type II collagenase for 30 minutes at 37°C, and the digestive fluid was absorbed. The retinal tissues were then transferred to solution A consisting of NaCl, KCl, CaCl, MgCl2 and mannitol, and the osmotic pressure was adjusted to 310mosmol. The retinal microvessels were cut into quadrants and placed in a glass-bottomed chamber. A coverslip was then placed over the retina, so that the microvessels adhered to the coverslip. The cells were sieved through two stainless steel mesh of 100 μm pore size, and the eluate was gathered.The eluate was centrifuged at 1500 r/m for 5 minutes at room temperature and the supernatant was removed.The precipitate after centrifugation was added to Dulbecco's modified Eagle's medium (DMEM) containing 100x103 U/L heparin sodium and fetal bovine serum. It was inoculated on a 24-well culture plate, and then placed in a 37°C, 5% CO2 incubator and periodically exchanged. Retinal vascular endothelial cells were observed under an inverted microscope before the medium was changed. The labeled hybrid cells were removed with a scraper. Following this, the cell viability was assessed using trypan blue staining.
The 4th generation rat RRVECs were inoculated on gelatin-coated coverslips at a density of 1x104 cells/ml and cultured for 24 h. After the cells were adherent, the coverslips were removed and immunofluorescence staining was used to identify the cells with vWF and anti-CD31 antibody at 4℃ over night. In the negative control group, PBS was used instead of the primary antibodies. Cy3 and FITC as the secondary antibodies were added to the working solution at 37°C for incubation, respectively. The color was observed under a fluorescence microscope, and images were collected. Cy3 emits green fluorescence and FITC emits red fluorescence as a positive expression.
Grouping of Cells.
The RRVECs were divided into normal control group, osmolarity control group, high glucose group and curcumin treatment group (High glucose +Curcumin).The glucose concentration in the normal control group was 5.5mmol/L. The osmotic control group had 5.5mmol/L glucose and 19.5mmol/L mannitol to a final concentration of 25mmol/L. In the high glucose group, the glucose was added at a concentration of 25mmol/L for 72h. In the treatment group was exposed to glucose at a concentration of 25 mmol/L for first 72 h and then treated with 30 μmol/L curcumin for 48h.
Morphological changes in RRVECs by transmission electron microscopy.
The cellular morphological changes in each group were detected by the transmission electron microscopy (TEM). The cells were collected via centrifugation (800rpm, 5minutes), and washed with PBS and fixed with 1% paraformaldehyde and then with 2% glutaraldehyde for 12hours. Afterwards, the fixed cells were treated with 1% osmium tetroxide for 3hours, dehydrated through an ethanol gradient, and embedded in Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined by the TEM (Tecnai, FEI). The EDX ChemSTEM analysis system was used to take TEM images at magnifications of 2500x.
Detection of intracellular superoxide by ROS Assay.
Flow cytometry was used to detect reactive oxygen species (ROS) levels in RRVEC.A single suspension of cells was prepared from each group of cells, inoculated evenly in a 6-well plate at the cell density of 2×10 5 cells/well, and cultured overnight at 37° C in 5% CO 2 saturation humidity. The cells were then digested with 0.25% trypsin without ethylenediaminetetraacetic acid. The digestion was stopped and the digested cells were centrifuged.The supernatant was discarded and the cells were rinsed twice with PBS. Following the instructions for the operation of the DCFH-DA cell ROS kit, the flow cytometry was performed.
Flow cytometry analysis.
Flow cytometry was used to analyze the apoptosis of RRVECs in each group. Each group of cells was cultured at 37° C in a 5% CO 2 incubator for 72 hours.A single suspension of cells was prepared from each sample, and incubated in the dark for 15 minutes at room temperature. Solutions of Annexin (5 μl) and propidium iodide (1μl) were added to cell suspension and incubated in the dark at room temperature for 15 minutes. After the incubation, 400 μl of annexin binding buffer was added, and finally analyzed by flow cytometry.
The expression of NF-κB p65 in RRVECs was detected using immunohistochemical staining described previously[]. The cells were seeded on a 6-well plate cover glass. After 24 hours, the serum-free medium was replaced and the culture was continued for 24 hours. The slides were removed,washed with PBS and rabbit anti-human NF-κB p65 polyclonal antibody was added. The procedure was performed according to the fungal avidin-peroxidase ligation immunohistochemistry kit. The cytoplasm showing yellow or brown-yellow staining was considered positive. Ten high-power field of each slice was observed and photographed under a microscope.The images were analyzed using a digital image analysis system. The relative expression level of the target protein was calculated by the average value of the positive cells.
Detection of NF-κB Activity by EMSA.
The Electrophoretic Mobility Shift Assay (EMSA) was performed using an NF-κB EMSA Kit (Thermo Scientific, USA). The nucleoproteins in the RRVEC were extracted using a Nucleoprotein and Cytoplasmic Protein Extraction Kit according to the manufacturer’s protocol. The following DNA sequences were synthesized for EMSA analysis:
consensus oligonucleotides of NF-κB p65: 5’-AGTTGAGGGGACTTTCCCAGG C-3’ 3’-TCAACTCCCCTGAAAGGGTCCG-5’. The NF-κB/DNA binding activity was determined with Light Shift Chemiluminescent EMSA kit. The detailed procedure of EMSA has been described previously[[26 27]].
The expression of NF-κBp65, phosphorylated NFκBp65, IKB-α, IL-1β, Bax and Bcl-2 in each group of RRVEC were detected by western blot. The total protein was extracted from the cells and the target proteins were detected. Protein samples and standard proteins diluted in PBS were respectively added to 96-well plates. Two parallel wells were set for each standard sample. Three parallel wells were set for the sample to be tested, and 20 μl samples was added to each well. Two parallel wells with PBS were blank controls. The liquid mixture of BCA protein with a ratio of 50:1 was added to each well in a 96-well plate, and incubated at 37° C for 30 minutes in the dark.
The microwell plate was read at a wavelength of 568nm with a microplate reader. The linear regression equation was calculated based on the standard protein concentration and the corresponding the value. According to the value of the protein sample, the regression equation was used to calculate the sample protein concentration. The extracted protein sample and five-time protein loading buffer were placed in boiling water for 10 minutes to perform protein denaturation. Electrophoresis and transfer were performed according to the literature method[]. The membrane was blocked and the primary antibody was added to incubate overnight at 4°C (GAPDH: 1:1000; NF-κBp65:1:10000; pNF-κBp65:1:1000; IKB-α:1:1000; IL-1β:1:1000; Bcl-2: 1:1000; Bax: 1:2000). After washing the membrane 5-6 times, the corresponding secondary antibody was added. After the membrane was washed, the films with chemiluminescence were developed, fixed, and rinsed. BandScan was used to analyze the film gray value. It was repeated 3 times.
Statistical software SPSS20.0 was used for statistical analysis. The data were distributed by the Shapiro-Wilk test, and the cell apoptosis rate was expressed as a percentage. Other experimental data were expressed as the mean ± standard deviation (`x±s); the average between groups was homogeneity using the variance by Levene test, and analysis of variance multiple comparisons(ANOVA) with Bonferroni’s multiple comparison test was performed to analyze the data in more than two groups of variables. P<0.05 was considered statistically significant.