ANA-12 Promotes Astrocyte Autophagy in Neuropathic Pain by Modulating TRKB/mTOR/c-JUN Axis

Abstract

Background: Neuropathic pain (NP) is a complex pain disorder caused by injury or dysfunction of the somatosensory nervous system. This study aimed to investigate the therapeutic effect of ANA-12 (a BNDF (brain-derived neurotrophic factor) inhibitor, N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl) amino] carbonyl] phenyl] benzo [b] thiophene-2-carboxamide, C₂₂H₂₁N₃O₃S, CAS No. 219766-25-3) on NP and its molecular mechanism. Methods: Rats were set into sham, model and ANA-12 groups (n = 10 mice/group). NP was induced in rats by sciatic nerve ligation. Astrocytes were treated with lipopolysaccharide (LPS, 100 ng/mL), ANA-12 (50 µM) and MHY1485 (10 µmol/L, C₁₇H₂₁N₇O₄, CAS No. 326914-06-1) to explore the role of ANA-12. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed. Rat spinal dorsal horn astrocytes were activated by LPS induction. Then, rats were treated with ANA-12 and mechanistic target of rapamycin (mTOR) agonists. Cell viability was monitored with cell counting kit 8 (CCK8). Immunofluorescence was used to test activated astrocytes and low complexity communications codec (LC3B) expression. Enzyme linked immunosorbent assay (ELISA) was conducted to measure interleukin (IL)-1β, IL-6 and tumour necrosis factor alpha (TNF-α) concentrations. The expression of autophagy and signaling pathway-related proteins and genes was analyzed by western blot and reverse transcription quantitative-polymerase chain reaction (RT-qPCR). Results: MWT and TWL of NP rats were significantly reduced compared to normal rats. The number of activated astrocytes increased in the spinal cord tissue of NP rats. The concentration of inflammatory factors and the expression of brain-derived neurotrophic factor (BNDF) and its specific receptor tropomyosin-receptor kinase B (TRKB) proteins increased in the spinal cord of NP rats. After ANA-12 treatment, astrocytes cell viability did not change significantly. Interestingly, ANA-12 treatment significantly reduced LPS-induced activation of astrocytes and pro-inflammatory factors compared to NP rats. Meanwhile, BNDF and TRKB proteins expression decreased, and LC3B and Beclin 1 genes and proteins expression increased after ANA-12 treatment in NP rats. mTOR agonists reversed the autophagy-promoting effect of ANA-12. Conclusions: Many astrocytes were abnormally activated in NP rats. ANA-12 inhibited inflammation and targeted mTOR to promote autophagy and reduce the activation of LPS-induced astrocytes. In summary, ANA-12 can alleviate NP.