Role of TLR4/NF-κB signaling pathway in hydrogen-rich water improving cognitive impairment induced by ionizing radiation in rats
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摘要:背景
在放射治疗过程中,射线杀伤肿瘤细胞的同时,也会不可避免地损伤正常组织细胞。
目的通过对大鼠全脑照射,研究Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路在富氢水改善电离辐射致认知功能损伤中的作用。
方法将15只雄性SD大鼠随机分为对照组、照射组(IR组)、富氢水干预组(IR+HRW组),每组5只。对照组不进行照射,每天灌胃给予纯化水(20 mL·kg−1);IR组和IR+HRW组进行单次20 Gy全脑照射,照前3天、照前10分钟、照后每天灌胃给予纯化水/富氢水(持续30 d,20 mL·kg−1)。每天观察大鼠一般状态,并在照射后第7天、第14天、第21天、第30天测定动物体重。于照射后第30天开展Morris水迷宫实验,检测大鼠学习记忆能力;结束后剖杀动物取材,采用HE染色检测海马病理学变化,酶联免疫吸附试验(ELISA)检测脑组织中谷胱甘肽(GSH)、丙二醛(MDA)、白介素-1β(IL-1β)、羟自由基水平,实时荧光定量PCR(qRT-PCR)、Western blotting检测大鼠海马中TLR4、NF-κB、NOD样受体蛋白-3(NLRP3)、半胱天冬酶1(Caspase 1)的mRNA、蛋白表达水平。
结果照射后,IR组大鼠出现头部脱毛、流涎等症状,IR+HRW组大鼠症状较轻。对照组和IR+HRW组未出现动物死亡情况,而IR组有1只大鼠死亡。在照射后第14天—第30天,IR+HRW组大鼠体重均有高于IR组的趋势,但差异无统计学意义(均
P >0.05)。Morris水迷宫结果表明,与IR组相比,除第3天外,第1天—第5天IR+HRW组大鼠逃避潜伏期均有缩短的趋势,但差异尚无统计学意义(均P >0.05),在第6天撤去平台后到达平台原来位置所用时间减少,穿越平台次数和在原平台象限停留时间增加(均P <0.05)。HE染色显示,IR+HRW组大鼠海马细胞数量轻度减少,排列整齐,未见明显的胞核深染及固缩现象。ELISA实验结果显示,IR+HRW组大鼠脑组织中MDA、羟自由基水平较IR组降低(均P <0.05),GSH含量有升高、IL-1β浓度有降低的趋势,但差异尚无统计学意义(均P >0.05)。qRT-PCR实验结果显示,IR+HRW组大鼠海马中TLR4 、Caspase 1 mRNA表达水平较IR组下降(均P <0.05),NF-κB 、NLRP3 mRNA表达水平有下降的趋势,但差异无统计学意义(均P >0.05)。Western blotting实验结果显示,IR+HRW组大鼠较IR组海马中TLR4、Caspase 1蛋白表达水平下降(均P <0.05),NF-κB p65、NLRP3蛋白表达水平有下降的趋势,但差异尚无统计学意义(均P >0.05)。结论富氢水可改善电离辐射导致的大鼠认知功能损伤,其机制可能与调控TLR4/NF-κB信号通路,抑制炎性因子,减弱氧化应激反应有关。
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关键词:
- 富氢水 /
- 电离辐射 /
- TLR4/NF-κB信号通路 /
- 认知功能损伤
Abstract:BackgroundIn the process of radiotherapy, when radiation kills tumor cells, it inevitably damages normal tissue cells.
ObjectiveTo investigate the role of Toll-like receptor 4 (TLR4)/nuclear factor−kappa B (NF-κB) signaling pathway in the improvement of cognitive impairment induced by ionizing radiation by hydrogen-rich water before and after whole brain irradiation in rats.
