Dihydroartemisinin Regulates Neuro-microglia to Relieve Neuropathic Pain

LIANG Tingjun; ZHANG Guoxin; LIU Ying; ZHU Chunyan; LIN Na

doi:10.13422/j.cnki.syfjx.20230440

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Dihydroartemisinin Regulates Neuro-microglia to Relieve Neuropathic Pain

更新时间：2023-04-13

- Dihydroartemisinin Regulates Neuro-microglia to Relieve Neuropathic Pain
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 10, Pages: 73-82(2023)
- 作者机构：
  
  1.福建中医药大学药学院，福州 350108
  2.中国中医科学院中药研究所，北京 100700
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230440
  CLC： R2-0;R33;R289;R745.1
- Received：01 February 2023，
  
  Published Online：10 March 2023，
  
  Published：20 May 2023
- 稿件说明：
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梁婷钧,张国鑫,刘莹等.双氢青蒿素调控神经-小胶质细胞缓解神经病理性疼痛[J].中国实验方剂学杂志,2023,29(10):73-82.

LIANG Tingjun,ZHANG Guoxin,LIU Ying,et al.Dihydroartemisinin Regulates Neuro-microglia to Relieve Neuropathic Pain[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(10):73-82.
梁婷钧,张国鑫,刘莹等.双氢青蒿素调控神经-小胶质细胞缓解神经病理性疼痛[J].中国实验方剂学杂志,2023,29(10):73-82. DOI： 10.13422/j.cnki.syfjx.20230440.

LIANG Tingjun,ZHANG Guoxin,LIU Ying,et al.Dihydroartemisinin Regulates Neuro-microglia to Relieve Neuropathic Pain[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(10):73-82. DOI： 10.13422/j.cnki.syfjx.20230440.

摘要

目的

该研究旨在L5脊神经结扎（SNL）模型及肿瘤坏死因子-

（TNF-

）海马连续注射模型中，评价双氢青蒿素（DHA）对海马神经损伤的干预效果；在原代培养的小胶质细胞-海马神经元中，确认DHA对小胶质细胞-海马神经元相互作用的调控模式。

方法

实验小鼠分为假手术（Sham）组、SNL组、DHA组（16 mg·kg

-1

），每组3只，用腺相关病毒［（连接钙调素依赖蛋白激酶Ⅱ（CaMKⅡ） dTomato AAV］标记海马CA3谷氨酸能神经元，并以免疫荧光染色追踪其向海马CA1、前额叶皮质（Frc）、前扣带回（ACC）、伏隔核（Nac）及杏仁核（BLA）的投射；实验小鼠分为Sham组、TNF-

海马连续注射模型组、DHA低、中、高剂量（DHA-L、DHA-M、DHA-H）组（4、8、16 mg·kg

-1

）及普瑞巴林组（25 mg·kg

-1

），每组4只，以高尔基染色统计海马CA1和CA3区锥体神经元形态；分别诱导海马原代神经元、小胶质细胞的持续激活，并进行细胞共培养给予DHA干预，以免疫荧光法统计细胞胞体面积和神经元初、次级树突棘内外的突触后密度蛋白95（PSD95）的表达量。

