THBS1 regulates the function and insulin sensitivity of HTR8/SVneo cells treated with high glucose through the RhoA/ROCK1 signaling pathway

Abstract

Aim

Gestational diabetes mellitus (GDM) increases the risk of maternal and fetal complications and impairs insulin sensitivity and placental function. This study aimed to explore the effect of thrombospondin-1 (THBS1) on HTR8/SVneo cell function and insulin sensitivity in GDM.

Methods

Placental tissues from pregnant women with GDM and normoglycemic pregnant women were collected, and HTR8/SVneo cells were exposed to normal and high glucose conditions. By overexpressing and knocking down THBS1, its effects on cell viability, migration, secretion of inflammatory factors, insulin signaling and glucose uptake were observed. qRT-PCR, Western blot, MTT, scratch test and ELISA were used for detection.

Results

Compared with the normal group, the expression of THBS1 in placenta tissue and HTR8/SVneo cells under high glucose conditions in the GDM group was significantly increased. THBS1 overexpression reduced cell viability and migration ability, increased the secretion of inflammatory factors, inhibited insulin signaling, and reduced glucose uptake; on the contrary, THBS1 knockdown significantly improved these indicators. In addition, the activation of the RhoA/ROCK pathway plays an important regulatory role in the effects of THBS1.

Conclusion

THBS1 impairs the function and insulin sensitivity of HTR8/SVneo cells by inhibiting insulin signaling and activating the RhoA/ROCK pathway. This indicates that THBS1 may play an important role in the pathogenesis of GDM and become a potential therapeutic target.

Introduction

Gestational diabetes mellitus (GDM) is one of the most common metabolic diseases during pregnancy worldwide, and its incidence continues to increase with changes in lifestyle and rising obesity rates [1, 2]. Currently, the global incidence of GDM is approximately 10–14%, and even reaches 20% in some areas [3]. In China, the incidence of GDM has also increased significantly in recent years, becoming a major public health issue affecting the health of pregnant women and newborns [4]. GDM not only increases the risk of pregnant women developing type 2 diabetes and cardiovascular disease in the future, but is also closely related to adverse pregnancy outcomes such as fetal macrosomia, premature birth, stillbirth, and neonatal respiratory distress syndrome [5, 6]. Its pathological mechanism is complex, mainly involving insulin resistance, inflammatory response and placental dysfunction [7, 8]. Therefore, in-depth study of the molecular mechanism of GDM and exploration of new therapeutic targets are of great significance to improving maternal and infant health.

At present, the treatment of GDM mainly includes diet control, exercise intervention and drug treatment, such as insulin or oral hypoglycemic drugs [9, 10]. These treatments can help control blood sugar levels in the short term and reduce acute pregnancy complications. However, existing treatments still have obvious shortcomings in long-term improvement of insulin sensitivity and prevention of long-term complications, especially in regulating placental function [11, 12]. For many patients, despite strict blood sugar control measures, insulin resistance persists, leading to placental dysfunction and affecting fetal growth and development [13, 14]. Therefore, there is an urgent need to explore new molecular mechanisms in order to develop more targeted treatments and improve the therapeutic effect of GDM.

Thrombospondin-1 (THBS1) is an extracellular matrix protein that has been recognized in recent years to play a key role in a variety of pathological processes [15]. Studies have shown that THBS1 plays an important regulatory role in insulin signaling, regulating cellular responses to insulin by affecting the activity of key insulin signaling proteins (such as IRS-1, AKT and GLUT4) [16, 17]. In addition, THBS1 is also involved in the migration and invasion of trophoblast cells, affecting the development and function of the placenta [18]. Studies have found that in a high-glucose environment, the expression of THBS1 is significantly increased, which may be a key mediator of insulin resistance and placental dysfunction. In GDM, overexpression of THBS1 may further aggravate insulin resistance and placental function damage by activating the RhoA/ROCK signaling pathway [19, 20]. Myosin phosphatase target subunit 1 (MYPT1), a downstream target of ROCK1, is phosphorylated upon ROCK1 activation and serves as a reliable marker for assessing the functional status of the RhoA/ROCK pathway [21]. Therefore, THBS1 may play an important regulatory role in the pathological process of GDM and is expected to become a potential therapeutic target.

This study aimed to explore its impact on cell function and insulin sensitivity in a high-glucose environment by regulating the expression of THBS1 in HTR8/SVneo cells. THBS1 may affect the occurrence of insulin resistance and placental dysfunction by regulating the insulin signaling pathway and trophoblast cell function. It aims to reveal the role of THBS1 in the pathological mechanism of GDM and provide theoretical basis and potential targets for the development of new GDM treatment strategies.

