Proteomic Study on the Effect of <italic style="font-style: italic">Wenshen Tongdu Formula </italic>（温肾通督方） on Spleen B Cells in Spinal Cord Injury Model Mice

SHI Lei; WU Chengjie; CHEN Sixian; PAN Yalan; WANG Lining; ZHAO Ruihua; WU Mao; MA Yong; GUO Yang

doi:10.13288/j.11-2166/r.2023.22.012

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Proteomic Study on the Effect of Wenshen Tongdu Formula （温肾通督方） on Spleen B Cells in Spinal Cord Injury Model Mice

更新时间：2023-11-10

- Proteomic Study on the Effect of Wenshen Tongdu Formula （温肾通督方） on Spleen B Cells in Spinal Cord Injury Model Mice
  增强出版
- Journal of Traditional Chinese Medicine Vol. 64, Issue 22, Pages: 2329-2338(2023)
- 作者机构：
  
  1.南京中医药大学中医学院·中西医结合学院，江苏省南京市栖霞区仙林大道138号，210023
  2.南京中医药大学骨伤修复与重建新技术实验室
  3.南京中医药大学无锡附属医院
- 作者简介：
- 基金信息：
- DOI：10.13288/j.11-2166/r.2023.22.012
  CLC：
- Received：24 May 2023，
  
  Revised：2023-08-30，
  
  Published：17 November 2023
- 稿件说明：
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石磊,吴承杰,陈思娴等.温肾通督方对脊髓损伤模型小鼠脾脏B细胞影响的蛋白组学研究[J].中医杂志,2023,64(22):2329-2338.

SHI Lei,WU Chengjie,CHEN Sixian,et al.Proteomic Study on the Effect of Wenshen Tongdu Formula （温肾通督方） on Spleen B Cells in Spinal Cord Injury Model Mice[J].Journal of Traditional Chinese Medicine,2023,64(22):2329-2338.
石磊,吴承杰,陈思娴等.温肾通督方对脊髓损伤模型小鼠脾脏B细胞影响的蛋白组学研究[J].中医杂志,2023,64(22):2329-2338. DOI： 10.13288/j.11-2166/r.2023.22.012.

SHI Lei,WU Chengjie,CHEN Sixian,et al.Proteomic Study on the Effect of Wenshen Tongdu Formula （温肾通督方） on Spleen B Cells in Spinal Cord Injury Model Mice[J].Journal of Traditional Chinese Medicine,2023,64(22):2329-2338. DOI： 10.13288/j.11-2166/r.2023.22.012.

摘要

目的

探讨温肾通督方对脊髓损伤的影响及可能机制。

方法

36只C57BL/6雌性小鼠随机分为假手术组、模型组、温肾通督方组，每组12只。模型组、温肾通督方组小鼠采用改良Allen's法建立小鼠脊髓损伤模型，假手术组仅进行脊髓暴露。温肾通督方组从术后第1天按50 g/（kg·d）给予温肾通督方溶液灌胃，假手术组及模型组给予生理盐水20 ml/（kg·d）灌胃，各组均连续灌胃14天。在术前及术后第1、7、14天，利用斜板试验及后肢运动功能（BMS）评分对小鼠运动功能进行评估；术后第3天通过肌电图及运动诱发电位检测小鼠神经电生理，测量各组小鼠脾脏长度，利用磁珠分选脾脏中的B细胞，通过蛋白组学技术分析蛋白差异表达情况，采用Western blot法检测脾脏B细胞中线粒体外膜转运孔蛋白20（Tom20）及下游剪切化半胱氨酸蛋白酶3（Cleaved Caspase-3）蛋白表达；术后第14天采用核磁共振观察小鼠脊髓恢复情况。

结果

与假手术组同时间比较，模型组在术后1、7、14天BMS主评分、副评分及斜板试验角度均降低，术后3天小鼠肌电图和运动诱发电位峰峰值降低、脾脏长度减少，脾脏B细胞中Tom20和Cleaved Caspase-3表达量升高（

＜0.05）。与模型组同时间比较，温肾通督方组小鼠术后14天时BMS副评分及术后7、14天时斜板试验角度升高，术后3天小鼠肌电图和运动诱发电位峰峰值升高、脾脏长度增加，Tom20和Cleaved Caspase-3表达量降低（

