A total of 200 Wistar rats (weight, 180–270; age, 8 weeks; female, 100, male, 100) were purchased from Junke Biological Co., Ltd. The female rats were fed with normal feed (control group, n=10 rats) or a high fat and high sucrose diet (GDM group, n=90 rats) for 8 weeks at 23–25°C with a relative humidity of 65–70% in a 12 h light/dark cycle. Subsequently, female rats that were in proestrus were caged with males overnight. The rats were identified as pregnant (day 0) according to the observed sperm or mucus plug by microscopy the following day. A total of 100 female rats were identified to be pregnant. The rats in the GDM group were injected intraperitoneally with streptozotocin (35 mg/kg, Sigma-Aldrich; Merck KGaA) to induce gestational diabetes. If the FBG level was stable at 13.5 mmol/l following 24 and 72 h, the GDM rat model was considered to have been successfully established (22). GDM rats were randomly divided into the following groups (n=10 per group): i) GDM group, no treatment; ii) GDM+vector group, 4 days following induction, rats were injected with miR-875-5p inhibitor negative control (NC, 10 µM vector) every 3 days via the tail vein; and iii) GDM+miR-875-5p inhibitor group, 4 days following induction, rats were injected with miR-875-5p inhibitor (10 µM vector) every 3 days via the tail vein. To further investigate the molecular mechanism of miR-875-5p and TXNRD1, the remaining GDM rats were randomly divided into five groups (n=10 per group): i) GDM, no treatment; ii) GDM+sh-NC; iii) GDM+sh-TXNRD1; iv) GDM+miR-875-5p inhibitor+sh-NC (100 µg pGPU6/GFP/Neo-shRNA-NC); and v) GDM+miR-875-5p inhibitor+sh-TXNRD1 (100 µg pGPU6/GFP/Neo-shTXNRD1). The sequences used for the inhibition of miR-875-5p or TXNRD1 were as follows: miR-875 inhibitor, 5′-CACCUGAUAAAACUGAGGUAUA-3′; inhibitor NC, 5′-AUCGACAGGGUUAACUCCACGA-3′; sh-TXNRD1 forward, 5′-CACCGCAATGATCTTGAGTTCTATGCGAACATAGAACTCAAGATCATTGC-3′ and reverse, 5′-AAAAGCAATGATCTTGAGTTCTATGTTCGCATAGAACTCAAGATCATTGC-3′; shRNA-NC forward, 5′-CACCGACATACTCTGTTAGTGTAGTCGAAACTACACTAACAGAGTATGTC-3′ and reverse, 5′-AAAAGACATACTCTGTTAGTGTAGTTTCGCACTACACTAACAGAGTATGTC-3′. All sequences were obtained from Guangzhou RiboBio Co., Ltd.

After 7 days, the rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg), followed by exsanguination 10 h after fasting. After anesthesia, blood (5 ml) was collected from the abdominal aorta for subsequent experiments. Blood, pancreatic islets and placental tissues of the rats were collected. After the blood samples were collected, they were stored overnight in the refrigerator at 4°C. Subsequently, the serum was collected by centrifugation at 4°C at 1,409 × g for 15 min and divided into 100 µl samples, which were stored at −80°C. Other tissues and organs were separated and stored at −80°C or fixed in 4% formaldehyde solution at room temperature for 48 h. The experimental process is presented in Fig. S1. All experimental procedures were approved by the Animal Experimental Ethics Committee of The First Hospital of Lanzhou University (approval no. LDYYLL2020-207).

Total RNA was extracted using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, and reverse transcribed into cDNA using miScript II RT Kit (Shanghai Beinuo Biotechnology Co., Ltd.) at 37°C for 20 min. miRNA was amplified using the miScript SYBR Green PCR Kit (Shanghai Beinuo Biotechnology Co., Ltd.). The other part of collected serum or pancreatic islet tissues was used to reverse transcribe into cDNA with the PrimeScript RT Reagent Kit (Takara Bio, Inc.) according to the manufacturer's protocol. mRNA was amplified using the SYBR Premix ExTaq (Takara Bio, Inc.). The thermocycling conditions were performed for qPCR as follows: Initial denaturation at 95°C for 3 min, followed by 45 cycles of 95°C for 5 sec, 60°C for 20 sec and 72°C for 15 sec. The Cq value of the target gene of each sample was normalized against U6 or GADPH, and the relative expression levels were measured using the 2^−ΔΔCq method (23). The primer sequences used were as follows: miR-875-5p forward, 5′-TATACCTCAGTTTTATCAGGTG-3′ and reverse, 5′-GCGGCCGCGTGCATAGCTTCTGTAAAGG-3′; TXNRD1 forward, 5′-CTTCCACGTACTGGGTCCAAATG-3′ and reverse, 5′-TCACCGACAGCGTTGTAAATATCTC-3′; CRP forward, 5′-CATCTGTGCCACCTGGGAGTC-3′ and reverse, AAGCCACCGCCATACGAGTC-3′; TNF-α forward, 5′-TGAGCACAGAAAGCATGATC-3′ and reverse, 5′-CATCTGCTGGTACCACCAGTT-3′; IL-6 forward, 5′-GGCCCTTGCTTTCTCTTCG-3′ and reverse, 5′-ATAATAAAGTTTTGATTATGT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and GADPH forward, 5′-TCACCATCTTCCAGGAGCGA-3′ and reverse, 5′- ACGCCAGTAGACTCCACG ACA-3′.