Methods

Modeling and grouping

According to the standards set by the International Association of Diabetes and Pregnancy Study Groups (IADPSG), placental tissues were collected from pregnant women who underwent cesarean section in our hospital and divided into gestational diabetes group (GDM group, n = 10) and normal blood sugar group (Normal group, n = 10). All subjects were full-term singleton pregnancies and met the following inclusion and exclusion criteria. Inclusion criteria: women aged 20–40 years with no history of pre-existing diabetes mellitus and who provided informed consent. Exclusion criteria: women with obesity (pre-pregnancy BMI ≥ 30 kg/m²), polycystic ovary syndrome (PCOS), or a history of GDM in previous pregnancies. Patients with hypertension, cardiovascular diseases, hepatic or renal dysfunction, other endocrine or infectious diseases were also excluded. The collected placental tissues were mechanically sheared and enzymatically processed to separate trophoblast cells. Trypsin (Guangzhou Yujia Biotechnology Co., Ltd, P4201-100 mg) and collagenase (Thermo Fisher Scientific, 17100017) were used in the enzymatic process to help separate placental trophoblast cells, which were then cultured in a medium containing fetal bovine serum (FBS) and specific growth factors to maintain cell activity and proliferation.

HTR-8a/SVneo cells, a human extravillous trophoblast cell line derived from the first-trimester placenta, were obtained from the American Type Culture Collection (ATCC, CRL-3271) and cultured in RPMI 1640 medium (Thermo Fisher Scientific, 11875093) supplemented with 10% fetal bovine serum (FBS, Gibco, A5670701) at 37 °C in a humidified incubator with 95% air and 5% CO₂. The cells were divided into the following groups according to the experimental requirements [22]: NG group (cultured in a normal glucose concentration of 5.5 mM for 48 h), HG group (cultured in a high glucose concentration of 25 mM for 48 h), HG + vector group (cultured in a high glucose concentration for 48 h after transfection with negative pcDNA3.1, served as a control for overexpression experiments), HG + oe-THBS1 group (cultured in a high glucose concentration for 48 h after transfection with pcDNA3.1-THBS1), HG + shNC group (cultured in a high glucose concentration for 48 h after transfection with negative shRNA, served as a negative control for knockdown experiments) and HG + sh-THBS1 group (cultured in a high glucose concentration for 48 h after transfection with THBS1 shRNA).

qRT-PCR

qRT-PCR was used to detect the expression level of THBS1 in placental tissues of the GDM group and the Normal group and in HTR8/SVneo cells of the NG group and the HG group. Total RNA was extracted using Trizol reagent (Guangzhou Yujia Biotechnology Co., Ltd, R0016), and RNA purity was tested by NanoDrop 2000 (Thermo Fisher Scientific, ND-2000) to ensure that the A260/A280 ratio was between 1.8 and 2.0. RNA was reverse transcribed into cDNA using a reverse transcription kit (Guangzhou Yujia Biotechnology Co., Ltd, D7153), and qRT-PCR amplification was performed on an ABI 7500 real-time quantitative PCR system with a reaction system of 20 µL, including SYBR Green qPCR Master Mix, specific primers, and cDNA template. The amplification program was pre-denaturation at 95 °C for 10 min, followed by 95 °C for 15 s and 60 °C for 30 s, for a total of 40 cycles. GAPDH was used as the internal reference gene, and the 2^−ΔΔCt method was used to calculate the relative expression of THBS1, and the expression of THBS1 in placental tissues and cells of each group was compared. All experiments were performed with three independent biological replicates, and each sample was analyzed in triplicate technical replicates to ensure accuracy and reproducibility.

Western blot

Western Blot was used to detect protein expression levels. Total cell protein was extracted with RIPA lysis buffer (Guangzhou Yujia Biotechnology Co., Ltd, P0013B), and protein concentration was determined by bicinchoninic acid assay (BCA). 30 µg of protein per well was separated by SDS-PAGE gel electrophoresis and transferred to PVDF membrane (Millipore, USA, IPVH00010). After blocking with 5% skim milk powder at room temperature for 1 h, THBS1 (Abcam, UK, ab267388), p-IRS-1 (Abcam, UK, ab313437), IRS-1 (Abcam, UK, ab131487), p-AKT (Abcam, UK, ab38449), AKT (Abcam, UK, ab8805), GLUT4 (Abcam, UK, ab188317), RhoA (Abcam, UK, ab187027), ROCK1 (Abcam, UK, ab134181), and p-MYPT1 (Abcam, UK, ab59203) antibodies were added and incubated overnight at 4 °C. The next day, the corresponding HRP-labeled secondary antibodies were added and incubated at room temperature for 1 h before protein bands were detected using ECL developer (ThermoFisher Scientific, 32106). β-actin was used as an internal reference for protein relative expression analysis. All experiments were conducted in three independent biological replicates, and protein detection was repeated at least three times for technical validation.

