[Mechanism of Spatholobi Caulis in inhibiting microglial activation to intervene neuropathic pain through TLR4/MyD88/NF-κB signaling].

Based on network pharmacology and molecular docking combined with animal experimental validation, this study aims to explore the mechanism of action of Spatholobi Caulis in the treatment of neuropathic pain(NP). Ultra-high-performance liquid chromatography Q-Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS) was used to identify the chemical constituents of Spatholobi Caulis, analyze the core drug-disease targets, and perform pathway enrichment and molecular docking validation. Forty-eight male Sprague Dawley(SD) rats were randomly divided into a sham group, a model group, high-dose, medium-dose, and low-dose groups of Spatholobi Caulis, and a positive control group of Pregabalin, with eight rats in each group. The NP model was established by chronic constriction injury(CCI) to the sciatic nerve of rats in all groups except the sham group in which only the sciatic nerve was exposed without ligation. Dosing was initiated after the behavioral measurements on the 5th day following modeling and was administered for 14 days. On one day before surgery, the 3rd and 5th days after CCI, and the 3rd, 7th, 11th, and 14th days after drug administration, the mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) of the experimental rats were measured by electronic Von Frey and a plantar test apparatus to assess changes in pain behavior. Immunohistochemistry was utilized to detect the expression of ionized calcium binding adapter molecule 1(Iba1) in spinal cord tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the level of interleukin-6(IL-6), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in spinal cord tissue, and Western blot and immunofluorescence were used to detect the expression of Toll-like receptor 4(TLR4), myeloid differentiation primary response 88(MyD88), nuclear factor kappaB(NF-κB) p65, phosphorylated NF-κB(p-NF-κB) p65, IL-6, IL-1β, TNF-α, and Iba1 proteins in spinal cord tissue. The results indicated that a total of 150 compounds were identified in the Spatholobi Caulis, including 61 flavonoids, 23 isoflavonoids, 11 organic oxides, 5 coumarins, 5 linear 1,3-diphenylpropanes, and 45 other types of compounds. Network pharmacology analysis revealed that for NP treatment with Spatholobi Caulis, epidermal growth factor receptor(EGFR), protein kinase B(AKT1), signal transducer and activator of transcription 3(STAT3), TNF, cysteine-aspartic acid protease-3(CASP3), mitogen-activated protein kinase 3(MAPK3), and TLR4 were the core targets, and the regulatory effect on the TLR signaling pathway was the key mechanism. Experimental verification results showed that compared with the sham group, the model group exhibited significantly decreased MWT and TWL(P<0.01) and significantly increased expressions of TLR4, MyD88, NF-κB p65, p-NF-κB p65, Iba1, IL-1β, TNF-α, and IL-6 proteins in spinal cord tissue(P<0.01). Compared with the model group, the high-dose group of Spatholobi Caulis displayed significantly increased MWT and TWL in NP rats(P<0.05) and down-regulated expressions of TLR4, MyD88, NF-κB p65, p-NF-κB p65, Iba1, IL-1β, TNF-α, and IL-6 proteins in spinal cord tissue(P<0.05). The molecular docking results showed that multiple components had a good affinity for TLR4 and MyD88, and buddleoside had the highest affinity for TLR4 and MyD88. In summary, Spatholobi Caulis can inhibit microglia activation and alleviate NP by modulating the TLR4/MyD88/NF-κB signaling pathway.