中国公共卫生  2019, Vol. 35 Issue (8): 1043-1045   PDF    
枸杞多糖对大鼠放射性脑损伤海马神经元凋亡的保护作用及其对PI3K/Akt/mTOR信号通路影响
王亿龙, 刘青峰    
中国医科大学附属第一医院肿瘤放射治疗科,沈阳 110001
摘要目的 了解枸杞多糖对放射性脑损伤海马神经元的保护作用并观察其与氧化应激和PI3K/Akt/mTOR信号通路的关系。方法 体内实验:SD大鼠,分为对照A(ConA)组、模型A(ModA)组和枸杞多糖A(LBPA)组,每组16只。LBPA组在照射前2周开始采用枸杞多糖50 mg/kg灌胃,ConA组和ModA组予以等剂量生理盐水灌胃,ConA组不予照射。水迷宫检测认知功能,海马组织行尼氏染色,TUNEL染色和检测氧化应激指标MDA、SOD、GSH-Px。体外实验(B):原代胎鼠海马神经元培养,分组为对照B(ConB)组,模型B(ModB)组,枸杞多糖B(LBPB)组。对ModB组和LBPB组进行放射性处理,ConB组不处理。MTT测细胞活性,annexin V-FITC/PI流式细胞仪检测细胞凋亡,Western blot检测PI3K、Akt、mTOR、Bcl-2、Bax、caspase-3蛋白表达水平。结果 与ConA组比较,LBPA组大鼠逃避潜伏期和空间探索时间明显缩短,海马神经元细胞形态改善显著,细胞凋亡率显著减少;LBPA组较ModA组MDA含量明显降低,SOD和GSH-Px活性明显升高;与ModB组比较,LBPB组细胞存活率增高,凋亡率降低,PI3K、pAkt、mTOR和Bcl-2表达明显增高,Bax和caspase-3的表达下降。结论 枸杞多糖能够抑制放射性脑损伤海马神经元的凋亡,其作用可能与PI3K/Akt/mTOR信号通路和氧化应激有关,具备神经保护作用。
关键词枸杞多糖     放射性脑损伤     海马神经元     PI3K/Akt/mTOR信号通路     氧化应激    
Effect of Lycium barbarum polysaccharide protecting against apoptosis of hippocampal neurons and on PI3K/Akt/mTOR signaling pathway in rats with radiation-induced brain injury
WANG Yi-long, LIU Qing-feng     
Department of Radiation Oncology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province 110001, China
Abstract: Objective To observe protective effect of Lycium barbarum polysaccharide (LBP) on hippocampal neuron injury induced by radiation in vivo and in vitro and to explore the associations of oxidative stress and PI3K/Akt/mTOR signaling pathway with the effect in rats and rat embryonic cells. Methods For in vivo experiment, Sprague-Dawley (SD) rats were divided into three groups (16 in each group): model group (ModA) with a single electron beam irradiation on brain at the dosage of 20 Gy and saline gavage, LBP group (LBPA) with intragastric administration of 50 mg/kg LBP two weeks before the irradiation, and control group (ConA) without irradiation but with saline gavage. Thirty days after the treatments, water maze test was used to test cognitive function of the rats; Nissl-stained hippocampal tissues were observed; TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method was been employed to detect apoptosis of hippocampal neurons; malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in hippocampal tissue homogenate were detected. For in vitro experiments, hippocampal neurons of primary fetal SD rats were cultured and also divided into three groups: model group (ModB) with single 30 Gy X-ray irradiation LBP group (LBPB) with the irradiation and LBP administration in culture fluid one hour before the irradiation at the concentration of 50 μg/ml, and control group(ConB) without the irradiation and LBP treatment. Cell activity was determined with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay; cell apoptosis was detected with annexin V-fluorescein/propidium iodide (FITC/PI) flow cytometry; and protein expressions of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), B-cell lymphoma/leukemia 2 (bcl-2), Bcl-2 associated X protein (Bax) and caspase-3 were detected with Western blot. Results Compared with those of the ModA rats, the escape latency period and space exploration time of LBPA rats were significantly shortened; the morphology of damaged hippocampal neurons was significantly improved; and the apoptosis rate was significantly reduced. The LBPA rats had significantly lower MDA content and significantly higher SOD and GSH-Px activity than the ModA rats (P < 0.01 for all). In comparison with the ModB cells, LBPB cells showed increased survival rate, decreased apoptosis rate, significantly increased expression of PI3K, pAkt, mTOR and bcl-2, and decreased expression of Bax and caspase-3 (all P < 0.01). Conclusion LBP can inhibit radiation-induced apoptosis of hippocampal neurons and the neuroprotective effect may be related to regulations of PI3K/Akt/mTOR signaling pathway and oxidative stress.
Key words: Lycium barbarum polysaccharide     radioactive brain injury     hippocampal neuron     PI3K/Akt/mTOR signaling pathway     oxidative stress    

