Isoquercitrin (purity >98%) was obtained from Shanghai Winherb Medical Technology Co., Ltd. Primary antibodies against Bax (cat. no. 2772), Bcl-2 (cat. no. 2870), C-caspase 3 (cat. no. 9661), T-caspase 3 (cat. no. 9662P), p53 (cat. no. 2524), phosphorylated (p)-p53 (Ser15; cat. no. 9284) and GAPDH (cat. no. 2118) were purchased from Cell Signaling Technology, Inc. Ubiquitin specific peptidase 10 (USP10) antibody was purchased from ProteinTech Group, Inc. (cat. no. 19374-1-AP). Pifithrin-β and Nutlin-3 were obtained from MedChemExpress. USP10 small interfering (si)RNA and negative control (NC) siRNA were purchased from Sangon Biotech Co., Ltd., and adenovirus (ad)USP10 and adGFP were purchased from OBiO Technology (Shanghai) Corp., Ltd.

The immortal HUVEC cell line was purchased from YRGene (cat. no. NC006). The cells were grown in DMEM (cat. no. C11995) containing 10% FBS and 1% penicillin-streptomycin solution (cat. no. 15140) (all Gibco; Thermo Fisher Scientific, Inc.) under standard conditions (5% CO₂ and 37°C). Before the experiment, cells were grown to 70-80% confluence and then synchronized in DMEM with 0.5% FBS for 12 h. Next, the cells were cultured with 10% FBS containing high D-glucose (30 mM; HG medium) or normal D-glucose (5 mM; NG medium) plus L-glucose (25 mM) to avoid the effects of osmotic imbalance in the cells as previously described (18-20). IQC (10, 50 or 100 µM) dissolved in PBS was added to NG or HG medium.

To inhibit p53 in cells exposed to glucotoxicity, the potent p53 inhibitor pifithrin-β (40 µM) was dissolved in DMSO (0.1% v/v) (21) and added to HUVECs stimulated with HG at 37°C for 24 h. For p53 activation, Nutlin-3 (5 µM) dissolved in DMSO (0.1% v/v), which can effectively inhibit the interaction of murine double minute 2 (MDM2)-p53 and therefore activate p53 (22), was added to HUVECs treated with HG in the presence of IQC (100 µM) at 37°C for 24 h.

To determine the cytotoxicity of IQC, the viability of HUVECs was tested using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.). HUVECs were seeded in 96-well plates and treated with different concentrations of IQC (0, 2.5, 10, 50, 100, 200, 300 or 500 µM) in NG medium (5 mM) for 24 h at 37°C. Subsequently, CCK-8 (10 µM) was added and incubated at 37°C for 4 h. The absorbance rate was measured at 450 nm using a microplate reader (Synergy HT; Agilent Technologies, Inc.).

Mitochondrial membrane potential was measured using the fluorescent JC-1 dye (Beyotime Institute of Biotechnology). Briefly, cells were grown in 6-well plates and treated with HG medium or NG medium with or without of IQC (10, 50 or 100 µM) at 37°C for 48 h. After the different treatments, the cells were incubated with 10 mM JC-1 for 20 min at 37°C in the dark, and then monitored using a fluorescence microplate microscope (magnification, ×200) (Eclipse 80i; Nikon Corporation) at 529 nm (monomeric form of JC-1, green) and at 590 nm (aggregate form of JC-1, red).

Cells were plated in 24-well plates and treated with HG medium or NG medium with or without IQC (10, 50 or 100 µM) at 37°C for 24 h. Subsequently, the cells were fixed in 4% paraformaldehyde for 10 min at room temperature and then apoptotic cells were detected using an ApopTag^® Plus Fluorescein In Situ Apoptosis Detection kit (EMD Millipore) according to the manufacturer's protocol. After the TUNEL assay treatment, images were captured under an Olympus DX51 fluorescence microscope (magnification, ×400) (Olympus Corporation).

Apoptosis was detected using an Annexin V-FITC/PI Apoptosis kit (cat. no. 556547; BD Biosciences). Cells were seeded in 6-well plates and treated with HG medium or NG medium with or without IQC (100 µM) at 37°C for 24 h. Subsequently, the cells were washed with cold PBS and resuspended in 1X binding buffer at a concentration of 1×10⁶ cells/ml. Annexin V-FITC and PI were added and incubated at room temperature for 15 min according to the manufacturer's instructions. Finally, the samples were analyzed by flow cytometry (CytoFLEX; Beckman Coulter, Inc.) within 1 h, and then the data were analyzed using CytExpert 2.0 (Beckman Coulter, Inc.).

Cells were fixed in 4% paraformaldehyde at room temperature for 10 min and permeabilized in 0.2% Triton X-100. Subsequently, 10% goat serum albumin (Invitrogen; Thermo Fisher Scientific, Inc.) was used to block the cells at room temperature for 1 h. The cells were incubated in a solution containing diluted anti-p53 (1:100) with or without anti-USP10 (1:100) overnight at 4°C and incubated with Alexa Fluor 488 (green) fluorescent secondary antibodies (1:200) at room temperature for 1 h (cat. no. A11008; Invitrogen; Thermo Fisher Scientific, Inc.). The nuclei were counterstained with DAPI at room temperature for 1 min. Images were captured under an Olympus DX51 fluorescence microscope (Olympus Corporation) (magnification, ×400) or an Olympus FV1200 confocal laser scanning microscope (magnification, ×1,000) (Olympus Corporation).

