Mechanism of acupuncture on cerebral ischemia-reperfusion injury via p53/SLC7A11/GPX4 signaling pathway in rat models

WANG Qi; HOU Ziwen; LIU Yaoyao; WEI Dan; KONG Qingjie; CHEN Xia

doi:10.13703/j.0255-2930.20240703-k0003

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Mechanism of acupuncture on cerebral ischemia-reperfusion injury via p53/SLC7A11/GPX4 signaling pathway in rat models

Study on Mechanism | 更新时间：2025-08-14

- Mechanism of acupuncture on cerebral ischemia-reperfusion injury via p53/SLC7A11/GPX4 signaling pathway in rat models
- Chinese Acupuncture & Moxibustion Vol. 45, Issue 8, Pages: 1099-1110(2025)
- 作者机构：
  
  天津中医药大学第一附属医院针灸科，天津　300193
  国家中医针灸临床医学研究中心，天津　300193
  天津中医药大学针灸推拿学院
  成都市新都区中医医院针灸科，四川成都　610500
- 作者简介：
- 基金信息：
- DOI：10.13703/j.0255-2930.20240703-k0003
  CLC：
- Received：03 July 2024，
  
  Online First：14 May 2025，
  
  Published：12 August 2025
- 稿件说明：
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WANG Qi, HOU Ziwen, LIU Yaoyao, et al. Mechanism of acupuncture on cerebral ischemia-reperfusion injury via p53/SLC7A11/GPX4 signaling pathway in rat models[J]. Chinese Acupuncture & Moxibustion, 2025, 45(8): 1099-1110.
DOI：

WANG Qi, HOU Ziwen, LIU Yaoyao, et al. Mechanism of acupuncture on cerebral ischemia-reperfusion injury via p53/SLC7A11/GPX4 signaling pathway in rat models[J]. Chinese Acupuncture & Moxibustion, 2025, 45(8): 1099-1110. DOI： 10.13703/j.0255-2930.20240703-k0003.

摘要

目的：

基于p53蛋白（p53）/溶质载体家族7成员11（SLC7A11）/谷胱甘肽过氧化物酶4（GPX4）信号通路探讨醒脑开窍针刺法对脑缺血再灌注损伤（CIRI）大鼠的神经保护作用及作用机制。

方法：

从102只雄性Wistar大鼠中随机选取20只作为假手术组，其余大鼠采用改良的颈外动脉进线栓法制备右侧大脑中动脉缺血再灌注损伤模型，剔除造模不成功和死亡大鼠各1只，将剩余大鼠随机分为模型组、激动剂组、针刺组及针刺+激动剂组，每组20只。针刺组及针刺+激动剂组予醒脑开窍针刺法干预，穴取“水

沟”、双侧“内关”、患侧“三阴交”，患侧“内关”“三阴交”连接电针，疏密波，频率2 Hz/15 Hz，电流1 mA，每日2次，每次20 min，连续7 d；激动剂组和针刺+激动剂组予腹腔注射p53激动剂COTI-2（15 mg/kg），每日1次，连续7 d。采用Zausinger六分法评估大鼠神经功能缺损程度；TTC染色法检测大鼠脑梗死体积百分比；HE染色法观察大鼠缺血侧脑组织形态；普鲁士蓝染色法检测大鼠缺血侧脑组织铁沉积量；透射电镜观察大鼠缺血侧皮质线粒体结构；比色法检测大鼠血清铁（Fe

）含量，硫代巴比妥酸法检测大鼠缺血侧海马丙二醛（MDA）含量，微板法检测大鼠缺血侧海马谷胱甘肽（GSH）含量；流式细胞术检测大鼠缺血侧皮质活性氧（ROS）平均荧光强度；实时荧光定量PCR法和Western blot法检测大鼠缺血侧海马GPX4、SLC7A11、p53 mRNA和蛋白表达。

