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.

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.