眼针干预脑缺血模型大鼠调节血管新生因子表达促进血管新生

Eye acupuncture can promote angiogenesis by regulating the expression of angiogenic factors in cerebral ischemia model rats

背景:眼针在临床应用中能够有效缓解脑缺血患者的症状,但其作用机制研究较少。miR-124-3p可能是改善缺血性脑损伤、促进血管生成的调节剂;氯化锂是Wnt/β-catenin通路的常用激活剂,具有抵抗神经损伤的能力。目的:观察眼针对脑缺血损伤模型大鼠缺血半暗带皮质脑组织中血管新生的影响及可能的作用机制。方法:108只SD大鼠随机分为假手术组、模型组、眼针组、体针组、miR-124-3p激动剂组和氯化锂组,每组18只。除假手术组外,其余各组运用改良线栓法建立大脑中动脉缺血再灌注损伤模型。大鼠造模后1 d,眼针组针刺大鼠眼周“肝区”“肾区”“上焦区”“下焦区”,体针组针刺大鼠“百会”穴,miR-124-3p激动剂组于左侧脑室给予miR-124-3p激动剂(20 nmol/mL),氯化锂组腹腔注射氯化锂溶液(4 mmol/kg);miR-124-3p激动剂组造模结束后给药一次,其他组每天干预1次,连续给药14 d。观察缺血半暗带脑组织病理学变化,实时荧光定量PCR检测大鼠脑组织中Wnt3a、β-连环蛋白(β-catenin)、血管内皮生长因子、血管生成素2及miR-124-3p mRNA表达水平,Western blot检测大鼠脑组织中Wnt3a、β-catenin、血管内皮生长因子、血管生成素2蛋白相对表达量。结果与结论:(1)造模后1 d及干预14 d,假手术组无神经功能障碍,模型组大鼠出现较为明显的神经功能障碍(P<0.05);(2)假手术组未出现脑缺血,模型组脑缺血损伤明显,经眼针干预后大鼠脑缺血面积明显减少(P<0.05);(3)与模型组比较,眼针组、体针组、miR-124-3p激动剂组和氯化锂组大鼠缺血半暗带皮质通路相关蛋白Wnt3a、β-catenin表达和miR-124-3p、Wnt3a、β-catenin基因表达上调(P<0.05),血管新生因子血管内皮生长因子、血管生成素2蛋白和基因表达水平上调(P<0.05);其中眼针组上述指标改善更为明显(P<0.05);(4)提示眼针可通过刺激缺血半暗带皮质区血管新生因子表达促进血管新生改善脑缺血大鼠神经功能,其作用机制可能是眼针通过调控miR-124-3p影响Wnt/β-catenin信号通路,调节血管新生因子表达和促进血管新生,进而改善脑缺血损伤。

BACKGROUND:Eye acupuncture can effectively alleviate the symptoms of patients with cerebral ischemia in clinical practice,but its mechanism of action is less studied.miR-124-3 p may be a regulator to improve ischemic brain injury and promote angiogenesis.Lithium chloride is a common activator of the Wnt/β-catenin pathway with the ability to resist nerve damage.OBJECTIVE:To observe the effect and possible mechanism of eye acupuncture on angiogenesis in the cerebral co rtex of ischemic penumbra in rats with cerebral ischemia injury.METHODS:108 Sprague-Dawley rats were randomly divided into sham operation group,model group,eye acupuncture group,body acupuncture group,mir-124-3 p agonist group and lithium chloride group,with 18 rats in each group.The middle cerebral artery ischemia-reperfusion injury model was established by using the modified thread occlusion method in all groups except for the sham operation group.The eye acupuncture group needled the "liver area", "kidney area", "upper focal area" and "lower focal area" around the eyes of rats,the body acupuncture group needled the "Baihui" point,the mir-124-3 p agonist group was given the miR-124-3 p agonist(20 nmol/m L) in the left ventricle,and the lithium chloride group was intraperitoneally injected with lithium chloride solution(4 mmol/kg).The miR-124-3 p group was administered once after modeling,and other groups were intervened once a day for 14 days.The pathological changes of the ischemic penumbra were observed.The expression levels of Wnt3 a,β-catenin,vascular endothelial growth fa ctor,angiopoietin 2 and miR-124-3 p mRNA in rat brain were detected by real-time fluorescence quantitative PCR.The relative expression levels of Wnt3 a,β-catenin,vascular endothelial growth factor,and angiopoietin 2 in rat brain were detected by western blot.RESULTS AND CONCLUSION:On the 1^(st) day after modeling and the 14^(th) day after intervention,there was no neurological dysfunction in the sham ope ration group but obvious neurological dysfunction in the model group(P <0.05).There was no cerebral ischemia in the sham operation group,but rats in the model group had obvious cerebral ischemia injury.After eye acupuncture intervention,the cerebral ischemia area of the rats was significantly reduced(P<0.05).Compared with the model group,the expression levels of Wnt3 a,β-catenin proteins and miR-124-3 p,Wnt3 a,β-catenin genes related to the cortical pathway in the ischemic penumbra of rats were up-regulated in the eye acupuncture group,body acupuncture group,miR-124-3 p agonist group and lithium chloride group(P <0.05),while the expression levels of vascular endothelial growth fa ctor and angiopoietin 2 proteins and genes were up-regulated(P <0.05).And the above indexes in the eye acupuncture group were improved more obviously(P <0.05).These findings suggest that eye acupuncture can promote angiogenesis and improve the neurological function of cerebral ischemic rats by stimulating the expression of angiogenic factors in the cortex of the ischemic penumbra.The mechanism of action may be that eye acupuncture affects Wnt/β-catenin signaling pathway by regulating miR-124-3 p,regulates the expression of angiogenic factors and promotes angiogenesis,thereby improving cerebral ischemia injury.