Objective Intervertebral disc degeneration (IDD) is the main cause of low back pain, which is closely related to an imbalance in extracellular matrix decomposition–anabolism mediated by immune inflammation. Duhuo Jisheng decoction (DHJSD) is effective in treating IDD, but its specific mechanism of action remains unclear and warrants further study. This study aimed to investigate the role of DHJSD in IDD treatment and its underlying mechanisms of action, providing potential therapeutic targets for IDD.
Methods High-performance liquid chromatography‒mass spectrometry (HPLC‒MS/MS) was employed to characterize the chemical composition of DHJSD. A Cell Counting Kit-8 (CCK-8) was used to detect the effects of DHJSD on the viability of pressure-treated nucleus pulposus (NP) cells. NP cells were randomly divided into three groups: control, pressure, and DHJSD groups. Western blotting was used to detect the expression of NLRP3, ASC, caspase-1, IL-1β, collagen II, aggrecan, Sox-9, MMP-3, MMP-13, and Adamts-4 in the aforementioned three groups of cells. An immunofluorescence assay was used to detect NLRP3 expression. Additional groups included the DHJSD + dimethyl sulfoxide (DMSO) and DHJSD + cyclosporine A groups. The expression of mitophagy-related factors, the NLRP3 inflammasome, and subsequent inflammatory reactions were detected by Western blotting and immunofluorescence. Network pharmacology and molecular docking were used to explore the key components and key genes of DHJSD involved in treating IDD. Finally, we verified the bioinformatics results using a rat tail acupuncture model of IDD.
Results DHJSD-containing serum improved NP cell viability; the optimal intervention concentration was 20% by volume, and the optimal intervention time was 24 h. Compared with the pressure group, the DHJSD group presented significantly decreased expression of NLRP3, ASC, caspase-1, IL-1β, MMP-3, MMP-13, Adamts-4, and p62, and significantly increased expression of collagen II, aggrecan, Sox-9, PINK1, Parkin, and LC3. However, upon the addition of a mitophagy inhibitor, the protective effect of DHJSD on NP cells was diminished. Network pharmacology and molecular docking studies revealed that HIF-1α may be the key target of DHJSD in the treatment of IDD. Imaging and histopathological results confirmed that DHJSD delayed IDD progression. Western blotting and immunohistochemistry revealed that DHJSD increased HIF-1α expression, regulated mitochondrial division and fusion, and inhibited the activation of the NLRP3 inflammasome and subsequent inflammatory reactions.
Conclusion DHJSD exerts a protective effect on NP cells by suppressing the activation of the NLRP3 inflammasome and subsequent imbalance in the extracellular matrix via mitophagy, which is potentially associated with the activation of the HIF-1α signaling pathway and the preservation of the metabolic balance of the mitochondria.
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Funding
The General Project of Hubei Natural Science Foundation(2025 AFB904)
The Traditional Chinese Medicine Research Project of Hubei Provincial Administration of Traditional Chinese Medicine(ZY2025Q033)
The Health Science and Technology Project of Hubei Province(WJ2025M037)
RIGHTS & PERMISSIONS
The Author(s), under exclusive licence to the Huazhong University of Science and Technology
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