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Title: Moxibustion preconditioning reduces inflammatory response in rats with cerebral ischemia-reperfusion injury by regulating PI3K / AKT / mTOR signaling pathway.
Author: Yu YY, Yang Y, Jiang J.
Journal: Zhen Ci Yan Jiu; 2024 Mar 25; 49(3):238-246. PubMed ID: 38500320.
Abstract:
OBJECTIVES: To observe the effect of moxibustion preconditioning on inflammatory response in rats with cerebral ischemia reperfusion injury (CIRI), so as to explore its mechanisms underlying improving CIRI. METHODS: Seventy-five male SD rats were randomly divided into sham operation, model, moxibustion preconditioning 3 days (Moxi 1), moxibustion preconditioning 5 days (Moxi 2) and moxibustion preconditioning 7 days (Moxi 3) groups, with 15 rats in each group. Moxibustion was applied at "Baihui"(GV20), "Dazhui"(GV14) and "Zusanli"(ST36) for 20 min once a day, totally for 3, 5 or 7 days. Thirty minutes after the last moxibustion treatment, the CIRI model was established by occlusion of the middle cerebral artery. The neurological deficit score was assessed by using Longa's method. The infarct size of the brain assessed after staining with 2% triphenyltetrazolium chloride (TTC). The morphological changes of cortical neurons were observed by HE staining. The contents of inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), S-100β protein (S-100β) and neuron-specific enolase (NSE) were detected by ELISA. The expression of phosphatidylinositol-3-kinase (PI3K), p-PI3K, protein kinase B (AKT) and mammalian target of rapamycin (mTOR) proteins in the ischemic cortex tissues were detected by immunohistochemistry and Western blot. RESULTS: Compared with the sham operation group, the neurological function score and the percentage of cerebral ischemic volume were increased (P<0.01). The contents of serum IL-1β, TNF-α, S-100β and NSE were significantly increased (P<0.01), while the protein expressions of PI3K, p-PI3K, AKT and mTOR in the cerebral cortex were significantly decreased (P<0.01) in the model group. Compared with the model group, the neurological function score and the percentage of cerebral ischemic volume were significantly decreased (P<0.01). The contents of serum IL-1β, TNF-α, S-100β and NSE were significantly decreased (P<0.01), and the expressions of PI3K, p-PI3K, AKT and mTOR proteins in the cerebral cortex were significantly increased (P<0.01) in three moxibustion groups. Compared with the Moxi 1 and Moxi 2 groups, the above indicators were significantly improved in rats of the Moxi 3 group (P<0.01, P<0.05). CONCLUSIONS: Moxibustion preconditioning can significantly improve the neurological function of rats after ischemia-reperfusion, inhibit serum inflammatory factors IL-1 β and TNF-α, inhibit brain tissue injury markers S-100β and NSE, which may be related to the activation of PI3K/AKT/mTOR signaling pathway. The protective effect of moxibustion preconditioning for 7 days on CIRI was better than that of 5 days and 3 days. 目的: 基于磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/AKT/mTOR）信号通路，探讨艾灸预处理对脑缺血再灌注损伤（CIRI）大鼠炎性因子的影响。方法: SD大鼠随机分为假手术组、模型组、艾灸预处理3 d组（艾灸1组）、艾灸预处理5 d组（艾灸2组）和艾灸预处理7 d组（艾灸3组），每组15只。各艾灸预处理组在造模前分别艾灸“百会”“大椎”“足三里”3、5、7 d，20 min/次，1次/d。末次艾灸干预20 min后，模型组和各艾灸组采用线栓法制备大脑中动脉阻塞模型。采用神经功能缺损评分评估大鼠脑神经损伤程度；TTC染色法观察脑梗死体积；HE染色法观察缺血区大脑皮层的形态变化；ELISA法检测血清中白细胞介素-1β （IL-1β）、肿瘤坏死因子-α（TNF-α）、S-100β蛋白（S-100β）、神经元特异性烯醇化酶（NSE）含量；免疫组织化学法和Western blot法检测缺血区大脑皮层PI3K、磷酸化（p）-PI3K、AKT、mTOR蛋白表达水平。结果: 与假手术组比较，模型组神经功能缺损评分、脑梗死体积明显升高（P<0.01），血清中IL-1β、TNF-α、S-100β、NSE含量明显升高（P<0.01），大脑皮层缺血区PI3K、p-PI3K、AKT、mTOR蛋白表达明显降低（P<0.01）。与模型组比较，各艾灸预处理组大鼠神经功能缺损评分、脑梗死体积下降（P<0.01），血清中IL-1β、TNF-α、S-100β、NSE含量降低（P<0.01），大脑皮层缺血区PI3K、p-PI3K、AKT、mTOR蛋白表达升高（P<0.01）。与艾灸1组和艾灸2组比较，艾灸3组血清中IL-1β、TNF-α、S-100β、NSE含量降低（P<0.05），大脑皮层缺血区PI3K、p-PI3K、AKT、mTOR蛋白表达明显升高（P<0.01，P<0.05）。结论: 艾灸预处理可明显改善CIRI大鼠的神经功能，抑制血清炎性因子IL-1β、TNF-α，抑制脑组织损伤标志物S-100β蛋白和NSE，可能与激活PI3K/AKT/mTOR信号通路有关。其中艾灸预处理7 d对CIRI的保护作用优于5 d和3 d。.

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