HUVECs were purchased from the American Type Culture Collection. Formononetin (purity, 98%) was purchased from Gracia Chemicals Pvt. Ltd. Tyrphostin AG 490 (AG490; a JAK2 inhibitor) was purchased from MedChemExpress LLC. Recombinant human interleukin (IL)-6 was purchased from Sigma-Aldrich (Merck KGaA). Anti-phosphorylated (p)-JAK2 (cat. no. ab32101), anti-JAK2 (cat. no. ab39636), anti-p-STAT3 (cat. no. ab76315), anti-STAT3 (cat. no. ab68153), anti-cleaved caspased-3 (cat. no. ab49822), anti-GAPDH (cat. no. ab70699) and a secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (cat. no. ab7090) were purchased from Abcam. IL-1β (cat. no. JLC6382) and intercellular adhesion molecule 1 (ICAM-1) ELISA kit (cat. no. JLC5547-96T) were purchased from Shanghai Jingkang Biological Engineering Co., Ltd. PCR materials and kits, including Transzol Up kit, TransScript II First-Strand cDNA Synthesis SuperMix kit and TransStart Top Green qPCR Super Mix kit, were purchased from Beijing Transgen Biotech Co., Ltd. MTT, 3-amino, 4-aminomethyl-2′,7′-difluorescein diacetate (DAF-FM DA) and a cell lysis buffer kit were obtained from Beyotime Institute of Biotechnology.

HUVECs were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Ltd.) supplemented with 5.5 mM glucose and 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.), 100 U/ml penicillin and 100 U/ml streptomycin. Cells were cultured at 37°C in an atmosphere containing 5% CO₂ and 95% humidity. Medium was replaced with fresh DMEM every day. Cells were passaged at 80% confluence.

HUVECs were seeded in 6-well-plates (6×10⁴ cells/well) and treated with glucose (5 and 25 mM), IL-6 (10 ng/ml), formononetin (2, 20 and 200 µM) and AG490 (80 µM) in combination for 24 h at 36°C. Total cellular protein was extracted using a protein extraction kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Total protein was quantified using a bicinchoninic protein assay kit. Subsequently, 25 µg protein was loaded into each lane and proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore). PVDF membranes were blocked using TBS containing 5% skimmed milk and 0.05% Tween 20 at 37°C for 3 h, followed by incubation with the primary antibody (1:300 dilution) at 4°C for 10 h and with secondary antibodies (1:1,000 dilution) at 37°C for 2.5 h. Protein bands were detected using BeyoECL Moon (Beyotime Institute of Biotechnology) and a ChemiDoc XRS system (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ software (1.8.0; National Institutes of Health).

HUVECs were seeded in 6-well-plates (6×10⁴ cells/well) and treated with glucose (5 and 25 mM), IL-6 (10 ng/ml), formononetin (2, 20 and 200 µM) or AG490 (80 µM) in combination for 24 h at 36°C. Subsequently, total RNA was extracted with the Transzol Up kit (Beijing Transgen Biotech Co., Ltd.), according to the manufacturer's protocol. A total of 5 µg RNA from each experimental group was used to synthesize the cDNA, according to manufacturer's protocol. Gene expression was determined using the TransStart Top SYBR Green qPCR Super Mix kit and analyzed using CFX manager 2.1 (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: Initial denaturation at 93°C for 4 min, followed by 33 cycles at 93°C for 16 sec and 60°C for 35 sec. The primer sequences were as follows: JAK2, forward, (F) 5′-TCTGGGGAGTATGTTGCAGAA-3′, reverse (R) 5′-AGACATGGTTGGGTGGATACC-3′; STAT3, F 5′-CAGCAGCTTGACACACGGTA-3′, R 5′-AAACACCAAAGTGGCATGTGA-3′; IL-1β, F 5′-AGCTACGAATCTCCGACCAC-3′, R 5′-CGTTATCCCATGTGTCGAAGAA-3′; ICAM-1, F 5′-ATGCCCAGACATCTGTGTCC-3′, R 5′-GGGGTCTCTATGCCCAACAA-3′; and β-actin, F 5′-CCTTATTCCGATCTACACAGAGC-3′ and R 5′-TATTCGGCGTAGGTCTGAGGG-3′. β-actin was used as the internal control (20).

