Ang-(1-7) was purchased from Sigma; Merck KGaA (Darmstadt, Germany) and stored at −20°C. The following 2′,7′-dichlorofluorescein diacetate (DCFH-DH), Hoechst 33258, 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imida-carbocyanine iodide (JC-1) and AG490 (an inhibitor of the STAT3/JAK2 pathway) were obtained from Sigma-Aldrich; Merck KGaA). The Cell Counting Kit-8 (CCK-8) was supplied by Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-p-STAT3 antibody (cat. no. SAB4300033), anti-total (t-)STAT3 antibody (cat. no. SAB4300708), anti-p-JAK2 antibody (cat. no. SAB4300124), anti-t-JAK2 antibody (cat. no. SAB4501599), anti-caspase-3 antibody (cat. no. C5737) anti-NADPH oxidase 4 (Nox4) antibody (cat. no. SAB4503153) and anti-endothelial nitric oxide synthase (eNOS) antibody (cat. no. N2643) were supplied by Cell Signaling Technology, Inc. (Boston, MA, USA), horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. KC-5A08) and a BCA protein assay kit were obtained from KangChen Biotech, Inc. (Shanghai, China). Enhanced chemiluminescence (ECL) solution was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). The reagents for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was obtained from Invitrogen; Thermo Fisher Scientific, Inc.).

The HUVECs were supplied by Sun Yat-sen University Experimental Animal Center (Guangzhou, China). The HUVECs were cultured in DMEM supplemented with 10% FBS under an atmosphere of 5% CO₂ and 37°C with 95% air.

To establish a model of HG-induced HUVEC injury, the cells were cultured in DMEM (5.5 mM glucose) for 12 h prior to the administration of 40 mM glucose (final concentration) for 24 h. The glucose concentration of the control group was 5.5 mM. To investigate the protective effect of exogenous Ang-(1-7) against HG (40 mM glucose)-induced injury, the cells were seeded at a density of 1×10⁴/ml and treated with HG in the presence or absence of Ang-(1-7) for 24 h at 37°C. In order to examine whether the STAT3/JAK2 pathway contributed to the protective effects of Ang-(1-7), and to further determine the mechanisms underlying the protective effects of Ang-(1-7), the HUVECs at a density of 1×10⁴/ml were co-treated with HG, 2 µmol/l Ang-(1-7) and 20 µmol/l AG490 (an inhibitor of the STAT3/JAK2 pathway), and incubated at 37°C.

HUVECs were transfected at 70% confluency using Lipofectamine transfection reagent (Life Technologies; Thermo Fisher Scientific, Inc.), with siRNA against NLRP3 (Ribo Biotechnology, Shanghai, China) or a physiologically irrelevant negative control siRNA. The siRNA sequences used in the present study were as follows: Sense, 5′-GCU UCA GCC ACA UGA CUU UTT-3′; and antisense, 5′-AAA GUC AUG UGG CUG AAG CTT-3′. Each siRNA was dissolved in nuclease-free water to achieve a final concentration of 20 µM. A total of 5 µl siRNA (20 µM) and 5 µl Lipofectamine were added to a 500 µl buffer system. The mixtures were kept at room temperature for 30 min to form complexes, and equal amounts were added into wells of a 6-well plate. The cultures were incubated at 37°C in a 5% CO₂ incubator. The medium was replaced after 12 h with DMEM without the presence of siRNA or transfection reagent. Cells were collected at 12 h for analyses.

Following the indicated treatments, the HUVECs were harvested using a cell scraper and lysed with cell lysis solution (Beyotime Institute of Biotechnology, Haimen, China), at 4°C for 30 min. The total proteins were quantified using the BCA protein assay kit. Loading buffer was added to the cytosolic extracts and boiled for 5 min. Equal quantities of supernatant from each sample (20 µg were fractionated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, following which the total proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk for 60 min in fresh blocking buffer containing 0.1% Tween20 in Tris-buffered saline (TBS-T) at room temperature, and incubated with either anti-p-STAT3 (1:1,000 dilution), anti-t-STAT3 (1:1,000 dilution), anti-Nox4 (1:1,000 dilution), anti-p-JAK2 (1:1,000 dilution), anti-t-JAK2 (1:1,000 dilution), anti-caspase-3 (1:1,000 dilution) and anti-eNOS (1:1,000 dilution) in freshly prepared TBS-T with 3% fat-free milk, overnight with gentle agitation at 4°C. The membranes were then washed for 15 min with TBS-T and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Kangchen Biotech, Inc.), at a 1:3,000 dilution, in TBS-T with 3% fat-free milk for 90 min at room temperature. The membranes were then washed three times with TBS-T for 15 min and the immunoreactive signals were visualized using an ECL detection. In order to quantify protein expression, the X-ray films were scanned and analyzed with ImageJ 1.47i software (National Institutes of Health, Bethesda, MA, USA). The experiment was repeated three times.

