MicroRNA-34a调控电针对缺血再灌注损伤大鼠反应性星形胶质细胞的影响

doi:10.3877/cma.j.issn.2095-123X.2023.03.002

目的

探讨电针"曲池"和"足三里"调控microRNA-34a（miR-34a）对缺血再灌注损伤大鼠缺血周边区反应性星形胶质细胞增殖的影响。

方法

40只雄性SD大鼠随机分为假手术组、模型组、电针组、电针+miR-34a抑制剂组和电针+二甲基亚砜（DMSO）组，每组8只。参照Koizumi方法，制备左侧局灶性大脑中动脉闭塞（MCAO）大鼠模型。术后第1天开始电针大鼠患侧肢体"曲池"、"足三里"穴30 min，1次/d，直至动物处死。比较各组大鼠在电针干预前后的神经行为学评分，同时在电针干预3 d后进行Rota-Rod旋转棒仪行为学检测，免疫荧光染色检测5-溴脱氧尿嘧啶核苷（Brdu）与神经胶质纤维酸性蛋白（GFAP）的共定位情况；逆转录实时定量PCR检测缺血周边区miR-34a相对表达量。

结果

电针干预3 d后，电针组和电针+DMSO组的神经功能缺损评分和在Rota-Rod旋转棒停留时间较模型组明显改善，差异具有统计学意义（P<0.05）。模型组miR-34a的表达较假手术组明显增加，电针组和电针+DMSO组高于模型组，差异具有统计学意义（P<0.05）。模型组和电针组大鼠缺血周围皮质区GFAP⁺/Brdu⁺细胞均表达增加，且电针组较模型组增加更明显，电针+miR-34a抑制剂组较电针组和电针+DMSO组表达降低，差异具有统计学意义（P<0.05）。

结论

电针能明显促进脑缺血再灌注大鼠缺血周围皮质区GFAP⁺/Brdu⁺的增殖，改善脑缺血大鼠的神经功能缺损及运动功能，可能是通过miR-34a调控。

关键词: 脑缺血再灌注, 电针, MicroRNA-34a, 星形胶质细胞

Objective

To explore the effect of electroacupuncture (EA) with Quchi and Zusanli acupoints on the regulation of microRNA-34a (miR-34a) on the proliferation of reactive astrocytes in the ischemic peripheral area of rats with cerebral ischemia-reperfusion injury.

Methods

A total of 40 male rats were randomly assigned into sham group, model group, EA group, EA+miR-34a inhibitor group and EA+dimethyl sulfoxide (DMSO) group, with 8 rats in each group. Preparation of a left focal middle cerebral artery ischemia-reperfusion injury (MCAO) rat model using the Koizumi method. EA was applied at ipsilateral Quchi and Zusanli after operation, 30 min once a day until animals were sacrificed. The neurological scores of rats in each group before and after the EA intervention were compared, the behavioral Rat Rota-Rod test was performed after 3 d of the EA intervention, and the co-location condition of glial fibrillary acidic protein (GFAP) and 5-bromodeoxyuridine (Brdu) was detected by immunofluorescence staining; Reverse transcription real-time quantitative PCR was used to detect the relative expression of miR-34a in the ischemic peripheral area.

Results

After 3 d of EA intervention, the neurological deficit symptoms and residence time on Rota-Rod rotating rod of MCAO rats were significantly improved compared to the EA group, with a statistically significant difference (P<0.05). The expression of miR-34a in the model group was significantly higher than that in the Sham surgery group, the expression of miR-34a in the EA group and the EA+DMSO group was higher than that in the model group, with a statistically significant difference (P<0.05). The expression of GFAP⁺/Brdu⁺ positive cells in the ischemic pericortical area of rats in both the model group and the EA group increased, and the increase was more significant in the EA group compared to the model group, the expression of GFAP⁺/Brdu⁺ positive cells in the EA+miR-34a inhibitor group was lower than that of the EA group and the EA+DMSO group, and the differences were statistically significant (P<0.05).

Conclusion

Electroacupuncture could promote the reactive astrocyte proliferation in ischemic peripheral areas by regulation of miR-34a expression.