MethodsFifteen male SD rats were randomly divided into three groups: control group, irradiated group (IR group), and hydrogen-rich water intervention group (IR+HRW group), with 5 rats in each group. The control group was not irradiated, but was given purified water (20 mL·kg−1) by gavage every day, while the IR group and the IR+HRW group were irradiated with a single dose of 20 Gy. Three days before, 10 min before, and 30 days after irradiation, purified water/hydrogen-rich water (20 mL·kg−1) was given by continuous gavage every day. The general condition of the rats was observed every day, and the body weight were measured on the 7th, 14th, 21st, and 30th days after irradiation. On the 30th day after irradiation, the learning and memory ability of the rats was tested by Morris water maze; the pathological changes of hippocampus were detected by hematoxylin-eosin (HE) staining after sacrificing the rats; the contents of glutathione (GSH), malondialdehyde (MDA), interleukin-1β (IL-1β), and hydroxyl radicals in brain tissues were detected by enzyme linked immunosorbent assay (ELISA); the mRNA and protein expression levels of TLR4, NF-κB, NOD-like receptor pyrin domain 3 (NLRP3), and cysteinyl aspartate specific proteinase 1 (Caspase 1) were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in the hippocampus of rats.
ResultsAfter irradiation, the rats in the IR group showed symptoms such as head hair removal and salivation, while the symptoms of the rats in the IR+HRW group were milder. No animal died in the control and the IR+HRW groups, while one rat died in the IR group. From day 14 to day 30 after irradiation, the body weight of the rats in the IR+HRW group tended to be higher than that in the IR group, but the difference was not statistically significant (
P >0.05). The Morris water maze results showed that the escape latency of the IR+HRW group was shortened compared with that of IR group from day 1 to day 5 except day 3, but the difference was not statistically significant (P >0.05). For the rats in the IR+HRW group, it took less time to reach the original location of the platform after removing the platform on day 6 and the number of crossing the platform and the residence time in the original platform quadrant increased (P <0.05). The HE staining showed that the number of hippocampal cells in the IR+HRW group was slightly reduced and