The HUVECs were seeded in 96-well plates at a concentration of 1×10⁴/ml, and incubated at 37°C, following which a CCK-8 assay was used to assess the cell viability of HUVECs. Following the indicated treatments, 10 µl CCK-8 solution at a 1/10 dilution was added to each well and the plate was incubated for 1.5 h in the incubator. The absorbance at 450 nm was determined using a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). The mean optical density (OD) of three wells in the indicated groups was used to calculate the percentage of cell viability according to the following formula: Cell viability (%) = (OD treatment group/OD control group) x 100%. The experiment was performed five times.

Apoptotic cell death was analyzed using Hoechst 33258 staining followed by photofluorography. Firstly, the HUVECs were plated in 35 mm dishes at a density of 1×10⁶ cells/well. Following the indicated treatments, the cells were fixed with 4% paraformaldehyde in 0.1 mol/l phosphate-buffered saline (PBS; pH 7.4) for 10 min at 4°C. The slides were washed 5 times with PBS. Following staining by incubation with 5 mg/ml Hoechst 33258 for 30 min, the cells were washed 5 times with PBS. Finally, the cells were visualized under a fluorescence microscope (Bx50-FLA; Olympus Corporation, Tokyo, Japan). Viable HUVECs exhibited a uniform blue fluorescence throughout the nucleus and normal nuclear size, whereas apoptotic HUVECs demonstrated condensed, distorted or fractured nuclei. The experiment was repeated 5 times.

Intracellular ROS generation was determined by the oxidative conversion of cell-permeable oxidation of DCF-DH to fluorescent DCF. The HUVECs were cultured on a slide with DMEM. Following the above treatments, the slides were washed twice with PBS. Subsequently, 10 µmol/l DCFH-DA solution in serum-free medium was added to the slides, and the cells were incubated at 37°C for a further 30 min. The slides were washed 5 times with PBS, and DCF fluorescence was measured over the entire field of vision using a fluorescence microscope connected to an imaging system (BX50-FLA; Olympus Corporation). The mean fluorescence intensity (MFI) from five randomly selected fields was measured using ImageJ 1.47i software and the MFI was used as an index of the level of ROS. The experiment was repeated 5 times.

The MMP was determined using the fluorescent dye, JC-1, a cell-permeable cationic dye, which preferentially enters mitochondria based on the highly negative MMP. Depolarization of MMP results in a loss of MMP from the mitochondria and a decrease in green fluorescence. The HUVECs were cultured on a slide with DMEM at a density of 1×10⁶ cells/well. Following the indicated treatments, the slides were washed 3 times with PBS; and the cells were incubated with 1 mg/l JC-1 at 37°C for 30 min in the incubator, washed briefly 3 times with PBS, and air-dried. The fluorescence was measured over the entire field of vision using a fluorescent microscope connected to an imaging system (BX50-FLA). The MFI of JC-1 from three randomly selected fields was analyzed using ImageJ 1.47i software, and the MFI was measured as an index of the levels of MMP. The experiment was repeated 3 times.

SOD activity was analyzed using an SOD assay kit. Following the indicated treatments, the HUVECs were washed using PBS and lysed in ice-cold 0.1 M Tris/HCl (pH 7.4) containing 0.5% Triton, 5 mmol/lβ-mercaptoethanol and 0.1 mg/ml phenylmethylsulfonyl fluoride. The lysates were clarified by centrifugation at 14, 000 × g at 4°C for 5 min and cell debris was discarded. SOD activity was detected using a commercial SOD Assay kit according to the manufacturer's protocol (Sigma-Aldrich; Merck KGaA). The absorbance values at 450 nm were measured using a microplate reader. The experiment was repeated 3 times.