Key words: Ischemia-reperfusion injury, Electroacupuncture, MicroRNA-34a, Glial fibrillary acidic protein

表1 4组大鼠治疗前后神经功能缺损评分比较（Mean±SE）

Tab.1 Comparison of neurobehavioral deficit scores before and after treatment in 4 groups (Mean±SE)

组别	只数	缺血再灌注2 h后	电针治疗3 d后
模型组	8	2.15±0.06	1.79±0.11
电针组	8	2.18±0.07	1.38±0.13^a
电针+miR-34a抑制剂组	8	2.09±0.06	1.73±0.11^b
电针+DMSO组	8	2.13±0.09	1.34±0.12^ac
F值		0.344	3.840
P值		0.794	0.014

表1 4组大鼠治疗前后神经功能缺损评分比较（Mean±SE）

Tab.1 Comparison of neurobehavioral deficit scores before and after treatment in 4 groups (Mean±SE)

组别	只数	缺血再灌注2 h后	电针治疗3 d后
模型组	8	2.15±0.06	1.79±0.11
电针组	8	2.18±0.07	1.38±0.13^a
电针+miR-34a抑制剂组	8	2.09±0.06	1.73±0.11^b
电针+DMSO组	8	2.13±0.09	1.34±0.12^ac
F值		0.344	3.840
P值		0.794	0.014

图1 4组大鼠在Rota-Rod旋转棒停留时间比率比较与模型组比较，^aP<0.05；与电针组比较，^bP<0.05；与电针+miR-34a抑制剂组比较，^cP<0.05；DMSO：二甲基亚砜

Fig.1 Comparison of the ratio of stay time on the Rota-Rod apparatus of 4 groups

表2 4组大鼠电针干预3 d后缺血周围皮质区miR-34a相对表达量（Mean±SE）

Tab.2 Relative expression levels of miR-34a in the ischemic pericortical area of rats in 4 groups after 3 d of electroacupuncture intervention (Mean±SE)

组别	只数	相对表达量
模型组	4	1.08±0.03
电针组	4	1.18±0.03^a
电针+miR-34a抑制剂组	4	0.88±0.05^b
电针+DMSO组	4	1.14±0.05^ac
F值		10.436
P值		<0.001

表2 4组大鼠电针干预3 d后缺血周围皮质区miR-34a相对表达量（Mean±SE）

Tab.2 Relative expression levels of miR-34a in the ischemic pericortical area of rats in 4 groups after 3 d of electroacupuncture intervention (Mean±SE)

组别	只数	相对表达量
模型组	4	1.08±0.03
电针组	4	1.18±0.03^a
电针+miR-34a抑制剂组	4	0.88±0.05^b
电针+DMSO组	4	1.14±0.05^ac
F值		10.436
P值		<0.001

图2 5组大鼠缺血周围皮质区GFAP⁺/Brdu⁺细胞免疫荧光双标表达结果红色：GFAP⁺细胞；绿色：Brdu⁺细胞；黄色：GFAP⁺/Brdu⁺共表达细胞；比例尺=100 mm；GFAP：胶质纤维酸性蛋白；Brdu：5-溴脱氧尿嘧啶核苷；DMSO：二甲基亚砜

Fig.2 Immunofluorescent double labeling of GFAP⁺/Brdu⁺ cells in the ischemic pericortical area of rats in 5 groups

表3 4组大鼠在缺血周围皮质区GFAP⁺/Brdu⁺细胞表达比较（Mean±SE）

Tab.3 Expression of GFAP⁺/Brdu⁺ cells in the ischemic pericortical area of rats in 4 groups (Mean±SE)

组别	只数	电针治疗3 d后
模型组	4	11.2±1.20
电针组	4	18.4±0.68^a
电针+miR-34a抑制剂组	4	13.4±1.08^b
电针+DMSO组	4	17.4±1.25^ac
F值		9.893
P值		0.001

表3 4组大鼠在缺血周围皮质区GFAP⁺/Brdu⁺细胞表达比较（Mean±SE）

Tab.3 Expression of GFAP⁺/Brdu⁺ cells in the ischemic pericortical area of rats in 4 groups (Mean±SE)

组别	只数	电针治疗3 d后
模型组	4	11.2±1.20
电针组	4	18.4±0.68^a
电针+miR-34a抑制剂组	4	13.4±1.08^b
电针+DMSO组	4	17.4±1.25^ac
F值		9.893
P值		0.001

