Sequential Stage-Specific Administration of Bushen-Tiaojing Decoctions II and III Ameliorates Mitochondrial Dysfunction in Ovarian Granulosa Cells of COH Mice by Regulating GRP75-Mediated MAM Homeostasis.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) offers a distinct therapeutic approach to infertility. Bushen-Tiaojing Decoctions II (BSTJ-II-D) and III (BSTJ-III-D; collectively, BSTJD) are clinical formulas developed by Professor Huilan Du according to the TCM theory of stage-specific treatment and are widely used in the clinical management of infertility. Previous studies have shown that sequential, stage-specific administration of these formulas can improve mitochondrial energy metabolism and increase pregnancy rates among infertile women undergoing in vitro fertilization and embryo transfer (IVF-ET). However, whether this sequential treatment improves mitochondrial function in ovarian granulosa cells (GCs) by preserving mitochondria-associated endoplasmic reticulum membrane (MAM) homeostasis remains unclear. AIM OF THE STUDY: Glucose-regulated protein 75 (GRP75) is a key tethering protein that maintains MAM homeostasis. This study aimed to determine whether sequential, stage-specific administration of BSTJ-II-D and BSTJ-III-D ameliorates mitochondrial dysfunction in ovarian GCs by regulating GRP75-mediated MAM homeostasis. MATERIALS AND METHODS: A controlled ovarian hyperstimulation (COH) mouse model was established, after which animals received BSTJ-II-D and BSTJ-III-D sequentially via oral gavage. A group treated with GRP75-IN-1, a novel small-molecule compound that targets and binds GRP75, was included as a control. MAM morphology, mitochondrial bioenergetic activity, and mitochondrial biogenesis were systematically evaluated. For the in vitro experiments, human KGN ovarian granulosa-like tumor cells were transduced with a GRP75-overexpressing lentiviral construct to clarify how elevated GRP75 expression affects MAM architecture and function and subsequently influences mitochondrial homeostasis. Protein levels of GRP75, voltage-dependent anion channel 1 (VDAC1), mitochondrial calcium uniporter (MCU), sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and nuclear factor erythroid 2-related factor 2 (Nrf2) were assessed in GCs. Cellular adenosine triphosphate (ATP) production, reactive oxygen species (ROS) levels, mitochondrial Ca2+ loading, and mitochondrial membrane potential (MMP) were determined using fluorescence-based assays. In addition, mitochondrial permeability transition pore (mPTP) opening was evaluated quantitatively by flow cytometric analysis. RESULTS: Sequential BSTJD treatment attenuated COH-induced aberrant GRP75 overexpression, restored MAM ultrastructural integrity, reduced mitochondrial oxidative stress (OS) and Ca2+ overload, and enhanced mitochondrial energy metabolism and biogenesis. Mechanistically, BSTJD increased the expression of SIRT1, PGC-1α, and Nrf2 while inhibiting activation of the PERK/ATF4 pathway, thereby alleviating endoplasmic reticulum (ER) stress and enhancing mitochondrial energy metabolism and biogenesis. GRP75-IN-1 reproduced the protective effects of BSTJD. Conversely, GRP75 overexpression in KGN cells disrupted MAMs and impaired mitochondrial function, further supporting GRP75 as a key mediator of the protective effects of BSTJD. CONCLUSION: Sequential BSTJD treatment preserves MAM homeostasis by suppressing aberrant GRP75 overexpression, thereby mitigating OS and mitochondrial Ca2+ overload. It also activates the SIRT1-PGC-1α-Nrf2 pathway to restore mitochondrial energy metabolism and biogenesis, ultimately ameliorating mitochondrial dysfunction in GCs. These findings provide new molecular evidence supporting the Bushen Tiaojing therapeutic approach for infertility.

Key Findings

• Sequential, stage-specific administration of Bushen-Tiaojing Decoctions II and III (BSTJ-II-D and BSTJ-III-D) improves mitochondrial function in ovarian granulosa cells by regulating GRP75-mediated mitochondria-associated endoplasmic reticulum membrane (MAM) homeostasis.
• Treatment with BSTJ-II-D and BSTJ-III-D enhances mitochondrial bioenergetic activity and mitochondrial biogenesis in a controlled ovarian hyperstimulation (COH) mouse model.
• Overexpression of GRP75 in human ovarian granulosa-like cells improves MAM architecture and mitochondrial homeostasis, indicating GRP75's key role in maintaining mitochondrial function.

Clinical Significance

Citation

Ding Xin, Wen Xiaoying, Xiong Wenyanet al.. Sequential Stage-Specific Administration of Bushen-Tiaojing Decoctions II and III Ameliorates Mitochondrial Dysfunction in Ovarian Granulosa Cells of COH Mice by Regulating GRP75-Mediated MAM Homeostasis. Journal of ethnopharmacology. 2026-Oct-10.