Diabetic retinopathy (DR) is the most common complication of diabetes and a major cause of new-onset blindness in the developed world. The present study aimed to examine the effect of kaempferol on high glucose-induced human retinal endothelial cells (HRECs)
Diabetic retinopathy (DR) is the most common complication of diabetes and the main cause of new-onset blindness in the developed world (
During the new blood vessel formation process, capillaries are formed when endothelial cells are stimulated to migrate, proliferate and invade the surrounding tissues (
Estrogen-related receptor α (ERRα) belongs to a nuclear receptor superfamily characterized by their high levels of sequence identity to estrogen receptors, and the primary role of ERRα is in energy metabolism (
In the present study, it was demonstrated for the first time that high glucose treatment increased the expression level of ERRα mRNA and protein in human retinal endothelial cells (HRECs). Luciferase reporter and
HRECs were purchased from ATCC (Manassas, VA, USA) and were cultured in a human microvascular endothelial medium (Cell Applications, Inc., San Diego, CA, USA) and were maintained at 37°C in a humidified 5% CO2 incubator. Experiments were performed using cells between passages 3 and 8.
For the time-dependence experiment, HRECs were treated with 30 mM glucose for 12, 24 or 48 h, and HRECs incubated in 5 mM normal glucose were used as a negative control. In the kaempferol treatment experiments, HRECs were divided into 5 mM normal glucose, 30 mM glucose, 30 mM glucose plus 10 µM kaempferol, and 30 mM glucose plus 30 µM kaempferol groups. The HRECs were split at 90% confluence and subcultured in 96-well plates or 6-well plates according to the appropriate assay conditions.
Kaempferol and XTC-790 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); pCMX-ERRα and pcDNA-PGC-1α plasmids and the respective plasmids (pCMX and pCDNA) used as negative controls were purchased from Generay Biotechnology Co., Ltd. (Shanghai, China). All transfections were conducted using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions.
Total RNA was isolated from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A TaqMan Reverse Transcription kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to prepare cDNA from the ERRα, VEGF, TSP-1 and ADAMTS-1 RNA. qPCR was performed using a SYBR Green PCR kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. GAPDH was used as an internal control. The primers for ERRα mRNA were: forward, 5′-TTCGGCGACTGCAAGCTC-3′ and reverse, 5′-CACAGCCTCAGCATCTTCAATG-3′; the primers for VEGF mRNA were: forward, 5′-TGCCATCCAATCGAGACCCTG-3′ and reverse, 5′-GGTGATGTTGGACTCCTCAGTG-3′; the primers for TSP-1 mRNA were: forward, 5′-GGTCGGCCTGCACTGTCACC-3′ and reverse, 5′-GGGGAAGCTGCTGCACTGGG-3′; the primers for ADAMTS-1 mRNA were: forward, 5′-CTCCGCCTGCACGCCTTTGA-3′ and reverse, 5′-ATCGCCATTCACGGTGCCGG-3′. Data were expressed as fold changes relative to GAPDH calculated based on the 2−∆∆Cq method (
HRECs were seeded in a 96-well plate (1×104 cells/well), incubated for 24 h, and then co-transfected with pGL3-ERRE-Luci (reporter; Promega Corporation, Madison, WI, USA) and pMCX-ERRα with or without pcDNA-PGC-1α plasmids.
HRECs (3×103) were seeded into each well of a 96-well plate and allowed to adhere for 24 h. When the cells were adherent to the bottom of the plate, they were cultured in serum-free medium for starvation for 24 h. The cells were then treated with glucose alone (5 and 30 mM) or 30 mM glucose with kaempferol (10 or 30 µM) for 24 h, and the proliferative activity was determined by Cell Counting kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. In brief, 10 µl CCK-8 was added to each well, and following incubation for 2 h, the absorbance at a wavelength of 450 nm was detected.
When the HRECs had grown to 90% confluence in 6-well plates, they were starved with serum-free medium for 12 h. When the HRECs were over-confluent, a 200-µl pipette tip was used to create a wound. The floating cells were removed by washing three times with sterile 1X phosphate-buffered saline. After this, the cells were incubated with serum-free media containing glucose alone (5 and 30 mM) or 30 mM glucose with kaempferol (10 or 30 µM) for 24 h, and then cultured in a 6-well plate at 37°C in an incubator with 5% CO2. The migration monolayer was photographed at 0, 12, 24 and 48 h. Photographic images of five fields were photographed for each well, and the migration distance was measured.
After thawing in a refrigerator at 4°C overnight, 60 µl Matrigel was added to a pre-cooled 96-well plate and then solidified by immediately placing the plate in a humidified CO2 incubator at 37°C for 30 min. HRECs that had been cultured with serum-free media containing glucose alone (5 and 30 mM) or 30 mM glucose with kaempferol (10 or 30 µM) for 24 h, were seeded immediately on the solidified Matrigel at a density of 1.5×104 cells/well. The plates were placed in a humidified atmosphere of 5% CO2 and 95% air at 37°C for 8 h. Images of the plates were captured, and the number of capillaries formed was qualitatively assessed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
HRECs were lysed in SDS lysis buffer containing protease inhibitor (Sigma-Aldrich; Merck KGaA), and the protein concentration was measured using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instructions. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane. The membrane was incubated with the following primary antibodies: Mouse anti-ERRα (1:1,500; sc-65718; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit anti-VEGF (1:2,000; no. 2463; Cell Signalling Technology, Inc., Boston, MA, USA), and mouse anti-β-actin (1:6,000; ab8226; Abcam, Cambridge, MA, USA) at 4°C overnight. Membranes were then incubated with the horseradish peroxidase-conjugated secondary antibodies (1:4,000; ab6728; Abcam) at room temperature for 1 h. After further incubation with the enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA), the membranes were exposed using a ChemoDoc XRS detection system (Bio-Rad Laboratories, Inc.). The band intensities were analysed by the Image Lab v.6.0 software (Bio-Rad Laboratories, Inc.).