Following fasting for 10 h, the blood samples of the rats in each group were collected from the abdominal aorta (5 ml). The blood samples were placed on blood glucose test strips to record FBG levels (Nova Biomedical Corporation). The detection of FINS was performed by enzyme linked immunosorbent assay (cat. no. hzA448Ra; Shanghai Huzhen Biological Technology Co., Ltd.). The HOMA-IR was calculated using the following formula: [FBG (mmol/l) × FINS (mU/l)] / 22.5. TG and HDL levels were determined by an BK-400 automatic biochemical analyzer (BioBase).

Total protein was isolated from pancreatic islet tissue using RIPA lysis buffer (Sigma-Aldrich; Merck KGaA). The concentration of total protein of the pancreatic islet tissue was determined using the Bradford method. Proteins (50 µg) were separated via SDS-PAGE on a 10% gel, and then transferred to PVDF membranes, which were blocked using 5% skimmed milk powder for 1 h at 4°C. The membranes were incubated with the following primary antibodies for 2 h at room temperature: Anti-C reactive protein (CRP; cat. no. ab207756; 1:1,000), TNF-α (cat. no. ab205587; 1:1,000), IL-6 (cat. no. ab208113; 1:1,000), TXNRD1 (cat. no. ab124954; 1:1,000) and GAPDH (cat. no. ab9485; 1:1,000) (all purchased from Abcam). Subsequently, membranes were incubated with the HRP-conjugated secondary antibody (cat. no. ab7090; 1:10,000; Abcam) for 1 h at room temperature. Chemiluminescence enhancement reagent (Thermo Fisher Scientific, Inc.) was added to react with the protein band at room temperature for 1 min. The results were analyzed using the ImageJ software (version 1.46r; National Institutes of Health).

Following blood collection, the pancreatic tissues were removed, cut into pieces and further homogenized. Following centrifugation at 1,917 × g for 5 min at 4°C, the supernatant was obtained. The activity of SOD and catalase (CAT), and the content of malondialdehyde (MDA) in the pancreatic tissues were determined by UV-visible spectrophotometry using assay kits (SOD, cat. no. S0086; CAT, cat. no. S0082; MDA, cat. no. S0131S; all purchased from Beyotime Institute of Biotechnology), according to the manufacturer's instructions. The OD value of each group was detected at 560, 405 or 532 nm for SOD activity, CAT activity and MDA content, respectively.

Following anesthesia of the animals with 1% sodium pentobarbital (50 mg/kg, abdominal injection), pancreatic and placental tissues were obtained. The tissues were fixed in 4% formaldehyde solution at room temperature for 48 h, embedded in paraffin (thickness of 5 µm) and stained with H&E. Hematoxylin staining was performed for 9 min, followed by eosin staining for 2 min. The pathophysiological changes of the pancreatic and placental tissues were observed by light microscopy (magnification, ×200).

INS-1 cells (American Type Culture Collection) were cultured in RPMI-1640 medium (containing 11.1 mM glucose; Thermo Fisher Scientific, Inc.), in the presence of 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 0.11 g/l sodium pyruvate, 50 µM β-mercaptoethanol, and 1% penicillin-streptomycin. The cells were maintained at 37°C with 5% CO₂. Cells were seeded into 96-well plates at a density of 40–60% and cultured in RPMI-1640 medium without penicillin-streptomycin for 24 h, and subsequently transfected with specific plasmids. miR-875-5p mimic (5′-UAUACCUCAGUUUUAUCAGGUG-3′; 50 nM; Guangzhou RiboBio Co., Ltd.) or scrambled miR-875-5p (5′-AUGCAAGGUCGGGGUAACCUCC-3′; 50 nM; Guangzhou RiboBio Co., Ltd.) were transfected into INS-1 cells with Lipofectamine^® 2000 for 48 h at 37°C (Invitrogen; Thermo Fisher Scientific, Inc.). Then, cells were collected and the expression of miR-875-5p was determined using RT-qPCR. The relationship between miR-875-5p and the 3′ untranslated region (UTR) of TXNRD1 was predicted using Starbase (starbase.sysu.edu.cn/). The interaction was further confirmed by performing dual-luciferase reporter assays. Mutant (MUT) 3′UTR of TRXR1 dual-luciferase reporter vectors (Zeye Bio-Web) were constructed using a Site-directed Mutagenesis Kit (cat. no. B639281; Sangon Biotech Co., Ltd.). Wild-type (WT) or MUT vectors and miR-875-5p mimic or scrambled miR-875-5p were co-transfected into the cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, luciferase activities were detected using a Luciferase Reporter Assay Kit (BioVision, Inc.) and a GloMax^® Discover Microplate Reader (Promega Corporation). Firefly luciferase activities were normalized to Renilla luciferase activities.

