电针足三里、内关穴抑制mTOR信号通路缓解大鼠脑缺血损伤作用的研究

doi:10.11958/20220727

天津医药 ›› 2022, Vol. 50 ›› Issue (11): 1146-1152.doi: 10.11958/20220727

电针足三里、内关穴抑制mTOR信号通路缓解大鼠脑缺血损伤作用的研究

张游(), 靳子言, 尹亚龙, 吴新贵^△()

广西医科大学第一附属医院中医科（邮编530021）

收稿日期:2022-05-11 修回日期:2022-06-24 出版日期:2022-11-15 发布日期:2022-11-11
通讯作者: 吴新贵 E-mail:843757818@qq.com;wxingui200061@aliyun.com
作者简介:张游（1995），男，硕士在读，主要从事脑血管病的针灸治疗方面研究。E-mail：843757818@qq.com
基金资助:
国家自然科学基金资助项目(81760885);广西自然科学基金资助项目(2017GXNSFDA198011);广西中医药管理局立项课题(GZZC14-44)

The effect of electroacupuncture at "Neiguan" and "Zusanli" points on inhibiting mTOR signaling pathway in alleviating cerebral ischemic injury in rats

ZHANG You(), JIN Ziyan, YIN Yalong, WU Xingui^△()

Department of Traditional Chinese Medicine, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China

Received:2022-05-11 Revised:2022-06-24 Published:2022-11-15 Online:2022-11-11
Contact: WU Xingui E-mail:843757818@qq.com;wxingui200061@aliyun.com

张游, 靳子言, 尹亚龙, 吴新贵. 电针足三里、内关穴抑制mTOR信号通路缓解大鼠脑缺血损伤作用的研究[J]. 天津医药, 2022, 50(11): 1146-1152.

ZHANG You, JIN Ziyan, YIN Yalong, WU Xingui. The effect of electroacupuncture at "Neiguan" and "Zusanli" points on inhibiting mTOR signaling pathway in alleviating cerebral ischemic injury in rats[J]. Tianjin Medical Journal, 2022, 50(11): 1146-1152.

摘要/Abstract

摘要：

目的探讨电针（EA）刺激足三里、内关穴对脑缺血大鼠的影响及对mTOR信号通路的调控作用。方法将75只大鼠按随机数字表法分为假手术（Sham）组、大脑中动脉阻塞（MCAO）模型3 d组、EA干预3 d组、MCAO模型7 d组、EA干预7 d组，每组15只。除Sham组外，其余组采用Longa线栓法构建永久性MCAO大鼠模型，EA干预组术后每日治疗内关和足三里穴20 min。于相应时间点对各组行神经功能缺损评分，采用2，3，5-三苯基四氮唑（TTC）染色分析脑梗死体积，HE和尼氏染色评估脑缺血后病理改变，Western blot法检测梗死侧大脑皮层中雷帕霉素靶蛋白（mTOR）通路相关因子蛋白激酶B（AKT）、磷酸化mTOR（p-mTOR）、总mTOR蛋白的表达，实时荧光定量PCR检测梗死侧大脑皮层中LC3B、Beclin-1和B淋巴细胞瘤-2相关蛋白X（Bax）mRNA的表达。结果与Sham组相比，MCAO模型组各时间点神经功能缺损评分升高（P<0.05），大脑皮层区出现大片坏死及炎性浸润，大量神经元坏死，细胞排列紊乱，完整神经元数量显著减少（P<0.05），LC3B、Beclin-1 mRNA表达显著降低（P<0.05），MCAO模型7 d组的Bax mRNA表达显著增加（P<0.05），并且mTOR磷酸化水平（p-mTOR/t-mTOR）显著高于MCAO模型3 d组（P<0.05）。与MCAO模型组相比，EA干预7 d组大鼠神经功能缺损评分降低，EA干预3 d组和7 d组脑梗死面积百分比均明显降低（P<0.05），大脑皮层区坏死灶显著减小，炎性浸润改善，细胞排列不规律，完整神经元数量显著增加（P<0.05）。与MCAO模型7 d组相比，EA干预7 d组LC3B、Beclin-1 mRNA表达显著增加，Bax mRNA表达显著降低，p-mTOR/t-mTOR水平显著降低（均P<0.05）。AKT蛋白在各组中表达无明显差异。结论 EA刺激足三里、内关穴对脑缺血性损伤具有神经保护作用，并诱导自噬发生，抑制凋亡反应，其机制可能与抑制mTOR信号通路活化有关。

