补阳还五汤对脑缺血再灌注损伤小鼠RhoA/ROCK通路的影响

邢烨锋; 赵武霞; 欧志杰; 张天艾; 胡玥

doi:10.14148/j.issn.1672-0482.2023.1006

补阳还五汤对脑缺血再灌注损伤小鼠RhoA/ROCK通路的影响

doi: 10.14148/j.issn.1672-0482.2023.1006

邢烨锋^{1, 2,},
赵武霞²,
欧志杰³,
张天艾²,
胡玥^2, ,

1.
南通市通州区中医院检验科, 江苏南通 226300
2.
南京中医药大学中医学院·中西医结合学院, 江苏南京 210023
3.
常熟市中医院神经内科, 江苏常熟 215516

基金项目:

国家自然科学基金面上项目 82204934

江苏省中医药科技发展计划项目 QN202001

高校大学生创新创业训练计划项目 202210315023Y

南通市科技局社会民生面上项目 MS1202104

详细信息

作者简介:
邢烨锋, 男, 副主任技师, E-mail: 54566435@qq.com

通讯作者:
胡玥, 女, 副教授, 主要从事中西医结合脑病学相关研究, E-mail: yuehu@njucm.edu.cn

中图分类号: R255.5
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- 被引次数: 0
出版历程
- 收稿日期: 2023-03-01
- 网络出版日期: 2023-11-10
- 发布日期: 2023-10-10

Effects of Buyang Huanwu Decoction on RhoA/ROCK Pathway after Cerebral Ischemia/Reperfusion Injury in Mice

XING Ye-feng^{1, 2
,},
ZHAO Wu-xia²,
OU Zhi-jie³,
ZHANG Tian-ai²,
HU Yue^{2
, ,}

1.
Laboratory Department, Nantong Tongzhou District Hospital of Chinese Medicine, Nantong 226300, China
2.
School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
3.
Neurology Department, Changshu Hospital of Chinese Medicine, Changshu 215516, China

摘要

摘要: 目的探讨补阳还五汤对脑缺血再灌注损伤小鼠小胶质细胞P2Y12受体介导的RhoA/ROCK炎症信号通路的影响。方法将C57BL/6J小鼠随机分为Sham组、MCAO组、BYHW-L(低剂量, 6.5 g·kg^-1)组、BYHW-H(高剂量, 26 g·kg^-1)组、BYHW-H+MRS2395组, 构建大脑中动脉闭塞模型(Middle cerebral artery occlusion, MCAO), 通过Longa生物学评分、TTC染色评估小鼠神经功能损伤情况; 通过免疫荧光双标染色法、qPCR方法检测小胶质细胞M1、M2表型极化情况; 通过Western blot法检测P2Y12受体介导的RhoA/ROCK通路蛋白的表达情况。结果与MCAO组相比, 补阳还五汤能改善脑缺血再灌注损伤小鼠的神经功能损伤(P＜0.05)和减小梗死体积(P＜0.05, P＜0.01), 增加小胶质细胞从M1型向M2型极化(P＜0.05, P＜0.01), 抑制P2Y12受体蛋白表达(P＜0.05, P＜0.01), 阻断P2Y12介导的RhoA/ROCK通路及通路下游的p38 MAPK磷酸化(P＜0.05, P＜0.01), 抑制炎症损伤(P＜0.05, P＜0.01)。加入P2Y12受体抑制剂MRS2395未阻断补阳还五汤改善神经功能损伤及抗炎作用。结论补阳还五汤可通过P2Y12受体介导的RhoA/ROCK炎症信号通路改善小鼠脑缺血再灌注后的脑损伤, 还可通过其他途径及通路发挥抗炎作用, 但具体机制有待进一步探索。
- 脑缺血再灌注损伤 /
- 补阳还五汤 /
- 小胶质细胞 /
- P2Y12受体 /
- RhoA/ROCK通路 /
- 炎症
Abstract: OBJECTIVE To investigate the effect of Buyang Huanwu Decoction on microglial P2Y12 receptor-mediated RhoA/ROCK inflammatory signaling pathway in mice with cerebral ischemia/reperfusion (I/R) injury. METHODS The C57BL/6J mice were randomly assigned to the Sham group, MCAO group, BYHW-L (low-dose, 6.5 g·kg^-1) group, BYHW-H (high-dose, 26 g·kg^-1) group, and BYHW-H+MRS2395 group. The middle cerebral artery occlusion (MCAO) model was constructed, and the neurological impairment of the mice was evaluated by Longa biological score and TTC staining. The phenotypic polarization of microglia towards M1 or M2 was detected by immunofluorescence staining and qPCR. The protein expression levels of factors in RhoA/ROCK pathway mediated by the P2Y12 receptor were detected by Western blot. RESULTS Compared with MCAO group, Buyang Huanwu Decoction could reduce the neurological impairment (P < 0.05) and infarct volume of I/R injured mice (P < 0.05, P < 0.01), increase the polarization of M1 to M2 in microglia cells (P < 0.05, P < 0.01), inhibit the expression of P2Y12 receptor protein (P < 0.05, P < 0.01), block the P2Y12-mediated RhoA/ROCK pathway and p38 MAPK phosphorylation in the downstream pathway (P < 0.05, P < 0.01), and inhibit the inflammatory damage (P < 0.05, P < 0.01). The P2Y12 receptor inhibitor MRS2395 did not block the anti-inflammatory effect of Buyang Huanwu Decoction. CONCLUSION Buyang Huanwu Decoction may help to improve brain function after cerebral I/R injury in mice through P2Y12-mediated RhoA/ROCK inflammatory signaling pathway, it may also play an anti-inflammatory role through other pathways, but the other mechanisms need further exploration.
- ischemia reperfusion injury /
- Buyang Huanwu Decoction /
- microglia /
- P2Y12 receptor /
- RhoA/ROCK pathway /
- inflammation

