基于PI3K/AKT/mTOR信号通路探讨化瘀通络灸促血管性痴呆大鼠髓鞘再生的作用机制

Mechanism of Action of Stasis-resolving and Collateral-dredging Moxibustion in Promoting Myelin Regeneration in Rats with Vascular Dementia:A Study Based on the Phosphoinositide 3 Kinase/Protein Kinase B/Mammalian Target of Rapamycin Signaling Pathway

目的观察化瘀通络灸对血管性痴呆(vascular dementia,VD)大鼠胼胝体磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的影响,探讨化瘀通络灸促VD大鼠髓鞘再生的作用机制。方法经Morris水迷宫筛选后,随机选取12只大鼠纳入假手术组,剩余大鼠复制VD模型成功后,随机分为模型组、艾灸组、艾灸+LY294002组,每组12只。艾灸组予以化瘀通络灸干预,艾灸+LY294002组在化瘀通络灸干预的基础上予以PI3K抑制剂LY294002腹腔注射,采用Longa评分法评价各组大鼠神经功能损伤程度,Morris水迷宫实验检测各组大鼠学习记忆能力,Western blot法检测各组大鼠PI3K/AKT/mTOR信号通路相关蛋白的表达水平,神经髓鞘固蓝染色法观察各组大鼠胼胝体髓鞘的形态,透射电子显微镜观察各组大鼠髓鞘超微结构。结果与假手术组比较,模型组和艾灸+LY294002组大鼠的Longa评分显著升高(P<0.05),逃避潜伏期显著延长(P<0.05),PI3K/AKT/mTOR通路相关蛋白表达水平显著降低(P<0.05),胼胝体内髓鞘纹理不清,排列混乱,边缘呈空泡或空网状改变,髓鞘线圈样结构离散,部分膨出和崩解,有髓神经轴突数量显著减少(P<0.05);与模型组和艾灸+LY294002组比较,艾灸组大鼠Longa评分显著下降(P<0.05),逃避潜伏期显著缩短(P<0.05),PI3K/AKT/mTOR通路相关蛋白表达水平显著提高(P<0.05),胼胝体内髓鞘结构有所恢复,排列整齐,边缘结构较为致密,有髓神经轴突数量显著增加(P<0.05)。结论化瘀通络灸可能通过激活PI3K/AKT/mTOR通路,修复VD大鼠损伤髓鞘并促进其重塑,恢复脑白质功能。

Objective To investigate the mechanism of action of stasis-resolving and collateral-dredging moxibustion in promoting myelin regeneration in rats with vascular dementia(VD)by observing its effect on the phosphoinositide 3 kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signaling pathway.Methods After screening by the Morris water maze test,12 rats were randomly selected as sham-operation group,and the remaining rats were randomly divided into model group,moxibustion group,and moxibustion+LY294002 group after successful modeling of VD,with 12 rats in each group.The rats in the moxibustion group were given stasis-resolving and collateral-dredging moxibustion,and those in the moxibustion+LY294002 group were given intraperitoneal injection of the PI3K inhibitor LY294002 in addition to stasis-resolving and collateral-dredging moxibustion.The Longa scoring system was used to evaluate the degree of neurovascular injury in rats;the Morris water maze test was used to evaluate the learning and memory abilities of rats;Western blot was used to measure the expression of proteins associated with the PI3K/AKT/mTOR signaling pathway;LFB myelin staining was used to observe the morphology of myelin in the corpus callosum of rats;transmission electron microscopy was used to observe the myelin ultrastructure of rats.Results Compared with the sham-operation group,the model group and the moxibustion+LY294002 group had significant increases in Longa score(P<0.05)and escape latency(P<0.05)and significant reductions in the expression levels of the proteins associated with the PI3K/AKT/mTOR signaling pathway(P<0.05);the myelin in the corpus callosum showed unclear texture,disordered arrangement,vacuolar changes at the edge,and fragmentation with partial protrusions and disintegration,as well as a significant reduction in the number of myelinated nerve axons(P<0.05).Compared with the model group and the moxibustion+LY294002 group,the moxibustion group had significant reductions in Longa score(P<0.05)and escape latency(P<0.05)and significant increases in the expression levels of the proteins associated with the PI3K/AKT/mTOR signaling pathway(P<0.05),with certain recovery of the structure of myelin in the corpus callosum,ordered arrangement,dense structure at the edge,and a significant increase in the number of myelinated nerve axons(P<0.05).Conclusion Stasis-resolving and collateral-dredging moxibustion can repair myelin damage,promote myelin remodeling,and restore white matter function by activating the PI3K/AKT/mTOR signaling pathway.