粒细胞集落刺激因子通过mTOR/p70S6K途径调节新生大鼠缺氧缺血性脑损伤后炎症反应

Granulocyte -colony stimulating factor exerts its neuroprotective effects by regulating inflammatory cascades via mTOR/p70SK6 signaling pathway when hypoxia -ischemia occurs in neonatal rats

目的观察粒细胞集落刺激因子（G-CSF）对新生大鼠缺氧缺血性脑损伤（HIBD）后炎症反应的影响及哺乳动物雷帕霉素靶蛋白/p70核糖体S6蛋白激酶（mTOR/p70S6K）信号通路在其中发挥的作用。方法7日龄新生SD大鼠90只按随机数字表法随机分为假手术组、缺氧缺血（HI）模型组、G-CSF组、雷帕霉素组及对照组，改良Rice法建立新生大鼠HIBD模型。HI前1 h，雷帕霉素组腹腔注射雷帕霉素250 μg/kg，对照组予等体积乙醇腹腔注射。HI 1 h后，G-CSF组、雷帕霉素组及对照组腹腔注射G-CSF 50 μg/kg，HI模型组及假手术组予等体积9 g/L盐水腹腔注射。HI 48 h后，Western blot检测脑组织中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-10及mTOR/p70S6K信号通路蛋白水平，尼氏染色评估海马CA1区及皮质神经元损伤情况，2，3，5-氯化三苯基四氮唑染色检测脑梗死面积。结果与HI模型组比较，G-CSF组及对照组脑梗死面积减少[（12.87±1.54）%、（11.90±1.31）%比（24.21±3.28）%]，尼氏染色阳性神经元计数增多[海马CA1区：（61.00±4.90）个/视野、（61.67±6.40）个/视野比（42.67±4.46）个/视野；皮质：（92.67±6.68）个/视野、（90.17±4.45）个/视野比（70.83±6.97）个/视野]，TNF-α及IL-1β水平降低（TNF-α：0.67±0.07、0.55±0.05比0.86±0.05；IL-1β：0.65±0.06、0.52±0.10比0.86±0.06），IL-10、p-mTOR/mTOR及p-p70S6K/p70S6K水平升高（IL-10：0.68±0.04、0.62±0.05比0.34±0.02；p-mTOR/mTOR：0.53±0.02、0.51±0.01比0.26±0.01；p-p70S6K/p70S6K：0.89±0.03、0.90±0.03比0.55±0.02），差异均有统计学意义（均P〈0.05）。与G-CSF组及对照组比较，雷帕霉素组脑梗死面积增加[（25.70±1.50）%]，尼氏染色阳性神经元计数减少[海马CA1区：（40.67±3.50）个/视野；皮质：（68.33±8.17）个/视野]，TNF-α及IL-1β水平升高（分别为0.97±0.06、0.98±0.10），IL-10、p-mTOR/mTOR及p-p70S6K/p70S6K水平降低（分别为0.21±0.02、0.30±0.01、0.55±0.01），差异均有统计学意义（均P〈0.05）。结论G-CSF可能通过上调mTOR/p70S6K信号通路抑制HIBD后的炎症反应。

ObjectiveTo investigate the role of granulocyte-colony stimulating factor （G-CSF） on the regulation of inflammatory cytokines in neonatal hypoxic-ischemic brain damage（HIBD） rat model, and to explore the possible mechanism involved in G-CSF neuroprotective effect via the mammalian target of Rapamycin/p70 ribosomal S6 protein kinase （mTOR/p70S6K） signaling pathway.MethodsA group of postnatal day 7 （P7） Sprague-Dawley rat pups （90 cases） were randomly divided into sham-operated group, hypoxia-ischemia（HI） group, G-CSF group, Rapamycin （RAP） group and control group, and the improved Rice method was used to establish a neonatal rat model of HIBD.One hour before HI induction, Rapamycin was administered intraperitoneally with a dose of 250 μg/kg, and the control group was given equal volume of ethanol injected intraperitoneally.One hour after HI, a dose of 50 μg/kg of G-CSF was injected intraperitoneally into the G-CSF group, Rapamycin group and control group.The same volume of normal saline was injected intraperitoneally into HI group and sham-operated group.Forty-eight hours after HI, Western blot was used to detect the protein levels of tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-10, and the mTOR/p70S6K signaling pathway in brain tissue.Neuron injury of the hippocampal CA1 region and the cortex was assessed by Nissl staining, and infarct volume detected by 2, 3, 5-triphenyltetrazolium chloride staining.ResultsThe G-CSF group and control group were associated with significantly reduced infarction volume compared to the HI group [（12.87±1.54）%, （11.90±1.31）% vs.（24.21±3.28）%], and the differences were statistically significant（P〈0.05）. There was an increased positive neuron cell number in the ipsilateral hemispheres of the hippocampal CA1 region in the G-CSF group and the control group [（61.00±4.90） cell/field and （61.67±6.40） cell/field] and cortex [（92.67±6.68） cell/field and （90.17±4.45） cell/field] compared with those in HI group [（42.62±4.46） cell/field and （70.83±6.97） cell/field], and the differences were all statistically significant （all P〈0.05）. The expression levels of TNF-α and IL-1β were significantly decreased in the G-CSF group and the control group, compared with those in HI group（TNF-α： 0.67±0.07, 0.55±0.05 vs.0.86±0.05; IL-1β： 0.65±0.06, 0.52±0.10 vs.0.86±0.06）, and the differences were all statistically significant （all P〈0.05）. There was increased expression levels of IL-10, p-mTOR/mTOR and p-p70S6K/p70S6K in the G-CSF group and the control group, compared with those in HI group （IL-10： 0.68±0.04, 0.62±0.05 vs.0.34±0.02; p-mTOR/mTOR： 0.53±0.02, 0.51±0.01 vs.0.26±0.01; p-p70S6K/p70S6K： 0.89±0.03, 0.90±0.03 vs.0.55±0.02）, and the differences were all statistically significant（all P〈0.05）. There was an increased infarct volume in Rapamycin group [（25.70±1.50）%], compared with the G-CSF group and the control group, and there were decreased number of positive neuron cell count in the hippocampal CA1 region [（40.67±3.50） cell/field] and cortex [（68.33±8.17） cell/field], increased expression levels of TNF-α and IL-1β （0.97±0.06 and 0.98±0.10, respectively）, decreased expression levels of IL-10, p-mTOR/mTOR and p-p70S6K/p70S6K （0.21±0.02, 0.30±0.01 and 0.55±0.01, respectively） in the Rapamycin group, and the differences were all statistically significant （all P〈0.05）.ConclusionsG-CSF may inhibit inflammatory responses after HIBD by up-regulating the mTOR/p70S6K signaling pathway in neonatal HI encephalopathy.