The HUVECs were seeded at 1×10⁴ cells/well and cultured in 96-well plates. Following the indicated treatments, the levels of IL-1β, IL-10, IL-12 and TNF-α in culture media were analyzed using ELISA according to the manufacturer's protocol. The experiments were repeated 5 times.

Total cellular RNA was extracted from cell cultures using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The extracted RNAs were DNase-treated with RQ1 RNase-Free DNase (Promega Corporation, Madison, WI, USA). First strand cDNA was prepared using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT; cat. no. 1701; Promega Corporation). The RT-qPCR analysis was performed using the Rotor-Gene™ SYBR Green PCR kit (Qiagen GmbH, Dusseldorf, Germany) and the QuantiNova SYBR Green PCR kit (Qiagen GmbH) on a Rotor-Gene 6000 Rotary Analyzer (Qiagen GmbH), and determined using Rotor-Gene 6000 software version 2.3.3 (Qiagen GmbH). For each assay, a total of 8 ng cDNA was added to a final reaction volume of 25 µl containing 1X Rotor-Gene SYBR Green PCR master mix or QuantiNova SYBR Green PCR master mix and 1 µM of each forward and reverse primer. Quantitative gene amplifications were performed using the following thermocycling conditions: Initial denaturation for 5 min at 95°C, 40 cycles of denaturation at 95°C for 5 sec, and annealing and extension at 60°C for 20 sec. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as endogenous control (or reference gene). A non-template reaction was used as a negative control. Three replicates were performed for all analysis.

The selection of the threshold intensity was set at a fixed intensity on the log-linear phase of the amplification curve for all the samples tested. Validation experiments, which included the generation of standard curves using a series of diluted cDNA samples, were performed to ensure primer efficiency, and target and reference gene amplification compatibility. Melt curve analysis and conventional agarose gel analysis were used alongside to verify the presence of a single amplicon. An interassay calibration scheme was used to minimize loading variation and to detect possible contamination with the inclusion of duplicate reactions and 'no-template' control, respectively, in each qPCR assay. Relative expression levels of the gene of interest were calculated using 2^−ΔΔCq method (27). All samples were normalized to GAPDH as the endogenous control (28).

The primers used were as follows: STAT3, forward 5′-CTT TGA GAC CGA GGT GTA TCA CC-3′; and reverse 5′-GGT CAG CAT GTT GTA CCA CAG G-3′; JAK2, forward 5′-CCG GAA TTC GCT TTG AGT CGG TTT CTC CGG TTC C-3′; and reverse 5′-TGC TCT AGA CCT CAT GCA GTC GCT GAA TAA GTC C-3′. GAPDH, forward, 5′CCA CCC ATG GCA AAT TCC ATG GCA3′; and reverse 5′TCT AGA CGG CAG GTC AGG TCC ACC3′.

All data are presented as the mean ± standard error of the mean. Differences between groups were analyzed using one-way analysis of variance with SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) software, and followed by an LSD post hoc comparison test. P<0.05 was considered to indicate a statistically significant difference.

The HUVECs were treated with different concentrations of glucose (10, 20, 30, 40, 50 and 60 mmol/l glucose) for 24 h, and it was found that glucose induced cell cytotoxicity (Fig. 1A). A glucose concentration of 40 mmol/l was considered a suitable concentration for use in the following experiments. To examine the cytoprotective effect of Ang-(1-7) against HG-induced cytotoxicity in HUVECs, a dose-response study with varying doses of Ang-(1-7) (0.5, 1, 2, 4, 6, 8 and 10 µM) was performed to calculate the cardioprotection dose of Ang-(1-7). As shown in Fig. 1B, exposure of the HUVECs to 40 mM glucose (HG) for 24 h induced cytotoxicity, as indicated by the decrease in cell viability. However, the cytotoxic effect of HG on HUVECs was markedly inhibited by pre-treatment of the cells with Ang-(1-7) for 30 min. The maximum inhibitory effect was observed with 2 µM Ang-(1-7). Alone, 10 µM Ang-(1-7) did not significantly alter the viability of the HUVECs. For this reason, the HUVECs were pre-treated with 2 µM Ang-(1-7) for 30 min prior to exposure to HG in all subsequent experiments.