MTT assay

MTT assay was used to detect the viability of HTR8/SVneo cells under different treatment conditions. The cells were seeded in 96-well plates (1 × 10^4 cells/well) and cultured for 24 h before grouping, including NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, HG + sh-THBS1 group and HG + sh-THBS1 + U46619 group. At 12, 24, 48 and 72 h after treatment, 20 µL of MTT solution (Guangzhou Yujia Biotechnology Co., Ltd, C0009S) was added to each well. After incubation for 4 h, the culture medium was aspirated and 150 µL of DMSO (Guangzhou Yujia Biotechnology Co., Ltd, ST038-100 ml) was added to dissolve the crystals. The absorbance value (OD value) was measured at a wavelength of 570 nm using an ELISA reader. At least 3 parallel experiments were performed under each experimental condition. The effects of different treatment conditions on cell viability were evaluated by comparing the OD values of each group of cells.

Scratch assay

The scratch assay was used to detect the migration ability of HTR8/SVneo cells under different treatment conditions. After the cells were seeded in a 6-well plate and cultured until the monolayer of cells was covered, a straight line was drawn in the center of the cell layer using a sterile 200 µL pipette tip [23]. The suspended cells were rinsed with PBS and then treated according to the experimental groups. The cell migration in the scratch area was photographed under a microscope at 0 h and 24 h, and the scratch closure area was calculated by image analysis software to quantify the cell migration ability of different treatment groups. Each experimental group was analyzed in three independent biological replicates, and scratch closure areas were measured in triplicate fields per well.

ELISA

The ELISA experiment was used to detect the levels of inflammatory factors TNF-α, IL-1β, IL-6 and IL-8 in the culture supernatant of HTR8/SVneo cells under different treatment conditions. After the cells were cultured to the logarithmic growth phase, they were treated according to the experimental groups. The cell culture supernatant was collected after 48 h of treatment and detected using a specific ELISA kit (Guangzhou Yujia Biotechnology Co., Ltd, PA008) according to the manufacturer’s instructions. The absorbance was read at a wavelength of 450 nm by an ELISA reader, and the concentrations of TNF-α, IL-1β, IL-6 and IL-8 in the supernatant of each group were calculated according to the standard curve to analyze the secretion differences of inflammatory factors under different treatment conditions. All measurements were performed using three independent biological replicates, with each sample analyzed in duplicate technical replicates according to the manufacturer’s protocol.

Glucose content determination

The cells were cultured in 6-well plates and treated with insulin (1 μm). After incubation for 48 h, the cells were treated with fasudil (HY-10341, MCE, USA; 10–50 μm) and cultured for 24 h. Then the cells were treated with 100 nm insulin for 30 min. The supernatant was collected and the glucose level was evaluated by a glucose determination kit (F006-1-1, Nanjing Jiancheng Bioengineering Institute, China). Experiments were conducted in three biological replicates, and each measurement was performed in duplicate technical replicates.

Data analysis

All experimental data were analyzed using GraphPad Prism 8.0.2 software. Data of each group are expressed as mean ± standard deviation (SD). Inter-group comparisons were performed using one-way analysis of variance (ANOVA), and subsequent multiple comparisons were performed using Tukey’s test.

Results

Expression of THBS1 in placenta tissue and cells treated with high glucose in GDM group

The expression levels of THBS1 in placenta tissues of GDM group and Normal group and HTR8/SVneo cells of HG group and NG group were detected by qRT-PCR and Western blot. The qRT-PCR results showed (Fig. 1A and C) that the mRNA expression level of THBS1 in the placenta tissue of the GDM group and cells of the HG group was significantly higher than that of the respective control groups (P < 0.01). Western blot analysis also showed (Fig. 1B and D) that the expression level of THBS1 protein in the placenta tissue of the GDM group and the cells of the HG group was significantly increased (P < 0.01). THBS1 was significantly up-regulated in both GDM placental tissue and high-glucose-treated HTR8/SVneo cells, suggesting its potential role in the pathogenesis of GDM.