Data were analyzed using GraphPad Prism software (version 8; GraphPad Software, Inc.). The data are presented as the mean ± SD. The comparisons among different groups were performed using ANOVA followed by Tukey's post hoc test. The unpaired Student's test was performed between two groups. P<0.05 was considered to indicate a statistically significant difference.

The miR-875-5p expression levels in GDM rats were initially analyzed by RT-qPCR. The results indicated that the serum miR-875-5p levels of GDM rats were significantly decreased compared with those of the control group (Fig. 1A). Subsequently, the functions of miR-875-5p were assessed through a series of experiments. miR-875-5p knockdown vectors were injected into GDM rats through the tail vein. Serum miR-875-5p levels were significantly reduced following transfection with the miR-875-5p inhibitor (Fig. 1B). Furthermore, the miR-875-5p inhibitor significantly decreased FBG and FINS levels in GDM rats compared with those in the GDM group (Fig. 1C). In addition, GDM-induced TC and TG levels were significantly decreased, whereas the level of HDL was increased in the miR-875-5p inhibitor group compared with the GDM group (Fig. 1D). The results implied that decreased miR-875-5p expression levels played protective roles in reducing FBG, IR and blood lipid levels in GDM rats.

CRP is a non-specific sensitive indicator of the inflammatory response that is closely associated with IR (24). CRP can inhibit the activity of the insulin receptor tyrosine kinase and induce the phosphorylation reaction of insulin receptor substrates (25). Therefore, the insulin synthesis and secretory mechanism are disrupted, which eventually lead to IR. TNF-α can enhance IR by directly inhibiting the expression of the glucose transporter T4 in adipocytes. IL-6 can reduce the sensitivity of the body to insulin and promote the secretion of growth hormones and glucocorticoids, which result in increased IR and BG levels (26,27). Therefore, the present study analyzed the expression levels of CRP, TNF-α and IL-6 in the pancreatic islet tissues in order to assess the effects of miR-875-5p on inflammation. The results indicated that miR-875-5p inhibition significantly reduced CRP, TNF-α and IL-6 expression levels in GDM rats (Fig. 2A and B). The induction of oxidative stress in the pancreatic tissues was evaluated by analysis of SOD, CAT and MDA levels (Fig. 2C). The results indicated that the enzyme activity of SOD was significantly increased following suppression of miR-875-5p levels in GDM rats compared with that of the GDM group (Fig. 2C). The levels of the anti-oxidative stress marker CAT were also significantly increased, whereas the concentration of MDA was decreased.

The pancreatic tissue presented normal morphology in the control group, whereas in the GDM group it demonstrated notable pathological changes, accompanied by varying degrees of inflammatory cell infiltration. The islets were markedly atrophic, with a reduced number of cells, sparse distribution and disordered arrangement. Following treatment of the animals with the miR-875-5p inhibitor, the pancreatic tissue lesions were notably decreased (Fig. 3A). In the control group, maturation of placental cell differentiation was noted, whereas the distribution was uniform and dense and the size of intercellular space was uniform and regular. The capillary blood vessels were evenly distributed and a large number of red blood cells were observed in the blood vessels. The cells in the placenta of the rats in the GDM group (Fig. 3B) were loosely distributed and disordered and the intercellular space was increased. Capillary distribution was reduced and the presence of red blood cells was rare. However, miR-875-5p inhibitor treatment markedly reduced placental injury in GDM rats.

Subsequently, the effects of miR-875-5p on TXNRD1 expression were evaluated. First, the expression of miR-875-5p was determined in INS-1 cells following transfection with a miR-875-5p mimic. A significant increase in the expression of miR-875-5p was observed in INS-1 cells with miR-875-5p mimic (Fig. 4A). Compared with the GDM+vector group, miR-875-5p inhibitor significantly upregulated the levels of TXNRD1 in the serum of GDM rats (Fig. 4B), indicating that miR-875-5p could regulate the expression of TXNRD1 in GDM rats. The StarBase database predicted that miR-875-5p could bind to the 3′UTR of TXNRD1, which was further confirmed using a dual-luciferase reporter gene assay. The luciferase activity in the TXNRD1 WT vector co-transfected with miR-875-5p mimic was significantly reduced compared with that of the vector group, whereas no differences were observed in the TXNRD1 MUT vectors co-transfected with miR-875-5p mimic or scrambled miR-875-5p (Fig. 4C).

Cells were transfected with sh-TXNRD1, which significantly suppressed the expression of TXNRD1 in the serum of GDM rats (Fig. 5A and B). Transfection with sh-TXNRD1 significantly reversed the effects of miR-875-5p inhibitor on glucose, FINS and HOMA-IR (Fig. 5C), inflammatory indicators (CRP, TNF-α and IL-6; Fig. 5D and E), and oxidative stress levels (SOD, CAT and MDA; Fig. 5F). In addition, TXNRD1 knockdown markedly altered the effects of miR-875-5p inhibitor on pancreatic and placental injury in GDM rats (Fig. 6A and B).