Protective Effect of GSK-3β/Nrf2 Mediated by Dimethyl Fumarate in Middle Cerebral Artery Embolization Reperfusion Rat Model
Authors: Li Yuan , Chu Lan , Liu Chunfeng , Zha Zongyi , Shu Yuanlu 
Published: November 2021

Aim:
This study investigated the protective effect of dimethyl fumarate (DMF) in rats
by mediating glycogen synthase kinase 3β (GSK-3β)/Nrf2 using the middle cerebral artery embolization
reperfusion (MCAO/R) rat model.

Background:
After an acute ischemic stroke (AIS), oxidative stress occurs. Dimethyl fumarate
(DMF), a nuclear factor-E2-related factor 2 (Nrf2) activator, approved by the US Food and Drug
Administration (FDA), was observed to regulate the Nrf2 pathway by acting as an anti-oxidative
stress agent; however, whether this agent is involved in inhibiting GSK-3β remains to be established.

Methods:
DMF model was used to explore the effects of GSK-3β on Nrf2 expression level, Nrf2-
ARE binding activity and Nrf2/ARE downstream expression level of anti-oxidant stress protein
in Cerebral ischemia-reperfusion injury (CIRI). 60 rats were randomly divided into Sham group,
MCAO/R group, solvent control group (DMSO group) and DMF treatment group, with 15 rats in
each group. The MCAO/R, DMSO and DMF groups were considered in the MCAO/R model using
the modified thread embolization method. In contrast, the Sham group was only anaesthetized and
disinfected, and tissue muscle was dissected without inserting suture emboli. DMF group was gavaged
with 45mg/kg per day of DMF, DMSO control group was gavaged with DMSO of equal volume,
while MCAO/R group was only modeled without any intragastric treatment. The rats were
treated seven days after the operation, and a neurological function Longa score was estimated. The
rats were sacrificed seven days later, and the infarct volume was assessed by TTC staining. Hematoxylin-
eosin (HE) staining was used to observe the pathological changes in rat brain tissue. Nissl
staining was used to observe the expression of neurons in the infarcted cortex. Western blotting (WB)
was used to observe the protein expression levels of GSK-3β, Nrf2, downstream heme oxygenase
1 (HO1) and NADPH quinone oxidoreductase 1 (NQO1) in four groups. The expression levels
of GSK-3β and Nrf2 in the four groups were observed by immunohistochemistry and immunofluorescence.

Results:
(1) The Longa score of the MCAO/R, DMSO and DMF groups was found to be higher
compared to the Sham group, indicating successful operation. The Longa score of the DMF group
was lower than that of the other three groups 4-7 days after surgery (P<0.05). (2) HE and Nissl
staining showed that the DMF group had lower neuron necrosis and higher gliosis compared to the
control groups. (3) TTC staining results showed that the infarct volume of the DMF group was significantly
smaller than the MCAO/R and DMSO groups. (4) Protein results showed that the
GSK-3β expression in the DMF group was lower than that in all groups, while the expression of Nrf2,
HO1 and NQO1 was higher compared to other groups.

Conclusion:
DMF can reduce neurological deficits and infarct size in the MCAO/R model. The
protective effect may be related to decreased GSK-3β expression and increased Nrf2 expression,
which may play a role in anti-oxidative stress.