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Based on the adiponectin receptor/5′-AMP-activated protein kinase catalytic subunit α-1/carnitine palmitoyltransferase 1b (AdipoR/AMPKα/Cpt1b) signaling axis, this study investigated the therapeutic mechanisms of Jianpi Huogu Formula (JPHGF) in alcohol-induced osteonecrosis of the femoral head (AONFH). A total of 56 Sprague-Dawley rats were randomly divided into a normal group, a model group, and a JPHGF group (5.0 g·kg-1). Except for the normal group, rats in the other two groups were administered 46% ethanol by gavage to establish the AONFH model, and JPHGF intervention was initiated in the treatment group at week 4 after model induction. After 4 and 8 weeks of intervention, samples were collected. Mechanical and cold pain thresholds were assessed using Von Frey filaments and the acetone drop test, respectively. Hindlimb muscle strength was evaluated by the inclined plane test. Micro-computed tomography (micro-CT) was used to determine bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb. Th), and other bone morphometric parameters. Hematoxylin-eosin (HE) staining was performed to observe histopathological changes. Transcriptomic sequencing was conducted to identify differentially expressed genes (DEGs) regulated by JPHGF in AONFH, followed by Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Western blot was used to detect the protein expression of AdipoR1, AdipoR2, phosphorylated AMPKα, and Cpt1b in femoral head tissues. The results showed that after 8 weeks of modeling, rats in the model group exhibited significantly decreased mechanical pain thresholds and increased cold sensitivity (P<0.05, P<0.01), indicating hyperalgesia. The maximum inclined angle was markedly reduced (P<0.01), suggesting weakened hindlimb muscle strength. The femoral head surface appeared rough, with enlarged dark-red cartilage areas and sparse, disrupted trabeculae; the proportion of empty lacunae and the number of adipocytes were significantly increased (P<0.01), while BMD, BV/TV, Tb. Th, and trabecular number (Tb. N) were significantly decreased (P<0.01). After 4 weeks of JPHGF intervention, mechanical and cold hyperalgesia were significantly alleviated (P<0.05), hindlimb strength was restored (P<0.01), and surface morphology of the femoral head was improved. After 8 weeks, trabecular structure was further restored, with decreased empty lacunae and reduced adipocyte numbers, and BMD, BV/TV, Tb. Th, and Tb. N significantly increased (P<0.05, P<0.01); cystic degeneration and collapse were markedly alleviated. Transcriptomic analysis revealed that JPHGF significantly regulated 231 key DEGs, which were mainly enriched in adipocytokine signaling pathways and closely associated with AdipoR1, AdipoR2, p-AMPKα, and Cpt1b. Western blot analysis showed that AdipoR1, AdipoR2, p-AMPKα, and Cpt1b expression levels were significantly downregulated in the AONFH model group compared with the normal group (P<0.01), but were markedly upregulated following JPHGF treatment (P<0.05, P<0.01). In summary, this study demonstrates that JPHGF exerts therapeutic effects against AONFH by activating the AdipoR/AMPKα/Cpt1b signaling axis, thereby improving bone marrow lipid homeostasis and restoring femoral head microarchitecture, effectively relieving pain and functional impairment. These findings provide new experimental evidence and molecular targets for the prevention and treatment of AONFH in clinical practice.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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