All statistical analysis was performed using GraphPad Prism 6 (GraphPad. Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± standard deviation; and differences among the treatment groups were compared by one-way analysis of variance followed by Dunnett's multiple comparison test, or unpaired t-test as appropriate. P<0.05 was considered to indicate a statistically significant difference.
RT-qPCR was conducted to evaluate the ERRα mRNA expression in HRECs following treatment with 30 mM glucose for 12, 24 and 48 h. The results showed that glucose (30 mM) significantly increased ERRα mRNA expression in a time-dependent manner (
Kaempferol has been shown to inhibit ERRα activity in cancer cells (
An
A wound scratching assay was conducted to examine whether kaempferol was able to modulate the glucose-induced migration of HRECs. The results showed that 30 mM glucose significantly accelerated the wound closure compared with that in the 5 mM glucose treatment group. Treatment with kaempferol (10 or 30 µM) significantly inhibited the migration of HRECs induced by 30 mM glucose compared with that in the 30 mM glucose group (
To examine the effect of glucose (30 mM) and kaempferol (10 or 30 µM) on angiogenesis, the tube formation of HRECs was evaluated by Matrigel assay. The results showed that 30 mM glucose significantly increased the number of capillary-like structures compared with that in the 5 mM glucose treatment group. HRECs treated with kaempferol (10 and 30 µM) had fewer capillary-like structures compared with the high glucose-treatment group (
To examine whether high glucose and kaempferol induce changes in VEGF, ADAMTS-1 and TSP-1 mRNA expression in HRECs, RT-qPCR was conducted. The expression of VEGF mRNA was significantly increased by treatment with 30 mM glucose compared with that with 5 mM glucose, and kaempferol (10 and 30 µM) significantly antagonized the glucose-induced increase in VEGF mRNA expression (
Western blotting results demonstrated that 30 mM glucose increased the expression of VEGF protein compared with that in the 5 mM glucose group, and kaempferol (10 and 30 µM) significantly antagonized the high glucose-induced increase in expression (
The present study demonstrated for the first time that high glucose treatment increases the expression of ERRα at the mRNA and protein levels in HRECs. Luciferase reporter and
The development of DR is largely attributed to high glucose levels, which generate cellular stress, cause injury to vascular pericytes and endothelial cells, and induce the development of abnormal capillaries (
ERRα has roles in energy metabolism, and various biosynthetic pathways, and is a key hypoxic growth regulator (
Angiogenesis is a complex and multistep process, and when the regulatory mechanism for angiogenesis is unbalanced, dysfunction may occur. VEGF is considered to mediate the abnormal angiogenesis that occurs in response to high glucose (
In conclusion, the findings of the present study suggest that kaempferol inhibits ERRα and reduces the high glucose-induced proliferation, migration and tube formation of HRECs via the regulation of pro- and anti-angiogenic factors. The results suggest that kaempferol is potentially useful as a drug to control the progression of DR. However,
The present study was supported by the Innovative Research Program of Shenzhen City (grant no. JYJ201304011).
High glucose (30 mM) increases the expression of ERRα. After HRECs had been treated with 30 mM glucose for 12, 24 and 48 h, (A) reverse transcription-quantitative polymerase chain reaction was performed to measure the expression level of ERRα mRNA and (B) western blotting was performed to determine the expression level of ERRα protein. All values represent the mean ± standard deviation (n=3). *P<0.05 and **P<0.01 vs. Con. HREC, human retinal endothelial cell; ERRα, estrogen-related receptor α; Con, control (5 mM glucose).
Kaempferol (10 and 30 µM) inhibits ERRα. HRECs were transfected with the plasmids pCMX-ERRα and/or pcDNA-PGC-1α together with reporter pGL3-ERRE-Luc and control
Kaempferol suppresses glucose (30 mM)-induced proliferation, migration and tube formation of HRECs. After HRECs had been treated with glucose (30 mM) alone or in combination with kaempferol (10 or 30 µM) for 24 h, (A) a CCK-8 assay was performed to assess cell proliferation, (B) wound scratch assay was performed to measure cell migration, and (C) Matrigel assay was performed to assess tube formation. All values represent the mean ± standard deviation (n=3). *P<0.05 vs. the 5 mM glucose group; #P<0.05 and ##P<0.01 vs. the 30 mM glucose group. HREC, human retinal endothelial cell; CCK-8, Cell Counting kit-8.
Effects of kaempferol on the expression of pro- and anti-angiogenic factors. After HRECs had been treated with glucose (30 mM) alone or in combination with kaempferol (10 or 30 µM) for 24 h, reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of (A) VEGF, (B) ADAMTS-1 and (C) TSP-1. All values represent the mean ± standard deviation (n=3). *P<0.05 vs. the 5 mM glucose group; #P<0.05 and ##P<0.01 vs. the 30 mM glucose group. HREC, human retinal endothelial cell; VEGF, vascular endothelial growth factor; ADAMTS-1, a disintegrin and metalloproteinase with thrombospondin motifs 1; TSP-1, thrombospondin 1.
Kaempferol (10 and 30 µM) inhibited glucose (30 mM)-induced VEGF expression. After HRECs had been treated with glucose (30 mM) alone or in combination with kaempferol (10 or 30 µM) for 24 h, western blotting was performed to determine the VEGF protein expression level. All values represent the mean ± standard deviation (n=3). *P<0.05 vs. the 5 mM glucose group; #P<0.05 vs. the 30 mM glucose group. HREC, human retinal endothelial cell; VEGF, vascular endothelial growth factor.