@article{WANG2026, 
author = {Lingling WANG and Xiaozhou JIA and Jing GE},
title = {Sestrin2 attenuates endoplasmic reticulum stress and inhibits trophoblast pyroptosis by activating the AMPK/mTOR pathway to improve placental injury in gestational diabetes mellitus},
year = {2026},
journal = {Journal of Army Medical University},
volume = {48},
number = {14},
pages = {1960-1972},
keywords = {gestational diabetes mellitus, endoplasmic reticulum stress, pyroptosis, Sestrin2, AMPK/mTOR pathway},
url = {https://www.sciopen.com/article/10.16016/j.2097-0927.202512036},
doi = {10.16016/j.2097-0927.202512036},
abstract = {ObjectiveGestational diabetes mellitus (GDM) can induce trophoblast pyroptosis through endoplasmic reticulum stress (ERS), leading to placental injury. Using in vivo and in vitro models of GDM, this study aims to investigate whether Sestrin2 attenuates ERS to inhibit trophoblast pyroptosis and thereby improve GDM-induced placental damage through AMPK/mTOR activation.Methods① In vitro experiments: HTR-8/SVneo cells were divided into normal glucose (NG, medium containing 5.5 mmol/L glucose for 48 h), isotonic control (MN, medium containing 5.5 mmol/L glucose plus 24.5 mmol/L mannitol for 48 h), and high glucose (HG, medium containing 30 mmol/L glucose for 48 h) groups. To modulate Sestrin2 expression, additional HG groups were established: HG+oe-NC (transfected with oe-NC control plasmid 24 h before HG treatment), HG+oe-Sestrin2 (transfected with oe-Sestrin2 overexpression plasmid 24 h before HG treatment), HG+si-NC (transfected with si-NC control 24 h before HG treatment), and HG+si-Sestrin2 (transfected with si-Sestrin2 specific siRNA 24 h before HG treatment). To intervene in ERS, the HG+4-PBA group (treated with 20 mmol/L 4-PBA 1 h before HG exposure) and the HG+si-Sestrin2+4-PBA group (transfected with si-Sestrin2 24 h before HG and treated with 20 mmol/L 4-PBA 1 h before HG) were included. To inhibit the AMPK pathway, the oe-Sestrin2+AMPK-IN-3 group (transfected with oe-Sestrin2 24 h before HG and treated with 12.5 μmol/L AMPK-IN-3 2 h before HG induction) was set. All cell experiments were independently repeated 3 times (n=3). ② In vivo experiments: Female C57BL/6J mice were divided into 4 groups (n=6): Control (normal chow), GDM model (high-fat/high-sugar diet for 1 month), GDM+oe-NC (high-fat/high-sugar diet for 1 month and injected with empty vector adeno-associated virus via tail vein on gestational day 8.5), and GDM+oe-Sestrin2 (high-fat/high-sugar diet for 1 month and injected with Sestrin2-overexpressing adeno-associated virus via tail vein on gestational day 8.5). Maternal body weight and blood glucose were recorded during pregnancy. Western blotting was performed to detect ERS markers (GRP78, CHOP, p-IRE1α, IRE1α, p-PERK, PERK), pyroptosis-related proteins (NLRP3, GSDMD-N, cleaved-caspase-1), and AMPK/mTOR pathway proteins (p-AMPK, AMPK, mTOR, p-mTOR). qPCR was applied to measure Sestrin2 mRNA level. ELISA was conducted to determine the levels of inflammatory cytokines (IL-18 and IL-1β). CCK-8 and lactate dehydrogenase (LDH) release assays were employed to evaluate cell viability and cytotoxicity. Co-immunoprecipitation (Co-IP) was utilized to analyze the interaction between Sestrin2 and GRP78. HE staining was used to observe placental histopathological changes.ResultsThe HG group showed significantly higher levels of ERS markers, pyroptosis-related proteins, LDH release rate, and inflammatory cytokines, and lower cell viability compared with the NG group (P<0.05), and all these changes were reversed by 4-PBA (P<0.05). Compared with the HG+oe-NC group, the HG+oe-Sestrin2 group exhibited increased Sestrin2 expression and cell viability, and decreased ERS markers, pyroptosis-related proteins, inflammatory cytokines, and LDH release rate (P<0.05). Opposite trends were observed in the HG+si-Sestrin2 group compared with the HG+si-NC group (P<0.05). 4-PBA partially counteracted the effects of si-Sestrin2 (P<0.05) but did not affect Sestrin2 expression (P>0.05). Regarding the AMPK/mTOR pathway, the HG group showed increased p-AMPK (P<0.05) and decreased p-mTOR (P< 0.05) than the NG group, while no difference was found between the MN and NG groups (P>0.05). Compared with the HG+oe-NC group, the HG+oe-Sestrin2 group had increased p-AMPK (P<0.05) and decreased p-mTOR (P<0.05). AMPK-IN-3 blocked the protective effects of oe-Sestrin2, as evidenced by significantly increased ERS markers, pyroptosis-related proteins, inflammatory cytokines, LDH release rate, and p-mTOR (P<0.05), and decreased p-AMPK and cell viability (P<0.05), without affecting Sestrin2 expression. Co-IP did not detect any interaction between Sestrin2 and GRP78. In vivo, compared with the Control group, the GDM group showed increased placental pathological score, body weight gain, and blood glucose levels (P< 0.05), as well as increased placental Sestrin2, p-AMPK, GRP78, CHOP, NLRP3, and GSDMD-N (P<0.05), and decreased p-mTOR (P<0.05). No significant differences were observed between the GDM and GDM+oe-NC groups. Compared with the GDM+oe-NC group, the GDM+oe-Sestrin2 group exhibited decreased placental pathological score, body weight gain, and blood glucose levels (P<0.05), decreased placental p-mTOR, GRP78, CHOP, NLRP3, and GSDMD-N (P<0.05), and increased placental Sestrin2 and p-AMPK (P<0.05).ConclusionSestrin2 attenuates high glucose-induced ERS in trophoblasts by activating the AMPK/mTOR pathway, thereby inhibiting pyroptosis and improving GDM-associated placental injury.}
}