黄芩苷通过PI3K/AKT/eNOS通路保护脑微出血大鼠神经损伤

doi:10.11958/20250606

天津医药 ›› 2025, Vol. 53 ›› Issue (5): 468-473.doi: 10.11958/20250606

黄芩苷通过PI3K/AKT/eNOS通路保护脑微出血大鼠神经损伤

钟文闻¹(), 邹正寿²^,^△(), 向庆伟³

1 黄石市妇幼保健院康复医学科（邮编435100）
2 黄石市中心医院神经内科
3 湖北省中医院老年病科

收稿日期:2025-02-18 修回日期:2025-03-13 出版日期:2025-05-15 发布日期:2025-05-28
通讯作者: △ E-mail：592965479@qq.com
作者简介:钟文闻（1989），女，主治医师，主要从事脑血管、神经变性疾病方面研究。E-mail：1158760034@qq.com
基金资助:
湖北省科学技术厅自然科学基金项目(2022CFD022)

Protective effect of baicalin on nerve injury in rats with cerebral microbleeds through PI3K/AKT/eNOS pathway

ZHONG Wenwen¹(), ZOU Zhengshou²^,^△(), XIANG Qingwei³

1 Department of Rehabilitation Medicine, Huangshi Maternal and Child Health Hospital, Huangshi 435100, China
2 Department of Neurology, Huangshi Central Hospital
3 Department of Geriatrics, Hubei Provincial Traditional Chinese Medicine Hospital

Received:2025-02-18 Revised:2025-03-13 Published:2025-05-15 Online:2025-05-28
Contact: △ E-mail：592965479@qq.com

钟文闻, 邹正寿, 向庆伟. 黄芩苷通过PI3K/AKT/eNOS通路保护脑微出血大鼠神经损伤[J]. 天津医药, 2025, 53(5): 468-473.

ZHONG Wenwen, ZOU Zhengshou, XIANG Qingwei. Protective effect of baicalin on nerve injury in rats with cerebral microbleeds through PI3K/AKT/eNOS pathway[J]. Tianjin Medical Journal, 2025, 53(5): 468-473.

摘要/Abstract

摘要：

目的探索黄芩苷通过磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）/内皮型一氧化氮合酶（eNOS）通路对脑微出血大鼠神经损伤的保护作用。方法通过侧脑室注射脂多糖（LPS）法构建脑微出血大鼠模型，并将其分为模型组、黄芩苷组（20 mg/kg）、LY294002组（PI3K抑制剂，10 mg/kg）、黄芩苷+LY294002组（20 mg/kg黄芩苷+10 mg/kg LY294002），同时选择未注射LPS的大鼠作为对照组。评估各组大鼠神经功能；氯化三苯基四氮唑（TTC）染色法评估各组大鼠脑梗死情况；苏木精-伊红（HE）染色观察各组大鼠脑组织病理形态；TUNEL法检测各组大鼠脑组织神经元凋亡情况；酶联免疫吸附试验（ELISA）检测各组大鼠脑组织肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1β及IL-17水平；Western blot法检测各组大鼠脑组织B细胞淋巴瘤-2（Bcl-2）、胱天蛋白酶（Caspase）-3、Bcl-2相关X蛋白（Bax）、PI3K、磷酸化（p）-AKT、AKT、p-eNOS、eNOS蛋白表达情况。结果与对照组比，模型组大鼠神经元稀疏，且细胞数量少、排列紊乱，神经功能评分、脑梗死面积、细胞凋亡率、Bax蛋白表达量、Caspase-3蛋白表达量及TNF-α、IL-1β、IL-17水平增加，而Bcl-2、PI3K、p-AKT、p-eNOS蛋白表达量降低（P<0.05）；与模型组比，黄芩苷组大鼠脑组织病理损伤明显改善，神经功能评分、脑梗死面积、细胞凋亡率、Bax蛋白表达量、Caspase-3蛋白表达量及TNF-α、IL-1β、IL-17水平降低，而Bcl-2、PI3K、p-AKT、p-eNOS蛋白表达量增加（P<0.05），但LY294002组大鼠脑组织损伤进一步加重，神经功能评分、脑梗死面积、细胞凋亡率、Bax、Caspase-3蛋白表达量及TNF-α、IL-1β、IL-17水平升高，而Bcl-2、PI3K、p-AKT及p-eNOS蛋白表达量减少（P<0.05）；在黄芩苷处理的基础上使用LY294002进行干预可逆转黄芩苷对脑微出血大鼠上述指标的改善作用（P<0.05）。结论黄芩苷可能通过激活PI3K/AKT/eNOS通路对脑微出血大鼠神经损伤发挥保护作用。

关键词: 黄芩苷, 脑出血, PI3K/AKT/eNOS, 神经损伤

Abstract:

