Objective

2

To explore the potential mechanism of electroacupuncture (EA) for vascular dementia (VD) using tandem mass tag (TMT) quantitative proteomics technology.

Methods

2

Among 80 male SPF SD rats

78 rats which met the selection criteria through the Morris water maze test were selected and randomly divided into a sham surgery group (18 rats) and a surgery group (60 rats). VD model was established by four-vessel occlusion (4-VO) method in the surgery group

and 36 rats with successful modeling were randomly assigned to a model group (18 rats) and an EA group (18 rats). Each group was further divided into three subgroups based on intervention duration

with each subgroup containing 6 rats. Seven days after model establishment

the EA group received EA intervention at left and right "Sishencong" (EX-HN 1) and bilateral "Fengchi" (GB 20)

with continuous wave at a frequency of 2 Hz and current intensity of 1 mA

daily for 30 min

with subgroups receiving EA for 7

14

or 21 d respectively. Cognitive function before and after interventions was assessed using Morris water maze. Proteomic analysis was conducted on the optimal EA subgroup and corresponding sham surgery and model subgroups

identifying differentially expressed proteins and analyzing them through bioinformatics. Differentially expressed target proteins was performed using parallel reaction monitoring (PRM) and Western blot techniques.

Results

2

Compared to the sham surgery group

the model group exhibited prolonged escape latency and reduced number of platform crossings (

P

<

0.01); compared with model group

the EA group showed reductions in escape latency and increased platform crossings after 7

14

and 21 days of intervention (

P

<

0.01

P

<

0.05). Compared to the 7 and 14-day intervention

the rats in the EA group of 21-day intervention showed the most significant improvements in reductions of escape latency and increased platform crossings (

P

<

0.01

P

<

0.05)

and was selected for further proteomic

PRM analyses

and Western blot validation. Compared to the sham surgery group

the model group displayed 71 differentially expressed proteins

with 50 up-regulated and 21 down-regulated proteins; compared to the model group

the EA group had 54 differentially expressed proteins

with 30 up-regulated and 24 down-regulated proteins. Functional enrichment and clustering analyses indicated that these proteins were pri

marily associated with cellular processes

metabolic processes

phagocytosis recognition

immune response

and regulation of extracellular matrix

etc. Enrichment was observed in the mammalian target of rapamycin (mTOR) signaling pathway and neurotrophic factors signaling pathways

involving glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase 2 (Map2k2)

with PRM and Western blot findings consistent with the proteomic results. Which meant that compared with the model group

the protein expression of GSK3β and Map2k2 of hippocampus was increased in the EA group (

P

<

0.01

P

<

0.05).

Conclusion

2

EA at "Sishencong" (EX-HN 1) and "Fengchi" (GB 20) could improve cognitive function in VD rats

with the mechanism involving multiple targets and pathways

potentially related to GSK3β

Map2k2 proteins

and the mTOR and neurotrophic factor signaling pathways.