电针调控microRNA-34a对缺血再灌注损伤大鼠内源性神经干细胞分化的影响

doi:10.3969/j.issn.1006-9771.2019.02.007

摘要/Abstract

摘要： 目的探讨电针曲池和足三里是否是通过调控microRNA-34a (miR-34a)促进缺血再灌注损伤大鼠缺血周边区神经干细胞分化为星形胶质细胞。方法将108只Sprague-Dawley大鼠随机分为假手术组、模型组和电针组，每组再分为3 d、7 d、14 d 3个时间点，每个时间点12只。另取16只大鼠随机分为电针+二甲基亚砜(DMSO)组(n = 8)和电针+miR-34a抑制剂组(n = 8)。构建大鼠大脑中动脉闭塞(MCAO)致局灶性脑缺血模型。电针组造模后第2天进行电针患侧肢体曲池(LI11)、足三里(ST36)，疏密波1/20 Hz，以肢体轻轻抖动为度，每次30 min，每天1次，共14 d。同时，造模后第2天至第14天各组大鼠腹腔注射5-溴代-2'-脱氧尿苷(BrdU)，每天2次，每次注射间隔8 h；DMSO溶解液、miR-34a抑制剂在造模前进行侧脑室注射；免疫荧光染色检测BrdU、巢蛋白(Nestin)与神经胶质纤维酸性蛋白(GFAP)的共定位情况；逆转录实时定量聚合酶链反应检测缺血周边区miR-34a相对表达量。结果电针组各时间点Longa神经行为学评分低于模型组(t > 2.084, P < 0.05)。模型组患侧肢体的爪印面积(右前爪、右后爪)、最大压强(右前爪、右后爪)与同时间假手术组比较均下降(P < 0.05)，模型组随着时间延长右后爪印面积增加(P < 0.05)。电针组患侧肢体的爪印面积(右前爪、右后爪)与同时间模型组比较增加(P <0.05)。电针干预3 d和7 d后，电针组患侧最大压强(右前爪、右后爪)与模型组比较无显著性差异(P > 0.05)；14 d后电针组大于模型组(P < 0.05)。电针干预3 d至7 d，电针组右后爪印面积与右前最大压强逐渐增加，一定程度上呈时间依赖性(P < 0.05)。模型组和电针组缺血周围区均表达Nestin+/GFAP+细胞和BrdU+/GFAP+细胞，且电针组3 d、7 d和14 d后Nestin+/GFAP+、BrdU+/GFAP+双阳性共标的细胞较模型组表达均增加(t > 3.292, P < 0.05)，且在7 d达到峰值。脑缺血7 d后模型组缺血周边区miR-34a的表达较假手术组明显增加(P < 0.01)；而电针组和+DMSO组miR-34a表达进一步升高(P < 0.05)；电针+miR-34a抑制组miR-34a表达和BrdU+/GFAP+细胞数较电针组均减少(P < 0.05)。结论电针曲池和足三里可以促进缺血再灌注损伤大鼠缺血周边区神经干细胞分化为星形胶质细胞，可能是通过调控miR-34a的表达。