[1]	Shehjar F, Maktabi B, Rahman ZA, et al. Stroke: molecular mechanisms and therapies: update on recent developments[J]. Neurochem Int, 2023, 162: 105458. DOI: 10.1016/j.neuint.2022.105458.
[2]	Li N, Wang X, Sun C, et al. Change of intestinal microbiota in cerebral ischemic stroke patients[J]. BMC Microbiol, 2019, 19(1): 191. DOI: 10.1186/s12866-019-1552-1.
[3]	Pekny M, Wilhelmsson U, Tatlisumak T, et al. Astrocyte activation and reactive gliosis-a new target in stroke?[J]. Neurosci Lett, 2019, 689: 45-55. DOI: 10.1016/j.neulet.2018.07.021.
[4]	Aswendt M, Wilhelmsson U, Wieters F, et al. Reactive astrocytes prevent maladaptive plasticity after ischemic stroke[J]. Prog Neurobiol, 2022, 209: 102199. DOI: 10.1016/j.pneurobio.2021.102199.
[5]	Hasel P, Rose IVL, Sadick JS, et al. Neuroinflammatory astrocyte subtypes in the mouse brain[J]. Nat Neurosci, 2021, 24(10): 1475-1487. DOI: 10.1038/s41593-021-00905-6.
[6]	Sofroniew MV, Vinters HV. Astrocytes: biology and pathology[J]. Acta Neuropathol, 2010, 119(1): 7-35. DOI: 10.1007/s00401-009-0619-8.
[7]	Tanaka K, Watase K, Manabe T, et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1[J]. Science, 1997, 276(5319): 1699-1702. DOI: 10.1126/science.276.5319.1699.
[8]	Kitchen P, Salman MM, Halsey AM, et al. Targeting aquaporin-4 subcellular localization to treat central nervous system edema[J]. Cell, 2020, 181(4): 784-799.e719. DOI: 10.1016/j.cell.2020.03.037.
[9]	徐如祥,高谋.脑损伤炎症反应与干细胞免疫调控研究[J].中华脑科疾病与康复杂志(电子版), 2019, 9(5): 257-261. DOI: 10.3877/cma.j.issn.2095-123X.2019.05.001.
[10]	Sims NR, Yew WP. Reactive astrogliosis in stroke: contributions of astrocytes to recovery of neurological function[J]. Neurochem Int, 2017, 107: 88-103. DOI: 10.1016/j.neuint.2016.12.016.
[11]	Verkhratsky A, Parpura V. Astrogliopathology in neurological, neurodevelopmental and psychiatric disorders[J]. Neurobiol Dis, 2016, 85: 254-261. DOI: 10.1016/j.nbd.2015.03.025.
[12]	Verkhratsky A, Ho MS, Vardjan N, et al. General pathophysiology of astroglia[J]. Adv Exp Med Biol, 2019, 1175: 149-179. DOI: 10.1007/978-981-13-9913-8_7.
[13]	Tao J, Xue XH, Chen LD, et al. Electroacupuncture improves neurological deficits and enhances proliferation and differentiation of endogenous nerve stem cells in rats with focal cerebral ischemia[J]. Neurol Res, 2010, 32(2): 198-204. DOI: 10.1179/174313209X414506.
[14]	Li G, Li X, Dong J, et al. Electroacupuncture ameliorates cerebral ischemic injury by inhibiting ferroptosis[J]. Front Neurol, 2021, 12619043. DOI: 10.3389/fneur.2021.619043.
[15]	Yin L, Tang T, Lin Y, et al. Functional connectivity of ipsilateral striatum in rats with ischemic stroke increased by electroacupuncture[J]. J Integr Neurosci, 2022, 21(6): 162. DOI: 10.31083/j.jin2106162.
[16]	Hong P, Jiang M, Li H. Functional requirement of dicer1 and miR-17-5p in reactive astrocyte proliferation after spinal cord injury in the mouse[J]. Glia, 2014, 62(12): 2044-2060. DOI: 10.1002/glia.22725.
[17]	Duan R, Wang SY, Wei B, et al. Angiotensin-(1-7) analogue AVE0991 modulates astrocyte-mediated neuroinflammation via lncRNA SNHG14/miR-223-3p/NLRP3 pathway and offers neuroprotection in a transgenic mouse model of Alzheimer's disease[J]. J Inflamm Res, 2021, 14: 7007-7019. DOI: 10.2147/JIR.S343575.
[18]	Tsai DY, Hung KH, Lin IY, et al. Uncovering microRNA regulatory hubs that modulate plasma cell differentiation[J]. Sci Rep, 2015, 5: 17957. DOI: 10.1038/srep17957.