Fig. 1.

THBS1 is upregulated in placental tissues of gestational diabetes and HTR8/SVneo cells treated with high glucose. (A) qRT-PCR was used to detect the expression level of THBS1 in placental tissues of GDM group and Normal group. (B) Western blot was used to detect the protein expression level of THBS1 in placental tissues of GDM group and Normal group. (C) qRT-PCR was used to detect the expression level of THBS1 in cells of NG group and HG group. (D) Western blot was used to detect the protein expression level of THBS1 in cells of NG group and HG group. Data were presented as mean ± SD (n = 10). **P < 0.01 vs. Normal or NG group

Effect of THBS1 on protein expression, cell viability and migration ability in HTR8/SVneo cells

To further evaluate the impact of THBS1 on HTR8/SVneo cell function in a high-glucose environment, the protein expression level, cell viability and migration ability of THBS1 in different treatment groups were detected.

Western blot analysis (Fig. 2A) showed that the protein expression level of THBS1 in cells in the HG group was significantly higher than that in the NG group (P < 0.01). Overexpression of THBS1 further elevated THBS1 protein levels relative to the HG + vector group (P < 0.01), while knockdown of THBS1 (HG + sh-THBS1 group) significantly reduced THBS1 expression compared to the HG + shNC grou (P < 0.01).

Fig. 2.

Downregulation of THBS1 promotes the viability and migration ability of HTR8/SVneo cells treated with high glucose. (A) Western blot was used to detect the protein expression level of THBS1 in the cells of NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group. (B) MTT was used to detect the viability of cells in NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group (12, 24, 48, and 72 h). (C) Scratch assay was used to detect the migration ability of cells in NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group. Data were presented as mean ± SD (n = 3). **P < 0.01 vs. NG group; ##P < 0.01 vs. HG + vector group; &&P < 0.01 vs. HG + shNC group

The MTT assay (Fig. 2B) showed that cell viability was significantly reduced in the HG group compared to the NG group (P < 0.01). THBS1 overexpression markedly decreased cell viability relative to the HG + vector group (P < 0.01), whereas THBS1 knockdown significantly enhanced cell viability compared to the HG + shNC group (P < 0.01).

Scratch assay results (Fig. 2C) revealed that the migration ability of cells was significantly reduced in the HG group compared to the NG group (P < 0.01). THBS1 overexpression further suppressed cell migration relative to the HG + vector group (P < 0.01), while THBS1 knockdown significantly restored migration ability compared to the HG + shNC group (P < 0.01).

These findings demonstrated that THBS1 overexpression significantly reduced cell viability and migration ability, while THBS1 knockdown significantly improved these indicators, suggesting that THBS1 plays an important role in regulating HTR8/SVneo cell function.

Effect of THBS1 on the secretion of inflammatory factors in HTR8/SVneo cells

To assess the role of THBS1 in regulating inflammatory cytokine secretion under high-glucose conditions, ELISA was performed to measure TNF-α, IL-1β, IL-6, and IL-8 levels in the cell culture supernatants of different treatment groups (Fig. 3).

Fig. 3.

Downregulation of THBS1 reduces the levels of inflammatory cytokines in HTR8/SVneo cells treated with high glucose. (A) ELISA was used to detect the levels of TNF-α in the cell culture supernatants of the NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group. (B) ELISA was used to detect the levels of IL-1β in the cell culture supernatants of the NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group. (C) ELISA was used to detect the levels of IL-6 in the cell culture supernatants of the NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group. (D) ELISA detection of IL-8 levels in the cell culture supernatant of NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group. Data were presented as mean ± SD (n = 3). **P < 0.01 vs. NG group; ##P < 0.01 vs. HG + vector group; &&P < 0.01 vs. HG + shNC group

As shown in Fig. 3, the levels of TNF-α, IL-1β, IL-6, and IL-8 were significantly increased in the HG group compared to the NG group (P < 0.01). Overexpression of THBS1 further enhanced the secretion of these cytokines relative to the HG + vector group (P < 0.01). In contrast, knockdown of THBS1 (HG + sh-THBS1 group) markedly reduced cytokine secretion compared to the HG + shNC group (P < 0.05).

These findings demonstrated that THBS1 overexpression significantly increased the secretion of inflammatory factors in HTR8/SVneo cells, while THBS1 knockdown reduced the secretion of these factors, suggesting the potential regulatory role of THBS1 in GDM-related inflammatory responses.