放射性脑损伤为头颅经放射治疗导致神经系统损害,多表现为进行性认知、记忆、理解、信息处理能力受损,影响患者生存质量。海马为大脑中最重要的脑区之一,与认知记忆密切相关,研究发现,放射性脑损伤认知受损程度与海马凋亡高度相关[12]。枸杞多糖(Lycium barbarum polysaccharides,LBP)是枸杞的主要有效成分,具有神经保护作用,能够改善神经病变和神经缺损[35];但LBP对放射性脑损伤的作用尚未见报道。为观察LBP对大脑放射性脑损伤海马神经元凋亡的保护作用,本研究拟从动物实验和细胞实验观察LBP对大脑放射性脑损伤和放射性损伤原代培养海马神经元的保护作用,并观察其与氧化应激和PI3K/Akt/mTOR信号通路的关系。

1 材料与方法 1.1 实验动物与材料

雄性SD大鼠体重180~200克8只每组16只,孕17天SD大鼠,SPF级,SCXK(京)2015-0036,北京维通利华公司采购。动物实验得到中国医科大学附属第一医院动物伦理委员会批准。TUNEL凋亡试剂盒(美国罗氏公司),尼氏染色试剂盒(北京雷根生物科技有限公司),MTT(美国Sigma公司),磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)、磷酸化丝氨酸/苏氨酸激酶(phosphorylated serine/threonine kinase,pAkt)、雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、B细胞淋巴瘤/白血病-2基因蛋白(B-cell lymphoma/leukemia 2,Bcl-2)、Bcl-2相关X蛋白基因蛋白(Bcl-2 associated X protein,Bax)、半胱氨酸蛋白酶-3(caspase-3)均购自英国Abcam公司,蛋白质定量试剂盒(Bradford法,P1510)、caspase-3活性测定试剂盒购自北京普利莱基因技术有限公司,谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)试剂盒购自南京建成生物工程研究所,LBP购于上海康舟真菌多糖公司。

1.2 实验仪器

– 80 ℃冰箱(MDF-U73V,日本SANYO公司),OLYPUS X71光学显微镜(日本索尼公司),Western blot系统(美国BIO-RAD公司),Morris水迷宫(XR-XM101,上海欣软信息科技有限公司),紫外分光光度计(UV-7504,上海欣茂仪器有限公司),细胞培养箱(美国Thermo Forma 公司)。

1.3 方法 1.3.1 体内实验(A)分组及模型建立

大鼠称重,4 % 水合氯醛腹腔注射(1ml/100g)麻醉,俯卧位固定于自制木板架上,直线加速器6MeV电子线对模型A(ModA)组、枸杞多糖A(LBPA)组大鼠行全脑单次20 Gy垂直照射,吸收剂量率为200 cGy/min,源皮距100 cm,在10 cm × 10 cm的限光筒铅模中,照射野大小为2.5 cm × 2.5 cm,照射野前界为双眼内眦连线,后界为双耳后连线。对照A(ConA)组大鼠麻醉后,放入相同环境,但不予照射。LBPA组在照射前2周采用枸杞多糖50 mg/kg灌胃,ConA组和ModA组予以等剂量生理盐水灌胃,至照射结束后30天行后续实验。

1.3.2 水迷宫实验

使用Morris水迷宫评价大鼠认知功能[5]。定位航行实验:将大鼠随机从4个象限入水,记录每只大鼠入水后2 min内到达隐蔽平台的时间,即为逃避潜伏期。若2 min内未到达平台,记录为潜伏期120 s。空间探索实验:实验第6天,移走平台,大鼠随机从任意入水点入水,观察大鼠穿过平台区域的时间,即为空间探索时间。

1.3.3 体内实验指标

大鼠给药结束后灌注固定:取脑内海马组织,低温冰冻切片,行尼氏染色,显微镜下观察,行TUNEL染色,计算细胞凋亡率;分离海马组织,组织匀浆;测定氧化应激指标MDA含量、GSH-PX和SOD活性。

1.3.4 体外实验(B)原代胎鼠海马神经元的培养

原代海马神经元取自孕17d SD 大鼠胚胎,镜下解剖大鼠海马,进行海马神经元的培养,细胞培养第7天,枸杞多糖B(LBPB)组中提前1h加入药物枸杞多糖,终浓度为50 μg/ml,对照B(ConB)组和模型B(ModB)组加入等体积细胞培养液。ModB组和LBPB组采用直线加速器,剂量率400cGy/min的6MV的X线照射,单次照射剂量为30Gy。照射后继续培养行下一步研究。

1.3.5 体外实验指标

细胞活性测定使用MTT方法,计算细胞存活率;流式细胞仪检测海马神经元细胞凋亡;Western blot 测定BAX、BCL-2、AMPK、pAMPK、mTOR蛋白的表达。

1.4 统计分析

采用SPSS18.0软件进行分析,采用方差分析进行多组间比较,P < 0.05为差异有统计学意义。

2 结 果 2.1 体内实验(A)枸杞多糖对放射性脑病大鼠海马CA3区神经元形态的影响

ConA组大鼠海马CA3神经元细胞层次丰富,排列整齐,包膜完整,形态正常,未见细胞坏死。ModA组大鼠海马神经元层次变少,细胞萎缩、排列稀疏,胞核变小甚至固缩,坏死细胞多见。LBPA组海马神经元细胞层次丰富,细胞形态规则,坏死细胞较少,较ModA组大鼠海马神经元形态改善显著。

