Augmented Cornus officinalis Levels in Liuwei Dihuang Decoction Inhibits Nucleus Pulposus Cell Pyroptosis to Enhance Therapeutic Efficacy Against Intervertebral Disc Degeneration

Abstract

Background: Intervertebral disc (IVD) degeneration (IVDD) is highly prevalent among the elderly population and stands as a leading cause of low back pain. Our prior studies have highlighted the therapeutic potential of Liuwei Dihuang decoction (LWDHD) and its component Cornus officinalis (CO)-derived compounds in alleviating IVDD and osteoarthritis, suggesting beneficial effects of CO in treating degenerative osteoarthropathies. However, uncertainty remains regarding the optimal CO dosage within LWDHD and its potential mechanism for effectively treating IVDD.

Objective: To ascertain the optimal dosage of CO within LWDHD for enhancing its therapeutic efficacy in treating IVDD, through a comparison of its effects across varied dosages using a mouse IVDD model.

Methods: Eight-week-old male C57BL/6J mice were subjected to a lumbar spine instability surgery to induce an IVDD model and received a modified LWDHD formulation containing varied dosages of CO (original dose of CO, or 5- or 10-time dose of CO (referred to as 1 × CO, 5 × CO, and 10 × CO)) for 8 weeks. The therapeutic efficacy on IVDD was evaluated through changes in lumbar spine function, histopathological morphology, extracellular matrix metabolism, nucleus pulposus cell viability, sensory nerve ingrowth, and nucleus pulposus (NP) cell pyroptosis.

Results: Augmenting CO levels in LWDHD led to a dose-dependent increase in the levels of CO-sourced active compounds in the plasma of mice. The modified LWDHD formulations, particularly the 5 × CO, exhibited a favorable pharmacological effect on lumbar function, structural integrity, ECM composition, NP cell viability, and sensory nerve ingrowth. Importantly, all 3 formulations notably mitigated NP cell pyroptosis by activating NRF2/KEAP1 pathway, with the 5 × CO formulation exhibiting superior efficacy. Additionally, a comprehensive score analysis indicated that 5 × CO formulation achieved the highest score.

Conclusion: These data underscore that elevating the dosage of CO to a specific threshold can enhance the effectiveness of LWDHD in treating IVDD.

Keywords: Cornus officinalis; intervertebral disc degeneration; liuwei dihuang decoction; nucleus pulposus cell; pyroptosis.

Figure 1

LWDHD formulations enhance lumbar function of IVDD mice. (A) Analysis and quantification of rear frequency test performed in IVDD mice after treatment with LWDHD formulation for 8 weeks. (B) Quantification of Von-Frey pain test. (C) Quantification of total distance, central motion time, and distance relative to the center in open field test of mice. Data are shown as mean  ±  SEM (n = 6). *Indicates a significant difference compared with the Sham group (**p < 0.01), ^#Means a significant difference compared with the Vehicle group (^#p < 0.05, ^##p < 0.01), ⁺Denotes a significant difference compared with the LWDHD group (⁺⁺p < 0.01).

Figure 2

LWDHD formulations alleviate IVDD progression in LSI surgery-induced IVDD mice. (A) HE staining of the IVDs of IVDD mice after treatment with LWDHD formulation for 8 weeks. Black arrows indicate ectopic bone formation. Blue arrows indicate tearing in annulus fibrosus. (B) Histological grading scores of IVD tissues in (A). (C and D) IHC analysis and corresponding quantification of AGGRECAN in IVDs of IVDD mice. Black arrows indicate high expression of AGGRECAN. (E–H) IHC analysis and corresponding quantification of COL2 (E and F) and MMP13 (G and H) in IVDs of mice. Black arrows indicate high expression of COL2 and MMP13. Data are shown as mean  ±  SEM (n = 6). *Denotes a significant difference compared with the Sham group (*p < 0.05, **p < 0.01), ^#Means a significant difference compared with the Vehicle group (^##p < 0.01), ⁺Indicates a significant difference compared with the LWDHD group (⁺p < 0.05, ⁺⁺p < 0.01), ^&Signifies a significant difference compared with the 5 × CO group (^&p < 0.05, ^&&p < 0.01).

Figure 3

LWDHD formulations mitigate NP cell viability and innervation in IVDs of IVDD mice. (A and B) TUNEL assay of the IVD section (A) and corresponding qualification (B) of the rate of positive cells in NP tissues of IVDD mice after treatment with LWDHD formulation for 8 weeks. DAPI stains nuclei (Blue). White arrows indicate high expression in the IVDs. (C–F) IF staining for Ki-67 (C and D) and TrkA expression (E and F) in IVDs of IVDD mice after 8 weeks of treatment with LWDHD formulation. White arrows indicate high expression in the IVDs. Data are shown as mean  ±  SEM (n = 6). *Denotes a significant difference compared with the Sham group (**p < 0.01), ^#Means a significant difference compared with the Vehicle group (^#p < 0.05, ^##p < 0.01), ⁺Indicates a significant difference compared with the LWDHD group (⁺⁺p < 0.01), ^&Means a significant difference compared with the 5 × CO group (^&&p < 0.01).

Figure 4

LWDHD formulations inhibit NP cell pyroptosis in IVDD mice. (A–D) IF staining and corresponding qualification of CASPASE1 (A and B), GSDMD (C and D) in IVDs of IVDD mice after treatment with LWDHD formulation for 2 weeks. White arrows indicate high expression of CASPASE1 and GSDMD in NP tissues. (E and F) IHC staining and corresponding qualification of IL-1β in IVDs of IVDD mice. Black arrows indicate high expression of IL-1β in NP tissues. Data are shown as mean  ±  SEM (n = 6). *Denotes a significant difference compared with the Sham group (**p < 0.01), ^#Means a significant difference compared with the Vehicle group (^##p < 0.01), ⁺Indicates a significant difference compared with the LWDHD group (⁺p < 0.05, ⁺⁺p < 0.01), ^&Means a significant difference compared with the 5 × CO group (^&&p < 0.01).

Figure 5

LWDHD formulations activate the NRF2 antioxidant pathway in IVDs of IVDD mice. (A and B) IF staining and corresponding qualification of NRF2 (S40) in IVDs of IVDD mice after treatment with LWDHD formulation for 1 week. White arrows indicate high expression of NRF2 (S40) in NP tissues. (C and D) IHC staining and corresponding qualification of KEAP1 in IVDs of IVDD mice. Black arrows indicate high expression of KEAP1 in NP tissues. (E–H) IF staining and corresponding qualification of p-I-κB (E and F) and p-P65 (G and H) in IVDs of IVDD mice. Data are shown as mean ±  SEM (n = 6). *Denotes a significant difference compared with the Sham group (**p < 0.01), ^#Means a significant difference compared with the Vehicle group (^#p < 0.05, ^##p < 0.01), ⁺Indicates a significant difference compared with the LWDHD group (⁺⁺p < 0.01), ^&Means a significant difference compared with the 5 × CO group (^&&p < 0.01).

Figure 6

Schematic illustration of the mechanism by which LWDHD formulation, containing varying dosages of CO, protects against IVDD progression through the inhibition of NP pyroptosis by activating the NRF2 pathway. The schematic illustration is drawn by Figdraw.