益气升清方调节HIF-1α/NLRP3信号通路对缺血性脑卒中大鼠神经元焦亡的影响

doi:10.11958/20231141

天津医药 ›› 2024, Vol. 52 ›› Issue (4): 350-355.doi: 10.11958/20231141

益气升清方调节HIF-1α/NLRP3信号通路对缺血性脑卒中大鼠神经元焦亡的影响

王月¹(), 权兴苗¹, 王玉², 宋春侠¹^,^△(), 邵月³, 徐立伟¹

1 承德医学院附属医院中医科（邮编067000）
2 承德市中医院针灸推拿科
3 承德医学院附属医院骨伤科

收稿日期:2023-08-01 修回日期:2023-10-30 出版日期:2024-04-15 发布日期:2024-04-19
通讯作者: ^△E-mail：scx-scx-001@163.com
作者简介:王月（1990），女，主治医师，主要从事中医学方面研究。E-mail：lucksong8@163.com
基金资助:
河北省中医药管理局科研计划项目(2022154)

Influence of Yiqi Shengqing recipe on neuron pyroptosis in ischemic stroke rats by regulating HIF-1α/NLRP3 signal pathway

WANG Yue¹(), QUAN Xingmiao¹, WANG Yu², SONG Chunxia¹^,^△(), SHAO Yue³, XU Liwei¹

1 Department of Traditional Chinese Medicine, the Affiliated Hospital of Chengde Medical University Chengde 067000, China
2 Department of Acupuncture and Massage, Chengde Hospital of Traditional Chinese Medicine
3 Department of Orthopedics and Traumatology, the Affiliated Hospital of Chengde Medical University

Received:2023-08-01 Revised:2023-10-30 Published:2024-04-15 Online:2024-04-19

王月, 权兴苗, 王玉, 宋春侠, 邵月, 徐立伟. 益气升清方调节HIF-1α/NLRP3信号通路对缺血性脑卒中大鼠神经元焦亡的影响[J]. 天津医药, 2024, 52(4): 350-355.

WANG Yue, QUAN Xingmiao, WANG Yu, SONG Chunxia, SHAO Yue, XU Liwei. Influence of Yiqi Shengqing recipe on neuron pyroptosis in ischemic stroke rats by regulating HIF-1α/NLRP3 signal pathway[J]. Tianjin Medical Journal, 2024, 52(4): 350-355.

摘要/Abstract

摘要：

目的探究益气升清方调节缺氧诱导因子-1α（HIF-1α）/NOD样受体热蛋白结构域相关蛋白3（NLRP3）信号通路对缺血性脑卒中大鼠神经元焦亡的影响。方法将SD大鼠随机分为假手术组（S组），模型组（M组），益气升清方低、中、高剂量组（3.465、6.930、13.860 g/kg）及益气升清方高剂量+HIF-1α激活剂DMOG组（13.860 g/kg益气升清方+40 mg/kg DMOG），每组15只。采用线栓法构建脑卒中模型。造模成功后进行神经功能缺陷评估；TTC染色评估脑梗死体积；ELISA法检测血清白细胞介素（IL）-1β、IL-18水平；HE染色检测缺血皮质区病理变化；TUNEL染色检测神经元凋亡；Western blot检测焦亡及HIF-1α/NLRP3通路相关蛋白表达。结果与S组比较，M组神经功能缺陷评分、梗死体积、IL-1β含量、IL-18含量、神经细胞凋亡率、胞膜穿孔蛋白D-N端（GSDMD-N）、胱天蛋白酶1（Caspase-1）、HIF-1α、NLRP3蛋白水平均上升（P<0.05）；与M组比较，低、中、高剂量益气升清方组神经功能缺陷评分、梗死体积、IL-1β含量、IL-18含量、神经细胞凋亡率、GSDMD-N、Caspase-1、HIF-1α、NLRP3蛋白水平均下调（P<0.05）；DMOG减弱了高剂量益气升清方对缺血性脑卒中大鼠神经元焦亡的改善作用。结论益气升清方可能通过下调HIF-1α/NLRP3信号通路减轻缺血性脑卒中大鼠神经元焦亡。

关键词: 卒中, 缺氧诱导因子1, α亚基, NLR家族, 热蛋白结构域包含蛋白3, 神经元, 细胞焦亡, 益气升清方

Abstract:

Objective To investigate the influence of Yiqi Shengqing recipe on neuronal pyroptosis in ischemic stroke rats by regulating hypoxia inducible factor-1α (HIF-1α)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) signal pathway. Methods SD rats were randomly divided into the sham operation group (S group), the model group (M group), the low, medium and high dose (3.465, 6.930 and 13.860 g/kg) Yiqi Shengqing recipe groups, and high-dose Yiqi Shengqing recipe+HIF-1α activator DMOG (13.860 g/kg Yiqi Shengqing recipe+40 mg/kg DMOG) group, 15 animals in each group. The stroke model was constructed by thread plug method. After successful modeling, neural function defects were evaluated including TTC staining for evaluating the volume of cerebral infarction, ELISA for detecting the serum interleukin (IL)-1β and IL-18 levels, HE staining for detecting pathological changes of ischemic cortex, TUNEL staining for detecting neuronal apoptosis and Western blot assay for detecting the expression of pyroprotein and HIF-1α/NLRP3 pathway protein. Results Compared with the S group, the neurological deficit score, infarct volume, IL-1β, IL-18 contents, apoptosis rate of nerve cells, GasderminD-N (GSDMD-N), Caspase-1, HIF-1α and NLRP3 protein levels were increased in the M group (P<0.05). Compared with the M group, the neurological defect score, infarct volume, IL-1β, IL-18 contents, neuronal apoptosis rate, GSDMD-N, Caspase-1, HIF-1α and NLRP3 protein levels were decreased in the low, medium and high dose Yiqi Shengqing recipe groups (P<0.05). DMOG attenuated the improvement effect of high-dose Yiqi Shengqing recipe on neuronal death in ischemic stroke rats. Conclusion Yiqi Shengqing recipe may reduce the neuronal pyroptosis in ischemic stroke rats by down-regulating HIF-1α/NLRP3 signal pathway.

