Tetrahydropalmatine ameliorates peripheral nerve regeneration by enhancing macrophage anti-inflammatory response

Peripheral nerve injury (PNI) is a common clinical problem that can result in partial or complete loss of sensory, motor, and autonomic functions. Tetrahydropalmatine (THP), a Corydalis yanhusuo-derived phytochemical alkaloid, possesses hypnotic, soothing, analgesic, and other effects, but little is known about the effect of THP on moderating peripheral nerve regeneration and its possible underlying mechanism of action.

In this study, we aim to elucidate the protective function of THP on PNI and further reveal the underlying pharmacological mechanisms.

PNI rats were in suit injection of THP solution at doses of 40 mg/kg for consecutive 3, 7, or 28 days, followed by harvesting the sciatic nerve tissues. The protective effect of THP on PNI was evaluated by electrophysiological test, transmission electron microscopy, immunofluorescence (IF), and western blotting (WB). Macrophage polarization, the expression of inflammatory-related genes and cytokines, and its upstream signaling pathways were detected by IF, WB, enzyme-linked immunosorbent assay (ELISA), mRNA-seq, and WB. In vitro, the Raw 264.7 cells were treated with lipopolysaccharide containing with/without THP. The degree of inflammatory activation and its potential pharmacological mechanism were measured by ELISA, qRT-PCR, IF staining, flow cytometry, and WB. Additionally, a pharmacological agonist or inhibitor was added to the cell medium to further identify the role of THP's potential pharmacological mechanism in regulating inflammatory response via IF and ELISA technology.

Using the sciatic nerve crush model, we found that THP significantly enhanced the rate of axonal growth and functional recovery, and altered macrophage subtype transformation from the M1/M0 phenotype into the M2 phenotype, inducing the secretion of large amounts of anti-inflammatory factors. Moreover, THP significantly increased the phosphorylation level of PI3K, AKT, GSK3β, and IκBa, and decreased the expression of TLR4 protein and NF-κB phosphorylation. Similarly, in vitro, THP also facilitated Raw 264.7 cell polarization to the M2 subtype under the condition of LPS stimulation. Meanwhile, the change of PI3K/AKT/GSK3β and TLR4/NF-κB signaling-related proteins in vitro was consistent with the results in vivo. Additionally, the THP-medicated anti-inflammatory effect on Raw 264.7 cells was partly eliminated when pharmacological intervention of these two signaling pathways.

THP has anti-inflammatory effects on facilitating M2-subtype macrophage polarization, which produces abundant anti-inflammatory cytokines to ameliorate peripheral nerve regeneration. Moreover, the potential mechanism of THP action may be intimately associated with activating the PI3K/AKT/GSK3β axis and inhibiting the TLR4/NF-κB pathway.