Ethnopharmacological relevance

Ruyi Zhenbao Pill (RYZBP) is a traditional Tibetan medicine that has been used for over 300 years in China to treat neurological diseases, specifically neuropathic pain (NP). However, its characteristics and mechanism of action in treating NP remains unclear.

Aim of the study

Based on animal experiments and transcriptomics to evaluate the characteristics and mechanism of RYZBP in treating NP.

Methods

Mice were divided into six groups using random assignment: sham-operation group, spinal nerve ligation (SNL) group, RYZBP low (0.65 g kg⁻¹), medium (1.30 g kg⁻¹), high (2.60 g kg⁻¹) doses groups, and positive drug pregabalin (PGB, 0.05 g kg⁻¹) group. Mice received intragastrical administered for 14 consecutive days. SNL and intrathecal injection models were employed. The analgesic effects were assessed using the Von Frey test, Acetone test, and Hot Plate test. L5 spinal dorsal horns were collected for transcriptomics on day 15. The potential signaling pathways and Hub genes of RYZBP to ameliorate NP were obtained through transcriptomics and network pharmacology. Molecular docking was utilized to evaluate the binding ability of candidate active ingredients with the Hub genes. Finally, western blot (WB) and immunofluorescence (IF) were used to validate the predicted targets.

Results

RYZBP demonstrated a dose-dependent alleviation of mechanical allodynia, cold and heat stimulus-induced pain in SNL mice. Transcriptomics analysis identified 24 differentially expressed genes, and pathway enrichment analysis revealed that the CXCL10-CXCR3 signal axis may be the primary biological pathway through which RYZBP relieve NP. Molecular docking test indicated that the active ingredient in RYZBP exhibit a strong affinity for the target protein CXCL10. WB and IF tests showed that RYZBP can significantly inhibit CXCL10 and CXCR3 and its downstream molecules expression in the spinal dorsal horn of SNL mice. Additionally, intrathecal injection of rmCXCL10 worsened pain hypersensitivity, while RYZBP was able to suppress the pain hypersensitivity response induced by rmCXCL10 and reduce the expression levels of CXCL10 and CXCR3 and its downstream molecules.

Conclusion

RYZBP had a significant analgesic effect on NP model, and this effect may be related to inhibiting the CXCL10-CXCR3 pathway in the spinal dorsal horn.