Integrated Network Pharmacology and Metabolomics to Reveal the Mechanisms of the Combined Intervention of Ligustrazine and Sinomenine in CCI-Induced Neuropathic Pain Rats

Neuropathic pain (NP) is a chronic pain resulting from injury or dysfunction of the nerves or spinal cord. Previous studies have shown that the combination of ligustrazine (LGZ) and sinomenine (SIN) exerts a synergistic antinociceptive effect in peripheral and central NP models. This study aims to evaluate the pharmacodynamic characteristics of LGZ and SIN in a chronic constriction injury (CCI)-induced NP models in rats, and to analyze its molecular regulatory mechanisms from the perspectives of network pharmacology and metabolomics. Firstly, a comprehensive analgesic evaluation including the mechanical withdrawal threshold test, cold allodynia test, and the incapacitance test was performed after 1, 2 and 3 days of intervention. The sciatic nerve histopathological changes were observed, and inflammatory factor analysis were conducted. Secondly, the pain-related targets of LGZ and SIN were systematically analyzed based on network pharmacology. The differential metabolites in the plasma and cerebrospinal fluid (CSF) were screened, and their content were quantified using LC-MS metabolomics technology. Finally, the key metabolic pathways with crucial role of LGZ and SIN in treating NP was identified from a joint analysis of the potential targets and the differential metabolites. The combination of LGZ and SIN significantly alleviated pain-like behaviors of CCI rats time- and dose-dependently, and the therapeutic effect was notably superior to that of LGZ or SIN alone. The combination of LGZ and SIN could improve pathological damage to the sciatic nerve and regulate the levels of inflammatory cytokine. Network pharmacology analysis revealed that LGZ and SIN have 6 shared pain-related targets, while LGZ have 16 distinct pain-related targets and SIN have 52 distinct pain-related targets. The metabolomics analyses revealed that 54 differential metabolites in plasma and 17 differential metabolites in CSF were associated with the combined intervention of LGZ and SIN. Finally, through integrated analysis of the core targets and differential metabolites, tyrosine metabolism, phenylalanine metabolism, and arginine and proline metabolism were identified as potential key metabolic pathways underlying the therapeutic effects of LGZ and SIN in CCI treatment. This study provides an integrated approach to elucidate the molecular mechanisms underlying the combined use of LGZ and SIN in the treatment of NP. Furthermore, the findings offer new experimental evidence to support the clinical application of LGZ and SIN in the treatment of NP.