Experimental animals

Three-month-old Sprague–Dawley rats sourced from the Animal Center of Soochow University were maintained in a constant-temperature environment following a 10:14 light–dark cycle, and were supplied with regular standard rat chow and water. The rats were housed in cages with a 1:1 sex ratio on clean bedding. On the subsequent day, the presence of a light yellow cone-shaped vaginal plug signified successful mating, denoting Gestational Day 1 (GD = 1). Random allocations were conducted to the control (CON) and high glucose and fat diet (HGF) groups of pregnant rats, with 40 rats in each group. The CON rats were provided with a standard diet (containing 52% carbohydrate, 22.1% protein, 5.3% fat, 1.0% sodium, 1.0% calcium, 0.9% phosphorus, 0.5% potassium, and 0.2% magnesium) and water, while the HGF rats were given the same diet except that it contained 42% kcal fat and high-sugar water (a 20% glucose solution) throughout the pregnancy. The dietary intake, water intake, and weight gain of the two groups of pregnant female rats were recorded on a daily basis.

At GD 21, a portion of the pregnant rats (N = 20 per group) were anesthetized using 3% isoflurane in order to obtain the fetuses. A total of 8 to 14 pups per litter were analyzed, with the immediate measurement of the placental index, litter size, and fetal body length and weight. The fetal thoracic aortas (TAs) were promptly isolated for vascular testing or histological analysis. For the subsequent offspring tests, the remaining 20 pregnant rats in each group were then provided with a regular diet and water and allowed to give birth spontaneously. After one month, the male and female pups were separated and given standard chow and water. They underwent testing at the ages of eight and sixteen weeks. The study (No: 2022476) was authorized by the Soochow University Animal Care and Use Committee, and all animal operations were carried out in compliance with the NIH Publication No. 85-23, 1996 and 2011 versions of the Guide for the Care and Use of Laboratory Animals.

Measurement of vascular constriction

Under isoflurane anesthesia, both pregnant rats and their offspring were euthanized via cervical dislocation. The TAs of fetal and adult offspring were promptly excised and immersed in physiological saline solution (with composition in mmol/L: NaCl 120.9, KCl 4.7, NaHCO3 14.9, KH2PO4 1.2, MgSO4·7H2O 1.7, CaCl2·2H2O 2.8, EDTA 0.025, glucose 5.0, HEPES 10.0; pH 7.4 at 4 °C), continuously gassed with 5% CO2 in O2. TA segments (2–3 mm) were threaded onto stainless steel wire with a diameter of 40 μm and mounted in an M series Myograph System (Radnoti LLC, Covina, CA) within a chamber where the saline solution was bubbled with 95% O2 and 5% CO2 for isometric tension (PowerLab 16/SP and Chart 5). The TA ring was adjusted to maintain the optimal passive force and allowed to equilibrate for one hour, as previously described [18, 19]. Before the addition of the drug, it was exposed to KCl (120 mmol/L) to verify the viability and integrity of the ring. Then the TA rings were washed 3 times with physiological saline solution and allowed to stabilize for 1 h. After complete stabilized, the rings were contracted with escalating concentrations of Angiotensin II (Ang II) (10−11 to 10−6 mol/L), 5-hydroxytryptamine (10−11 to 10−4 mol/L), Phenylephrine (10−11 to 10−4 mol/L), PDBu (PKC activator, 10−11 to 10−5 mol/L), BayK8644 (L-type calcium channel agonist, 10−11 to 10−5 mol/L). Losartan (AT1R antagonist, 10−5 mol/L), PD123319 (AT2R antagonist, 10−5 mol/L), nifedipine (NIFE, L-type calcium channels antagonist, 10−5 mol/L), GF109203X (GF; PKC antagonist, 10−6 mol/L), 2APB (IP3 receptor inhibitor, 10−5 mol/L), Y27632 (Rho/rho-kinase inhibitor, 10−5 mol/L), Apocynin (NOXs inhibitor, 10−5 mol/L), Vas2870 (NOX2 inhibitor, 10−5 mol/L), Tempol (an SOD [superoxide dismutase] mimetic, 10−5 mol/L), or AS1949490 (SHIP2 inhibitor, 10−5 mol/L) were used to pretreat the segments for 30 min prior to the application of Ang II [19, 20]. All drugs and chemicals were procured from Sigma-Aldrich.

Cell culture and treatments

Smooth muscle cells (SMCs) were isolated from the TAs as our previous studies [19, 20]. The TAs were promptly excised, freed from the surrounding tissues, and placed in physiological saline. The endothelial cells of the TAs were removed by gentle swabbing, then cut into pieces and digested with 0.2% collagenase type 2 for the release of SMCs. After digestion, the samples were vortexed vigorously to resuspend the cells, and then collected and cultured in DMEM containing fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 mg/mL) was added and incubated at 37 °C in 95% air and 5% CO2. SMCs were identified by co‐staining with α‐smooth muscle actin (α-SMA) and cultured in 10 cm2 dishes, with passages conducted every 2 to 3 days, for utilization between passages 3 and 5. In the study of the SHIP2 inhibitor (AS1949490), SMCs were inoculated and grown for 2 days in the presence or absence of AS1949490 [10–6 mol/L]. In the SHIP2 knockdown assays, siRNA transfection was performed using Lipofectamine 2000 (Invitrogen), and 48 h later, the cells were harvested for mRNA, protein, and DNA extraction. siRNA duplexes were synthesized by Genepharma (GenePharma, China) and are listed in Table 1.