结果：

与假手术组比较，模型组神经功能缺损评分降低（

0.01）；脑梗死体积百分比升高（

0.01）；脑组织出现大片细胞坏死，细胞核固缩深染，空泡样改变；缺血侧皮质及海马铁沉积百分比增加（

0.01）；线粒体膜密度增加，线粒体嵴断裂或减少，外膜破裂；Fe

、MDA含量和ROS平均荧光强度升高（

0.01），GSH含量降低（

0.01）；GPX4、SLC7A11 mRNA和蛋白表达降低（

0.01），p53 mRNA和蛋白表达升高（

0.01）。与模型组比较，激动剂组神经功能缺损评分降低（

0.05）；脑梗死体积百分比升高（

0.01）；脑组织病理性损伤进一步加重；缺血侧皮质及海马铁沉积百分比增加（

0.01）；线粒体数量减少，膜密度增加，线粒体嵴断裂或减少，外膜破裂；Fe

、MDA含量和ROS平均荧光强度升高（

0.01，

0.05），GSH含量降低（

0.05）；GPX4、SLC7A11 mRNA和蛋白表达降低（

0.01，

0.05），p53 mRNA和蛋白表达升高（

0.01）。与模型组比较，针刺组及针刺+激动剂组神经功能缺损评分升高（

0.01，

0.05）；针刺组脑梗死体积百分比降低（

0.01）；针刺组及针刺+激动剂组脑组织病理性损伤减轻；针刺组及针刺+激动剂组缺血侧皮质及海马铁沉积百分比减少（

0.01）；针刺组及针刺+激动剂组线粒体结构相对清晰，线粒体嵴轻度断裂或减少；针刺组Fe

、MDA含量和ROS平均荧光强度降低（

0.01），GSH含量升高（

0.01），针刺+激动剂组ROS平均荧光强度降低（

0.01）；针刺组及针刺+激动剂组GPX4、SLC7A11 mRNA表达升高（

0.01），p53 mRNA表达降低（

0.01，

0.05），针刺组GPX4、SLC7A11蛋白表达升高（

0.05，

0.01），p53蛋白表达降低（

0.01），针刺+激动剂组p53蛋白表达降低（

0.05）。与激动剂组比较，针刺+激动剂组神经功能缺损评分升高（

0.01）；脑梗死体积百分比降低（

0.01）；脑组织病理性损伤减轻；缺血侧皮质及海马铁沉积百分比减少（

0.01）；线粒体结构相对清晰，线粒体嵴轻度断裂或减少；Fe

、MDA含量和ROS平均荧光强度降低（

0.01），GSH含量升高（

0.05）；GPX4、SLC7A11 mRNA和蛋白表达升高（

0.01，

0.05），p53 mRNA和蛋白表达降低（

0.01）。与针刺+激动剂组比较，针刺组神经功能缺损评分升高（

0.05）；脑梗死体积百分比降低（

0.05）；脑组织病理性损伤减轻；皮层及海马铁沉积百分比减少（

0.01）；线粒体形态趋于正常；Fe

、MDA含量和ROS平均荧光强度降低（

0.05），GSH含量升高（

0.01）；GPX4、SLC7A11 mRNA和蛋白表达升高（

0.01，

0.05），p53 mRNA和蛋白表达降低（

0.01，

0.05）。

结论：

醒脑开窍针刺法可减轻CIRI大鼠神经损伤，其机制可能为通过p53/SLC7A11/GPX4信号通路抑制神经细胞铁死亡。

Abstract

Objective

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

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

) 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

Compared with the sham-operated group

the model group exhibited the decrease in neurological deficit score (

0.01)

and the increase in cerebral infarction volume percentage (

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 (

0.01)

mitochondrial membrane density increased

the cristae was broken or reduced

and the outer membrane ruptured. The levels of Fe

and MDA

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

0.01) and the level of GSH was red

uced (

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

0.01)

while that of p53 rose (

0.01). When compared with the model group

in the agonist group

the neurological deficit score was reduced (

0.05)

the percentage of infarction volume was higher (

0.01)

the histopathological damage was further exacerbated

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

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

and MDA

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

0.01

0.05) and the level of GSH was reduced (

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

0.01

0.05)

while that of p53 was elevated (

0.01). Besides

in comparison with the model group

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

0.01

0.05)

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

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 (

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

and MDA

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

0.01) and the level of GSH was higher (

0.01) in the acupuncture group. The mean flu

orescence intensity of ROS were dropped (

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

0.01) and that of p53 was reduced (

0.01

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

0.05

0.01) and that of p53 was dropped (

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

0.05). When compared with the agonist group

in the acupuncture + agonist group

neurological deficit score increased (

0.01)

the percentage of cerebral infarction volume decreased (

0.01)

the pathological brain tissue damage was reduced

the percentage of iron deposition in cortex and hippocampus decreased (

0.01)

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

and MDA

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

0.01)

while the level of GSH increased (

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

0.01

0.05)

and that of p53 reduced (

0.01). In comparison with the acupuncture + agonist group

in the acupuncture group

the neurological deficit score increased (

0.05)

the percentage of cerebral infarction volume decreased (

0.05)

the pathological brain tissue damage was alleviated

the percentage of iron deposition in cortex and hippocampus decreased (

0.01)

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

and MDA

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

0.05)

while the level of GSH rose (

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

0.01

0.05)

and that of p53 reduced (

0.01

0.05).

Conclusion

Xingnao Kaiqiao

acupuncture can alleviate neurological damage in CIRI rats

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

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