关键词: 缺血再灌注损伤, 电针, microRNA-34a, 神经干细胞, 分化, 大鼠

Abstract: Objective To investigate whether electroacupuncture (EA) at Quchi (LI11) and Zusanli (ST36) acupoints may regulate microRNA-34a (miR-34a) to promote neural stem cells differentiation in ischemic peripheral areas in rats with cerebral ischemia-reperfusion injury or not. Methods A total of 108 rats were randomly assigned into sham group, model group and EA group, and each group was divided into three subgroups (three days, seven days and 14 days), with twelve rats in each subgroup. Besides, 16 rats were randomly divided into EA+dimethyl sulfoxide (DMSO) group and EA+miR-34a inhibitor group, with eight rats in each group. The middle cerebral artery occlusion (MCAO) model was induced for focal cerebral ischemia in rats. EA group was electroacupunctured at the ipsilateral Quchi and Zusanli acupoints on the second day. The dilatational wave was 1/20 Hz, 30 minutes every time, once a day for seven days, totally. At the same time, 5-Bromo-2′-Deoxyuridine (BrdU) was intraperitoneally injected twice a day, with an 8-hours interval. The DMSO and miR-34a inhibitor were injected into the lateral ventricle before modeling. The co-location condition was evaluated by immunofluorescence. The expression of miR-34a in ischemic peripheral areas was detected by reverse transcription real-time quantitative polymerase chain reaction. Results The Longa's score was lower in EA group than in the model group (t > 2.084, P < 0.05). At the same time points, the paw print areas (right forepaw, right hind paw) and maximum pressures (right forepaw, right hind paw) of the affected limbs decreased in the model group than in the sham group (P < 0.05), and the paw print area of right hind paw gradually increased in the model group (P < 0.05); the paw print areas (right forepaw and right hind paw) of the affected limbs improved in EA group, compared with the model group (P < 0.05); and there was no significant difference in the maximum pressure of the affected limbs three days and seven days after electroacupuncture (P > 0.05); however, it was higher in EA group than in the model group 14 days after electroacupuncture (P < 0.05). And the paw print area of the right hind paw and the maximum pressure of the right forepaw gradually increased in EA group three days and seven days after electroacupuncture, which was in time-dependent manner (P < 0.05). The Nestin+/GFAP+ and BrdU+/GFAP+ cells expressed in ischemic peripheral areas both in the model group and EA group. And the Nestin+/GFAP+ and BrdU+/GFAP+ double positive cells increased in EA group compared to the model group three days, seven days and 14 days after electroacupuncture (t > 3.292, P < 0.05), and they reached peak seven days after electroacupuncture. The expression of miR-34a in ischemic peripheral areas was higher in the model group than in the sham group seven days after modeling (P < 0.01), however, the expression of miR-34a further increased in EA+DMSO group after electroacupuncture (P < 0.05). After injection of miR-34a inhibitor, the expression of miR-34a and BrdU+/GFAP+ cells was lower in EA+miR-34a inhibitor group than in EA group (P < 0.05). Conclusion Electroacupuncture at Quchi and Zusanli acupoints could promote the neural stem cells differentiation in ischemic peripheral areas by regulation of miR-34a expression.

Key words: ischemia-reperfusion injury, electroacupuncture, microRNA-34a, neural stem cells, differentiation, rats

中图分类号:

R743.3

柳维林, 郑薏, 金婷婷, 张宇豪, 施丹, 陈立典, 陶静. 电针调控microRNA-34a对缺血再灌注损伤大鼠内源性神经干细胞分化的影响[J]. 《中国康复理论与实践》, 2019, 25(2): 162-171.

LIU Wei-lin, ZHENG Yi, JIN Ting-ting, ZHANG Yu-hao, SHI Dan, CHEN Li-dian, TAO Jing. Effect of Electroacupuncture on Neural Stem Cell Differentiation via Regulating MicroRNA-34a in Rats with Cerebral Ischemia-reperfusion Injury[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2019, 25(2): 162-171.