arranged neatly, without obvious nuclear hyperchromatic and pyknotic phenomenon. The ELISA results showed that the MDA and hydroxyl radical levels were decreased in the IR+HRW group compared with the IR group (P <0.05), the GSH content was increased, and the IL-1β concentration was decreased, but the differences were not statistically significant (P >0.05). The results of qRT-PCR showed that the mRNA expression levels ofTLR4 andCaspase 1 in the hippocampus of the IR+HRW group were decreased compared with the IR group (P <0.05), and the mRNA expression levels ofNF-κB andNLRP3 were also decreased, but the differences were not statistically significant (P >0.05). The results of Western blotting showed that the expression levels of TLR4 and Caspase 1 protein in the hippocampus of the IR+HRW group were decreased compared with the IR group (P <0.05), and the expression levels of NF-κB p65 and NLRP3 protein were also decreased, but the differences were not statistically significant (P >0.05).ConclusionHydrogen-rich water can improve cognitive impairment induced by ionizing radiation in rats, and its mechanism may be related to regulating TLR4/NF-κB signaling pathway, inhibiting inflammatory factors, and attenuating oxidative stress.
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放射治疗是目前广泛应用于中枢神经系统肿瘤的治疗方法,临床应用时在对肿瘤组织提供有效的照射剂量前提下,也兼顾对健康组织产生尽可能小的损伤。但即使采取了一定的保护措施,也不能完全避免正常脑组织的损伤,接受放射治疗后存活期超过6个月的患者中,有很大一部分会出现认知损伤,影响其日常生活质量[1-2],并且这种衰退是持续的、渐进的。虽然辐射引起认知功能损伤的重要性和影响因素备受关注,但其机制尚未完全阐明,尚无有效的预防或治疗措施[3]。
电离辐射通过电离与激发体液中的水分子,产生具有毒性的羟自由基[4],刺激大量内源性活性氧(reactive oxygen species, ROS)产生,主要影响线粒体呼吸链和机体氧化还原系统[5]。随着ROS的持续激活和增加,形成氧化应激,导致人体的抗氧化系统遭到破坏[6]。研究表明电离辐射引起脑损伤的机制涉及细胞死亡、神经发生损伤、氧化应激、血管损伤、脱髓鞘和炎症反应等[7-8]。氢气已被证明是一种理想的具有抗氧化功能的物质,可以中和羟自由基,对变态反应性疾病、炎症反应性疾病及全身多个脏器疾病均有一定程度的疗效[9]。氢气的辐射防护作用机制与清除羟自由基等自由基有关,同时与其抗炎、抗凋亡的特性以及可调节细胞内信号的基因表达间接相关[10]。氢气有易燃易爆性,由呼吸摄入氢气在使用上有一定局限性。而将氢气溶解于水中,制成饱和富氢水溶液,拓展了氢气的安全使用途径。饮用富氢水是摄入氢的有效方式之一。本课题组在前期研究发现,富氢水对电离辐射引起的认知功能损伤具有一定的保护作用,可能的作用机制涉及富氢水可减少神经细胞凋亡,并通过调节脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)-酪氨酸激酶受体B(tyrosine kinase receptor B, TrkB)信号通路保护新生神经元,改善动物的认知功能[11-13]。