To detect the effect of formononetin on cellular viability, an MTT assay was performed. HUVECs were seeded in a 96-well plate (1×10⁴ cells/well) and were treated with glucose (5 and 25 mM), formononetin (2, 20 and 200 µM), AG490 (80 µM) and mannitol (25 mM) in combination for 24 h at 36°C. After 24 h treatment, 20 µl MTT solution (5 mg/ml) was added into each well for 4 h at 36°C. DMSO (150 µl/well) was used to dissolve the formazan crystals. Cell viability was measured using a scanning multiwell spectrophotometer at a wavelength of 570 nm.

HUVECs were seeded in 6-well-plates (6×10⁴ cells/well) and treated with glucose (5 and 25 mM), formononetin (2, 20 and 200 µM) and AG490 (80 µM) in combination for 24 h at 36°C. Subsequently, IL-1β and ICAM-1 protein concentrations in the cell supernatants were analyzed using ELISA kits, according to the manufacturer's protocol.

HUVECs were seeded into 6-well-plates (6×10⁴ cells/well) and treated with glucose (5 and 25 mM), formononetin (2, 20 and 200 µM) and AG490 (80 µM) in combination for 24 h at 36°C. Subsequently, cells were washed twice with PBS and incubated with 5 µM DAF-FM DA (NO synthase assay kit; Beyotime Institute for Biotechnology) at 37°C for 25 min. After washing with buffer solution to remove the probe, NO was detected using an Olympus fluorescence microscope (IX51S8F-3; Olympus Corporation) at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. ImageJ software (1.8.0; National Institutes of Health) was used to analyze fluorescence intensity.

60 Male Sprague-Dawley rats (weight, 180–200 g) were purchased from The Animal Experiment Center of Wuhan University and were housed according to the institutional guidelines (temperature, 25°C; relative humidity, 50%-60; light/dark cycle, 12 h) with free access to food and water. All experimental procedures and protocols were approved by The Institutional Animal Care and Use Committee of the Institute for Endocrinology, Xiangyang First People's Hospital. After acclimation for 10 days, rats were divided into six groups (n=10/group): i) Control group; ii) high glucose group; iii) 4 mg/kg formononetin group; iv) 40 mg/kg formononetin group; v) 400 mg/kg formononetin group; and vi) 8 mg/kg AG490 group. Drugs were delivered through intragastric administration every day. Untreated rats in the control group were fed a standard diet (NIH-41). The other five groups were fed a high glucose diet [10% lard oil, 20% sucrose, 2.5% egg yolk powder, 0.5% cholesterol, 0.5% sodium cholate and 66.5% standard diet (w/w)]. The rats were sacrificed after 12 weeks of treatment. Thoracic aortas form the rats were dissected to examine thoracic aortic function. For the thoracic aortic function analysis, isolated thoracic aortas in each group were treated with chilled Krebs-Henseleit (K-H) solution (pH 7.4). The composition of the solution was as follows: 117.9 mM NaCl, 4.7 mM KCl, 2.6 mM MgCl₂, 3.3 mM CaCl₂, 25.0 mM NaHCO₃, 1.2 mM KH₂PO₄ and 11.0 mM glucose. Aortas were isolated, and after removing tissue debris, aortic rings (length, 4–7 mm) were separated from the aortas. The aortic rings were equilibrated for 1 h at 37°C with a resting tension of 1.5 g. Subsequently, the aortic rings were incubated with high K⁺ (60 mM) solution for 20 min at 37°C to induce maximal contraction. Then, vascular contraction induced by phenylephrine (10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ mol/l; Sigma-Aldrich; Merck KGaA) for 20 min at 37°C was recorded using a force-displacement transducer connected to a polygraph. Phenylephrine was added cumulatively until the maximum response was detected. A cumulative dose-response curve was calculated, and, after addition of phenylephrine, the maximum response was determined when the dose-response curve reached a plateau. Contraction responses were calculated using the following formula: Contraction response = [(contraction tension-resting tension)/resting tension] ×100%. Before determining the degree of relaxation, the aortic rings were washed three times with K-H solution every 20 min and recontraction was induced with 10⁻⁶ mol/l phenylephrine. The cumulative dose-response curves for acetylcholine (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ and 10⁻⁵ mol/l; Sigma-Aldrich; Merck KGaA) for 20 min at 37°C were calculated to evaluate endothelial relaxation. Relaxant response was calculated as the percentage decrease of the contraction amplitude induced by phenylephrine before the application of acetylcholine.