As shown in Fig. 1C, exposure of the HUVECs to HG for 24 h induced cytotoxicity, which led to a decrease in cell viability. However, this decreased cell viability was markedly repressed by pre-treatment with 2 µM Ang-(1-7) or 20 µM AG490. Alone, neither 2 µM Ang-(1-7) or 20 µM AG490 affected the viability of HUVECs.

To observe the effects of JAK2 siRNA on the expression of the JAK2/STAT3 pathway, the HUVECs were treated with JAK2 siRNA. As shown in Fig. 2, JAK2 siRNA significantly inhibited the expression levels of p-JAK2 (Fig. 2A and B) and p-STAT3 (Fig. 2C and D). In addition, JAK2 siRNA attenuated the HG-induced decrease in cell viability in the HUVECs, as shown in Fig. 2E.

The present study also examined the effects of HG on the STAT3/JAK2 pathway, including the phosphorylation of STAT3 and JAK2. As shown in Fig. 3A–D, the effect of glucose on the HUVECs were examined. The cells were exposed to the indicated concentrations (20, 30, 40, 50 and 60 mM) of glucose for 24 h, and exposure to glucose significantly upregulated the expression levels of (Fig. 3A and C) pSTAT3, peaking at 60 mM glucose. However, the expression of t-STAT3 was not affected by the indicated concentrations of glucose. Based on these results, the effect of time on the expression of p-STAT3 was examined. As shown in Fig. 3B and D, following exposure of HUVECs to 40 mM glucose for the indicated times (3, 6, 12, 18, 24, 36 and 48 h), the expression levels of p-STAT3 were significantly upregulated, reaching a peak at 36 h, whereas the expression of t-STAT3 remained unchanged. Similarly, exposure of the cells to 40 mM glucose increased the expression levels of p-JAK2, as shown in the dose-response experiment (Fig. 3E) and time-response experiment (Fig. 3F), respectively. The mRNA expression levels of STAT3 (Fig. 3G) and JAK2 (Fig. 3H) were also markedly increased.

To observe effects of Ang-(1-7) on the activation of the JAK2/STAT3 pathway induced by HG, the HUVECs were pre-treated with 2 µM Ang-(1-7) for 30 min, prior to exposure to HG for 24 h. As shown in Fig. 4, exposure of the cells to 40 mM glucose significantly increased the expression levels of p-STAT3 (Fig. 4A and B) and p-JAK2 (Fig. 4A and C). However, the increased phosphorylation of the JAK2/STAT3 pathway was reduced by pre-treatment with 2 µM Ang-(1-7).

The present study also examined the effects of HG on the expression levels of caspase-3 and eNOS in HUVECs. As shown in Fig. 5A and E, The HUVECs were exposed to (A) the indicated concentrations (20, 30, 40, 50 and 60 mM) of glucose for 24 h, or (B) with 40 mM glucose for the indicated durations (3, 6, 12, 18, 24, 36 and 48 h). (C) Exposure to different glucose concentrations markedly increased the expression levels of caspase-3, peaking at 60 mM glucose. Based on these results, the effect of time on the expression was examined. As shown in Fig. 5D, when the HUVECs were exposed to 40 mM glucose for the indicated durations (3, 6, 12, 18, 24, 36 and 48 h), the expression levels of caspase-3 were significantly upregulated, reaching a peak at 12 and 18 h. By contrast, exposure of the cells to HG decreased the expression levels of eNOS, as shown in the dose-response experiment (Fig. 5E) and time-response experiment (Fig. 5F), respectively.

Ang-(1-7) and AG490 downregulate the increased expression of caspase-3 and upregulate the decreased expression of eNOS induced by HG in HUVECs. To observe the effects of Ang-(1-7) and AG490 on the increased expression level of caspase-3 and upregulating the decreased expression level of eNOS induced by HG, the HUVECs were pre-treated with 2 µM Ang-(1-7), as shown in Fig. 6A–C, or 20 µM AG490 for 30 min (Fig. 6D–F), prior to exposure to HG for 24 h. As shown in Fig. 6, exposure of the cells to 40 mM glucose significantly increased the expression levels of caspase-3 (Fig. 6A, C, D and F). However, the increased expression level of caspase-3 was reduced by pre-treatment with 2 µM Ang-(1-7) (Fig. 6A and B) or 20 µM AG490 (Fig. 6D and F) for 30 min prior to exposure to HG for 24 h. Secondly, the exposure of cells to 40 mM glucose significantly decreased the expression levels of eNOS (Fig. 6A, B, D and E). However, the decreased expression levels of eNOS were upregulated following pre-treatment with 2 µM Ang-(1-7) (Fig. 6A and B) or 20 µM AG490 (Fig. 6D and E) for 30 min prior to exposure to HG for 24 h.