2.2 体内实验(A)枸杞多糖对放射性脑病大鼠海马神经元细胞凋亡的影响

ConA组凋亡细胞数量少见,凋亡率(8.7 ± 1.86)%;ModA组凋亡细胞显著增加(42.8 ± 4.95)%,与ConA组相比具有统计学差异(P < 0.01);LBPA组凋亡率(19.4 ± 1.93)%,与ModA组比较差异有统计学意义( P < 0.01)。

2.3 体内实验(A)枸杞多糖放射性脑病大鼠海马MDA含量及SOD、GSH-Px活性的影响(表1
表 1 不同组别MDA含量及SOD、GSH-Px活性($\bar x \pm s$)

与ConA组比较,ModA组MDA含量明显升高(P < 0.05)、SOD( P < 0.05)和GSH-Px( P < 0.05)活性明显降低;与ModA组比较,LBPA组MDA含量明显降低( P < 0.05)、SOD( P < 0.05)和GSH-Px( P < 0.05)活性明显升高。

2.4 体外实验(B)各组原代胎鼠海马神经元细胞的存活率及凋亡率

ConB组海马神经元细胞生长良好;ModB组海马神经元出现大量凋亡及坏死细胞,细胞出现萎缩,生长稀疏,活力下降;LBPB组海马神经元出现部分坏死及凋亡细胞,但较ModB组明显改善。ConB组、ModB组、LBPB组胎鼠海马神经元细胞存活率分别为100 %、(38.8 ± 14.4)%、(77.2 ± 13.6)%;ModB组较ConB组存活率明显下降(P < 0.01),LBPB组较ModB组存活率明显上升( P < 0.01)。ConB组、ModB组、LBPB组凋亡率分别为(6.8 ± 4.08)%、(44.5 ± 3.87)%、(26.6 ± 7.27)%;ModB组凋亡率较ConB组明显增加( P < 0.01),LBPB组较ModB组明显降低( P < 0.01)。

2.5 体外实验(B)各组原代胎鼠海马神经元细胞PI3K/Akt/mTOR/BAX/Bcl-2蛋白表达(图1
图 1 枸杞多糖对放射性损伤海马神经元凋亡蛋白的影响

ModB组Bax、capsase-3表达较ConB组增加,PI3K、Akt、mTOR、Bcl-2表达较ConB组降低。LBPB组Bax、capsase-3表达较ModB组降低,PI3K、Akt、mTOR、Bcl-2表达较ModB组增加。

3 讨 论

放射性脑损伤是头颈部肿瘤患者放疗后的并发症之一,常导致患者认知功能障碍,影响生活质量。如何防治放射性脑损伤亦成为重要议题。枸杞多糖是枸杞主要的有效成分。近年来,多项研究显示,枸杞多糖对帕金森病[6]、癫痫[7]、脑缺血[89]、视网膜损伤[1011]等均有保护作用,枸杞多糖还具有抗神经系统肿瘤的作用[12]。本研究显示,枸杞多糖在体内实验能够减轻放射性脑损伤大鼠的认知功能障碍,抑制海马神经元的凋亡;在体外实验能够减少放射性脑损伤模型海马神经元细胞的凋亡率,提高存活率,提示枸杞多糖对放射性脑损伤大鼠具备神经保护作用。神经细胞经放射性照射后胞体内出现大量氧自由基及炎性介质,同时线粒体功能受到放射性损伤,氧自由基清除减少,导致自由基大量蓄积,诱发炎性反应,促进细胞凋亡,此为放射性脑损伤的重要原因之一[1];MDA、SOD和GSH-Px是氧化应激的标志性物质。枸杞多糖具有抗氧化应激作用[1314];本研究显示,与模型组相比,LBP组MDA含量显著降低,SOD和GSH-Px活性显著升高,提示枸杞多糖能够改善放射性脑损伤的氧化应激反应。PI3K/Akt/mTOR 信号通路在放射性脑损伤过程神经细胞存活、凋亡过程中发挥重要的生物学功能[15]。放射性损伤造成线粒体膜电位的下降甚至破坏线粒体,进而抑制PI3K/Akt/mTOR信号通路,诱导细胞凋亡[1]。Caspases是细胞凋亡程序执行者,其表达的升高可促进细胞凋亡。Bax是促凋亡基因,Bcl-2是抑制凋亡基因,两者广泛分布于线粒体内膜、核膜、细胞内质网等。多项研究显示,枸杞多糖能够通过激活PI3K/Akt/mTOR信号通路改善脑缺血再灌注的神经缺损症状[16],减轻神经细胞凋亡[17]。本研究显示,枸杞多糖能够抑制放射性脑损伤大鼠海马的氧化应激,同时能够增强氧糖剥夺/复氧海马神经元细胞的PI3K/Akt/mTOR信号通路的表达,升高Bcl-2表达,降低Bax和caspase-3的表达,上述作用与其神经保护功能相关。

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