Down-regulation of THBS1 improves insulin sensitivity of HTR8/SVneo cells treated with high glucose

To explore the effect of THBS1 on insulin sensitivity of HTR8/SVneo cells, glucose uptake measurement and insulin signaling pathway-related proteins were detected.

As shown in Fig. 4A, glucose uptake was significantly reduced in the HG group compared to the NG group (P < 0.01). Overexpression of THBS1 (HG + oe-THBS1 group) further decreased glucose uptake compared to the HG + vector group (P < 0.01). In contrast, knockdown of THBS1 (HG + sh-THBS1 group) markedly increased glucose uptake relative to the HG + shNC group (P < 0.01).

Fig. 4.

Downregulation of THBS1 improves the insulin sensitivity of HTR8/SVneo cells treated with high glucose. (A) After the cells in the NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group were treated with 100 nM insulin for 30 min, the glucose uptake levels were measured. (B) Western blot was used to detect the expression levels of p-IRS-1, IRS-1, p-AKT, AKT, and GLUT4 proteins in the cells in the NG group, HG group, HG + vector group, HG + oe-THBS1 group, HG + shNC group, and HG + sh-THBS1 group. Data were presented as mean ± SD (n = 3). **P < 0.01 vs. NG group; ##P < 0.01 vs. HG + vector group; &&P < 0.01 vs. HG + shNC group

Western blot analysis (Fig. 4B) revealed that compared with the NG group, the HG group exhibited a significant increase in p-IRS-1 levels and a significant decrease in IRS-1, p-AKT, and GLUT4 protein levels (P < 0.01), with no notable change in total AKT levels. THBS1 overexpression resulted in a further increase in p-IRS-1 and reduced expression of IRS-1, p-AKT, and GLUT4 compared to the HG + vector group (P < 0.01). Conversely, THBS1 knockdown led to a significant reduction in p-IRS-1 and restored IRS-1, p-AKT, and GLUT4 levels compared to the HG + shNC group (P < 0.01).

These findings indicated that THBS1 overexpression inhibited the insulin sensitivity of HTR8/SVneo cells, while THBS1 knockdown significantly improved insulin sensitivity, indicating that THBS1 may affect the cell response to insulin by regulating the insulin signaling pathway in a high-glucose environment.

Down-regulation of THBS1 inhibits the activation of RhoA/ROCK1 pathway in HTR8/SVneo cells treated with high glucose

Western blot analysis analyzed the protein expression levels of RhoA, ROCK1 and p-MYPT1 in HTR8/SVneo cells in different treatment groups. The results showed (Fig. 5) that the protein expression of RhoA, ROCK1 and p-MYPT1 in cells in the HG group was significantly higher than that in the NG group (P < 0.05). Compared with the HG + vector group, the expression levels of RhoA, ROCK1 and p-MYPT1 in the HG + oe-THBS1 group were significantly increased (P < 0.05); while the HG + sh-THBS1 group significantly reduced the expression levels of these proteins. expression ( P < 0.05).

Fig. 5.

Downregulation of THBS1 inhibits RhoA/ROCK1 pathway in HTR8/SVneo cells treated with high glucose

These findings indicated that upregulation of THBS1 promoted the activation of the RhoA/ROCK1 signaling pathway, while its downregulation inhibited the activation of this signaling pathway, indicating that THBS1 may affect the biological functions of cells through the RhoA/ROCK1 pathway.

Western blot was used to detect the protein expression levels of RhoA, ROCK1, and p-MYPT1 in NG, HG, HG + vector, HG + oe-THBS1, HG + shNC, and HG + sh-THBS1 groups. Data were presented as mean ± SD (n = 3). **P < 0.01 vs. NG group; ##P < 0.01 vs. HG + vector group; &&P < 0.01 vs. HG + shNC group.

THBS1 regulates the viability and migration ability of HTR8/SVneo cells treated with high glucose through the RhoA/ROCK1 signaling pathway

In order to clarify whether THBS1 regulates the viability and migration ability of HTR8/SVneo cells treated with high glucose through the RhoA/ROCK1 pathway, Western blot, MTT and scratch assays were used to analyze.

Western blot results (Fig. 6A) showed that the protein expression levels of RhoA, ROCK1 and p-MYPT1 in cells in the HG group were significantly higher than those in the NG group (P < 0.01); the protein expression levels in the HG + sh-THBS1 group were higher than those in the HG + shNC group. group was significantly lower (P < 0.01); while the protein expression level of the HG + sh-THBS1 + U46619 group was significantly higher than that of the HG + sh-THBS1 group (P < 0.05).

