[Gandou Bushen decoction ameliorates ovarian injury in murine hepatolenticular degeneration via the AGE/RAGE/NF-κB signaling pathway].

OBJECTIVES: To investigate the protective effect and underlying mechanism of Chinese herbal medicine Gandou Bushen decoction (GBD) on ovarian injury in murine hepatolenticular degeneration (HLD) model.

METHODS: The chemical constituents of GBD were analyzed using liquid chromatography-mass spectrometry (LC-MS). Forty female C3He-Atp7btx-J mice (6-week-old) were randomly divided into model, penicillamine (positive control), low-dose GBD, and high-dose GBD groups. Ten DL syngeneic female mice served as the normal control group. Body and ovarian weights were measured to calculate the ovarian coefficient. Ovarian copper content was detected by complexometric colorimetry. Histopathological and ultrastructural changes were observed by hematoxylin-eosin staining and transmission electron microscopy, respectively. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, and progesterone were measured by enzyme-linked immunosorbent assay (ELISA). RNA sequencing was performed to identify differentially expressed genes, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A copper overloaded cell model was established in ovarian granulosa cells (iCell-0114a) by treating them with copper sulfate. Cells were divided into normal control, model control, and low-, medium-, and high-dose GBD groups. The mRNA expressions of FSH receptor (FSHR), steroidogenic acute regulatory protein (StAR), insulin-like growth factor-1 (IGF-1), receptor for advanced glycation end products (RAGE), and nuclear factor κB (NF-κB) were detected by quantitative reverse transcription polymerase chain reaction. The levels of TNF-α, IL-1β, and IL-6 were measured by ELISA. Superoxide dismutase (SOD) activity was measured using WST-1 assay. Reactive oxygen species (ROS) levels were measured using DCFH-DA fluorescence, and mitochondrial membrane potential was assessed using JC-1 staining coupled with flow cytometry. Protein expression of B-cell lymphoma 2 protein (BCL-2), BCL-2-associated X protein (BAX), caspase-3, advanced glycation end products (AGE), RAGE, and NF-κB was determined by Western blotting.

RESULTS: A total of 1465 chemical components were identified in GBD. Compared with the normal control group, the model group showed decreased body weight, ovarian weight, and ovarian coefficient (all P<0.01). GBD treatment alleviated tissue copper deposition (both P<0.01), improved ovarian histomorphology and ultrastructure, and increased serum levels of FSH, LH, estradiol, and progesterone (all P<0.01). RNA sequencing identified 507 differentially expressed genes. KEGG enrichment analysis indicated that the mechanism underlying GBD's protective effects primarily involved the AGE/RAGE/NF-κB signaling pathway. In copper-overloaded granulosa cells, GBD dose-dependently increased the mRNA expression of FSHR, StAR, and IGF-1, reduced the levels of TNF-α, IL-1β, and IL-6, increased SOD activity, and decreased ROS levels (all P<0.01). The medium- and high-dose GBD groups showed a lower percentage of cells with mitochondrial depolarization (both P<0.01). All GBD dose groups showed decreased expression of BAX and caspase-3 (all P<0.05), while the medium- and high-dose groups showed increased BCL-2 expression (all P<0.05). Furthermore, medium and high doses of GBD reduced the protein expression of AGE, RAGE, and NF-κB (all P<0.05), and all doses downregulated the mRNA expression of RAGE and NF-κB (all P<0.01).

CONCLUSIONS: GBD ameliorates ovarian injury in HLD, and its mechanism of action is associated with the suppression of the AGE/RAGE/NF-κB signaling pathway.