雷帕霉素对劳力型热射病大鼠下丘脑损伤的保护作用及其机制  

Protective effect of rapamycin on hypothalamus injury in exertional heat stroke rats and its mechanism

作　　者：宣律 张玉想 王佳兴 顾焱 任卫东[3] 孙正中 马运亚 Xuan Lv;Zhang Yu-Xiang;Wang Jia-Xing;Gu Yan;Ren Wei-Dong;Sun Zheng-Zhong;Ma Yun-Ya(Graduate School of Hebei North University,Zhangjiakou,Hebei 075000,China;Department of Critical Care Medicine,the Eighth Medical Center of Chinese PLA General Hospital,Beijing 100091,China;Department of Endocrinology,the First Affiliated Hospital of Hebei North University,Zhangjiakou,Hebei 075000,China)

机构地区：[1]河北北方学院研究生学院,河北张家口075000 [2]解放军总医院第八医学中心重症医学科,北京100091 [3]河北北方学院附属第一医院内分泌科,河北张家口075000

出　　处：《解放军医学杂志》2023年第8期903-912,共10页Medical Journal of Chinese People's Liberation Army

基　　金：军事医学创新工程项目(18CXZ023);解放军总医院第八医学中心重点项目(2021ZD002,2016ZD-008)。

摘　　要：目的探讨雷帕霉素(Rapa)对劳力型热射病大鼠下丘脑损伤的保护作用及其机制。方法80只Wistar雄性大鼠随机分为对照组、Rapa组、劳力型热射病(EHS)组及EHS+Rapa组,每组20只。Rapa组及EHS+Rapa组大鼠分别于建模前连续4 d腹腔注射Rapa(1 mg/kg,1次/d),对照组及EHS组给予同体积生理盐水。将EHS组及EHS+Rapa组大鼠置于温度(39.5±0.3)℃、湿度55%±5%的气候舱中跑步,建立EHS模型;对照组及Rapa组大鼠置于相同室温下,进行与EHS组同等强度的跑步。在模型建立过程中,观察各组大鼠的一般状态,测量核心温度并绘制大鼠生存曲线(n=11)。EHS组及EHS+Rapa组大鼠于入舱80 min后,从舱中取中,并观察300 min。对各组大鼠实施麻醉,取腹主动脉血及下丘脑等组织,HE及尼氏染色观察下丘脑的组织病理学改变;免疫荧光法测定神经元凋亡情况;Western blotting法检测大鼠下丘脑组织中哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(pmTOR)、自噬效应蛋白(Beclin-1)、泛素结合蛋白(p62)及自噬标志物微管相关蛋白1轻链3(LC3)的表达,并计算pmTOR/mTOR及LC3-Ⅱ/LC3-Ⅰ比值。采用酶联免疫吸附试验(ELISA)检测大鼠血清中神经元特异性烯醇化酶(NSE)、脑活性肽100β蛋白(S100β)、白细胞介素-6(IL-6)及肿瘤坏死因子-α(TNF-α)的表达水平。结果在入舱后80 min时,与对照组相比,EHS组的核心温度明显升高;与Rapa组相比,EHS+Rapa组的核心温度明显升高(P<0.001)。与EHS组相比,EHS+Rapa组大鼠的生存率升高(P<0.01)。下丘脑组织HE及尼氏染色显示,EHS组大鼠下丘脑神经元病理损伤更重,而EHS+Rapa组大鼠下丘脑神经元病理损伤较EHS组明显减轻(P<0.001)。免疫荧光检测结果显示,EHS组大鼠下丘脑组织中细胞凋亡率较对照组显著增高,而与EHS组相比,EHS+Rapa组大鼠下丘脑组织中细胞凋亡率显著降低(P<0.05)。Western blotting结果显示,与对照组相比,EHS组大鼠下丘脑组织中pmTOR/mTOObjective To investigate the protective function and mechanism of rapamycin on the hypothalamus injury of rats with exertional heat stroke.Methods Eighty male Wistar rats were randomly divided into four groups:control group,rapamycin group(Rapa group),exertional heat stroke group(EHS group),and exertional heat stroke+rapamycin group(EHS+Rapa group),with 20 rats in each group.The rats in Rapa group and EHS+Rapa group were injected intraperitoneally with Rapa(1 mg/kg,once a day)for four consecutive days before modeling.The rats in control and EHS groups were treated with the same dose of 0.9%normal saline.In EHS group and EHS+Rapa group,the rats ran in a climate chamber with a temperature range of(39.5±0.3)℃and a humidity range of(55%±5%).After successful modeling,the rats were removed from the climate chamber for cooling at room temperature.The animals in control and Rapa groups ran at the same intensity and room temperature as EHS group.During the establishment of the model,we monitored the general state,measured core temperature,and profiled the survival curve of the rats in each group(11 rats randomly selected from each group).The rats in EHS group and EHS+Rapa group were removed from the chamber after modeling of 80 min,and after 300 min observation,each group of rats was anesthetized.Then we collected the abdominal aorta blood and hypothalamus.The histopathological changes in the hypothalamus were analyzed by HE and Nissl staining.Immunofluorescence was used to determine the apoptosis of hypothalamus.Western blotting was used to detect the expression of mammalian target of rapamycin(mTOR)and phosphorylated mTOR(pmTOR),autophagy effector protein(Beclin-1),ubiquitin-binding protein(p62)and autophagy marker microtubule-associated protein 1 light chain 3(LC3)in the hypothalamus of rats.We calculated the ratios of pmTOR/mTOR and LC3-Ⅱ/LC3-Ⅰ.We measured the expression levels of neuron-specific enolase(NSE),brain active peptide 100βprotein(S100β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in a

关 键 词：中暑 下丘脑 雷帕霉素 自噬 

分 类 号：R58[医药卫生—内分泌]