Objective To explore the protective effect of baicalin on nerve damage in rats with cerebral microbleeds through the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) pathway. Methods The rat model of cerebral microbleeds was established by intraventricular injection of lipopolysaccharide (LPS), and rats were separated into the model group, the baicalin group (20 mg/kg), the LY294002 group (PI3K inhibitor, 10 mg/kg) and the baicalin+LY294002 group (20 mg/kg baicalin and 10 mg/kg LY294002). Rats without LPS injection were served as the control group. The nerve function was evaluated in five groups of rats. Triphenyltetrazolium chloride (TTC) staining was used to evaluate the cerebral infarction status. Hematoxylin-eosin (HE) staining was used to observe the pathological morphology of brain tissue of rats in each group. TUNEL method was used to detect neuronal apoptosis in brain tissue. Enzyme linked immunosorbent assay (ELISA) was performed to measure tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-17 (IL-17) in brain tissue of rats in each group. In addition, Western blot assay was used to measure B-cell lymphoma 2 (Bcl-2), Caspase-3, Bcl-2 associated X protein (Bax), PI3K, phosphorylated (p)-AKT, AKT, p-eNOS and eNOS proteins in brain tissue of rats. Results Compared with the control group, neurons in the model group was sparse, with fewer cells and disordered arrangement, the nerve function score, infarct area, TNF-α, IL-1β, IL-17, apoptosis rate, Bax and Caspase-3 proteins were increased, and Bcl-2, PI3K, p-AKT and p-eNOS proteins were decreased (P<0.05). Compared with the model group, the baicalin group showed clear improvement in brain pathological damage, the nerve function score, infarct area, apoptosis rate, Bax and Caspase-3 proteins, TNF-α, IL-1β and IL-17 were decreased, while protein expressions of Bcl-2, PI3K, p-AKT and p-eNOS were increased (P<0.05). However, the brain tissue damage of the LY294002 group was further aggravated, and the nerve function score, infarct area, TNF-α, IL-1β, IL-17, apoptosis rate and the expression of apoptosis proteins Bax and Caspase-3 were increased, the Bcl-2, PI3K, p-AKT and p-eNOS proteins were decreased (P<0.05). Intervention with LY294002 on basis of baicalin treatment could reverse the improvement effect of baicalin on the above indicators in rats with cerebral microbleeds (P<0.05). Conclusion Baicalin may exert a protective effect on nerve damage in rats with cerebral microbleeds by activating PI3K/AKT/eNOS pathway.

Key words: baicalin, cerebral hemorrhage, PI3K/AKT/eNOS, nerve damage

中图分类号:

R743

图/表 8

图1 各组大鼠脑梗死面积的TTC染色结果

Fig.1 TTC staining results of cerebral infarction area of rats in each group

图2 各组大鼠脑组织HE染色结果（×400）

Fig.2 HE staining results of brain tissue in each group (×400)

图3 各组大鼠脑组织神经元凋亡的TUNEL染色结果（×200）

Fig.3 TUNEL staining results of neuronal apoptosis in rat brain tissue in each group (×200)

图4 各组大鼠脑组织凋亡蛋白Bax、Caspase-3、Bcl-2表达情况 A：对照组；B：模型组；C：黄芩苷组；D：LY294002组；E：黄芩苷+LY294002组。

Fig.4 Expression of apoptosis proteins Bax, Caspase-3 and Bcl-2 in brain tissue of rats in each group

表1 各组大鼠脑组织神经元凋亡率及凋亡相关蛋白表达水平比较（n=5,$\bar{x}±s$）

Tab.1 Comparison of neuronal apoptosis rate and apoptosis-related protein expression levels in rat brain tissue between five groups

组别	细胞凋亡率/%	Bax	Caspase-3	Bcl-2
对照组	2.94±0.37	0.31±0.03	0.27±0.03	0.95±0.09
模型组	32.41±3.65^a	0.90±0.08^a	0.92±0.09^a	0.37±0.03^a
黄芩苷组	8.83±0.94^b	0.42±0.04^b	0.39±0.04^b	0.86±0.07^b
LY294002组	49.56±5.72^b	1.26±0.10^b	1.35±0.12^b	0.10±0.02^b
黄芩苷+LY294002组	31.02±2.96^c	0.87±0.08^c	0.88±0.09^c	0.41±0.04^c
F	161.497^＊＊	148.093^＊＊	144.464^＊＊	200.425^＊＊

表2 各组大鼠脑组织TNF-α、IL-1β、IL-17水平比较（n=5,$\bar{x}±s$）

Tab.2 Comparison of levels of TNF-α, IL-1β and IL-17 in brain tissue of rats between five groups

组别	TNF-α/（ng/L）	IL-1β/（ng/L）	IL-17/（μg/L）
对照组	24.63±2.89	21.54±2.36	18.47±2.51
模型组	65.48±6.33^a	78.25±6.73^a	52.82±5.03^a
黄芩苷组	29.85±3.16^b	26.39±3.24^b	23.61±2.54^b
LY294002组	86.73±6.81^b	97.42±7.92^b	68.93±5.78^b
黄芩苷+LY294002组	62.57±6.06^c	71.26±6.87^c	49.85±4.72^c
F	120.233^＊＊	162.874^＊＊	119.551^＊＊

图5 各组大鼠脑组织PI3K/AKT/eNOS通路蛋白表达情况 A：对照组；B：模型组；C：黄芩苷组；D：LY294002组；E：黄芩苷+LY294002组。

Fig.5 PI3K/AKT/eNOS pathway protein expression in brain tissue of rats in each group

表3 各组大鼠脑组织PI3K/AKT/eNOS通路相关蛋白表达量比较（n=5,$\bar{x}±s$）

Tab.3 Comparison of PI3K/AKT/eNOS pathway related protein expression in brain tissue of rats between five groups

组别	PI3K	p-AKT	p-eNOS
对照组	1.34±0.12	0.95±0.08	0.91±0.09
模型组	0.47±0.04^a	0.39±0.03^a	0.36±0.03^a
黄芩苷组	1.19±0.11^b	0.91±0.08^b	0.83±0.07^b
LY294002组	0.21±0.02^b	0.16±0.02^b	0.12±0.01^b
黄芩苷+LY294002组	0.52±0.05^c	0.43±0.04^c	0.39±0.03^c
F	194.460^＊＊	191.115^＊＊	189.044^＊＊

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黄芩苷通过PI3K/AKT/eNOS通路保护脑微出血大鼠神经损伤

Protective effect of baicalin on nerve injury in rats with cerebral microbleeds through PI3K/AKT/eNOS pathway

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