目的 探讨天麻素通过PI3K/AKT信号通路对新生大鼠缺氧缺血性脑损伤(HIBD)后活化小胶质细胞介导的炎症反应的作用机制。方法 将39只3日龄新生SD大鼠随机分为假手术组(sham，n=9)、缺氧缺血模型组(HIBD，n=15)、天麻素处理组(HIBD+G，n=15)。采用Western blotting检测HIBD后TNF-α、IL-1β、IL-10和TGF-β1蛋白的表达;网络药理学筛选天麻素治疗HIBD的潜在作用靶点;Western blotting检测HIBD和氧糖剥夺(OGD)诱导活化的小胶质细胞中PI3K/AKT信号通路的表达;CCK8检测PI3K/AKT通路特异性抑制剂LY294002对BV-2小胶质细胞的细胞毒性作用;RT-qPCR检测LY294002干预后天麻素对TNF-α和TGF-β1的mRNA水平的影响。结果 Western blotting显示，与HIBD组相比，天麻素降低缺血侧胼胝体区TNF-α和IL-1β的蛋白表达(P<0。05)，促进IL-10和TGF-β1的蛋白表达(P<0。05);网络药理学显示，PI3K/AKT信号通路显著富集，且天麻素与PI3K之间具有较好的结合能力;Western blotting显示，与HIBD组、OGD组相比，天麻素促进PI3K和AKT的磷酸化水平(P<0。05);CCK8结果显示LY294002在0～120 μmol/L浓度范围内对BV-2小胶质细胞没有细胞毒性作用;RT-qPCR结果显示，与对照组相比，OGD组TNF-α的mRNA水平升高，TGF-β1的mRNA水平降低(P<0。05);天麻素干预后降低TNF-α的mRNA水平，升高TGF-β1的mRNA水平(P<0。05);LY294002处理后TNF-α的mRNA水平进一步升高，TGF-β1的mRNA水平进一步降低(P<0。05);而LY294002与天麻素联合用药后TNF-α和TGF-β1的mRNA水平无明显变化。结论 天麻素能抑制HIBD后活化小胶质细胞介导的炎症反应，其作用机制与PI3K/AKT信号通路有关。
Gastrodin improves microglia-mediated inflammatory response after hypoxic-ischemic brain damage in neonatal rats via PI3K/AKT pathway
Objective To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxic-ischemic brain damage(HIBD)in neonatal rats.Methods Thirty-nine 3-day-old SD rats were randomly divided into sham group,HIBD group and gastrodin treatment group.Western blotting was used to detect the expressions of TNF-α,IL-1β,IL-10 and TGF-β1 in the corpus callosum of the rats.The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis.The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation(OGD)were detected with Western blotting.The effects of LY294002(a specific inhibitor of the PI3K/AKT pathway)and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR.Results In the neonatal rats with HIBD,gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum.Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K.Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD.In BV-2 cells with OGD,gastrodin obviously suppressed OGD-induced increase of TNF-α and reduction of TGF-β1 mRNA expressions,and this effect was strongly attenuated by LY294002 treatment.Conclusion Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.
hypoxic-ischemic brain damagegastrodininflammatory responseoxygen and sugar deprivationmicrogliaPI3K/AKT
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