β-sitosterol alleviates neuropathic pain by affecting microglia polarization through inhibiting tlr4/nf-κb signaling pathway

Neurology Biochemistry Life Sciences Immunology Pharmacy Medicine Cell Biology
β-sitosterol tlr4 nf-κb gmi-r1 inflammation sciatica

β-Sitosterol Alleviates Neuropathic Pain by Inhibiting the TLR4/NF-κB Signaling Pathway

Background

Neuropathic pain is a common and difficult-to-treat chronic pain condition in clinical practice, with complex and not fully understood etiology. Research has shown that neuroinflammation is one of the main causes of chronic neuropathic pain. Microglia are resident immune cells in the central nervous system that, under normal conditions, participate in neuronal development and maintenance by secreting neurotrophic factors. However, when the neural microenvironment homeostasis is disrupted by external stimuli, microglia undergo polarization, producing two different phenotypes: the classically activated M1 (pro-inflammatory) phenotype and the alternatively activated M2 (anti-inflammatory) phenotype. After nerve injury, M1 polarization of microglia leads to enhanced inflammatory responses and induces neuropathic pain. Therefore, regulating microglial polarization has become a new strategy for alleviating neuropathic pain.

The TLR4/NF-κB signaling pathway is a well-known classical inflammatory signaling pathway. Activation of TLR4 and its downstream molecules such as MyD88, IκB kinase (IKK), and NF-κB can promote the expression of pro-inflammatory factors, thereby exacerbating neuroinflammation. Thus, inhibiting the activation of the TLR4/NF-κB signaling pathway may be an effective method to reduce neuroinflammation.

β-Sitosterol is a plant-derived compound that is commonly found in daily diets and has fewer side effects. Studies have shown that β-sitosterol not only lowers cholesterol but also has significant anti-inflammatory effects. Recent research has confirmed that β-sitosterol reduces inflammatory responses by inhibiting the TLR4/NF-κB signaling pathway. Based on this background, this study explored the potential role and mechanism of β-sitosterol in alleviating neuroinflammation and neuropathic pain.

Research Source

This article was jointly published by researchers Zheng Yachun, Zhao Jiaji, Chang Shiquan, et al. from Jinan University, mainly exploring the regulatory effects of β-sitosterol on microglial polarization and neuropathic pain. The paper was published in the Journal of Neuroimmune Pharmacology in 2023.

Research Methods

Experimental Design and Procedures

This study used a combination of in vivo and in vitro experiments to investigate the mechanism of action of β-sitosterol. The specific experimental procedures are as follows:

  1. In vivo experiments

    • Experimental animals and grouping: 32 8-week-old male Sprague-Dawley (SD) rats were selected as research subjects and randomly divided into four groups: sham operation group, CCI model group, ibuprofen group, and β-sitosterol group, with 8 rats in each group.
    • Construction of sciatic nerve injury model: The chronic constriction injury (CCI) method was used to construct the sciatic nerve pain model, including transverse cutting of the skin and muscle, followed by four consecutive sutures on the sciatic nerve with 4.0 sutures, with each suture spaced about 1 mm apart.
    • Drug treatment: Starting one day after surgery, corresponding drugs were administered by gavage. The ibuprofen group received ibuprofen solution (10 mg/kg/bid); the β-sitosterol group received β-sitosterol solution (50 mg/kg/bid); the sham operation group and model group received saline (0.9%, 6 ml/kg/bid) for 21 consecutive days.
    • Behavioral experiments: The von Frey test was used to evaluate mechanical pain sensitivity in SD rats.

  2. In vitro experiments

    • Cell culture and grouping: GMI-R1 microglial cells were cultured in vitro and grouped. Groups included control group, LPS group, LPS+TAK-242 group, LPS+JSH-23 group, LPS+β-sitosterol group, and β-sitosterol group.
    • Cell viability assay: The CCK8 assay was used to determine the effect of different concentrations of β-sitosterol on cell viability, and an appropriate concentration was selected for subsequent experiments.
    • Real-time quantitative PCR (qRT-PCR): mRNA expression of related genes in each group of cells was detected.
    • Flow cytometry: Detection of microglial polarization markers CD32 (M1 marker) and CD206 (M2 marker).
    • Immunofluorescence detection: Immunofluorescence labeling and quantitative analysis of M1 and M2 marker expression levels in microglia.
    • Western Blot detection: Detection of related protein expression levels in each group of cells.

  3. Data analysis

    • GraphPad Prism 8.0.2 software was used to process and analyze all experimental data. One-way analysis of variance (ANOVA) was used to compare differences between groups, with the significance level set at P < 0.05.

Main Results

  1. Behavioral experiment results

    • Results showed that mechanical pain sensitivity significantly increased in CCI group rats after surgery, while pain sensitivity gradually returned to normal levels in the ibuprofen and β-sitosterol groups.

  2. Expression of inflammatory factors in serum

    • ELISA results showed that levels of pro-inflammatory factors IL-1β and IL-8 were significantly elevated in the serum of CCI group rats, while levels of pro-inflammatory factors significantly decreased and anti-inflammatory factor IL-4 levels significantly increased after 21 days of β-sitosterol treatment.

  3. HE staining of sciatic nerve

    • HE staining showed disordered sciatic nerve structure, neuronal loss, nuclear pyknosis, and inflammatory infiltration in CCI group rats. After β-sitosterol treatment, nerve fiber structure returned to normal, and nuclear pyknosis significantly improved.

  4. Expression of TLR4/NF-κB signaling pathway-related proteins

    • WB and immunohistochemistry results showed that the CCI model activated the TLR4/NF-κB signaling pathway, upregulated pro-inflammatory factor expression, and promoted microglial activation, while β-sitosterol effectively inhibited these changes.

  5. Cell experiment results

    • CCK8 results determined the optimal concentration of β-sitosterol to be 40 μM.
    • qRT-PCR results showed that β-sitosterol significantly downregulated LPS-induced pro-inflammatory factor expression and upregulated anti-inflammatory factor expression.
    • Flow cytometry and immunofluorescence results indicated that β-sitosterol reduced the proportion of M1 polarization and increased the proportion of M2 polarization in microglia by inhibiting the TLR4/NF-κB signaling pathway.

Conclusion

This study demonstrates that β-sitosterol can alleviate neuropathic pain by regulating microglial polarization and inhibiting the TLR4/NF-κB signaling pathway, thereby reducing neuroinflammation. This provides theoretical foundation and experimental evidence for β-sitosterol as a new drug for treating neuropathic pain.

Research Highlights

  1. β-Sitosterol significantly alleviates neuropathic pain: This study is the first to systematically demonstrate the effectiveness of β-sitosterol in alleviating neuropathic pain.
  2. Reveals potential mechanisms: A series of in vivo and in vitro experiments verified that β-sitosterol affects microglial polarization by regulating the TLR4/NF-κB signaling pathway.
  3. Safety analysis: HE staining results showed that β-sitosterol has no obvious hepatorenal toxicity, indicating good prospects for clinical application.
  4. Innovative methods: Combining in vivo behavioral tests and in vitro molecular biology techniques to comprehensively explore the mechanism of drug action.