Progesterone (PG) is a C21 steroid hormone that is synthesised from cholesterol via the pregnenolone pathway (Kolatorova, Vitku, Suchopar, Hill, & Parizek, 2022). In humans, PG is mainly produced in the ovaries and placenta, and also in the adrenal cortex, testes, and brain at much lower levels (Kolatorova et al., 2022, Sundström-Poromaa et al., 2020, Taraborrelli, 2015). Functionally, PG is essential for regulating the menstrual cycle and maintaining pregnancy. During the luteal phase of the menstrual cycle, PG prepares the endometrium for the potential implantation of an embryo. When pregnancy occurs, PG level remains elevated to support the pregnancy and prevent uterine contraction that may lead to miscarriage (Devall and Coomarasamy, 2020, Piette, 2020). In addition to its reproductive functions, progesterone also has significant effects on other systems, such as neuroprotection, immune and inflammatory regulation, and bone metabolism. PG has been reported to be involved in the processes of emotion and anxiety by influencing neuronal excitability (Standeven, McEvoy, & Osborne, 2020). PG has anti-inflammatory and immunomodulatory effects in vivo, showing therapeutic potential in autoimmune and inflammatory diseases, such as rheumatoid arthritis, endometriosis, and miscarriage (Fedotcheva, Fedotcheva, & Shimanovsky, 2022). Through its action as an osteoblast receptor and antagonist of glucocorticoid receptor, PG contributes to the maintenance of bone density, especially in postmenopausal women (Prior, 2018, Zhong et al., 2017). Notably, PG antagonises the effects of oestrogen in the normal menstrual cycle, and its deficiency in perimenopausal period disrupts this balance (oestrogen dominance and progesterone resistance), leading to endometrial hyperplasia, subsequent abnormal uterine bleeding (AUB), and even endometrial carcinoma (MacLean & Hayashi, 2022). Clinically, PG is widely used to prevent and treat perimenopausal symptoms, including AUB, depressive symptoms, vasomotor disturbances, etc. (Gordon et al., 2018; Hipolito Rodrigues & Gompel, 2021; Jewson, Purohit, & Lumsden, 2020; Nicula & Costin, 2015; Prior et al., 2023). Overall, the multifaceted roles of PG in various physiological processes emphasise its importance in the body.

RNA sequencing (RNA-seq), a high-throughput sequencing method, enables the quantitative assessment of transcriptional signatures across diverse biological contexts (Wang, Gerstein, & Snyder, 2009). The molecular mechanisms of PG are complex, involving a series of regulatory genes and signalling pathways. Based on RNA-seq, the underlying mechanisms of action of PG in inflammatory response have been uncovered to some extent. For example, PG and cAMP synergistically inhibit inflammatory regulators, including STAT3/6, OCT1/7, and CEBPA/B in myometrial cells of pregnant women (Stanfield et al., 2019). PG ameliorates retinal inflammation in experimental autoimmune uveitis by regulating Th17/Treg imbalance, the Id2/Pim1 axis, and the IL-23/Th17/GM-CSF signalling pathway (Liu et al., 2023). PG inhibits the activation of NLRP1/NLRP3 inflammasomes and the TLR4-MyD88-NF-κB signalling pathway in monocytes from pregnant women with pre-eclampsia (Matias et al., 2021). Additionally, RNA-seq analysis also contributes to the discovery of further mechanisms of action of PG in many other diseases, such as cervical insufficiency (Lan et al., 2020), breast cancer (Yadav et al., 2022), endometrial cancer (Saito-Kanatani et al., 2015), glioblastoma (Chen et al., 2024), and behavioural disorder (Liang et al., 2024). However, RNA-seq analysis on PG-related bone metabolism is rarely reported.

Periodontitis is a prevalent disease characterised by microbial-associated and host-mediated inflammation, leading to the loss of periodontal attachment (Tonetti, Greenwell, & Kornman, 2018). The interactions between pathogenic bacteria and host’s immune response are the main pathogenesis of periodontitis (Slots, 2017). More and more evidence has determined that the smoking, obesity, diabetes, physical activity, dietary habits, genetic factors, etc., are risk factors for periodontitis, and these factors should also be considered in the classification (Darby, 2022; Tonetti et al., 2018). Notably, hormonal fluctuations in women, especially PG deficiency, contribute to the progression of periodontitis (Li and Wang, 2018, Shu et al., 2008). As a bone-trophic factor positively associated with bone formation, PG has a potential for the treatment of periodontitis. Yuan et al. have reported that PG promotes the osteoblastic differentiation of human periodontal ligament cells (Yuan et al., 2010). Our recent study also showed that PG inhibits inflammatory alveolar bone loss in perimenopausal women, contributing to the remission of periodontitis (Man et al., 2025). However, the underlying mechanisms of action of PG on alveolar bone are still unclear. In the present study, primary human periodontal mesenchymal stem cells (hPDLSCs) were isolated and then treated with lipopolysaccharide (LPS) to simulate periodontitis in vitro. Under an inflammatory environment, relevant molecular mechanisms of PG on the osteogenesis of hPDLSCs were explored by RNA-seq. In addition, BUB1 and UBE2C are both mitotic regulators associated with osteogenic differentiation (Yoshida et al., 2024; H. Zhang et al., 2024). These two key hub proteins are speculated to be involved in the mechanisms of action of PG on osteogenesis. The aim of this study is to reveal the potential molecular mechanisms by which PG attenuates the inhibitory effects of LPS on the osteogenic differentiation of hPDLSCs, and to identify key target genes involved in this process.