Key words: stroke, hypoxia-inducible factor 1, alpha subunit, NLR family, pyrin domain-containing 3 protein, neurons, pyroptosis, Yiqi Shengqing recipe

中图分类号:

R285.5

图/表 9

图1 各组大鼠脑组织TTC染色图

Fig.1 TTC staining of brain tissue of rats in each group

表1 各组大鼠神经功能缺陷评分及梗死体积比较（$\bar{x}±s$）

Tab.1 Comparison of neurological deficit score and infarct volume of rats between six groups

组别	神经功能缺陷评分/分（n=15）	梗死体积/% （n=5）
S组	0.00±0.00	0.00±0.00
M组	2.93±0.26^a	17.03±1.70^a
L-Med组	2.13±0.35^b	12.87±1.45^b
M-Med组	1.20±0.41^bc	8.97±0.83^bc
H-Med组	0.47±0.52^bcd	5.00±0.05^bcd
H-Med+DMOG组	2.13±0.35^e	13.21±1.43^e
F	90.734^＊＊	67.926^＊＊

表2 各组大鼠血清IL-1β、IL-18水平比较（n=15，ng/L，$\bar{x}±s$）

Tab.2 Comparison of serum IL-1β and IL-18 levels of rats between six groups

组别	IL-1β	IL-18
S组	12.34±1.34	22.22±2.88
M组	50.76±5.56^a	57.43±5.95^a
L-Med组	42.45±4.65^b	46.31±4.62^b
M-Med组	30.34±3.26^bc	35.43±3.54^bc
H-Med组	21.23±2.17^bcd	24.00±3.13^bcd
H-Med+DMOG组	43.64±4.15^e	49.24±4.22^e
F	225.615^＊＊	174.083^＊＊

图2 各组大鼠脑组织缺血皮质区病理变化（HE染色，×200）

Fig.2 Pathological changes of cerebral ischemic cortex in each group of rats (HE staining, ×200)

图3 各组大鼠缺血皮质区神经细胞凋亡（TUNEL染色，×200）

Fig.3 Apoptosis of neurons in ischemic cortex of rats in each group (TUNEL staining, ×200)

图4 Western blot检测缺血皮质区GSDMD-N、Caspase-1蛋白表达情况 A—F依次为S组、M组、L-Med组、M-Med组、H-Med组、H-Med+DMOG组。

Fig.4 Expression of GSDMD-N and Caspase-1 protein in ischemic cortex by Western blot assay

表3 各组大鼠脑组织GSDMD-N、Caspase-1表达水平比较（n=5，$\bar{x}±s$）

Tab.3 Comparison of GSDMD-N and Caspase-1 expression levels in brain tissue of rats between six groups

组别	GSDMD-N/GAPDH	Caspase-1/GAPDH
S组	0.53±0.05	0.93±0.06
M组	1.35±0.16^a	2.65±0.28^a
L-Med组	1.13±0.13^b	2.19±0.24^b
M-Med组	0.91±0.10^bc	1.73±0.21^bc
H-Med组	0.68±0.06^bcd	1.21±0.13^bcd
H-Med+DMOG组	1.15±0.12^e	2.25±0.22^e
F	39.689^＊＊	52.596^＊＊

图5 Western blot检测缺血皮质区HIF-1α、NLRP3蛋白表达情况 A—F依次为S组、M组、L-Med组、M-Med组、H-Med组、H-Med+DMOG组。

Fig.5 Expression of HIF-1α and NLRP3 protein in ischemic cortex by Western blot assay

表4 各组大鼠脑组织HIF-1α、NLRP3表达水平比较（n=5，$\bar{x}±s$）

Tab.4 Comparison of HIF-1α and NLRP3 expression levels in brain tissue of rats between six groups

组别	HIF-1α/GAPDH	NLRP3/GAPDH
S组	1.13±0.13	1.03±0.11
M组	2.44±0.29^a	2.34±0.24^a
L-Med组	2.03±0.23^b	1.95±0.21^b
M-Med组	1.62±0.16^bc	1.47±0.15^bc
H-Med组	1.21±0.12^bcd	1.05±0.11^bcd
H-Med+DMOG组	2.11±0.22^e	2.15±0.24^e
F	34.092^＊＊	46.356^＊＊

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益气升清方调节HIF-1α/NLRP3信号通路对缺血性脑卒中大鼠神经元焦亡的影响

Influence of Yiqi Shengqing recipe on neuron pyroptosis in ischemic stroke rats by regulating HIF-1α/NLRP3 signal pathway

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