Astragaloside IV regulates FOXM1 deubiquitination to ameliorate trophoblast damage caused by high glucose

Abstract

Background: Gestational diabetes mellitus (GDM) is a common metabolic complication during pregnancy that poses significant risks to both the pregnant woman and her fetus. Astragaloside IV (Ast IV) belongs to the class of triterpenoid saponins and exhibits important physiological roles in various aspects, including antidiabetic, antioxidant, and antiviral effects. The main objective of this study is to investigate the effects of Ast IV on trophoblast damage caused by high glucose (HG) and its underlying mechanism of action.

Methods: Cell viability was determined by the CCK8 assay. The levels of oxidative stress in cells were determined by lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) kits. Ferroptosis in cells was assessed by the iron content kit. Gene expression levels were detected by real-time quantitative reverse transcription PCR (qRT-PCR) and western blot. The protein stability of Forkhead box protein M1 (FOXM1) was determined by the cycloheximide (CHX) assay. The ubiquitination level of FOXM1 was detected by the immunoprecipitation assay.

Results: Ast IV alleviated the inhibitory effect of HG on the proliferation of HTR-8/SVneo cells and reduced HG-induced oxidative stress and ferroptosis. Ast IV was able to decrease the ubiquitination of FOXM1, thereby ensuring the stability of its expression. The overexpression of FOXM1 significantly mitigated the inhibitory effect of HG on the viability of HTR-8/SVneo cells and concurrently decreased the occurrence of HG-induced oxidative stress and ferroptosis processes. Conversely, knockdown of FOXM1 diminished the protective effect of Ast IV on HTR-8/SVneo cells.

Conclusions: Ast IV ameliorates HG-induced trophoblast injury by modulating deubiquitination of FOXM1, which provides a new insight into the treatment of GDM.

Keywords: Astragaloside IV; Deubiquitination; Forkhead box protein M1; Gestational diabetes mellitus; Hyperglycemic induction.

Fig. 1

Ast IV enhances the proliferative capacity and attenuates oxidative stress in HTR-8/SVneo cells under HG environment. HTR-8/SVneo cells were divided into five groups: Control, HG, HG + Ast IV 10 µM, HG + Ast IV 20 µM, and HG + Ast IV 40 µM. A: CCK8 assay was used to assess the viability of the cells. B: The amount of LDH in HTR-8/SVneo cells was detected by colourimetric assay. C: Colourimetric assay was used to detect the level of MDA in HTR-8/SVneo cells. D: Fluorescence assay was used to detect content of ROS in HTR-8/SVneo cells. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns indicated insignificant. Sample size description: The number of replicates for each experiment was 3 (n = 3)

Fig. 2

Ast IV is able to alleviate the HG-induced ferroptosis process in HTR-8/SVneo cells. HTR-8/SVneo cells were divided into five groups: Control, HG, HG + Ast IV 10 µM, HG + Ast IV 20 µM, and HG + Ast IV 40 µM. A: Colorimetric assay was used to detect the level of iron in HTR-8/SVneo cells. B-F: Western blot was used to demonstrate the expression levels of GXP4, SLC7A11, FTH1, and ALOX12 in HTR-8/SVneo cells. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns indicated insignificant. Sample size description: The number of replicates for each experiment was 3 (n = 3)

Fig. 3

Ast IV reduces ubiquitination of FOXM1 and thus stabilizing its protein expression. HTR-8/SVneo cells were divided into five groups: Control, HG, HG + Ast IV 10 µM, HG + Ast IV 20 µM, and HG + Ast IV 40 µM. A: The mRNA expression level of FOXM1 in HTR-8/SVneo cells was detected by qRT-PCR. B: Western blot was used to assess the expression level of FOXM1 in HTR-8/SVneo cells. C: CHX assay was used to demonstrate the protein stability of FOXM1 in HTR-8/SVneo cells. D: The ubiquitination level of FOXM1 in HTR-8/SVneo cells was checked by immunoprecipitation assay. ** P < 0.01, *** P < 0.001, and ns indicated insignificant. Sample size description: The number of replicates for each experiment was 3 (n = 3)

Fig. 4

Overexpression of FOXM1 attenuates the damage caused by HG to HTR-8/SVneo cells. HTR-8/SVneo cells were divided into four groups: Control, HG, HG + pc-DNA, and HG + pc-DNA-FOXM1. A: Western blot was used to detect the expression level of FOXM1 in HTR-8/SVneo cells. B: The viability of the HTR-8/SVneo cells was assessed through CCK8 assay. C: Colourimetric assay was used to demonstrate the LDH content of the HTR-8/SVneo cells. D: The MDA level of the HTR-8/SVneo cells was detected by colourimetric assay. E: Fluorescence assay was used to detect ROS content in HTR-8/SVneo cells. F: The iron content in HTR-8/SVneo cells was detected through colourimetric assay. G: Western blot was used to demonstrate the expression levels of GXP4, SLC7A11, FTH1, and ALOX12 in HTR-8/SVneo cells. * P < 0.05, ** P < 0.01, and *** P < 0.001. Sample size description: The number of replicates for each experiment was 3 (n = 3)

Fig. 5

Knockdown of FOXM1 counteracts the effects of Ast IV. HTR-8/SVneo cells were divided into five groups: Control, HG, HG + Ast IV, HG + Ast IV + si-NC, and HG + Ast IV + si-FOXM1. A: Western blot was used detect the expression level of FOXM1 in HTR-8/SVneo cells. B: CCK8 assay was used to test the viability of the HTR-8/SVneo cells. C: The content of LDH in HTR-8/SVneo cells was checked through colourimetric assay. D: Colourimetric assay was used to detect MDA content in HTR-8/SVneo cells. E: The ROS content in HTR-8/SVneo cells was tested by fluorescence assay. F: Colourimetric assay was used to detect iron content in HTR-8/SVneo cells. G: Western blot was used to detect the expression levels of GXP4, SLC7A11, FTH1, and ALOX12 in HTR-8/SVneo cells. * P < 0.05, ** P < 0.01, and *** P < 0.001. Sample size description: The number of replicates for each experiment was 3 (n = 3)