Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway

Xing-Yong Chen; Shao-Fen Wan; Nan-Nan Yao; Ze-Jing Lin; Yan-Guang Mao; Xiao-Hua Yu; Yin-Zhou Wang

doi:10.1186/s40779-021-00356-x

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Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway

RESEARCH | Updated：2023-02-28

- Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway
- Military Medical Research Vol. 9, Issue 4, Pages: 404-418(2022)
- Affiliations：
  
  1.Department of Neurology, Fujian Provincial Hospital, Shengli Clinical Medical College of Fujian Medical University, No. 134, Dongjie, Gulou District, Fuzhou 350001, China
  2.Fujian Academy of Medical Science, Fuzhou 350001, China
  3.Key Testing Laboratory of Fujian Province, Fuzhou 350001, China
- Author bio：
  
  * cxyong77@163.com;
  wphd@163.com
- Funds：
- DOI：10.1186/s40779-021-00356-x
  CLC：
- Published：2022-08
- Accepted：
Scan QR Code
Chen XY, Wan SF, Yao NN, Lin ZJ, Mao YG, Yu XH, et al. Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway. Mil Med Res. 2021;8(1):62.
DOI：

Chen XY, Wan SF, Yao NN, Lin ZJ, Mao YG, Yu XH, et al. Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway. Mil Med Res. 2021;8(1):62. DOI： 10.1186/s40779-021-00356-x.

摘要

Abstract

Background:

Disruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However

the relationship between the immunoproteasome LMP2 and the BBB remains unclear.

Methods:

Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO

the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions

in vitro

. The RNA interference-mediated knockdown of LMP2 or

-catenin was analysed

in vivo

and

in vitro

. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence

Western blotting and cell migration were quantified using the software ImageJ (Version 1.53). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test.

Results:

Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins [occludin

claudin-1 and zonula occludens (ZO-1)] in the MCAO/R group compared with the sham group (

0.001). However

inhibition of the immunoproteasome LMP2 restored the expression of these proteins

resulting in higher levels of occludin

claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (

0.001). In addition

inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g.

the quantity of extravasated EB: LMP2-shRNA group (58.54±7.37) μg/g

. control-shRNA group (103.74±4.32) μg/g

0.001]

and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R.

In vitro

experiments

OGD/R induced marked upregulation of LMP2

proapoptotic protein Bax and cleaved caspase-3

and downregulation of occludin

claudin-1

ZO-1 and Bcl-2

as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs

compared with the control group under normal culture conditions (

0.001). However

silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins

and reduced the proliferation and migration of RBMVECs following OGD/R

compared with the control-siRNA group (

0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.

Conclusions:

This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury

and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/

-catenin signalling pathway under ischemic conditions.

关键词

Keywords

references

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