Effects of <italic style="font-style: italic">Naotai Formula</italic> （脑泰方） on Cerebral White Matter Lesions and Wnt/β-Catenin Pathway in Corpus Callosum of Rats with Cerebral Small Vessel Disease Model

WANG Xiangyuan; MEI Zhigang; Ge Jinwen

doi:10.13288/j.11-2166/r.2024.20.012

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Effects of Naotai Formula （脑泰方） on Cerebral White Matter Lesions and Wnt/β-Catenin Pathway in Corpus Callosum of Rats with Cerebral Small Vessel Disease Model

更新时间：2024-10-12

- Effects of Naotai Formula （脑泰方） on Cerebral White Matter Lesions and Wnt/β-Catenin Pathway in Corpus Callosum of Rats with Cerebral Small Vessel Disease Model
- Journal of Traditional Chinese Medicine Vol. 65, Issue 20, Pages: 2135-2144(2024)
- 作者机构：
  
  1.湖南中医药大学中西医结合学院，湖南省长沙市岳麓区学士路300号， 410208
  2.三峡大学健康医学院
  3.湖南省中医药研究院
- 作者简介：
- 基金信息：
- DOI：10.13288/j.11-2166/r.2024.20.012
  CLC：
- Received：10 June 2024，
  
  Revised：2024-08-15，
  
  Published：17 October 2024
- 稿件说明：
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王香元,梅志刚,葛金文.脑泰方对脑小血管病模型大鼠脑白质病变及胼胝体Wnt/β-catenin通路的影响[J].中医杂志,2024,65(20):2135-2144.

WANG Xiangyuan,MEI Zhigang,Ge Jinwen.Effects of Naotai Formula （脑泰方） on Cerebral White Matter Lesions and Wnt/β-Catenin Pathway in Corpus Callosum of Rats with Cerebral Small Vessel Disease Model[J].Journal of Traditional Chinese Medicine,2024,65(20):2135-2144.
王香元,梅志刚,葛金文.脑泰方对脑小血管病模型大鼠脑白质病变及胼胝体Wnt/β-catenin通路的影响[J].中医杂志,2024,65(20):2135-2144. DOI： 10.13288/j.11-2166/r.2024.20.012.

WANG Xiangyuan,MEI Zhigang,Ge Jinwen.Effects of Naotai Formula （脑泰方） on Cerebral White Matter Lesions and Wnt/β-Catenin Pathway in Corpus Callosum of Rats with Cerebral Small Vessel Disease Model[J].Journal of Traditional Chinese Medicine,2024,65(20):2135-2144. DOI： 10.13288/j.11-2166/r.2024.20.012.

摘要

目的

从无翅型MMTV整合位点家族成员（Wnt）/β连环蛋白（β-catenin）通路探讨脑泰方对脑小血管病白质病变的作用及其机制。

方法

将10只WKY大鼠作为假手术组，30只自发性高血压大鼠（SHR）随机分为模型组、脑泰方低剂量组、脑泰方高剂量组，每组10只。除假手术组外，其余各组大鼠采用D-半乳糖背部皮下注射56天造模，并在造模第29天进行双侧颈总动脉狭窄术低灌注28天构建复合危险因素脑小血管病大鼠模型。脑泰方低、高剂量组大鼠手术当天分别给予脑泰方9、27 g/（kg·d）灌胃，假手术组和模型组给予10 ml/（kg·d）纯水灌胃，各组大鼠灌胃4周。给药前及给药后每周监测大鼠的血压；造模第50天开始连续6天进行水迷宫实验；术前及术后、给药后激光散斑检测脑血流量变化。最后一次灌胃次日取材，卢卡斯快蓝（LFB）和透射电镜观察大鼠胼胝体髓鞘损伤程度，ELISA法检测大鼠胼胝体白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、白细胞介素10（IL-10）、肿瘤坏死因子β（TNF-β）水平，免疫荧光法检测大鼠胼胝体中髓鞘碱性蛋白（MBP）表达水平和离子化钙结合适配分子1（IBA1）、神经胶质抗原2（NG2）、细胞增殖标志物Ki67抗原（Ki67）/NG2、2'

3'-环核苷酸3'-磷酸二酯酶（CNPase）表达；Western Blot法检测胼胝体中MBP、无翅型MMTV整合位点家族成员3a（Wnt3a）、磷酸化糖原合成酶激酶3β（p-GSK-3β）、糖原合成酶激酶3β（GSK-3β）、磷酸化β连环蛋白（p-β-catenin）、β-catenin表达，RT-qPCR法检测胼胝体中GSK-3β、β-catenin mRNA表达。

结果

与假手术组比较，模型组大鼠各时间点收缩压升高，水迷宫实验中穿越平台次数、目标象限停留时间百分比减少，第4天和第5天逃避潜伏期明显延长，术后及给药后大鼠脑血流量下降（

＜0.05或

＜0.01）；LFB染色及透射电镜观察可见胼胝体髓鞘损伤明显；胼胝体中TNF-α和IL-1β含量增加，TNF-β含量降低，MBP荧光强度和蛋白表达降低，IBA1阳性细胞数增加，Wnt3a和p-GSK-3β/GSK-3β升高，p-β-catenin/β-catenin降低，GSK-3β mRNA表达降低，β-catenin mRNA表达升高（

