白芷在神经病理性疼痛中对MrgprD-TRPA1通路的调控作用分析

Analysis of the regulatory effect of Angelica dahurica on the MrgprD-TRPA1 pathway in neuropathic pain

目的本研究拟通过建立坐骨神经慢性缩窄损伤(chronic constriction injury,CCI)小鼠模型,分析和探讨白芷在神经病理性疼痛中的镇痛效果及其对MrgprD-TRPA1信号通路的调控作用。方法无菌外科手术结扎缠绕30只小鼠坐骨神经制备CCI小鼠模型;VonFrey实验检测白芷对小鼠机械刺激疼痛行为学变化,热辐射实验评估白芷对小鼠热痛觉过敏情况;Western Blot、免疫荧光、RT-PCR检测白芷对小鼠MrgprD和TRPA1蛋白表达水平、DRG阳性神经元数量、MrgprD和TRPA1 mRNA水平的影响;通过对HEK293细胞分别单转染和共转染MrgprD、TRPA1质粒后的钙成像实验,分析荧光信号强度差异性。结果共成功制备了25只CCI小鼠模型,造模率达到83.33%(25/30);白芷灌胃的CCI小鼠机械性阈值和缩足潜伏时间均显著大于对照组(P<0.05);白芷灌胃的CCI小鼠中MrgprD和TRPA1蛋白表达水平均显著低于对照组(P<0.05);白芷灌胃的CCI小鼠DRG中MrgprD和TRPA1阳性神经元的数量显著低于对照组(P<0.05);白芷灌胃的CCI小鼠中MrgprD和TRPA1 mRNA相对表达水平均显著低于对照组(P<0.05);共转染MrgprD和TRPA1质粒的HEK293细胞中荧光强度显著高于单转染和对照组(P<0.05)。结论本研究通过探究白芷在CCI小鼠模型中镇痛的效果,证明了MrgprD-TRPA1是神经病理性疼痛的重要作用靶点,揭示了白芷可以通过调控MrgprD-TRPA1信号转导通路来抑制神经病理性疼痛程度,这为后续开发新型临床镇痛药物及镇痛机制的深入研究奠定了基础。

Objective To analyze and explore the analgesic effect of Angelica dahurica in neuropathic pain and its regulatory effect on the Mas-related G-protein coupled receptor member D(MrgprD)-transient receptor potential ankyrin 1(TRPA1)signaling pathway,using a mouse model of sciatic nerve chronic constriction injury(CCI).Methods A CCI mouse model was prepared by sterile surgical ligation and wrapping of the sciatic nerve in 30 mice.Pain-related behavioral changes induced by mechanical stimulation were detected by the VonFrey method,and the thermal hyperalgesic effects of Angelica dahurica were evaluated by thermal radiation experiments.The effects of Angelica dahurica on the protein expression levels MrgprD and TRPA1,the number of dorsal root ganglion(DRG)positive neurons,and mRNA levels of MrgprD and TRPA1 in mice were detected by Western Blot,immunofluorescence,and reverse transcription-polymerase chain reaction,respectively.Differences in fluorescence signal intensity in HEK293 cells after single transfection and co-transfection with MrgprD and TRPA1 plasmids,respectively,were analyzed by calcium imaging experiments.Results A total of 25 CCI mouse models were successfully prepared,with a modeling rate of 83.33%(25/30).The mechanical threshold and foot retraction latency were significantly higher in CCI mice treated with Angelica dahurica compared with the control group(P<0.05).Expression levels of MrgprD and TRPA1 proteins were significantly lower in CCI mice treated with Angelica dahurica than in the control group(P<0.05).The number of MrgprD-and TRPA1-positive neurons in the DRG was significantly lower group(P<0.05)and the mRNA levels of MrgprD and TRPA1 were also significantly lower in CCI mice treated with Angelica dahurica than in the control group(P<0.05).The fluorescence intensity was significantly higher in HEK293 cells co-transfected with MrgprD and TRPA1 plasmids than in single-transfected and blank control cells(P<0.05).Conclusions This study demonstrated that the MrgprD-TRPA1 pathway is an important target for neuropathic pain,and indicated that Angelica dahurica can inhibit neuropathic pain by regulating this signal transduction pathway.These result provide a foundation for further research on the development of new clinical analgesic drugs and analgesic mechanisms.