Objective

2

To explore the neuroprotective effect and underlying mechanism of

Xingnao Kaiqiao

acupuncture (acupuncture for regaining consciousness and opening orifices) in the rat models of cerebral ischemia-reperfusion injury (CIRI) based on the p53 protein (p53)/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling pathway.

Methods

2

Of 102 male Wistar rats

20 rats were randomly collected as a sham-operation group. Using a modified external carotid artery filament insertion method

CIRI models were prepared by occluding the middle cerebral artery in the rest rats. After modeling and excluding 1 non-successfully modeled rat and 1 dead one

the other modeled rats were randomized into a model group

an agonist group

an acupuncture group

and an acupuncture + agonist group

20 rats in each one.

Xingnao Kaiqiao

acupuncture therapy was delivered in the rats of the acupuncture group and the acupuncture + agonist group. The acupoints included "Shuigou" (GV26)

bilateral "Neiguan" (PC6)

and "Sanyinjiao" (SP6) on the affected side. Electroacupuncture was attached to "Neiguan" (PC6

) and "Sanyinjiao" (SP6) on the affected side

with dense-disperse wave

a frequency of 2 Hz/15 Hz and intensity of 1 mA. The intervention was delivered twice daily

20 min each time and for 7 consecutive days. In the agonist group and acupuncture+agonist group

p53 agonist

COTI-2 was intraperitoneally injected (15 mg/kg)

once daily for 7 consecutive days. Neurological deficit was evaluated using Zausinger's six-point scale. Cerebral infarction volume was quantified by triphenyl tetrazolium chloride (TTC) staining. Histopathological changes were observed using hematoxylin-eosin (HE) staining. Iron deposition was assessed by Prussian blue staining. Mitochondrial ultrastructure in the ischemic cortex was examined under transmission electron microscopy (TEM). Serum iron (Fe

2+

) was measured with chromometry. Malondialdehyde (MDA) and glutathione (GSH) levels in the ischemic hippocampus were determined using thiobarbituric acid and microplate assays

respectively. The mean fluorescence intensity of reactive oxygen species (ROS) in the ischemic cortex was analyzed by flow cytometry. The mRNA and protein expression of GPX4

SLC7A11

and p53 in the ischemic hippocampus were evaluated using quantitative real-time PCR (qRT-PCR) and Western blotting

respectively.

Results

2

Compared with the sham-operated group

the model group exhibited the decrease in neurological deficit score (

P

<

0.01)

and the increase in cerebral infarction volume percentage (

P

<

0.01). The changes of brain tissue were presented in extensive cellular necrosis

pyknotic and deeply-stained nuclei

and vacuolar degeneration. The iron deposition was elevated in cortex and hippocampus (

P

<

0.01)

mitochondrial membrane density increased

the cristae was broken or reduced

and the outer membrane ruptured. The levels of Fe

2+

and MDA

as well as the mean flourscence intensity of ROS were elevated (

P

<

0.01) and the level of GSH was red

uced (

P

<

0.01). The mRNA and protein expression of GPX4 and SLC7A11 was reduced (

P

<

0.01)

while that of p53 rose (

P

<

0.01). When compared with the model group

in the agonist group

the neurological deficit score was reduced (

P

<

0.05)

the percentage of infarction volume was higher (

P

<

0.01)

the histopathological damage was further exacerbated

and the percentage of iron deposition increased in the cortex and hippocampus (

P

<

0.01). The mitochondrial quantity decreased

the membrane density increased

the mitochondrial cristae were broken or reduced

and the outer membrane was ruptured. The levels of Fe

2+

and MDA

as well as the mean flourscence intensity of ROS were higher (

P

<

0.01

P

<

0.05) and the level of GSH was reduced (

P

<

0.05). The mRNA and protein expression of GPX4 and SLC7A11 decreased (

P

<

0.01

P

<

0.05)

while that of p53 was elevated (

P

<

0.01). Besides

in comparison with the model group

the neurological deficit score was higher in the acupuncture group and the acupuncture + agonist group (

P

<

0.01

P

<

0.05)

the percentage of cerebral infarction volume was lower in the acupuncture group (

P

<

0.01)

the pathological damage of brain tissue was alleviated in the acupuncture group and the acupuncture + agonist group

and the percentage of iron depositiondecreased in the cortex and hippocampus (

P

<

0.01). The mitochondrial structure was relatively clear

the mitochondrial cristae were fractured or reduced mildly in the acupuncture group and the acupuncture + agonist group. The levels of Fe

2+

and MDA

as well as the mean flourscence intensity of ROS were lower (

P

<

0.01) and the level of GSH was higher (

P

<

0.01) in the acupuncture group. The mean flu

orescence intensity of ROS were dropped (

P

<

0.01) in the acupuncture + agonist group. The mRNA expression of GPX4 and SLC7A11 was elevated (

P

<

0.01) and that of p53 was reduced (

P

<

0.01

P

<

0.05) in either the acupuncture group or the acupuncture + agonist group; the protein expression of GPX4 and SLC7A11 rose (

P

<

0.05

P

<

0.01) and that of p53 was dropped (

P

<

0.01) in the acupuncture group; and the protein expression of p53 was also lower in the acupuncture + agonist group (

P

<

0.05). When compared with the agonist group

in the acupuncture + agonist group

neurological deficit score increased (

P

<

0.01)

the percentage of cerebral infarction volume decreased (

P

<

0.01)

the pathological brain tissue damage was reduced

the percentage of iron deposition in cortex and hippocampus decreased (

P

<

0.01)

the mitochondrial structure was relatively clear and the cristae broken or reduced slightly; the levels of Fe

2+

and MDA

as well as the mean fluorescence intensity of ROS were dropped (

P

<

0.01)

while the level of GSH increased (

P

<

0.05); the mRNA and protein expression of GPX4 and SLC7411 was elevated (

P

<

0.01

P

<

0.05)

and that of p53 reduced (

P

<

0.01). In comparison with the acupuncture + agonist group

in the acupuncture group

the neurological deficit score increased (

P

<

0.05)

the percentage of cerebral infarction volume decreased (

P

<

0.05)

the pathological brain tissue damage was alleviated

the percentage of iron deposition in cortex and hippocampus decreased (

P

<

0.01)

the mitochondrial structure was normal in tendency; the levels of Fe

2+

and MDA

as well as the mean fluorescence intensity of ROS were reduced (

P

<

0.05)

while the level of GSH rose (

P

<

0.01); the mRNA and protein expression of GPX4 and SLC7411 was elevated (

P

<

0.01

P

<

0.05)

and that of p53 reduced (

P

<

0.01

P

<

0.05).

Conclusion

2

Xingnao Kaiqiao

acupuncture can alleviate neurological damage in CIRI rats

which is obtained probably by inhibiting ferroptosis through p53/SLC7A11/GPX4 pathway.