Toll样受体(Toll-like receptor, TLR)是先天性免疫应答的模式识别受体[14],可被与细胞损伤产物相关的分子模式激活[15]。研究表明,包括TLR4在内的一些TLR亚型在脑内广泛存在,并在脑损伤后炎症调节中发挥重要作用[16-17]。TLR4是TLRs家族的关键成员之一,与炎症反应过程密切相关。核因子-κB(nuclear factor-kappa B, NF-κB)是位于TLR4下游的炎症因子,可被其激活[18-19]。TLR4/NF-κB是经典的炎症反应信号转导通路,NF-κB一旦被激活,就会诱导产生大量的炎性细胞因子,导致炎症反应,从而引起更多的细胞氧化应激。NF-κB的激活可导致Nod样受体蛋白-3(Nod-like receptor pyrin domain 3, NLRP3)炎症小体的mRNA转录增加,激活NLRP3炎症小体,进而激活和募集半胱天冬酶1(cysteinyl aspartate specific proteinase 1, Caspase 1),释放白介素-1β(interleukin-1β, IL-1β)等炎症因子,引起细胞焦亡。有研究指出NLRP3炎症小体通路的激活和细胞焦亡可导致神经元损伤、认知功能障碍,甚至死亡。通过降低TLR4、NF-κB、IL-1β等细胞因子的表达,可限制级联扩大的炎症反应,减少NLRP3激活导致的脑组织进一步损伤[20]。富氢水能否通过调控TLR4/NF-κB信号通路来改善电离辐射引起的大鼠认知功能损伤未见相关报道,故本实验从氧化应激、炎症角度研究TLR4/NF-κB信号通路在富氢水改善电离辐射致认知功能损伤中的作用。
1. 对象与方法
1.1 实验动物
雄性SD大鼠15只,体重250~300 g,8周龄。购自北京维通利华实验动物技术有限公司(合格证号:110011220104118961),饲养于中国辐射防护研究院动物实验中心[SYXK(晋)2018-0005]。本实验已通过中国辐射防护研究院放医所实验动物管理和使用伦理委员会伦理审查(受理编号:CIRP-IACUC-(R)2021015),实验严格遵循国家有关实验动物管理和使用的规定。
1.2 主要试剂与仪器设备
谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)、羟自由基试剂盒(中国南京建成),IL-1β试剂盒(中国北京永辉),总RNA抽提试剂盒(中国上海生工),反转录试剂盒、SYBR Green qPCR Mix试剂盒(中国北京莫纳),二喹啉甲酸(BCA)蛋白质浓度测定试剂盒(中国上海雅酶),TLR4抗体、NF-κB p65抗体(美国Affinity),NLRP3抗体、Caspase 1抗体、辣根过氧化物酶(HRP)标记山羊抗兔抗体、HRP标记山羊抗鼠抗体(英国Abcam)。
直线加速器(Elekta Synergy,瑞典Elekta),富氢水杯(ZFFQ-500,中国中辐核仪),便携式溶解氢测定仪(ENH-1000,日本Trustlex),全波长酶标仪(Multiskan GO,美国Thermo Fisher),实时荧光定量PCR(quantitative real-time polymerase chain reaction, qRT-PCR)仪(Quantstudio 7 Flex,美国Applied Biosystems)。
1.3 动物分组及处理
所有大鼠适应性喂养1周后,按照体重随机分成对照组、照射组(IR组)、富氢水干预组(IR+HRW组),每组5只,共15只。对照组不进行照射,每天灌胃给予纯化水(20 mL·kg−1);IR组和IR+HRW组进行单次20 Gy全脑照射,照前3天、照前10分钟、照后每天灌胃给予纯化水/富氢水(持续30 d,20 mL·kg−1)。
富氢水采用富氢水杯制备,使用便携式溶解氢测定仪测定其浓度为0.5 mmol·L−1,现制现用。照射条件:在室温下用直线加速器传输的6 MeV电子束以3 Gy·min−1的剂量率对IR组和IR+HRW组大鼠进行单次20 Gy全脑照射。Morris水迷宫实验后,用2.5%戊巴比妥钠麻醉剖杀动物,收集大鼠的脑组织和海马,于−80 ℃保存。
1.4 体重测定
在照射后第7天、第14天、第21天、第30天测定各组动物体重变化情况。
1.5 Morris水迷宫实验
Morris水迷宫实验于照射后第30天进行。(1)定位航行实验。在正式实验开始前1天,允许大鼠在未放置平台的水池中自由游泳2 min以熟悉实验环境。然后将水池划分为8个方向(东、南、西、北、东南、西南、西北、东北)进行实验,在东北方向放置一个平台,之后大鼠连续5 d接受训练,每天从4个方向(西南、北、东、东南)将大鼠面向池壁放入池中,追踪游泳路线,记录从入水到爬上平台(所有肢体都在平台上)的时间,视为逃避潜伏期。大鼠在平台上停留10 s后,放回笼中。若2 min内未找到平台,将其引至平台,停留10 s,记录其逃避潜伏期为120 s。计算每天4次逃避潜伏期的平均值。(2)空间探索实验。在定位航行实验后进行,即在第6天撤去平台,在西南方向将大鼠面向池壁放入池中,记录大鼠在2 min内的游泳轨迹并进行分析。记录大鼠到达原平台位置所用时间、穿过平台位置的次数、在原平台象限停留时间,以检测大鼠的空间记忆能力。
1.6 HE染色
取新鲜大鼠脑组织,置于4%多聚甲醛固定后,进行石蜡包埋,切成厚度为4 μm的切片,然后用苏木精和伊红染色。光镜下观察脑组织病理学改变。
1.7 酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)
取大鼠脑组织。使用GSH、MDA、IL-1β、羟自由基试剂盒,按照说明书检测GSH、MDA、IL-1β、羟自由基水平。
1.8 qRT-PCR
分离大鼠海马。称量30~50 mg海马,加入1 mL TRIzon充分研磨离心,氯仿抽提,取水相,异丙醇沉淀,乙醇清洗,晾干沉淀后加入30 μL RNase-Free Water充分溶解RNA,以提取大鼠海马RNA,按照反转录试剂盒说明书将RNA反转录为cDNA,根据SYBR Green qPCR Mix试剂盒说明书检测mRNA表达水平。以β-actin为内参,对目标基因进行归一化处理,并应用2−ΔΔCt法来计算每组TLR4、NF-κB、NLRP3、Caspase 1 mRNA的相对表达,引物序列见表1。