Fig. 6.

THBS1 can regulate the viability and migration ability of HTR8/SVneo cells treated with high glucose through the RhoA/ROCK1 pathway. (A) Western blot was used to detect the protein expression levels of RhoA, ROCK1, and p-MYPT1 in the NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group. (B) MTT was used to detect the viability of the cells in the NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group (12, 24, 48, and 72 h). (C) Scratch assay was used to detect the migration ability of the cells in the NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group. Data were presented as mean ± SD (n = 3). **P < 0.01 vs. NG group; ##P < 0.01 vs. HG + shNC group; &&P < 0.01 vs. HG + sh-THBS1 group

The results of the MTT experiment (Fig. 6B) showed that the cell viability of the HG group was significantly lower than that of the NG group (P < 0.01); the cell viability of the HG + sh-THBS1 group was significantly higher than that of the HG + shNC group (P < 0.01); and the HG + sh-THBS1 + U46619 group was significantly lower than that of the HG + sh-THBS1 group (P < 0.01).

Scratch assay results (Fig. 6C) showed that the migration ability of cells in the HG group was significantly reduced compared to the NG group (P < 0.01); THBS1 knockdown significantly enhanced cell migration relative to the HG + shNC group (P < 0.05); while U46619 treatment again reversed this effect, significantly reducing cell migration compared to the HG + sh-THBS1 group (P < 0.01).

These findings indicated that downregulation of THBS1 significantly enhanced cell viability and migration ability, while the RhoA/ROCK1 pathway activator U46619 partially reversed this effect, suggesting that THBS1 may affect the function of high glucose-treated cells through the RhoA/ROCK1 pathway.

THBS1 regulates the secretion of inflammatory cytokines in HTR8/SVneo cells treated with high glucose through the RhoA/ROCK1 pathway

ELISA analysis (Fig. 7) showed that the levels of TNF-α, IL-1β, IL-6 and IL-8 in the cell culture supernatant were significantly increased in the HG group compared with the NG group (P < 0.01). Notably, knockdown of THBS1 led to a marked reduction in the secretion of these inflammatory cytokines compared with the HG + shNC group (P < 0.01). However, treatment with the RhoA/ROCK1 activator U46619 reversed this effect, resulting in significantly higher levels of TNF-α, IL-1β, IL-6, and IL-8 compared to the HG + sh-THBS1 group (P < 0.01).

Fig. 7.

THBS1 can regulate the levels of inflammatory cytokines in high glucose-treated HTR8/SVneo cells through the RhoA/ROCK1 pathway. (A) ELISA was used to detect the levels of TNF-α in the cell culture supernatants of the NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group. (B) ELISA was used to detect the levels of IL-1β in the cell culture supernatants of the NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group. (C) ELISA was used to detect the levels of IL-6 in the cell culture supernatants of the NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group. (D) ELISA detection of IL-8 levels in the cell culture supernatant of NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group. Data were presented as mean ± SD (n = 3). **P < 0.01 vs. NG group; ##P < 0.01 vs. HG + shNC group; &&P < 0.01 vs. HG + sh-THBS1 group

These findings indicated that THBS1 knockdown suppressesd the secretion of inflammatory factors in cells treated with high glucose, while the RhoA/ROCK1 pathway activator U46619 reversed this effect. This suggests that THBS1 may regulate the inflammatory response of HTR8/SVneo cells under high glucose environment through the RhoA/ROCK1 signaling pathway.

THBS1 regulates glucose uptake and insulin signaling in HTR8/SVneo cells treated with high glucose through the RhoA/ROCK1 pathway

To further evaluate whether THBS1 affects glucose uptake and insulin signaling in HTR8/SVneo cells treated with high glucose through the RhoA/ROCK1 pathway, glucose uptake assay and Western blot analysis were performed to analyze the expression of insulin signaling pathway-related proteins.

As shown in Fig. 8A, glucose uptake was significantly reduced in the HG group compared with the NG group (P < 0.01). Downregulation of THBS1 markedly increased glucose uptake compared to the HG + shNC group, while treatment with the RhoA/ROCK1 pathway activator U46619 significantly decreased glucose uptake relative to the HG + sh-THBS1 group (P < 0.01).

Fig. 8.