SPSS version 24.0 (IBM Corp.) was used for statistical analysis. The Shapiro–Wilk normality test was performed, and all variables were found to be normally distributed. Data are presented as the mean ± SEM from three experiments. One-way analysis of variance followed by Tukey's test was used to compare multiple groups. P<0.05 was considered to indicate a statistically significant difference.

High glucose levels can activate the JAK/STAT signaling pathway in HUVECs (13). Therefore, the effects of formononetin on the JAK/STAT signaling pathway were investigated in HUVECs. Treatment with 25 mM glucose for 24 h significantly increased the protein expression levels of p-JAK2 and p-STAT3 in HUVECs (P<0.01; Fig. 1A and C). Similarly to the JAK2 inhibitor AG490, treatment with 20 and 200 µM formononetin significantly decreased the protein expression levels of p-JAK2 and JAK2 mRNA expression (P<0.05; Fig. 1A and B). Furthermore, 20 and 200 µM formononetin significantly inhibited the protein expression levels of p-STAT3, a protein downstream to JAK2, and STAT3 mRNA expression (P<0.05; Fig. 1C and D). Additionally, the JAK/STAT signaling agonist IL-6 was used to examine the pharmacological effects of formononetin. Even when HUVECs were cotreated with high glucose and IL-6, formononetin was able to significantly inhibit p-JAK2 and p-STAT3 protein levels (P<0.01; Fig. 2A and C), in addition to the mRNA expression levels of JAK2 and STAT3 (P<0.01; Fig. 2B and D). Collectively, the present results suggested that formononetin, similarly to AG490, was able to inhibit the JAK/STAT signaling pathway in HUVECs treated with high glucose.

After investigating the role of formononetin in the JAK/STAT signaling pathway in HUVECs, the effects of formononetin on HUVEC viability were examined under high glucose conditions. Following high glucose stress, the viability of HUVECs was decreased to 67% (Fig. 3A). There was no significant difference in cellular viability between the 25 mM mannitol group and the 5 mM glucose-control group, suggesting that the decreased viability in high glucose was not related to osmotic pressure. However, formononetin increased cellular viability under high glucose stress in a dose-dependent manner (P<0.05). Furthermore, formononetin could significantly decrease the protein expression levels of active caspase-3 in HUVECs treated with high glucose or cotreated with high glucose and IL-6 (P<0.05; Fig. 3B and C). In order to investigate the inhibitory effects of formononetin on inflammation, the expression levels of IL-1β and ICAM-1 were investigated by ELISA and qPCR. The glucose-induced protein and mRNA expression levels of IL-1β and ICAM-1 were decreased after formononetin treatment, also in the presence of IL-6 (P<0.05; Figs. 4 and 5). Collectively, formononetin may protect against endothelial damage, at least in part, through the JAK/STAT signaling pathway.

Endothelial function was examined to determine whether formononetin could improve endothelial function. High glucose stress decreased NO production, which is associated with endothelial dysfunction (21). In vitro, NO levels were significantly restored following treatment with 20 and 200 µM formononetin, and 80 µM AG490 under 25 mM glucose conditions (P<0.01; Fig. 6). Additionally, in HUVECs cotreated with 25 mM glucose and 10 ng/ml IL-6, formononetin (200 µM) still exhibited a protective role in NO production. Additionally, the contraction response to phenylephrine was investigated in thoracic aortic rings isolated from rats fed a high-glucose diet (Fig. 7). Compared with the control group (rats fed a standard diet), aortic rings isolated from rats in the high glucose group exhibited a significantly reduced maximal contractile response induced by phenylephrine and a significantly decreased maximal relaxation response induced by acetylcholine. Following formononetin administration at 4, 40 and 400 mg/kg, contraction and relaxation of aortic rings were significantly improved (P<0.05). The JAK2 inhibitor AG490 at 80 mg/kg exhibited similar effects to formononetin at 400 mg/kg. Collectively, in vivo and in vitro results suggested that formononetin improved endothelial function under high glucose stress through the JAK/STAT signaling pathway.