Objective

2

To observe the effects of

Shugan Tiaoshen

acupuncture (acupuncture for smoothing liver

qi

and regulating the mind) on hippocampal neural mitophagy and oxidative stress-related factors in the rats with chronic inflammatory pain and depression comorbidity (CIPDC)

and its protective effect on hippocampal neurons; and explore the underlying mechanism of this acupuncture therapy for alleviating CIPDC.

Methods

2

Of 60 Sprague-Dawley (SD) rats

12 rats were randomly selected as the control group. CIPDC models were established by subcutaneous injection with complete Freund's adjuvant (CFA) into the plantar region of the left hind paws of the rest rats. Forty-eight successfully modeled rats were assigned into a model group

an electroacupuncture (EA) group

an inhibitor group and an EA + inhibitor group

12 rats in each one. In the EA group

EA was applied to "Yintang" (GV24

+

)

"Baihui" (CV20)

and bilateral "Hegu" (LI4) and "Taichong" (LR3). Electric stimulation was delivered at "Hegu" (LI4) and "Taichong" (LR3) unilaterally

with disperse-dense wave

a frequency of 2 Hz/10 Hz

and a current of 1 mA. EA w

as operated for 30 min daily. In the inhibitor group

autophagy inhibitor

3-methl adenine (3-MA) was injected intraperitoneally. In the EA + inhibitor group

EA was delivered after intraperitoneal injection with 3-MA. The intervention of each group was operated once daily and for 14 consecutive days. Pain threshold was assessed using paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PTWL). Depressive-like behaviors were evaluated using the sucrose preference test

open-field test

and forced swimming test. Hematoxylin and eosin (HE) staining was used to observe the pathological morphology of hippocampal neurons. Fluoro-Jade B staining was performed to assess hippocampal neuronal injury and degeneration. Transmission electron microscopy (TEM) was employed to examine the ultrastructure of hippocampal mitochondria. Hippocampal reactive oxygen species (ROS) expression was detected using flow cytometry. The content of malondialdehyde (MDA) and superoxide dismutase (SOD) in the hippocampus were measured using a colorimetric method. Western blot analysis was conducted to detect the protein expression of sequenstering protein 1 (p62)

PTEN induced putative kinase 1 (PINK1)

Parkin protein (Parkin)

Beclin1

and microtubule-associated protein 1A/1B 1ight chain3 (LC3) in the hippocampus. Real-time PCR was used to detect the mRNA expression of p62

PINK1

Parkin and Beclin1.

Results

2

Compared with the control group

in the model group

PWMT

PTWL

sucrose preference rate

total distance traveled in the open field

and time spent in the center area decreased (

P

<

0.05)

while the immobility time increased (

P

<

0.05); the mitochondria was ruptured and swollen and the internal crista were broken or disappeared; the content of ROS and MDA elevated (

P

<

0.05) and SOD reduced (

P

<

0.05); the protein and mRNA expression of hippocampal p62

PINK1

Parkin and Beclin 1

as well as the protein expression

of LC3 were not different statistically (

P

>

0.05). Compared with the model group

in the EA group

PWMT

PTWL

sucrose preference rate

total distance traveled in the open field

and time spent in the center area increased (

P

<

0.05)

while the immobility time decreased (

P

<

0.05); hippocampal neurons were rounded and neatly arranged

with the number of degenerated neurons declined (

P

<

0.05); the mitochondrial morphology was relatively normal

and the bilayer membrane structure intact

and autophagosomes visible; the content of ROS and MDA reduced (

P

<

0.05) and SOD elevated (

P

<

0.05); the protein and mRNA expression of hippocampal p62 was declined (

P

<

0.05)

and the protein and mRNA expression of PINK1

Parkin and Beclin 1 was elevated (

P

<

0.05)

and the protein expression of LC3 rose (

P

<

0.05). When compared with the EA group

in the EA + inhibitor group

PWMT

PTWL

sucrose preference rate

total distance traveled in the open field

and time spent in the center area were reduced (

P

<

0.05)

while the immobility time was higher (

P

<

0.05); hippocampal neurons were more disarranged

with increased nuclear membrane damage and more degenerated neurons (

P

<

0.05); the damage in mitochondrial bilayers and cristae was obvious; the content of ROS and MDA higher (

P

<

0.05) and SOD lower (

P

<

0.05); the protein and mRNA expression of hippocampal p62 was elevated (

P

<

0.05)

and protein and mRNA expression of PINK1

Parkin and Beclin 1 was reduced (

P

<

0.05)

and the protein expression of LC3 decreased (

P

<

0.05).

Conclusion

2

Shugan Tiaoshen

acupuncture presents a protective effect on CIPDC by enhancing hippocampal neuronal mitophagy through regulating the PINK1/Par

kin pathway in the model rats.