THBS1 can regulate the insulin sensitivity of HTR8/SVneo cells treated with high glucose through the RhoA/ROCK1 pathway. (A) After the cells in the NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group were treated with 100 nM insulin for 30 min, the glucose uptake levels were measured. (B) Western blot was used to detect the expression levels of p-IRS-1, IRS-1, p-AKT, AKT, and GLUT4 proteins in the cells in the NG group, HG group, HG + shNC group, HG + sh-THBS1 group, and HG + sh-THBS1 + U46619 group. Data were presented as mean ± SD (n = 3). **P < 0.01 vs. NG group; ##P < 0.01 vs. HG + shNC group; &&P < 0.01 vs. HG + sh-THBS1 group

Western blot results (Fig. 8B) showed that the protein expression level of p-IRS-1 in cells in the HG group was significantly higher than that in the NG group (P < 0.01), while the expression levels of IRS-1, p-AKT and GLUT4 proteins were significantly higher than those in the NG group (P < 0.01). Knockdown of THBS1 significantly restored the levels of p-IRS-1, IRS-1, p-AKT and GLUT4 (P < 0.01) compared to the HG + shNC group. However, activation of the RhoA/ROCK1 pathway with U46619 reversed these effects, leading to reduced expression of these proteins in the HG + sh-THBS1 + U46619 group (P < 0.01).

These results indicated that THBS1 knockdown enhanced glucose uptake and restores insulin signaling in HTR8/SVneo cells under high-glucose conditions, while activation of the RhoA/ROCK1 pathway partially abolished these beneficial effects, suggesting that THBS1 modulated glucose metabolism and insulin sensitivity via the RhoA/ROCK1 signaling pathway.

Discussion

This study demonstrates that THBS1 plays a critical regulatory role in GDM-related cellular dysfunctions by activating the RhoA/ROCK1 signaling pathway. We found that THBS1 expression was significantly upregulated in placental tissues from GDM patients and in HTR8/SVneo cells treated with high glucose. Functionally, THBS1 overexpression reduced cell viability, migration ability, and glucose uptake, while promoting inflammatory cytokine secretion and insulin resistance. In contrast, THBS1 knockdown improved these cellular functions. To further confirm whether these effects are mediated through the RhoA/ROCK1 pathway, we used the RhoA activator U46619, which reversed the beneficial effects of THBS1 knockdown, indicating that THBS1 modulates cell function and insulin sensitivity through the RhoA/ROCK1 pathway. These findings suggest that THBS1 contributes to GDM pathogenesis by regulating key processes such as trophoblast function, inflammatory responses, and insulin signaling.

Previous studies have identified THBS1 as a key regulator of insulin resistance in various metabolic diseases, including obesity and type 2 diabetes [24]. It has been shown that THBS1 impairs insulin signaling by inhibiting IRS-1, AKT, and GLUT4, which are critical components of the insulin signaling pathway [25]. Consistent with these findings, our study showed that THBS1 overexpression in HTR8/SVneo cells decreased glucose uptake by reducing the expression of p-IRS-1, p-AKT, and GLUT4, while THBS1 knockdown improved these indicators, thereby enhancing glucose uptake and insulin sensitivity. These results confirm the negative regulatory role of THBS1 in insulin sensitivity, further supporting its involvement in GDM pathology. Notably, our study is the first to demonstrate that THBS1 regulates glucose uptake and insulin signaling through the RhoA/ROCK1 pathway, highlighting the potential cross-talk between cytoskeletal remodeling and insulin resistance mechanisms [26]. This finding provides new insights into the molecular mechanisms underlying insulin resistance in GDM.

In addition to impairing insulin signaling, we found that THBS1 overexpression significantly decreased cell viability and migration ability in HTR8/SVneo cells, which are important for proper placental development. These effects were also reversed by knocking down THBS1, suggesting that THBS1 plays a role in trophoblast dysfunction under high-glucose conditions. This observation aligns with previous studies showing that the RhoA/ROCK1 pathway regulates cytoskeletal dynamics, cell motility, and migration in various cell types [27, 28]. The inhibition of trophoblast migration and viability by THBS1 may contribute to abnormal placental development and function in GDM, highlighting ts potential as a biomarker for predicting placental dysfunction and a therapeutic target for restoring normal trophoblast function.