关键词: 脑缺血, 电针, 穴, 足三里, 穴, 内关, 自噬, Beclin-1蛋白, bcl-2相关X蛋白质, TOR丝氨酸-苏氨酸激酶

Abstract:

Objective To investigate the effects of electroacupuncture (EA) stimulation at “Zusanli” and “Neiguan” points on cerebral ischemia and the mTOR signaling pathway in rats. Methods A total of 75 rats were divided into the sham group, the middle cerebral artery occlusion (MCAO) model 3-d group, the EA intervention 3-d group, the MCAO model 7-d group, and the EA intervention 7-d group by random number table method, with 15 rats in each group. Except for the sham group, the permanent MCAO rat model was constructed using the Longa thread embolization method. The EA intervention group was treated at "Neiguan" and "Zusanli" points for 20 min daily after surgery. The neurological deficits were scored at the corresponding time points in each group. 2,3,5-triphenyltetrazolium (TTC) staining was used to analyze the cerebral infarct volume. HE and Nissler staining were used to assess the post-ischemic pathological changes. Western blot assay was used to detect the expression levels of mammalian target of rapamycin (mTOR) pathway-related factors protein kinase B (AKT), p-mTOR and t-mTOR protein in cerebral cortex on the infarct side. Real-time fluorescence quantitative PCR was used to detect the expression levels of LC3B, Beclin-1 and B lymphocytoma-2-associated protein X (Bax) mRNA in cerebral cortex on the infarct side. Results Compared with the sham group, neurological deficit scores at all time points increased in the MCAO group (P<0.05), large necrotic and inflammatory infiltrates in cortical areas, massive neuronal necrosis, disturbed cell arrangement and significantly reduced intact neurons (P<0.05). There were significantly decreased LC3B and Beclin-1 mRNA expression (P<0.05), and Bax mRNA expression was significantly increased in the MCAO model 7-d group (P<0.05). The phosphorylation level of mTOR (p-mTOR/t-mTOR) was significantly higher in the MCAO model 7-d group than that in the MCAO model 3-d group (P<0.05). Compared with the MCAO group, the neurological deficit score was significantly reduced in the EA intervention 7-d group, and the percentage of brain infarct volume was significantly reduced in the EA intervention 3-d group and 7-d group (P<0.05). The necrotic foci were significantly reduced in cortical area, the inflammatory infiltration was improved, the cell arrangement was inregular and the number of intact neurons was significantly increased (P<0.05). Compared with the MCAO model 7-d group, LC3B and Beclin-1 mRNA expression levels were significantly increased (P<0.05), and Bax mRNA, p-mTOR/t-mTOR expression levels were significantly decreased (P<0.05) in the EA intervention 7-d group (P<0.05). There was no significant difference in AKT protein expression between the different groups. Conclusion EA stimulation at “Zusanli” and “Neiguan” points has a neuroprotective effect on cerebral ischemic injury and induces autophagy and inhibits apoptotic responses, which may be related to the inhibition of mTOR signaling pathway activation.

Key words: brain ischemia, electroacupuncture, POINT ST36 (ZUSANLI), POINT PC6 (NEIGUAN), autophagy, Beclin-1, bcl-2-associated X protein, TOR serine-threonine kinases

中图分类号:

R743，R245

图/表 9

表1 引物序列

Tab.1 Primer sequences

基因名称	引物序列（5'→3'）	产物大小（bp）
LC3B	上游：AACACAGCCACCTCTCGACCT	125
	下游：ACACAACCCACACACGGCAG	125
Beclin-1	上游：AGGAGTTGCCGTTGTACTGTTCT	178
	下游：GTGTCTTCAATCTTGCCTTTCTCC	178
Bax	上游：GGGTGGTTGCCCTTTTCTACTT	104
	下游：GAAGTCCAGTGTCCAGCCCAT	104
β-actin	上游：TGCTATGTTGCCCTAGACTTCG	240
	下游：GTTGGCATAGAGGTCTTTACGG	240

