Gengnianshou Formula Inhibits Estrogen Deficiency Induced Obesity via an HSPA5-Associated Phosphatidylcholine (PC) Regulatory Pathway: Ethnopharmacological Relevance and Mechanistic Insights from Network Pharmacology and Multi-Omics

Ethnopharmacological relevance Gengnianshou Formula (GNS), a traditional Chinese medicine formula composed of 9 herbs, has long been used for regulating menopausal syndromes based on the TCM theory. However, its mechanistic basis for ameliorating perimenopausal obesity (a common estrogen deficiency-induced metabolic disorder) remains unelucidated. Aim of the study This study aimed to investigate the anti-obesity effects of GNS under estrogen-deficient conditions and to explore the potential involvement of HSPA5 and phosphatidylcholine (PC) metabolism using network pharmacology and multi-omics approaches. Materials and methods An ovariectomized (OVX) rat model was established using female Sprague-Dawley rats, which were randomly assigned to six groups (n = 6 per group) to evaluate the therapeutic effects of GNS. Network pharmacology analysis was conducted to identify potential targets. Transcriptomics and lipidomics analyses were integrated to explore molecular alterations. In vitro experiments using human adipose-derived stem cells (hADSCs) under estrogen-deprived conditions were performed to functionally validate the role of HSPA5 through siRNA-mediated knockdown, followed by Oil Red O staining and gene expression analysis. Results The OVX model successfully induced obesity, as evidenced by significant increases in body weight, visceral and subcutaneous white adipose tissue (vWAT/sWAT) volume, and adipocyte hypertrophy. GNS dose-dependently attenuated these OVX-induced alterations of body weight gain, vWAT/sWAT accumulation, and adipocyte hypertrophy in rats, and normalized perturbed serum lipid profiles (TG, LDL-C, HDL-C). Network pharmacology identified HSPA5 as a potential key target. Multi-omics analysis revealed significant alterations in lipid metabolism, particularly in PC-related species, which were associated with HSPA5 expression changes. Functional experiments demonstrated that silencing HSPA5 attenuated lipid accumulation and downregulated adipogenic markers, including PPARγ and C/EBPα, under estrogen-deficient conditions. Conclusion These findings support a functional role of HSPA5 in regulating adipogenesis, while alterations in PC metabolism may represent associated downstream metabolic changes. GNS ameliorates estrogen deficiency-induced obesity, potentially involving HSPA5 and PC metabolism. This study provides integrative evidence supporting HSPA5 as a functional regulator in this context, while highlighting a possible link to PC metabolic remodeling.