参考文献

[1] Wu K J, Yu S, Lee J Y, et al. Improving neurorepair in stroke brain through endogenous neurogenesis-enhancing drugs [J]. Cell Transplant, 2017, 26(9): 1596-1600.
[2] Reeve R L, Yammine S Z, De Veale B, et al. Targeted activation of primitive neural stem cells in the mouse brain [J]. Eur J Neurosci, 2016, 43(11): 1474-1485.
[3] Abrahams J M, Lenart C J, Tobias M E. Temporal variation of induction neurogenesis in a rat model of transient middle cerebral artery occlusion [J]. Neurol Res, 2009, 31(5): 528-533.
[4] Shen C C, Yang Y C, Chiao M T, et al. Characterization of endogenous neural progenitor cells after experimental ischemic stroke [J]. Curr Neurovasc Res, 2010, 7(1): 6-14.
[5] Shimada I S, Le Comte M D, Granger J C, et al. Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke [J]. J Neurosci, 2012, 32(23): 7926-7940.
[6] Kim Y R, Kim H N, Ahn S M, et al. Electroacupuncture promotes post-stroke functional recovery via enhancing endogenous neurogenesis in mouse focal cerebral ischemia [J]. PLoS One, 2014, 9(2): e90000.
[7] 唐强,徐志华,白晶. 电针对大鼠脑梗死后内源性神经干细胞增殖迁移的影响[J]. 针灸临床杂志, 2008, 24(8): 49-51.
[8] Zhang Q, Zhang K, Zhang C, et al. MicroRNAs as big regulators of neural stem/progenitor cell proliferation, differentiation and migration: a potential treatment for stroke [J]. Curr Pharm Des, 2017, 23(15): 2252-2257.
[9] Lodygin D, Tarasov V, Epanchintsev A, et al. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer [J]. Cell Cycle, 2008, 7(16): 2591-2600.
[10] Tsai D Y, Hung K H, Lin I Y, et al. Uncovering microRNA regulatory hubs that modulate plasma cell differentiation [J]. Sci Rep, 2015, 5: 17957.
[11] 李忠仁. 实验针灸学[M]. 北京:中国中医药出版社, 2003: 360.
[12] Rastogi V, Santiago-Moreno J, Doré S. Ginseng: a promising neuroprotective strategy in stroke [J]. Front Cell Neurosci, 2015, 8: 457.
[13] Shichita T, Ito M, Yoshimura A. Post-ischemic inflammation regulates neural damage and protection [J]. Front Cell Neurosci, 2014, 8: 319.
[14] Longa E Z, Weinstein P R, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke, 1989, 20(1): 84-91.
[15] Parkkinen S, Ortega F J, Kuptsova K, et al. Gait impairment in a rat model of focal cerebral ischemia [J]. Stroke Res Treat, 2013, 2013: 410972.
[16] 谢西梅,安军明,黄琳娜,等. 针刺对缺血性中风患者运动功能障碍影响的随机对照研究[J]. 针灸临床杂志, 2012, 28(12): 15-18.
[17] Lan L, Tao J, Chen A, et al. Electroacupuncture exerts anti-inflammatory effects in cerebral ischemia-reperfusion injured rats via suppression of the TLR4/NF-kappaB pathway [J]. Int J Mol Med, 2013, 31(1): 75-80.
[18] 钟艺华,唐显军,李光勤,等. 不同穴组电针对局灶性脑梗死大鼠神经行为学及Neurocan表达的影响[J]. 中国康复理论与实践, 2013, 19(5): 444-447.
[19] Zheng J, Zhang P, Li X, et al. Post-stroke estradiol treatment enhances neurogenesis in the subventricular zone of rats after permanent focal cerebral ischemia [J]. Neuroscience, 2013, 231: 82-90.
[20] Hermann D M, Chopp M. Promoting brain remodelling and plasticity for stroke recovery: therapeutic promise and potential pitfalls of clinical translation [J]. Lancet Neurol, 2012, 11(4): 369-380.
[21] Jin K, Minami M, Lan J Q, et al. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat [J]. Proc Natl Acad Sci U S A, 2001, 98(8): 4710-4715.
[22] Tashiro A, Makino H, Gage F H. Experience-specific functional modification of the dentate gyrus through adult neurogenesis: a critical period during an immature stage [J]. J Neurosci, 2007, 27(12): 3252-3259.
[23] Shimada I S, Borders A, Aronshtam A, et al. Proliferating reactive astrocytes are regulated by Notch-1 in the peri-infarct area after stroke [J]. Stroke, 2011, 42(11): 3231-3237.
[24] Shen C C, Yang Y C, Chiao M T, et al. Characterization of endogenous neural progenitor cells after experimental ischemic stroke [J]. Curr Neurovasc Res, 2010, 7(1): 6-14.
[25] Liu X Y, Zhou X Y, Hou J C, et al. Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway [J]. Acta Pharmacol Sin, 2015, 36(4): 421-428.
[26] Fang M, Yuan Y, Rangarajan P, et al. Scutellarin regulates microglia-mediated TNC1 astrocytic reaction and astrogliosis in cerebral ischemia in the adult rats [J]. BMC Neurosci, 2015, 16: 84.
[27] Lehner B, Sandner B, Marschallinger J, et al. The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survival [J]. Cell Tissue Res, 2011, 345(3): 313-328.
[28] 叶晓雪,新贵,黄元平,等. 电针与手针对脑梗死大鼠脑星形胶质细胞影响的比较研究[J]. 广西中医药, 2014, 37(3): 71-74.
[29] 张业贵,龚鑫,李怀斌. 电针对脑缺血再灌注大鼠齿状回Nestin、GFAP表达的影响[J]. 中国组织化学与细胞化学杂志, 2014, 23(2): 148-153.
[30] Tarantino C, Paolella G, Cozzuto L, et al. miRNA 34a, 100, and 137 modulate differentiation of mouse embryonic stem cells [J]. FASEB J, 2010, 24(9): 3255-3263.
[31] 郑洁,韩明利,陈晓丹,等. miR-34a过表达对小鼠脑皮质神经干细胞中巢蛋白表达的影响[J]. 中国生物制品学杂志, 2013, 26(7): 945-948.
[32] Morgado A L, Xavier J M, Dionísio P A, et al. MicroRNA-34a modulates neural stem cell differentiation by regulating expression of synaptic and autophagic proteins [J]. Mol Neurobiol, 2015, 51(3): 1168-1183.
[33] Aranha M M, Santos D M, Xavier J M, et al. Apoptosis-associated microRNAs are modulated in mouse, rat and human neural differentiation [J]. BMC Genomics, 2010, 11: 514.