富氢液对创伤性颅脑损伤大鼠大脑皮质水通道蛋白1表达的影响

Effects of hydrogen-rich water on the expression of aquaporin 1 in the cerebral cortex of rat with traumatic brain injury

目的探讨富氢液对大鼠创伤性颅脑损伤（TBI）后脑水肿及水通道蛋白1（AQP1）表达的影响。方法将90只成年雄性SD大鼠按随机数字表法分为假手术（Sham）组、TBI模型组和富氢液干预组，每组30只。采用颅脑撞击法制备TBI模型；Sham组只开颅窗、骨蜡封闭缝合，不撞击。富氢液干预组于制模后经腹腔注射富氢液5mL／kg，Sham组和TBI组注射等量生理盐水，均每日1次，共5d。各组分别于术后6、12、24、48h和5d取6只大鼠进行神经损伤严重度评分（NSS）；取大脑皮质，光镜下观察脑组织病理学变化；采用免疫组化染色，镜下观察大脑皮质AQP1阳性表达；采用实时荧光定量反转录-聚合酶链反应（RT—PCR）检测大脑皮质AQP1 mRNA表达；采用蛋白质免疫印迹试验（Western Blot）检测大脑皮质AQPl蛋白表达。结果①Sham组各时间点NSS评分均为0分；TBI组NSS评分随时间延长呈升高趋势，24h达峰值后逐渐降低；富氢液干预后可明显降低NSS评分，24h与TBI组比较最为明显（分：9．83±2．78比13．50±2．42，P〈0．05）。②光镜下显示，TBI组大鼠6h大脑皮质神经细胞排列即明显紊乱，24h脑水肿和出血最为严重，之后水肿逐渐消退；各时间点软脑膜处AQPl阳性表达明显增加，以24h时最为明显。富氢液干预组术后12h～5d脑组织病理学改变均较TBI组明显减轻；软脑膜处AQPl阳性表达较TBI组明显减少。③与Sham组比较，TBI组脑组织AQP1的mRNA和蛋白表达均随时间延长逐渐升高，并于24h达峰值[AQP1 mRNA（2-△△Ct）：7．50±0．26比1，AQP1蛋白（灰度值）：1．986±0．110比0．336±0．034，均P〈0．05]，之后逐渐下降；富氢液干预可明显下调脑组织AQP1的mRNA和蛋白表达[24hAQP1 mRNA（2-△△Ct）：5．40±0．21比7．50±0．26，24hAQP1蛋白（灰度值）：1．246±0．137比1．986±0．110，均P〈0．05]。结论TBI大鼠大脑皮质AQP1 mRNA和蛋白表达上调，可能参与了TBI后脑水肿的病理生理过程；早期腹腔注射富氢液可能通过下调AQP1的表达，减轻TBI后脑水肿，从而起到脑保护作用。

Objective To investigate the effect of hydrogen-rich water on cerebral edema and aquaporin 1 （AQP1） expression in rats with traumatic brain injury （TBI）. Methods Ninety male Sprague-Dawley （SD） rats were randomly divided into sham operation group, TBI model group, hydrogen-rich water treatment group （H group）, with 30 rats in each group. TBI model was reproduced by weight dropping method. The skulls of rats in sham operation group underwent only craniotomy without direct hit and with bone wax sealed suture. 5 mL/kg of hydrogen-rich water injection was given intraperitoneally after model reproduction in H group, and equal amount of normal saline was given in sham and TBI groups, once a day for both groups for 5 days. Six rats from each group were sacrificed at 6, 12, 24, 48 hours and 5 days after evaluating neurological severity scores （NSS）. The cerebral cortex was harvested, and the pathological changes in morphology of brain tissue were observed with light microscope. The positive expression of AQP1 in cerebral cortex was observed with immunohistochemistry by light microscopy, the AQP1 mRNA expression in cerebral cortex was determined by real-time fluorescent quantization reverse transcription-polymerase chain reaction （RT-PCR）, and the AQP1 protein expression in cerebral cortex was determined by Western Blot. Results （1） All rats in sham operation group had a NSS of zero at each time point. NSS of TBI group was obviously raised with time prolongation, and peaked at 24 hours followed by a lower tendency, while the score in H group was significantly lower than that of TBI group, and the difference was the most obvious at 24 hours as compared with TBI group （9.83±2.78 vs. 13.50±2.42, P 〈 0.05）. （2） It was shown by light microscope that in the TBI group there were pathological changes in cerebral cortex, including obvious irregular arrangement of nerve cells, cerebral edema, obvious bleeding, especially at 24 hours, then the cerebral edema became vanished gradually; and the positive expression of AQP1 in the pia mater at all the time points in the TBI group was significantly increased, and it was most obvious at 24 hours. Compared with TBI group, the pathological changes at time points of 12 hours to 5 days in H group was significantly lessened, and the positive expression of AQP1 in the cerebral pia mater was reduced obviously. （1） Compared with sham operation group, the mRNA and protein expressions of AQP1 in cerebral cortex in TBI group were significantly elevated, peaked at 24 hours [AQP1 mRNA （2-△△Ct）： 7.50±0.26 vs. 1, AQP1 protein （gray value）： 1.986±0.110 vs. 0.336±0.034, both P 〈 0.05], then they gradually declined. The mRNA and protein expressions of AQP1 in cerebral cortex were significantly decreased after hydrogen-rich water treatment [24-hour AQP1 mRNA （2-△△Ct）： 5.40±0.21 vs. 7.50±0.26, 24-hour AQP1 protein （gray value）： 1.246±0.137 vs. 1.986±0.110, both P 〈 0.05]. Conclusions The up-regulation of AQP1 mRNA and protein in rats＇ cerebral cortex after TBI perhaps participates in edema formation which might be involved in the pathophysiology of cerebral edema in TBI. Early treatment with an intraperitoneally injection of hydrogen-rich water is capable of attenuating the extent of TBI-induced up-regulation of AQP1 mRNA and protein, alleviating cerebral edema, and achieving its protective effects.