表2 各组大鼠脑缺血性损伤后不同时间点神经功能缺损评分比较 (n=15,分,$\bar{x}\pm s$)

Tab.2 Comparison of neurological deficit scores at different time points after ischemic brain injury between the three groups of rats

组别	术后1 d	术后3 d	术后7 d
Sham组	0.00±0.00	0.00±0.00	0.00±0.00
MCAO模型组	2.70±0.48^a	2.50±0.52^a	2.30±0.48^a
EA干预组	2.80±0.42^a	2.60±0.51^a	1.60±0.51^b
F	184.135^＊＊	119.571^＊＊	83.400^＊＊

表3 各组大鼠脑梗死面积百分比及完整神经元数量的比较 (n=5, $\bar{x}\pm s$)

Tab.3 Comparison of percentage of cerebral infarction area and number of intact neurons between the five groups of rats

组别	脑梗死面积百分比（%）	神经元数量（个/视野）
Sham组	—	542.00±43.13
MCAO模型3 d组	68.32±5.78	245.27±48.84^a
EA干预3 d组	52.91±0.47^b	356.13±82.28^b
MCAO模型7 d组	60.25±2.86	406.27±39.40^a
EA干预7 d组	36.15±1.66^c	500.87±36.23^c
F	84.053^＊＊	75.273^＊＊

图1 TTC染色检测各组脑梗死面积

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

图2 各组大鼠梗死侧大脑皮层区病理形态学比较（HE染色，×200）红色箭头表示血管改变，黑色箭头表示神经元改变。

Fig.2 Comparison of pathomorphological changes of cortical areas on the infarct side of rats between the five groups (HE staining, ×200)

图3 各组大鼠梗死侧大脑皮层区神经元形态学比较（尼式染色，×200）

Fig.3 Comparison of neuronal morphology in the cortical area of the infarct side of rats between the five groups (Nissl staining, ×200)

表4 各组大鼠脑组织AKT、p-mTOR和t-mTOR蛋白相对表达水平比较 (n=5, $\bar{x}\pm s$)

Tab.4 Comparison of relative expression levels of AKT, p-mTOR and t-mTOR proteins in rat brain tissues between the five groups

组别	AKT	p-mTOR/t-mTOR	t-mTOR
Sham组	2.07±1.14	1.15±0.40	0.87±0.32
MCAO模型3 d组	2.89±0.70	0.65±0.22^a	1.40±0.26
EA干预3 d组	2.33±0.73	0.69±0.30	1.10±0.33
MCAO模型7 d组	2.74±0.59	1.49±0.40^b	1.02±0.93
EA干预7 d组	2.11±0.90	0.63±0.21^c	0.97±0.32
F	0.972	7.015^＊＊	2.470

图4 蛋白印迹分析显示各组AKT、p-mTOR和t-mTOR蛋白的表达 A~E分别为Sham组、MCAO模型3 d组、EA干预3 d组、MCAO模型7 d组和EA干预7 d组。

Fig.4 Western blot analysis showed the expressions of AKT, p-mTOR and t-mTOR in the five groups

表5 各组大鼠脑组织不同时间点LC3B、Beclin-1和Bax mRNA表达量比较(n=5,$\bar{x}\pm s$)

Tab.5 Comparison of LC3B, Beclin-1 and Bax mRNA expressions in rat brain tissue at different time points between the five groups

组别	LC3B	Beclin-1	Bax
Sham组	1.00±0.00	1.00±0.00	1.00±0.00
MCAO模型3 d组	0.21±0.00^a	0.21±0.01^a	0.57±0.08^a
EA干预3 d组	0.19±0.02	0.16±0.00	0.53±0.06
MCAO模型7 d组	0.62±0.23^a	0.17±0.01^a	1.58±0.21^a
EA干预7 d组	1.68±0.09^c	0.76±0.03^c	1.30±0.10^c
F	142.452^＊＊	2 229.827^＊＊	78.799^＊＊

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电针足三里、内关穴抑制mTOR信号通路缓解大鼠脑缺血损伤作用的研究

The effect of electroacupuncture at "Neiguan" and "Zusanli" points on inhibiting mTOR signaling pathway in alleviating cerebral ischemic injury in rats

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