HTML全文

图 1 补阳还五汤对MCAO小鼠脑梗死体积的影响(x±s, n=6)

Figure 1. The effect of BYHW on infract volume of MCAO mice(x±s, n=6)

下载: 全尺寸图片幻灯片

图 2 补阳还五汤对MCAO小鼠小胶质细胞极化的影响

注: A.小胶质细胞的M1型标记物CD16;B.小胶质细胞的M2型标记物CD206;与Sham组比较, ^##P＜0.01, ^###P＜0.001;与MCAO组比较, ^*P＜0.05, ^{* *}P＜0.01。x±s, n=6。

Figure 2. The effect of BYHW on the polarization of microglia of MCAO mice

下载: 全尺寸图片幻灯片

图 3 补阳还五汤对MCAO小鼠小胶质细胞表型标志物mRNA表达的影响

注: 与Sham组比较, ^#P＜0.05, ^##P＜0.01, ^###P＜0.001;与MCAO组比较, ^*P＜0.05, ^{* *}P＜0.01。x±s, n=6。

Figure 3. The effect of BYHW on the mRNA level of polarization makers of microglia of MCAO mice

下载: 全尺寸图片幻灯片

图 4 补阳还五汤对小胶质细胞P2Y12受体表达的影响

注: 与Sham组比较; ^##P＜0.01;与MCAO组比较, ^{* *}P＜0.01。x±s, n=6。

Figure 4. The effect of BYHW on the expression of microglial P2Y12

下载: 全尺寸图片幻灯片

图 5 补阳还五汤对P2Y12介导的RhoA/ROCK相关蛋白表达的影响

注: 与Sham组比较; ^###P＜0.001;与MCAO组比较, ^*P＜0.05, ^{* *}P＜0.01。x±s, n=6。

Figure 5. The effect of BYHW on the protein expression of P2Y12 mediated RhoA/ROCK pathway

下载: 全尺寸图片幻灯片

表 1 qPCR引物序列

Table 1. Primer sequences of qPCR

基因名称	引物序列(5′→3′)
CD206-F	ATTCCGGTCGCTGTTCAACT
CD206-R	AACGGAGATGGCGCTTAGAG
Arg-1-F	GGACATCGTGTACATCGGCT
Arg-1-R	GTAGCCGGGGTGAATACTGG
TGF-β-F	AGGGCTACCATGCCAACTTC
TGF-β-R	CCACGTAGTAGACGATGGGC
IL-10-F	GCTCAGCACTGCTATGTTGC
IL-10-R	TGTTGTCCAGCTGGTCCTTC
CD16-F	AAAAGCCTCATCTCGCACCA
CD16-R	CCGCTTAGTGGTCTGAAGCA
CD86-F	AGACATGTGTAACCTGCACCAT
CD86-R	CTGCGCCCAAATAGTGTTCG
GAPDH-F	TGGTGCTGAGTATGTCGTGG
GAPDH-R	GGTTCACACCCATCACAAAC

下载: 导出CSV

表 2 各组神经功能评分(x±s, n=6)

Table 2. Neurological function score of each group (x±s, n=6)

组别	0 h	24 h	48 h	72 h	7 d
Sham	0	0	0	0	0
MCAO	3.76±0.34	2.79±0.53	2.52±0.49	2.02±0.78	1.67±0.46^##
BYHW-L	3.70±0.35	2.72±0.34	2.32±0.16	1.81±0.46	1.40±0.28
BYHW-H	3.68±0.64	2.68±0.61	2.25±0.57	1.65±0.38	1.26±0.49^*
BYHW-H+MRS2395	3.72±0.56	2.66±0.62	2.16±0.46	1.57±0.29	1.23±0.62^**
注: 与Sham组比较, ^##P＜0.01;与MCAO组比较, ^P＜0.05, ^{ *}P＜0.01。

下载: 导出CSV

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