针刺调控miR-128-3p对卒中大鼠神经功能及Nrf2表达影响

Effect of Acupuncture Modulation of MiR-128-3p on Neural Functions and Nrf2 Expression in Stroke Rats

目的观察针刺对卒中大鼠的神经功能改善作用,并初步探讨其机制。方法建立脑缺血再灌注大鼠模型,72只SD大鼠随机分为模型组、激动剂组、激动剂阴性对照组、针刺组、针刺+激动剂组,每组12只,另取12只SD大鼠设为假手术组,针刺组、针刺+激动剂组大鼠于双侧阳陵泉穴及曲池穴行针,激动剂组、激动剂阴性对照组、针刺+激动剂组大鼠分别以5 mL/kg剂量尾静脉注射miR-128-3p agomir、miR-128-3p agomir阴性对照,假手术组、模型组和针刺组尾静脉注射等剂量生理盐水,持续7 d。对各组大鼠进行神经功能缺损评分,以苏木素-伊红(HE)染色检测各组大鼠皮质组织病理变化,以酶联免疫吸附法检测血清中枢神经特异性蛋白(S100β)、神经元特异性烯醇化酶(NSE)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平,以实时荧光定量PCR检测各组大鼠脑皮质组织miR-128-3p、红系衍生的核转录相关因子(Nrf2)mRNA水平,以Western blot法检测脑皮质组织中Nrf2蛋白表达情况。结果与假手术组相比,模型组大鼠脑皮质神经元呈现细胞核收缩变小、细胞坏死、数量减少等病理损伤;神经功能缺损评分,血清中β、NSE、IL-6及TNF-α水平,脑皮质组织miR-128-3p水平均明显升高(P<0.05);脑皮质组织Nrf2 mRNA及蛋白水平明显降低(P<0.05)。与模型组相比,针刺组大鼠脑皮质神经元病理损伤减轻;神经功能缺损评分,血清中S100β、NSE、IL-6及TNF-α水平,脑皮质组织miR-128-3p水平降低(P<0.05);脑皮质组织Nrf2 mRNA及蛋白水平升高(P<0.05)。激动剂组大鼠脑皮质神经元病理损伤加重;神经功能缺损评分,血清中S100β、NSE、IL-6及TNF-α水平,脑皮质组织miR-128-3p水平升高(P<0.05);脑皮质组织Nrf2 mRNA及蛋白水平降低(P<0.05)。激动剂阴性对照组大鼠无明显变化(P>0.05)。与针刺组比较,针刺+激动剂组大鼠脑皮质神经元病理损伤加重;神经功能缺损评分,血清中S100β、NSE、IL-6及TNF-α水平,脑皮质组织miR-128-3p水平升高(P<0.05);脑皮质组织Nrf2 mRNA及蛋白水平降低(P<0.05)。与激动剂组比较,针刺+激动剂组大鼠脑皮质神经元病理损伤减轻;神经功能缺损评分,血清中S100β、NSE、IL-6及TNF-α水平,脑皮质组织miR-128-3p水平降低(P<0.05);脑皮质组织Nrf2 mRNA及蛋白水平升高(P<0.05)。结论针刺可能通过下调miR-128-3p表达,促进Nrf2表达,减轻卒中大鼠炎症损伤,改善其神经功能。

Objective To investigate the improving effect of acupuncture on neural functions in stroke rats and preliminarily explore its mechanism.Method A rat model of cerebral ischemia and reperfusion was made.Seventy-two SD rats were randomized to model,agonist,agonist negative control,acupuncture and acupuncture+agonist groups,with 12 rats in each group.Another 12 SD rats constituted a sham operation group.The acupuncture and acupuncture+agonist groups of rats received acupuncture at bilateral points Yanglingquan and Quchi.The agonist,agonist negative control and acupuncture+agonist groups received caudal vein injection of miR-128-3p agomir 5 ml/kg.The miR-128-3p agomir negative control,sham operation and acupuncture groups received caudal vein injection of the same volume of normal saline.All the procedures lasted seven days.Neural deficits were scored in various groups of rats.Pathological changes in cortical tissues examined by hematoxylin-eosin(HE)staining.Serum CNS specific protein(S100 calcium binding protein B,S100b),neuron-specific enolase(NSE),interleukin-6(IL-6)and tumor necrosis factor-a(TNF-a)levels were measured by enzyme-linked immunosorbent assay.Cerebral cortex miR-128-3p and nuclear factor erythroid 2-related factor 2(Nrf2)mR NA levels were measured by real-time fluorescent quantitative PCR various groups of rats.Nrf2 expression in cerebral cortex was determined by Western blot.Result Cortical neurons had pathological injuries such as contracted and reduced cell nuclei,cellular necrosis and decreased cell number,and the neural deficit score,serum S100b,NSE,IL-6 and TNF-a levels and cerebral cortex miR-128-3p levels increased significantly(P<0.05)and cerebral cortex Nrf2 mRNA and protein levels decreased significantly(P<0.05)in the model group of rats compared with the sham operation group.Compared with the model group,cortical neurons had minor pathological injuries,and the neural deficit score,serum S100b,NSE,IL-6 and TNF-a levels and cerebral cortex miR-128-3p levels decreased(P<0.05)and cerebral cortex Nrf2 mRNA and protein levels increased(P<0.05)in the acupuncture group of rats;cortical neurons had severer pathological injuries,and the neural deficit score,serum S100b,NSE,IL-6 and TNF-a levels and cerebral cortex miR-128-3p levels increased(P<0.05)and cerebral cortex Nrf2 mRNA and protein levels decreased(P<0.05)in the agonist group of rats;there were no obvious changes in the agonist negative control group of rats(P>0.05).Compared with the acupuncture group,cortical neurons had severer pathological injuries,and the neural deficit score,serum S100b,NSE,IL-6 and TNF-a levels and cerebral cortex miR-128-3p levels increased(P<0.05)and cerebral cortex Nrf2 mRNA and protein levels decreased(P<0.05)in the acupuncture+agonist group of rats.Compared with the agonist group,cortical neurons had minor pathological injuries,and the neural deficit score,serum S100b,NSE,IL-6 and TNF-a levels and cerebral cortex miR-128-3p levels decreased(P<0.05)and cerebral cortex Nrf2 mR NA and protein levels increased(P<0.05)in the acupuncture+agonist group of rats.Conclusion Acupuncture may reduce inflammatory injury and improve neural functions in stroke rats by down-regulating miR-128-3p expression and promoting Nrf2 expression.