Total RNA was extracted from HUVECs using TRIzol^® reagent (cat. no. 15596-026; Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed to cDNA with a Transcriptor First Strand cDNA Synthesis kit (cat. no. 04896866001; Roche Diagnostics), according to the manufacturer's instructions. Subsequently, Light Cycler 480 SYBR Green Master Mix (cat. no. 04887352001; Roche Diagnostics) was used for qPCR analysis, according to the manufacturer's instructions. PCR thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 5 sec and 72°C for 15 sec. Data were analyzed using the 2^−ΔΔCq method (23). The following primer sequences were used: Bax forward, 5′-TCA TGG GCT GGA CAT TGG AC-3′ and reverse, 5′-GCG TCC CAA AGT AGG AGA GG-3′; Bcl-2 forward, 5′-AAC ATC GCC CTG TGG ATG AC-3′ and reverse, 5′-GAC TTC ACT TGT GGC CCA GAT-3′; p53 forward, 5′-AAG TCT AGA GCC ACC GTC CA-3′ and reverse, 5′-CAA TCC AGG GAA GCG TGT CA-3′; and GAPDH forward, 5′-CAT CAC CAT CTT CCA GGA GCG AGA-3′ and reverse, 5′-TGC AGG AGG CAT TGC TGA TGA TCT-3′. The mRNA expression levels of Bax, Bcl-2 and p53 were normalized to those of GAPDH.

Western blotting was used to detect the protein expression levels of Bax, Bcl-2, C-caspase 3, T-caspase 3, p-p53, p53 and GAPDH. Briefly, cells were lysed with RIPA lysis buffer, and then protein concentrations were evaluated using a BCA protein assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.). Total protein (50 µg/lane) was electrophoresed using 10-12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% milk and 0.1% Tween-20 in 1X TBS for 1 h at room temperature with gentle shaking, and then incubated overnight at 4°C with primary antibodies (1:1,000) against Bax, Bcl-2, C-caspase 3, T-caspase 3, p-p53, p53 and GAPDH. After incubation with horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. no. A21020; Abbkine Scientific Co., Ltd.) at 37°C for 1 h, the immunoblots were detected with ECL reagents and scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences). Densitometry was analyzed using Image Lab software (v3.0; Bio-Rad Laboratories, Inc.).

To knock down USP10 expression, HUVECs were seeded in 6-well plates, grown in antibiotic-free HG medium for 48 h and then transfected with USP10 siRNA (siUSP10; position 1583-1601; 5′-CCACCTGATGAAGTTCATT-3′) and non-targeting NC siRNA using Lipofectamine^® 6000 (Beyotime Institute of Biotechnology) at a final concentration of 100 nM at 37°C for 6 h according to the manufacturer's instructions. The medium was then replaced with fresh culture medium with or without IQC (100 µM) for another 48 h at 37°C before subsequent experiments.

For overexpression of USP10, the adenovirus-mediated overexpression system containing full-length USP10 cDNA (adUSP10) and the NC adenovirus vector (adGFP) were used. Cells were infected with adUSP10 or adGFP at a multiplicity of infection (MOI) of 50 at 37°C for 24 h according to the manufacturer's instructions, and then the medium was replaced with fresh culture medium with or without IQC (100 µM) at 37°C for another 48 h before subsequent experiments. The efficiency of USP10 siRNA and adUSP10 was assessed by western blotting, as aforementioned.

All experiments were repeated at least three times. Images were quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.). Data are presented as the mean ± SEM and were analyzed using SPSS 24.0 software (IBM Corp.). Normal distribution detection was determined using a one-sample Kolmogorov-Smirnov test. Differences between groups were analyzed by one-way ANOVA and Tukey's post-hoc test if the data were homoscedastic. Alternatively, Tamhane's T2 test was used. P<0.05 was considered to indicate a statistically significant difference.

To evaluate whether IQC affected the survival of HUVECs during the experiments, HUVECs were treated with 0, 2.5, 10, 50, 100, 200, 300 or 500 µM IQC in NG medium for 24 h. The CCK-8 assay results revealed that at concentrations of 2.5, 10, 50 or 100 µM IQC did not affect the viability of HUVECs, while the higher concentrations of IQC (200, 300 and 500 µM) slightly decreased cell viability (Fig. 1). No significant differences in cell viability were observed among these groups. Thus, 10, 50 and 100 µM IQC were chosen for subsequent experiments.

