- Authors: Wei X. et al.
- Year: 2021
- Journal: J Neuroinflammation 18 96
- Applications: in vitro / shRNA plasmid / jetOPTIMUS
- Cell type: PC-12
- The PC-12 cells were seeded in 6-well plates at a concentration of 4 × 105 cells per well and cultured at 37 °C for 24 h to reach 70–80% confluent. - Then, cells were washed with PBS three times and incubated with jetOPTIMUS® (2μg DNA and 2 μL reagent in 200 μL jetOPTIMUS® buffer) following the manufacturer’s protocol. - Four hours later, the medium was changed to an antibiotic-free culture medium. - After transfection for 24 hours, we observed transfection efficiency by using a fluorescent microscope. ASIC3 shRNA knockdown efficiency was confirmed by RT-PCR and western blotting analysis.
Background: Postherpetic neuralgia (PHN) is a devastating complication after varicella-zoster virus infection. Brain-derived neurotrophic factor (BDNF) has been shown to participate in the pathogenesis of PHN. A truncated isoform of the tropomyosin receptor kinase B (TrkB) receptor TrkB.T1, as a high-affinity receptor of BDNF, is upregulated in multiple nervous system injuries, and such upregulation is associated with pain. Acid-sensitive ion channel 3 (ASIC3) is involved in chronic neuropathic pain, but its relation with BDNF/TrkB.T1 in the peripheral nervous system (PNS) during PHN is unclear. This study aimed to investigate whether BDNF/TrkB.T1 contributes to PHN through regulating ASIC3 signaling in dorsal root ganglia (DRGs). Methods: Resiniferatoxin (RTX) was used to induce rat PHN models. Mechanical allodynia was assessed by measuring the paw withdrawal thresholds (PWTs). Thermal hyperalgesia was determined by detecting the paw withdrawal latencies (PWLs). We evaluated the effects of TrkB.T1-ASIC3 signaling inhibition on the behavior, neuronal excitability, and inflammatory response during RTX-induced PHN. ASIC3 short hairpin RNA (shRNA) transfection was used to investigate the effect of exogenous BDNF on inflammatory response in cultured PC-12 cells. Results: RTX injection induced mechanical allodynia and upregulated the protein expression of BDNF, TrkB.T1, ASIC3, TRAF6, nNOS, and c-Fos, as well as increased neuronal excitability in DRGs. Inhibition of ASIC3 reversed the abovementioned effects of RTX, except for BDNF and TrkB.T1 protein expression. In addition, inhibition of TrkB.T1 blocked RTX-induced mechanical allodynia, activation of ASIC3 signaling, and hyperexcitability of neurons. RTX-induced BDNF upregulation was found in both neurons and satellite glia cells in DRGs. Furthermore, exogenous BDNF activated ASIC3 signaling, increased NO level, and enhanced IL-6, IL-1β, and TNF-α levels in PC-12 cells, which was blocked by shRNA-ASIC3 transfection. Conclusion: These findings demonstrate that inhibiting BDNF/TrkB.T1 reduced inflammation, decreased neuronal hyperexcitability, and improved mechanical allodynia through regulating the ASIC3 signaling pathway in DRGs, which may provide a novel therapeutic target for patients with PHN.