Jiang Yongsheng, Cao Jianye, Li Rui, Yu Jia, Peng Yan, Huang Qiong, Zuo Wei, Chen Junyue
Xiangshan Hospital of Wenzhou Medical University, Ningbo, Zhejiang 315700, P.R. China.
Wenzhou Medical University, Wenzhou 325035, China.
Int Immunopharmacol. 2025 Feb 6;147:114000. doi: 10.1016/j.intimp.2024.114000. Epub 2025 Jan 6.
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.
周围神经损伤（PNI）是一个常见的临床问题，可导致感觉、运动和自主神经功能部分或完全丧失。四氢巴马汀（THP）是一种从延胡索中提取的植物化学生物碱，具有催眠、安神、镇痛等作用，但关于THP对周围神经再生的调节作用及其潜在作用机制知之甚少。
在本研究中，我们旨在阐明THP对PNI的保护作用，并进一步揭示其潜在的药理机制。
对PNI大鼠连续3、7或28天腹腔注射40mg/kg剂量的THP溶液，随后采集坐骨神经组织。通过电生理测试、透射电子显微镜、免疫荧光（IF）和蛋白质免疫印迹法（WB）评估THP对PNI的保护作用。通过IF、WB、酶联免疫吸附测定（ELISA）、mRNA测序和WB检测巨噬细胞极化、炎症相关基因和细胞因子的表达及其上游信号通路。在体外，用含或不含THP的脂多糖处理Raw 264.7细胞。通过ELISA、qRT-PCR、IF染色、流式细胞术和WB测量炎症激活程度及其潜在的药理机制。此外，在细胞培养基中添加药理激动剂或抑制剂，以通过IF和ELISA技术进一步确定THP潜在药理机制在调节炎症反应中的作用。
使用坐骨神经挤压模型，我们发现THP显著提高了轴突生长速率和功能恢复，并改变了巨噬细胞亚型从M1/M0表型向M2表型的转变，诱导大量抗炎因子的分泌。此外，THP显著提高了PI3K、AKT、GSK3β和IκBa的磷酸化水平，并降低了TLR4蛋白的表达和NF-κB磷酸化。同样，在体外，THP在LPS刺激条件下也促进了Raw 264.7细胞向M2亚型的极化。同时，体外PI3K/AKT/GSK3β和TLR4/NF-κB信号相关蛋白的变化与体内结果一致。此外，当对这两条信号通路进行药理干预时，THP对Raw 264.7细胞的抗炎作用部分被消除。
THP具有促进M2亚型巨噬细胞极化的抗炎作用，可产生丰富的抗炎细胞因子以改善周围神经再生。此外，THP的潜在作用机制可能与激活PI3K/AKT/GSK3β轴和抑制TLR4/NF-κB通路密切相关。