＜0.05）。蛋白组学结果显示，温肾通督方组与模型组相比共有100个差异蛋白，其中37个上调，63个下调。GO富集显示差异蛋白主要在氧结合、外源性凋亡负反馈、锌离子相应、氧输送等方面发挥作用。KEGG富集显示差异蛋白主要集中在代谢途径、亨廷顿病、氧化磷酸化等通路。亚细胞定位显示差异蛋白与线粒体相关。术后14天磁共振成像显示，假手术组小鼠脊髓结构完整，节段清晰，脊髓信号无异常；模型组小鼠脊髓损伤区域低信号区域增加，脊髓明显变细；温肾通督方组损伤节段有明显凹陷，但较模型组结构要完整。

结论

温肾通督方可能通过调节免疫功能促进脊髓损伤修复，其机制可能与抑制脾脏B细胞焦亡等相关。

Abstract

Objective

To explore the effects and possible mechanism of

Wenshen Tongdu Formula

（温肾通督方， WTF） on spinal cord injury.

Methods

Thirty-six C57BL/6 female mice were randomly divided into sham operation group， model group and WTF group， with 12 mice in each group. The spinal cord injury model was established in the model group and the WTF group using the modified Allen's method， while in the sham operation group the spinal cord was only exposed. Since the 1st day after surgery， 50 g/（kg·d） of WTF solution was given to the WTF group by gavage， while 20 ml/（kg·d） of normal saline was given to the sham operation and model group by gavage， all for 14 days. Before surgery and on the 1st， 7th， and 14th days after surgery， the motor function of the mice was evaluated using the inclined plane test and hind limb motor function score （by BMS）. On the 3rd day after surgery， the nerve electrophy-siology was detected through electromyography and motor evoked potential； the spleen length was measured， and B cells in the spleen were sorted by magnetic beads； the differential expression of proteins were detected through proteomics technology； and the protein expression of mitochondrial outer membrane transport porin 20 （Tom20） and downstream cleaved caspase-3 in spleen B cells were measured using Western blotting. On the 14th day after surgery， MRI was used to observe the recovery of the spinal cord.

Results

Compared to those in the sham operation group at the same time， the BMS scores and subscores and the inclined plane test angle in the model group were reduced on the 1st， 7th and 14th days after surgery； the peak value of electromyogram and motor evoked potential were reduced， and the spleen length was shortened， while the expression of Tom20 and cleaved caspase-3 increased in splenic B cells increased （

＜0.05）. Compared to those in the model group at the same time， the BMS subscores on the 14th day and the angle of the inclined plane test on the 7th and 14th days after surgery increased in the WTF group； the peak value of electromyography and motor evoked potential， as well as the length of spleen increased， and the expression of Tom20 and cleaved caspase-3 decreased （

＜0.05）. The proteomics results showed that there were 100 differential proteins in the WTF group versus the model group， of which 37 were up-regulated and 63 were down-regulated. GO enrichment analysis showed that differential proteins mainly played their roles in oxygen binding， exogenous apoptosis negative feedback， zinc ion response， and oxygen transport. KEGG enrichment analysis showed that differential proteins were mainly concentrated in metabolic pathways， Huntington's disease， oxidative phosphorylation and other pathways. Subcellular localization showed that differential proteins were associated with mitochondria. Magnetic resonance imaging on the 14th day after surgery showed that the spinal cord structure of the mice in the sham operation group was intact， and the segments were clear， with normal spinal cord signal； the low signal area in the spinal cord injury area increased in the model group， and the spinal cord became significantly thinner； the injured segment had obvious depression in the WTF group， but the structure was more complete than that in the model group.

Conclusion

WTF may promote spinal cord injury repair by regulating immune function， and its mechanism may be related to inhibiting pyroptosis of spleen B cells.

关键词

Keywords

references

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Proteomic Study on the Effect of Wenshen Tongdu Formula （温肾通督方） on Spleen B Cells in Spinal Cord Injury Model Mice

DOI：10.13288/j.11-2166/r.2023.22.012

摘要

Abstract

关键词

Keywords

references