＜0.05或

＜0.01）。与模型组比较，脑泰方高剂量组大鼠胼胝体髓鞘损伤明显改善，上述各指标均显著改善（

＜0.05或

＜0.01）。与脑泰方低剂量组比较，脑泰方高剂量组胼胝体髓鞘损伤改善，收缩压降低，脑血流量及胼胝体中MBP荧光强度及蛋白表达，NG2、CNPase阳性细胞数，p

-β-catenin/β-catenin、GSK-3β mRNA表达均升高，Wnt3a表达、p-GSK-3β/GSK-3β及β-catenin mRNA表达降低（

＜0.05或

＜0.01）。

结论

高剂量脑泰方可显著改善脑小血管病白质病变，其机制可能与抑制Wnt/β-catenin信号通路，减轻脑内炎性微环境，促进少突胶质前体细胞分化成为少突胶质细胞有关。

Abstract

Objective

To explore the effects and mechanisms of

Naotai Formula

（脑泰方） against white matter lesions associated with cerebrovascular disease via Wnt/β-catenin pathway.

Methods

Ten WKY rats were used as sham surgery group， and 30 spontaneously hypertensive rats （SHRs） were randomly divided into model group， low-and high-dose

Naotai Formula

group， with 10 rats in each group. In addition to the sham surgery group， rats in each group were modelled by dorsum subcutaneous injection of D-galactose for 56 days， by performing bilateral common carotid artery stenosis on the 29th day of modelling with low perfusion for 28 days to construct the rat models of cerebral small-vessel disease with composite risk factors. The rats in the low- and high-dose

Naotai Formula

groups were given 9 and 27 g/（kg·d） of

Naotai Formula

by gavage on the day after surgery， and 10 ml/（kg·d） of pure water by gavage in the sham surgery group and the model group， with all rats in each group gavaged for 4 weeks. Blood pressure of rats was monitored weekly before and after medication administration； water maze experiments were performed for 6 consecutive days from the 50th day of modelling； and changes in cerebral blood flow were detected by laser scattering preoperatively， postoperatively， and after medication administration. The samples were taken on the next day after the last gavage， and the extent of myelin damage in the rat corpus callosum was observed by Lucas fast blue （LFB） and transmission electron microscopy， and the levels of interleukin 1β （IL-1β）， tumour necrosis factor α （TNF-α）， interleukin 10 （IL-1

0）， and tumour necrosis factor β （TNF-β） were detected by ELISA， and the levels of expression of myelin basic protein （MBP） and ionized calcium binding adaptor molecule 1 （IBA1）， neuroglial antigen 2 （NG2）， cell proliferation marker Ki67 antigen （MBP）， and cell proliferation marker Ki67 antigen （Ki67A）/NG2， 2'，3'- cyclic-nucleotide 3'-phosphodiesterase （CNPase） expression were measured by immunofluorescence. MBP in the corpus callosum， wingless MMTV integration site family member 3a （Wnt3a）， phosphorylated glycogen synthase kinase 3β （p-GSK-3β）， glycogen synthase kinase 3β （GSK-3β）， phosphorylated β-catenin （p-β-catenin）， β-catenin expression were detected by western blotting， and GSK-3β， β-catenin mRNA expression in the corpus callosum were detected by RT-qPCR.

Results

Compared with the sham surgery group， rats in the model group had elevated systolic blood pressure at all time points， decreased the number of traversing platforms and the percentage of time spent in the target quadrant in the water maze experiments， significantly prolonged the evasion latency on days 4 and 5， and decreased cerebral blood flow in the postoperative period and after medication administration （

＜0.05 or

＜0.01）； significant myelin sheath damage in the corpus callosum was seen by LFB staining and transmission electron microscopy； the callus had significantly higher levels of TNF-α and IL-1β content increased， TNF-β content decreased， MBP fluorescence intensity and protein expression decreased， the number of IBA1-positive cells increased， Wnt3a and p-GSK-3β/GSK-3β values increased， p-β-catenin/β-catenin values decreased， GSK-3β mRNA expression decreased， and β-catenin mRNA expression increased （

＜0.05 or

＜0.01）. Compared with the model group， the myelin sheath damage of corpus callosum in rats in the high-dose

Naotai Formula

groups was significantly improved， and all of the above indexes were significantly improved （

＜0.05 or

＜0.01）. Compared with the low-dose

Naotai Formula

group， myelin damage in the corpus callosum of the high-dose

Naotai Formula

group was improved， systolic blood pressure reduced， cerebral blood flow and MBP fluorescence intensity and protein expression in the corpus callosum， the number of positive cells for NG2 and CNPase， the values of p-β-catenin/β-catenin and GSK-3β mRNA expression increased， and the expression of Wnt3a， p-GSK-3β/ GSK-3β values and β-catenin mRNA expression decreased （

＜0.05 or

＜0.01）.

Conclusions

High-dose

Naotai Formula

can significantly improve white matter lesions in cerebral small vessel disease， and the mechanism may be related to the inhibition of the Wnt/β-catenin signalling pathway， reducing the inflammatory microenvironment in the brain， and promoting the differentiation of oligodendrocyte precursor cells into oligodendrocytes.

关键词

Keywords

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Effects of Naotai Formula （脑泰方） on Cerebral White Matter Lesions and Wnt/β-Catenin Pathway in Corpus Callosum of Rats with Cerebral Small Vessel Disease Model

DOI：10.13288/j.11-2166/r.2024.20.012

摘要

Abstract

关键词

Keywords

references