Tian Sheng, Wu Lanxiang, Zheng Heqing, Cheng Zhijuan, Liu Jingjing, Liu Mingxu, Yu Xinping, Tu Jianglong, Wu Wei
Department of Neurology, the Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China.
Laboratory of Neurological Diseases, Nanchang, China.
J Headache Pain. 2025 Jul 22;26(1):165. doi: 10.1186/s10194-025-02109-w.
Microglia pyroptosis, a newly identified form of inflammatory cell death, is involved in the development of neuropathic pain (NP). Botulinum toxin type A (BTX-A) has been shown to be effective in relieving NP, but the mechanisms involved have not been clarified.
A mice model of NP was established with chronic constriction injury (CCI) method. The expression levels of key molecules and the extent of microglia pyroptosis were assessed using RT-qPCR, western blot, ELISA and immunofluorescence. Moreover, lipopolysaccharide (LPS) was used in vitro to induce pyroptosis of microglia to explore the potential molecular mechanisms of BTX-A.
In a mice model of NP, BTX-A administration increased the pain threshold and decreased the Cblb protein expression level, consistent with the results of in vitro experiments. Functional experiments and mouse models were respectively used to evaluate the severity of microglia pyroptosis. The results showed that BTX-A inhibited microglia pyroptosis through Cblb protein. Subsequently, mass spectrometry (MS) analysis and immunoprecipitation were conducted to identify proteins interacting with Cblb. The results identified Pdlim1 was a potential interacting partner of Cblb, which regulats the ubiquitination of Pdlim1. Mechanically, Cblb binds to the PDZ and LIM domains of Pdlim1 and then targets Pdlim1 at K244 for ubiquitination and proteasome-mediated degradation. Pdlim1 knockdown lentiviral plasmid was constructed and stable Pdlim1 knockdown microglial cell lines were established for rescue experiments. The results demonstrated that BTX-A suppresses microglia pyroptosis via Pdlim1/NF-κB signaling axis. Finally, intrathecal injection of adeno-associated virus overexpressing Cblb was used in rescue experiments. The results confirmed that BTX-A attenuates neuropathic pain via the Cblb/Pdlim1/NF-kB signaling axis.
This study demonstrates that BTX-A suppresses the activity of Cblb, thereby reducing Pdlim1 protein degradation, inhibiting the NF-kB pathway, and ultimately mitigating microglia pyroptosis. Our findings suggest that Cblb could serve as a novel therapeutic target for BTX-A in the treatment of NP.
小胶质细胞焦亡是一种新发现的炎症性细胞死亡形式，参与神经性疼痛（NP）的发生发展。A型肉毒杆菌毒素（BTX-A）已被证明可有效缓解NP，但其作用机制尚未阐明。
采用慢性缩窄损伤（CCI）法建立NP小鼠模型。运用RT-qPCR、蛋白质免疫印迹法、酶联免疫吸附测定（ELISA）和免疫荧光法评估关键分子的表达水平及小胶质细胞焦亡程度。此外，体外使用脂多糖（LPS）诱导小胶质细胞焦亡，以探究BTX-A的潜在分子机制。
在NP小鼠模型中，给予BTX-A可提高疼痛阈值并降低Cblb蛋白表达水平，这与体外实验结果一致。分别采用功能实验和小鼠模型评估小胶质细胞焦亡的严重程度。结果表明，BTX-A通过Cblb蛋白抑制小胶质细胞焦亡。随后，进行质谱（MS）分析和免疫沉淀以鉴定与Cblb相互作用的蛋白质。结果确定Pdlim1是Cblb的潜在相互作用伴侣，其调节Pdlim1的泛素化。从机制上讲，Cblb与Pdlim1的PDZ和LIM结构域结合，然后将Pdlim1在K244处靶向泛素化并通过蛋白酶体介导降解。构建Pdlim1敲低慢病毒质粒并建立稳定的Pdlim1敲低小胶质细胞系用于拯救实验。结果表明，BTX-A通过Pdlim1/NF-κB信号轴抑制小胶质细胞焦亡。最后，在拯救实验中使用鞘内注射过表达Cblb的腺相关病毒。结果证实，BTX-A通过Cblb/Pdlim1/NF-κB信号轴减轻神经性疼痛。
本研究表明，BTX-A抑制Cblb的活性，从而减少Pdlim1蛋白降解，抑制NF-κB途径，并最终减轻小胶质细胞焦亡。我们的研究结果表明，Cblb可作为BTX-A治疗NP的新治疗靶点。