表 1 引物序列(5'→3')Table 1. Sequence of primers (5'→3')基因名称(Gene) 正向引物(Forward prime) 反向引物(Reverse primer) TLR4 TATCCAGAGCCGTTGGTGTA CCCACTCGAGGTAGGTGTTT NF-κB CCACTGTCAACAGCAGATGG TTCTTCTCACTGGAGGCACC NLRP3 TCCTGCAGAGCCTACAGTTG TCAGCTCAGGCTTTTCCTCC Caspase 1 AACACATTGAAGTGCCCAAGC ACTCCTTGTTTCTCTCCACGG β-actin CGTTGACATCCGTAAAGACCTC TAGGAGCCAGGGCAGTAATCT 1.9 Western blotting
取大鼠海马组织20~30 mg,提取组织蛋白,使用BCA法进行定量。根据蛋白分子量不同,设定不同条件进行电泳及转膜,室温下使用5%脱脂奶粉封闭2 h,加入一抗(稀释比例分别为:TLR4,1∶500;NF-κB p65,1∶800;NLRP3,1∶800;Caspase 1,1∶2000;β-actin,1∶20000)4 ℃摇床孵育、过夜,用三乙醇胺缓冲盐水溶液+吐温(TBST)漂洗3次,每次10 min,分别加入HRP标记的山羊抗鼠抗体和山羊抗兔抗体(稀释比例为1∶5000),37 ℃孵育1 h,然后用TBST漂洗3次,每次10 min,洗脱后加入超敏发光液进行显像。用Image J 1.52a软件对蛋白条带图进行灰度值的分析。内参蛋白为β-actin,以TLR4、NF-κB p65、NLRP3、Caspase 1与β-actin的灰度值之比分析各蛋白的相对表达水平。
1.10 统计学分析
采用SPSS 22.0软件对数据进行统计分析,Graphpad prism 8.0作图。体重与定位航行实验结果比较采用重复测量方差分析,LSD法两两分析;其余经方差齐性检验后采用单因素ANOVA方差分析,LSD法两两分析。检验水准α=0.05。
2. 结果
2.1 一般状态和体重变化
实验期间,对照组大鼠发育正常,IR组大鼠出现头部脱毛(3/5,于照射后第7天出现)、流涎(5/5,于照射后第4天出现),IR+HRW组大鼠头部脱毛(2/5,于照射后第7天出现)、流涎(5/5,于照射后第5天出现)症状较IR组略有减轻。对照组和IR+HRW组大鼠未出现死亡情况,而IR组在照射后第14天有1只大鼠死亡。
IR组大鼠体重在照射后第14天、第21天、第30天均低于对照组(P<0.05),IR+HRW组大鼠体重在照射后第14天、第21天低于对照组(P<0.05),在照射后第30天有低于对照组的趋势,但差异无统计学意义(P>0.05)。在照射后第14天—第30天,IR+HRW组大鼠体重有高于IR组的趋势,但差异尚无统计学意义(P>0.05)(图1)。
2.2 学习记忆能力变化
随着大鼠训练天数的增加,所有组别的大鼠逃避潜伏期都逐渐缩短。每个时间点各组间的逃避潜伏期显示,除第3天外,第1天—第5天IR+HRW组大鼠相对于IR组逃避潜伏期均有缩短的趋势,但差异尚无统计学意义(P>0.05)(图2)。与IR组相比,IR+HRW组大鼠在第6天撤去平台后到达平台原来位置所用时间减少,穿越平台次数和在原平台象限停留时间增加(P<0.05)(图3)。
图 3 照射后不同组别大鼠Morris水迷宫实验结果(n=5)A:首次到达原平台位置所用时间;B:穿越平台次数;C:原平台象限停留时间。*:P<0.05,**:P<0.01。Figure 3. Morris water maze experiment results of rats in different groups after irradiation (n=5)A: First arrival time at the original platform; B: Platform crossing times; C: Time to stay in the original platform quadrant. *: P<0.05, **: P<0.01.2.3 海马病理学变化
镜下可见:对照组大鼠海马CA3区细胞形态正常,排列整齐;IR组大鼠海马细胞数量中度减少,排列紊乱,部分神经元染色加深,胞核固缩;IR+HRW组大鼠海马细胞数量轻度减少,排列整齐,未见明显的胞核深染及固缩现象(图4)。
2.4 脑组织中炎症因子、氧化应激指标变化
与IR组相比,IR+HRW组大鼠脑组织中MDA、羟自由基水平降低(P<0.05),GSH含量有升高、IL-1β浓度(质量浓度)有降低的趋势,但差异尚无统计学意义(P>0.05)(图5)。
图 5 照射后不同组别大鼠炎症因子、氧化应激指标变化(n=5)A:GSH;B:MDA;C:IL-1β;D:羟自由基。**:P<0.01,***:P<0.001,****:P<0.0001。Figure 5. Changes in inflammatory factors and oxidative stress indexes of rats in different groups after irradiation (n=5)A: GSH; B: MDA; C: IL-1β; D: Hydroxyl radical. **: P<0.01, ***: P<0.001, ****: P<0.0001.2.5 海马中TLR4、NF-κB、NLRP3、Caspase 1 mRNA表达变化