[19]	Juenemann M, Yeniguen M, Schleicher N, et al. Impact of bubble size in a rat model of cerebral air microembolization[J]. J Cardiothorac Surg, 2013, 8: 198. DOI: 10.1186/1749-8090-8-198.
[20]	王龙,金保哲,张新中.脑血流监测对大鼠脑缺血模型制备的评价作用[J].中国脑血管病杂志, 2017, 14(5): 254-260. DOI: 10.3969/j.issn.1672-5921.2017.05.007.
[21]	Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1): 84-91. DOI: 10.1161/01.str.20.1.84.
[22]	李忠仁.实验针灸学[M].北京:中国中医药出版社, 2004.
[23]	Chen J, Sanberg PR, Li Y, et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats[J]. Stroke, 2001, 32(11): 2682-2688. DOI: 10.1161/hs1101.098367.
[24]	Rabinstein AA. Update on treatment of acute ischemic stroke[J]. Continuum (Minneap Minn), 2020, 26(2): 268-286. DOI: 10.1212/CON.0000000000000840.
[25]	Chen C, Li M, Lin L, et al. Clinical effects and safety of edaravone in treatment of acute ischaemic stroke: a meta-analysis of randomized controlled trials[J]. J Clin Pharm Ther, 2021, 46(4): 907-917. DOI: 10.1111/jcpt.13392.
[26]	Liu G, Ma HJ, Hu PP, et al. Effects of painful stimulation and acupuncture on attention networks in healthy subjects[J]. Behav Brain Funct, 2013, 7(9): 23. DOI: 10.1186/1744-9081-9-23.
[27]	Berman BM, Langevin HM, Witt CM, et al. Acupuncture for chronic low back pain[J]. N Engl J Med, 2010, 363(5): 454-461.
[28]	Zhang ZJ, Wang XM, McAlonan GM. Neural acupuncture unit: a new concept for interpreting effects and mechanisms of acupuncture[J]. Evid Based Complement Alternat Med, 2012, 2012: 429412. DOI: 10.1155/2012/429412.
[29]	Li SS, Hua XY, Zheng MX, et al. Electroacupuncture treatment improves motor function and neurological outcomes after cerebral ischemia/reperfusion injury[J]. Neural Regen Res, 2022, 17(7): 1545-1555. DOI: 10.4103/1673-5374.330617.
[30]	Hol EM, Pekny M. Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system[J]. Curr Opin Cell Biol, 2015, 32: 121-130. DOI: 10.1016/j.ceb.2015.02.004.
[31]	Robel S, Berninger B, Götz M. The stem cell potential of glia: lessons from reactive gliosis[J]. Nat Rev Neurosci, 2011, 12(2): 88-104. DOI: 10.1038/nrn2978.
[32]	Liu Z, Li Y, Cui Y, et al. Beneficial effects of gfap/vimentin reactive astrocytes for axonal remodeling and motor behavioral recovery in mice after stroke[J]. Glia, 2014, 62(12): 2022-2033. DOI: 10.1002/glia.22723.
[33]	Fineberg SK, Datta P, Stein CS, et al. MiR-34a represses Numbl in murine neural progenitor cells and antagonizes neuronal differentiation[J]. PLoS One, 2012, 7(6): e38562. DOI: 10.1371/journal.pone.0038562.
[34]	Fan F, Sun A, Zhao H, et al. MicroRNA-34a promotes cardiomyocyte apoptosis post myocardial infarction through down-regulating aldehyde dehydrogenase 2[J]. Curr Pharm Des, 2013, 19(27): 4865-4873. DOI: 10.2174/13816128113199990325.
[35]	Lanjakornsiripan D, Pior BJ, Kawaguchi D, et al. Layer-specific morphological and molecular differences in neocortical astrocytes and their dependence on neuronal layers[J]. Nat Commun, 2018, 9(1): 1623. DOI: 10.1038/s41467-018-03940-3.

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Effect of electroacupuncture on the expression of reactive astrocyte via regulating microRNA-34a in rats with cerebral ischemia-reperfusion injury

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Effect of electroacupuncture on the expression of reactive astrocyte via regulating microRNA-34a in rats with cerebral ischemia-reperfusion injury