As shown in Fig. 7Aa and b, exposure of HUVECs to 40 mM glucose for 24 h induced typical apoptosis, which was manifested as the nuclear condensation and fragmentation condensation of chromatin, and the shrinkage of nuclei and apoptotic bodies. However, pre-treatment of the cells with 2 µM Ang-(1-7) for 30 min prior to exposure to HG for 24 h mitigated the HG-induced increase in the number of cells undergoing apoptosis (Fig. 7Ac). In addition, pre-conditioning of the cells with 20 µM AG490 for 30 min prior to exposure to HG for 24 h also ameliorated the HG-induced apoptosis of cardiac cells (Fig. 7Ad). Alone, 2 µM Ang-(1-7) (Fig. 7Ae) or 20 µM AG490 (Fig. 7Af) did not significantly alter the number of apoptotic cells. Quantification of results is shown in Fig. 7B.

The results demonstrated that oxidative stress contributed to HG-induced HUVEC injury. As shown in Figs. 8 and 9, exposure of the HUVECs to 40 mM glucose for 24 h resulted in oxidative stress, as evidenced by an increase in the generation of ROS (Fig. 8Ab and B), a decrease in SOD activity (Fig. 9A) and increases in the expression level of Nox4 in the dose-response experiment (20, 30, 40, 50 and 60 mM glucose; Fig. 9B and C) and time-response experiment (3, 6, 12, 18, 24, 36 and 48 h; Fig. 9D and E). However, pre-treatment of the cells with 2 µM Ang-(1-7) for 30 min prior to exposure to HG for 24 h mitigated the HG-induced increase in ROS generation (Fig. 8Ac and B), increased the HG-induced decrease in SOD activity (Fig. 9A) and decreased the expression level of Nox4 (Fig. 9F and G). To determine whether the JAK2/STAT3 pathway was involved in HG-induced oxidative stress, the HUVECs were pre-treated cells with 20 µM AG490 for 30 min prior to exposure to HG for 24 h. The resulting data showed that pre-treatment of the cells with 20 µM AG490 for 30 min prior to exposure to HG for 24 h decreased the generation of ROS (Fig. 8Ad and B), upregulated SOD activity (Fig. 9A) and reduced the expression levels of Nox4 (Fig. 9F and G) in the HUVECs. Treatment with Ang-(1-7) or AG490 alone did not alter the basal level of ROS, activity of SOD or expression level of Nox4 in the HUVECs.

It was shown that the exposure of HUVECs to 40 mM glucose for 24 h elicited mitochondrial damage, as manifested by the dissipation of MMP (Fig. 10Aa and b). The dissipation of MMP was reduced by pre-treatment of the cells with 2 µM Ang-(1-7) for 30 min prior to exposure to HG for 24 h (Fig. 10Ac), which demonstrated that Ang-(1-7) protected the HUVECs against HG-induced mitochondrial damage. Similarly, pre-treatment of HUVECs with 20 µM AG490 for 30 min prior to exposure to HG for 24 h attenuated the HG-induced dissipation of MMP (Fig. 10Ad). The quantitative results are shown in Fig. 10B.

As shown in Fig. 11, the levels of IL-1β (Fig. 11A), IL-10 (Fig. 11B), IL-12 (Fig. 11C) and TNF-α (Fig. 11D) were markedly increased in the HG-induced HUVECs, compared with those in the control group (P<0.01). However, these increased levels of IL-1β, IL-10, IL-12 and TNF-α were significantly suppressed by pre-treatment of the HUVECs with 2 µM Ang-(1-7) for 30 min prior to exposure to HG for 24 h. This suggested an inhibitory effect of Ang-(1-7) on the production of pro-inflammatory cytokines, including IL-1β, IL-10, IL-12 and TNF-α, induced by HG. Similarly, pre-treatment of the HUVECs with 20 µM AG490 for 30 min prior to exposure to HG for 24 h decreased the enhanced production of IL-1β, IL-10, IL-12 and TNF-α.