结果

与Sham组比较，SNL组海马CA3谷氨酸能神经元向CA1区、Frc及ACC的投射减少（

0.01），而向Nac及BLA的投射升高（

0.01）；与SNL组比较，DHA组海马CA3谷氨酸能神经元向CA1区、Frc及ACC的投射升高（

0.01），而向Nac及BLA的投射显著降低（

0.01）。与Sham组比较，TNF-

海马连续注射模型组小鼠CA1锥体神经元树突棘密度及树突分支数量均显著减少（

0.01）；与TNF-

海马连续注射模型组比较，DHA-M、DHA-H组海马CA1和CA3锥体神经元树突棘密度及树突分支数明显增多（

0.05，

0.01）；与DHA-M组比较，DHA-H组海马CA1锥体神经元树突总长度显著增多（

0.01），DHA-L组CAI神经元树突总长度和CA3神经元树突基部总长度则显著减少（

0.01）。与空白组比较，甘氨酸组和谷氨酸组细胞胞体面积显著增大（

0.01）；与甘氨酸组和谷氨酸组比较，甘氨酸+谷氨酸组细胞胞体面积显著升高（

0.01）；与谷氨酸组比较，谷氨酸+DHA组细胞胞体面积显著减少（

0.01）；与甘氨酸+谷氨酸组比较，甘氨酸+谷氨酸+DHA组细胞胞体面积显著减少（

0.01）；与谷氨酸+DHA组比较，甘氨酸+谷氨酸+DHA组细胞胞体面积减少（

0.05）。与空白组比较，谷氨酸组细胞胞体面积显著升高（

0.01）；与谷氨酸组比较，谷氨酸+DHA各剂量组细胞胞体面积显著降低（

0.01）。与空白组比较，静息原代小胶质细胞+甘氨酸组神经元初、次级树突棘内、外PSD95表达量显著升高（

0.01）；与静息原代小胶质细胞+甘氨酸组比较，活化原代小胶质细胞+甘氨酸组神经元的初、次级树突棘内、外PSD95表达量显著降低（

0.01）；与活化原代小胶质细胞+甘氨酸组比较，活化原代小胶质细胞+甘氨酸+DHA组中神经元的初、次级树突棘内、外PSD95表达量显著升高（

0.01）；与活化原代小胶质细胞+DHA组比较，活化原代小胶质细胞+甘氨酸+DHA组神经元初、次级树突棘内、外PSD95表达量显著升高（

0.01）。

结论

DHA对NP病理状态下，海马小胶质细胞及TNF-

过表达导致的锥体神经元损伤具有显著的修复效果，而该修复与DHA对神经元-小胶质细胞的双重抑制密切相关。

Abstract

Objective

To evaluate the intervention effect of dihydroartemisinin （DHA） on hippocampal nerve injury in L5 spinal nerve ligation （SNL） model and tumor necrosis factor-

（TNF-

） hippocampal continuous injection model. In primary cultured microglia-hippocampal neurons， the regulatory pattern of DHA on microglia-hippocampal neuronal interactions was confirmed.

Method

The experimental animals were divided into Sham group， SNL group， and DHA group （16 mg·kg

-1

）， with 3 mice in each group. The hippocampal CA3 glutamatergic neurons were labeled with adeno-associated virus ［Calmodulin-dependent protein kinase Ⅱ（CaMKⅡ） dTomato AAV］， and their contributions to the hippocampal CA1， prefrontal cortex （Frc）， anterior cortex （ACC）， projections of nucleus accumbens （Nac）， and Basolateral Amygdala （BLA） were traced by immunofluorescence staining. The experimental animals were divided into a Sham group， a TNF-

hippocampus continuous injection model group， DHA-L， DHA-M， and DHA-H groups （4， 8， 16 mg·kg

-1

）， and pregabalin group （25 mg·kg

-1

）， with 4 mice in each group. The morphology of pyramidal neurons in the hippocampal CA1 and CA3 regions was counted by Golgi staining. The continuous activation of hippocampal primary neurons and microglia was induced， DHA intervention was given by co-culture， and the cell soma area and the expression of postsynaptic density protein 95 （PSD95） inside and outside the primary and secondary dendritic spines of neurons were counted by immunofluorescence.

Result

Compared with the Sham group， the projection of CA3 glutamatergic neurons to CA1 region， Frc， and ACC in the SNL group was significantly reduced （

0.01）， while the projection to Nac and BLA was significantly increased （

0.01）. As compared with the SNL group， the projection of hippocampal CA3 glutamatergic neurons to CA1 region， Frc， and ACC was significantly increased in the DHA group （

0.01）， while the projection to Nac and BLA was significantly reduced （

0.01）. Golgi staining results showed that as compared with the Sham group， the density of dendritic spines and the number of dendritic branches in the CA1 and CA3 pyramidal neurons in the TNF-

hippocampal continuous injection model group were significantly reduced （

0.01）. As compared with the TNF-

hippocampal continuous injection model， the density of dendritic spines and the number of dendritic branches in hippocampal CA1 and CA3 pyramidal neurons in the DHA-M and DHA-H groups were significantly increased （

0.05，

0.01）. Compared with DHA-M group， the total dendrite length of CA1 pyramidal neurons in hippocampus in DHA-H group was significantly increased （

0.01）， while the total dendrite length of CA1 neurons and the total dendrite base length of CA3 neurons in DHA-L group was significantly decreased （

0.01）. Compared with the blank control group， the cell soma area of the glycine group and glutamate group increased significantly （

0.01）. As compared with the glycine group and glutamate group， the cell area of the glycine + glutamate group was significantly increased （

0.01）， and as compared with the glutamate group， the cell soma area of the glutamate + DHA group was significantly reduced （

0.01）. As compared with the glycine acid + glutamate group， the cell soma area of the glycine + glutamate + DHA group was significantly reduced （

0.01）， and as compared with the glutamate + DHA group， the cell soma area of the glycine + glutamate + DHA group was also significantly reduced （

0.05）. Compared with the blank control group， the cell soma area of the glutamate group was significantly increased （

0.01）. As compared with the glutamate group， the cell soma area of the glutamate + DHA-L， glutamate + DHA-M， and glutamate + DHA-H groups was significantly reduced （

0.01）. As compared with the blank control group， the expression of the resting primary microglia + glycine group in primary and secondary dendritic internal and external postsynaptic density protein 95 （PSD95） was significantly increased （

0.01）. As compared with the resting primary microglia + glycine group， the expression of PSD95 in the primary and secondary dendritic spinous and external neurons of the activated primary microglia + glycine group was significantly reduced （

0.01）. As compared with the activated primary microglia + glycine group， the expression of PSD95 in the primary and secondary dendritic spinous and external neurons in the activated primary microglia + glycine + DHA group was significantly increased （

0.01）. As compared with the activated primary microglia + DHA group， the expression of PSD95 in the primary and secondary dendritic spines and outside neurons in the activated primary microglia + glycine + DHA group was significantly increased （

0.01）.

Conclusion

DHA has a significant repair effect on vertebral neuronal damage caused by hippocampal microglia and TNF-

overexpression in NP pathology， and this repair is closely related to the dual inhibition of neuronal-microglia by DHA.

关键词

Keywords

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