针刺调控miR-10a改善VD大鼠认知并减轻神经炎症的研究

Study on acupuncture regulating miR-10a to improve cognition and reduce neuroinflammation in VD rats

关键词:  针刺  血管性痴呆  认知功能  miR-10a  神经炎症

Key words:  acupuncture  vascular dementia  cognitive function  miR-10a  neuroinflammation

摘要:

[目的] 探讨针刺通过调控Micro RNA 10a（miR-10a）改善血管性痴呆（VD）大鼠认知功能并减轻神经炎症的效应及机制。[方法] 采用双侧颈总动脉夹闭法制作VD大鼠模型，并分为假手术组、VD组、针刺组和各miR干预组，针刺组予以“三焦针法”进行治疗；各miR干预组给予相应的miR-10a空白溶剂或抑制剂。采用莫里斯（Morris）水迷宫评价大鼠的认知能力；实时荧光定量（RT-qPCR）法测定miR-10a表达；蛋白质印迹（Western blot）法检测核因子-κB（NF-κB）通路相关蛋白NF-κB p65、磷酸化NF-κBp65的表达。酶联免疫吸附实验（ELISA）法测定肿瘤坏死因子（TNF-α）及白细胞介素6（IL-6）水平。[结果] 与假手术组相比，模型组在水迷宫隐蔽平台实验中的逃避潜伏期显著延长（P<0.01）；在空间探索实验中穿越原平台位置的次数减少、首次穿越原平台所需时间延长（P<0.01）；而针刺组对上述指标具有改善作用，与模型组相比差异显著（P<0.01）。此外，与假手术组相比，模型组海马miR-10a表达下降，NF-κB t-p65和p-p65蛋白及TNF-α、IL-6水平上升（P<0.05或P<0.01）；针刺治疗后上述指标出现相反变化，与模型组相比差异显著（P<0.05或P<0.01）。转染miR-10a抑制剂后发现，抑制剂组海马组织中NF-κB t-p65和p-p65蛋白及TNF-α、IL-6水平较模型组上升（P<0.05或P<0.01）；针刺治疗能提高miR-10a抑制剂空白溶剂组大鼠的认知功能，降低其NF-κB及TNF-α、IL-6水平，但在抑制剂组中却未发现上述作用。双荧光素酶报告基因实验发现miR-10a可直接作用于NF-κB并抑制其表达。[结论] 针刺能改善VD大鼠的认知功能，其机制部分与调节miR-10a表达，进而抑制缺血脑组织炎症损伤有关。

Abstract:

[Objective] To explore the effect and mechanism of acupuncture on improving cognitive function and reducing neuroinflammation in vascular dementia(VD) rats by regulating miR-10a. [Methods] The VD rat model was established by clamping bilateral common carotid arteries and divided into sham operation, VD, acupuncture and miR intervention groups. The acupuncture group was treated with "sanjiao acupuncture". Each miR intervention group was given corresponding miR-10a inhibitor NC or inhibitor. Morris water maze was used to evaluate the cognitive ability of rats;the expression of miR-10a was detected by RT-PCR;NF-κB total p65 and phosphorylated p65 expression were detected by Western blot;TNF-α and IL-6 levels were determined by ELISA. [Results] Compared with the sham operation group, the escape latency of the model group in the water maze hidden platform test was significantly prolonged (P<0.01). In the space exploration test, the number of times to cross the original platform position decreased, and the time required to cross the original platform for the first time increased(P<0.01). The acupuncture group had an improvement effect on the above indicators, which was significantly different from the model group(P<0.01). In addition, compared with sham operation group, the expression of miR-10a in hippocampus of model group decreased, and NF-κB t-p65 and p-p65 proteins and TNF-α, IL-6 level increased (P<0.05 or P<0.01).After acupuncture treatment, the above indexes showed opposite changes, which were significantly different from the model group(P<0.05 or P<0.01). After transfection with miR-10a inhibitor, increased levels of NF-κB t-p65 and p-p65 proteins and TNF-α, IL-6 was found in the hippocampus of the inhibitor group when compared with the model group(P<0.05 or P<0.01).Aacupuncture treatment could improve the cognitive function of rats in miR-10a inhibitor NC group and reduce the NF-κB protein and TNF-α, IL-6 level, but no such effect was found in the inhibitor group. Double luciferase reporter gene experiment found that miR-10a can directly act on NF-κB and inhibit its expression. [Conclusion] Acupuncture can improve the cognitive function of VD rats. Its mechanism is partly related to regulating the expression of miR-10a, and then inhibiting the inflammatory damage of ischemic brain tissue.