Furthermore, our study demonstrated that THBS1 upregulates inflammatory cytokine secretion in HTR8/SVneo cells, including TNF-α, IL-1β, IL-6 and IL-8, which are well-known mediators of inflammation in GDM [29]. THBS1 overexpression significantly increased the secretion of these inflammatory factors, while THBS1 knockdown reduced their levels. The RhoA activator U46619 reversed the anti-inflammatory effects of THBS1 knockdown, confirming that THBS1 regulates inflammatory responses through the RhoA/ROCK1 pathway. This finding is critical because inflammation plays a central role in GDM pathophysiology, contributing to both insulin resistance and placental dysfunction [30, 31]. Therefore, our study expands the understanding of THBS1’s role by linking its effects on inflammation, insulin signaling, and trophoblast function, all of which are central to GDM pathology.

The dual regulatory role of THBS1 in insulin resistance and inflammation suggests that it may serve as a therapeutic target for GDM treatment. Current GDM therapies primarily focus on controlling blood glucose levels through dietary interventions and pharmacological agents [32]. However, these treatments have limited effects on reducing insulin resistance and improving placental function, which are critical for preventing adverse pregnancy outcomes. Our findings indicate that targeting THBS1 or inhibiting the RhoA/ROCK1 pathway could provide a novel therapeutic approach that addresses both metabolic and inflammatory dysfunctions in GDM. Of note, monoclonal antibodies and CD47 antagonists targeting THBS1 have been explored in other diseases and shown promising effects in modulating pathological inflammation and improving tissue function [33]. These therapeutic strategies could be investigated in preclinical models of GDM to assess their feasibility and efficacy in improving maternal and fetal outcomes.

Although this study reveals the role of THBS1 in GDM, several limitations should be acknowledged. Firstly, the results were derived from in vitro cell models, which may not fully recapitulate the complex pathophysiology of GDM in vivo. Secondly, the relatively small sample size of placental tissues may limit the generalizability of our findings. Although statistically significant differences were observed, larger cohorts would help to validate the robustness of these results and improve statistical power. Therefore, future studies should employ animal models and larger clinical cohorts to confirm the relevance of these findings in real-world scenarios. Additionally, assessing the potential of THBS1 as a biomarker for predicting GDM-related placental dysfunction, and evaluating the therapeutic efficacy of THBS1-targeted interventions (e.g., monoclonal antibodies or small-molecule inhibitors), would strengthen the translational significance of this work. Moreover, it is likely that other signaling pathways, such as PI3K/AKT and NF-κB, may also contribute to THBS1-mediated cellular dysfunctions [34]. Upstream regulators such as hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-β (TGF-β) have also been implicated in the control of THBS1 expression under metabolic stress [35]. Investigating these pathways and their interactions with THBS1 may provide a more comprehensive understanding of its role in GDM.

In summary, this study provides novel insights into the role of THBS1 in GDM by demonstrating that it regulates trophoblast function, inflammation, and insulin sensitivity through the RhoA/ROCK1 pathway. These findings highlight the potential of THBS1 as a therapeutic target for improving both metabolic and inflammatory dysfunctions in GDM. Future research should focus on in vivo validation and explore the development of small-molecule inhibitors targeting THBS1 or its downstream pathways. Additionally, the potential role of THBS1 in other metabolic disorders such as type 2 diabetes and obesity warrants further investigation to expand its therapeutic potential beyond GDM.

Conclusion

This study identified THBS1 as a key regulator of trophoblast cell function, inflammatory responses, and insulin sensitivity in HTR8/SVneo cells under high glucose conditions via RhoA/ROCK1 signaling. Targeting THBS1 could offer a novel therapeutic approach for managing GDM by improving insulin sensitivity and reducing inflammation, highlighting its potential clinical significance.

Abbreviations

GDM

Gestational diabetes mellitus

IADPSG

International Association of Diabetes and Pregnancy Study Groups

FBS

Fetal bovine serum

BCA

Bicinchoninic acid assay

ANOVA

Analysis of variance

SD

Standard deviation

Author contributions

Y.C.: Conceptualization; Data curation; Formal analysis; Investigation; Validation; Visualization; Writing - original draft; Writing - review & editing. J.T.: Data curation; Formal analysis; Investigation; Methodology; Resources; Writing - review & editing. A.L.: Formal analysis; Investigation; Methodology; Resources; Writing - review & editing. Q.Z.: Conceptualization; Formal analysis; Supervision; Visualization; Writing - review & editing.

Funding

This study did not receive any funding in any form.

Data availability

The data used to support the findings of this study are available from the corresponding author upon request.

Declarations

Ethics approval and consent to participate

This study was approved by the Medical Ethics Committee of the Wujin Hospital Affiliated with Jiangsu University(2024-SR-112) and conducted in accordance with the Declaration of Helsinki (as revised in 2013). All subjects signed the consent form before participation in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.