Since apoptosis serves an important role in HG-induced injury, the effect of IQC on the apoptosis of endothelial cells was examined. First, apoptosis was analyzed through TUNEL staining. TUNEL positive cells (green) were considered as apoptotic cells. As shown in Fig. 2B, the number of green spots in HUVECs was markedly increased under HG stress. When HUVECs were treated with 10, 50 or 100 µM IQC and HG, the green spots were markedly decreased (Fig. 2B). To further confirm the anti-apoptotic effect of IQC, JC-1 staining was used to examine the mitochondrial membrane potential. Green fluorescence indicates depolarized mitochondrial membrane potential, while red fluorescence indicates normal mitochondrial membrane potential. The ratio of red fluorescence to green fluorescence represents the degree of mitochondrial depolarization. To some extent, a decreased fluorescence ratio indicates increased apoptosis. The present results revealed that HG resulted a significant decrease in the fluorescence ratio in HUVECs compared with NG (Fig. 2A). By contrast, the ratio gradually increased after treatment with IQC (Fig. 2A). Furthermore, Annexin V-FITC/PI staining was used to investigate the effects of IQC on apoptosis stimulated by HG. The apoptotic rates in the NG, NG+IQC, HG and HG+IQC groups were 7.350±0.457, 7.163±0.709, 31.900±1.920 and 21.820±0.753%, respectively (Fig. 2E), consistent with the aforementioned TUNEL staining results. The results demonstrated that HG significantly increased the apoptotic rate of HUVECs compared with the NG group; however, apoptosis was significantly decreased in the HG+IQC treatment group compared with in the HG group (Fig. 2E).

Next, the protein expression levels of apoptotic markers, including Bax, Bcl-2 and C-caspase3, were analyzed. Compared with the cells treated with NG, western blotting analysis revealed that HG increased the protein levels of Bax and C-caspase3, and decreased the protein levels of Bcl-2, and these effects were attenuated by IQC treatment in a dose-dependent manner (Fig. 2C). Similarly, RT-qPCR results revealed that the change in the mRNA expression levels of Bax and Bcl-2 were consistent with those observed by western blotting analysis (Fig. 2D). Overall, the current results indicated that IQC may have a critical effect on HG-induced apoptosis of HUVECs.

To investigate the underlying molecular mechanism of the effect of IQC on endothelial apoptosis, the effects of the p53 signaling pathway under HG stress in the absence or presence of IQC were explored. Western blotting results revealed that HG significantly induced p53 activation in HUVECs and that this effect was inhibited by IQC (Fig. 3A). Immunofluorescence staining indicated that HG exposure significantly enhanced p53 protein expression in the nucleus and that this effect was inhibited by IQC administration (Fig. 3B). To determine whether the protective effect of IQC was dependent on p53, cells were treated with a p53 inhibitor (pifithrin-β) or a p53-MDM2 antagonist (Nutlin-3) for 24 h. Next, the protein expression levels of p53, Bax, Bcl-2, and C-caspase 3 were measured. Under HG conditions, the protein levels of p53 and Bax were decreased, while the expression levels of Bcl-2 were increased in pifithrin-β-treated cells compared with the IQC-treated group (Fig. 3D). Meanwhile, Nutlin-3 and IQC co-treatment significantly increased the protein expression levels of p53, Bax and C-caspase 3, and decreased Bcl-2 expression compared with in the IQC group under HG conditions (Fig. 3D). Similar trends were observed from the RT-qPCR results for Bax and Bcl-2 mRNA expression (Fig. 3C). Overall, the current results suggested that IQC partially attenuated HG-induced apoptosis of endothelial cells in a p53-dependent manner.

As shown in Fig. 4A, IQC did not affect the mRNA expression levels of p53 in response to HG, indicating that IQC may control p53 expression by modulating p53 protein stability. Since phosphorylation is a well-known post-translational modification that regulates p53 stability, the phosphorylation of p53 was examined. Western blotting results revealed that p53 and p-p53 (Ser15) protein expression was significantly enhanced in the HG group compared with in the NG group. IQC treatment partially suppressed their expression levels under HG conditions compared with those in the HG group (Fig. 4B). In addition to phosphorylation, ubiquitination is involved in p53 protein stability, and it has been proven that USP10, an enzyme that mediates the deubiquitination of p53, specifically participates in the proteasomal degradation of p53 (24). Subsequently, the interaction between p53 and USP10 was demonstrated using immunofluorescence co-localization. Immunofluorescence images revealed that HG caused USP10 to translocate into the nucleus, while IQC treatment promoted the nuclear export of USP10 (Fig. 5A). Moreover, p53 expression in the nucleus was increased under HG stress, which was consistent with the findings shown in Fig. 3B.

To further confirm that the change in p53 expression induced by IQC was dependent on USP10, USP10 siRNA and adUSP10 were used to knock down and overexpress USP10, respectively. As shown in Fig. 5B, USP10 silencing significantly decreased p53 and USP10 protein expression in the siUSP10 group compared with that in the control group, and IQC treatment caused a further decrease in their expression levels in USP10-silenced HUVECs. By contrast, USP10 overexpression significantly promoted the HG-induced expression levels of USP10 and p53, and IQC treatment in adUSP10 HUVECs upregulated their expression levels compared with in the adGFP+IQC group (Fig. 5C).

Overall, the current results indicated that IQC exhibited a protective effect by regulating the phosphorylation of p53 and the nuclear export of USP10 in HG-induced apoptosis.