Table 1 Primers for siRNA related to SHIP2 in this studyImmunofluorescence staining

Immunofluorescence staining was employed for histological examination of the TAs. TA specimens of fetal and adult offspring were obtained, freed from the surrounding tissues, and fixed in 4% paraformaldehyde. Slices (2–4 μm) were sectioned using a Leica CM1850 microtome, adhered to glass slides, and permeabilized for antibody infiltration. Non-specific adsorption sites were blocked with 5% bovine serum albumin, thereafter specimens were incubated with primary antibodies against NOX2, SHIP2, or α-SMA, followed by fluor-conjugated secondary antibodies and 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen). Imaging was conducted using a Nikon E600 microscope equipped with a DMX1200 camera. Wall thickness was measured from images of 10 TAs each on the outer and luminal sides per group, via Image-Pro 6.0 software.

Immunofluorescence staining was also utilized to assess the expression of NOX2 and SHIP2 in primary isolated SMCs. The cells were subjected to a 20-min fixation in 4% paraformaldehyde solution in phosphate buffer saline (PBS) at room temperature, followed by a 20-min permeabilization stage with 1% Triton X-100 in PBS at 4 °C. After being washed with PBST (PBS with 0.05% Triton X-100), the cells were blocked with 5% bovine serum albumin in PBST for 1 h at 37 °C prior to overnight incubation at 4 °C with primary antibodies (dilution of 1:500 for NOX2 or SHIP2). Subsequently, the cells were washed and incubated with fluor-conjugated secondary antibodies and DAPI for 1 h at 37 °C. Images were captured using a Nikon E600 microscope with a DMX1200 camera after the final PBST washing.

Quantitative real-time PCR and western blot

Total RNA was extracted from the TAs or cultured SMCs using Trizol reagent and quantified with a NanoDrop 2000C spectrophotometer. Total RNA was reverse transcribed into first-strand cDNA using the First-Strand cDNA Synthesis Kit (Takara, Dalian, China). Quantitative real-time PCR (qRT-PCR) was performed on an iQ5 Real-Time PCR Detection System (Bio-Rad) with SYBR Green PCR Master Mix (Applied Biosystems, USA), and the gene expression was calculated using the ΔΔCt method with GAPDH normalization. Regarding miRNA, miRNAs were isolated through the miRcute miRNA Isolation Kit (Tiangen, Beijing, China) and reverse transcribed with the miRcute Plus miRNA First-Strand cDNA Kit (Tiangen, Beijing, China), with U6 serving as the normalization control. The primer sequences are presented in Table 2. Experiments were replicated three times with independent samples, as previously described [19, 21].

Table 2 The primers or sequence used in this study

To extract total protein from the TA or cultured SMCs, cell lysis was conducted using a whole cell lysate buffer containing 50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, and 10 mmol/L EDTA, along with a protease inhibitor cocktail (#sc-29130, Santa Cruz Biotechnology). Western blot was used to assess NOX2 and SHIP2 protein levels in the TAs or cultured SMCs, normalized to β-actin. The antibodies used in the study were as follows: NOX2 (SAB4300724, dilution 1:1000), SHIP2 (2730S, dilution 1:1000), and β-actin (A5441, dilution 1:3000), all of which were obtained from Sigma-Aldrich or Cell Signaling Technology. The experiments were conducted 3 to 5 times using separate lysate preparations, in accordance with the methods described [21, 22].

Determination of reactive oxygen species (ROS) levels

The generation of ROS in the TAs or cultured SMCs was assessed using the fluorescent labeled dihydroethidium (DHE, product #S0063, Beyotime, China). DHE, serving as a fluorescent indicator for ROS, was added to the samples at a concentration of 10−5 mol/L and incubated in a lighttight, moistened container at 37 °C for a half-hour. Following incubation, the samples were rinsed with PBS and then fixed with 4% paraformaldehyde for 20 min. The fluorescence intensity was subsequently recorded using a Nikon E600 microscope equipped with a DMX1200 digital camera (excitation at 535 nm; emission at 610 nm). Quantification of the ROS levels was performed using Image-Pro Plus 6.0 software.

Luciferase reporter assay

The dual luciferase reporter assay was carried out to investigate the direct interaction between miR-325-3p and the 3'UTR of SHIP2. We constructed a luciferase reporter gene in the PmirGLO vector (Promega, USA), which contains the wild-type (SHIP2 wt) or mutant binding site (SHIP2 mut) of the SHIP2 3' UTR fragments. The recombinant plasmids PmirGLO-SHIP2 wt and pmirGLO-SHIP2 mut, along with miR-325-3p mimics, negative control (NC), and empty vector were respectively transfected into SMCs using Lipofectamine 2000 (Invitrogen, USA). After 48 h, the SMCs cell lysates were added to firefly luciferase or sea horse luciferase in accordance with the protocols supplied by the manufacturer, and the luciferase activity was quantified by the Dual Luciferase Reporter Assay System (#RG027, Beyotime, China).

Bioinformatics analysis

Candidate miRNAs targeting the gene were mainly predicted by the miRDB database (https://mirdb.org/index.html). miRDB is an online database for miRNA target prediction and functional annotation, and it is currently widely utilized. Taking SHIP2 (also known as INPPL1) as an example, by inputting the species of Rat and the gene name in the "Search by gene target" search option, candidate miRNAs that regulate the expression of SHIP2 can be searched for and sorted from high to low according to the "Target Score". In this study, we selected five candidate miRNAs with relatively high scores (miR-184, miR-186-5p, miR-205, miR-325-3p, and miR-331-3p) as the candidate miRNAs that regulate the expression of SHIP2 to conduct subsequent experiments.

Statistical analysis

The data are expressed as the average plus or minus the standard error of the mean (SEM). For concentration-dependent responses, we employed two-way analysis of ANOVA with subsequent Bonferroni post-hoc tests using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Additional data were assessed via Student’s t-tests or one-way ANOVA, depending on the nature of the data. Statistical specifics are detailed within the figure legends, and a significance threshold of P < 0.05 was applied.