qRT-PCR结果显示,与IR组相比,IR+HRW组大鼠海马中TLR4、Caspase 1 mRNA表达水平下降(P<0.05),NF-κB、NLRP3 mRNA表达水平有下降的趋势,但差异无统计学意义(P>0.05)(图6)。
2.6 海马中TLR4、NF-κB p65、NLRP3、Caspase 1蛋白表达变化
Western blotting结果显示,与IR组相比,IR+HRW组大鼠海马中TLR4、Caspase 1蛋白表达水平下降(P<0.05),NF-κB p65、NLRP3蛋白表达水平有下降的趋势,但差异尚无统计学意义(P>0.05)(图7)。
3. 讨论
放射治疗是治疗原发性和继发性脑肿瘤最有效的方法之一。大脑曾被认为是具有辐射抗性的组织器官,但现在已证实大脑是放疗敏感器官之一,部分和全脑放射治疗的标准治疗方法也可引起不同程度脑组织损伤[21-22]。脑肿瘤患者在放疗后一般预后较好,超过一半的患者可以存活6个月以上。然而,其中50%~90%的患者会伴随有放射性脑损伤,特别是认知改变[23],主要表现为学习、记忆和空间信息处理能力受损[3],严重影响患者的生存质量。本实验Morris水迷宫实验结果发现与IR组比较,IR+HRW组大鼠逃避潜伏期缩短,在第6天撤去平台后到达平台原来位置所用时间减少,平台穿越次数和在平台原来位置停留时间增加,提示富氢水对辐射导致的大鼠认知功能损伤有一定的缓解作用。通过ELISA检测脑组织中GSH、MDA、羟自由基等水平发现富氢水可以防止组织过氧化,减轻氧化应激程度,减少氧化损伤。
Dong等[24]通过实验得出大黄素甲醚通过抑制TLR4/NF-κB通路保护神经元细胞,使其免受脑缺血再灌注损伤。霍瑞卿等[25]研究得出化浊解毒活血通络方具有改善脑缺血再灌注损伤的作用,其机制可能与降低脂多糖含量,调控TLR4/NF-κB信号通路有关。He等[26]研究得出预防性电针可减轻空间学习记忆的损伤,其机制可能与抑制TLR4/NF-κB信号通路有关。本研究使用qRT-PCR和Western blotting进行检测,发现照射后大鼠海马中TLR4、NF-κB、NLRP3、Caspase 1的mRNA和蛋白表达上调或有上调的趋势,富氢水干预后上述指标表达下调或呈下调的趋势,提示电离辐射可能通过TLR4/NF-κB介导的细胞焦亡参与放射性脑损伤的发生发展,同时也提示富氢水可通过干预TLR4/NF-κB信号通路改善电离辐射引起的认知功能损伤。
本研究有一定的局限性。其他一些炎性小体如NLRP1b、黑素瘤缺乏因子2、NOD样受体家族凋亡抑制蛋白、NLRP6、NLRP9b也有报道可引发细胞焦亡[27],但本研究仅关注NLRP3介导的焦亡。因此,将在后续研究中探索更多电离辐射所致脑细胞焦亡的可能机制。
本研究证实电离辐射可能通过TLR4/NF-κB信号通路引起脑细胞焦亡,进而损伤认知功能,而富氢水可能通过干预TLR4/NF-κB信号通路减轻电离辐射所致脑损伤,改善认知功能,其机制与抑制炎症因子的表达,减弱氧化应激有关。
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图 3 照射后不同组别大鼠Morris水迷宫实验结果(n=5)
A:首次到达原平台位置所用时间;B:穿越平台次数;C:原平台象限停留时间。*:P<0.05,**:P<0.01。
Figure 3. Morris water maze experiment results of rats in different groups after irradiation (n=5)
A: First arrival time at the original platform; B: Platform crossing times; C: Time to stay in the original platform quadrant. *: P<0.05, **: P<0.01.
图 5 照射后不同组别大鼠炎症因子、氧化应激指标变化(n=5)
A:GSH;B:MDA;C:IL-1β;D:羟自由基。**:P<0.01,***:P<0.001,****:P<0.0001。
Figure 5. Changes in inflammatory factors and oxidative stress indexes of rats in different groups after irradiation (n=5)
A: GSH; B: MDA; C: IL-1β; D: Hydroxyl radical. **: P<0.01, ***: P<0.001, ****: P<0.0001.
表 1 引物序列(5'→3')
Table 1 Sequence of primers (5'→3')
基因名称(Gene) 正向引物(Forward prime) 反向引物(Reverse primer) TLR4 TATCCAGAGCCGTTGGTGTA CCCACTCGAGGTAGGTGTTT NF-κB CCACTGTCAACAGCAGATGG TTCTTCTCACTGGAGGCACC NLRP3 TCCTGCAGAGCCTACAGTTG TCAGCTCAGGCTTTTCCTCC Caspase 1 AACACATTGAAGTGCCCAAGC ACTCCTTGTTTCTCTCCACGG β-actin CGTTGACATCCGTAAAGACCTC TAGGAGCCAGGGCAGTAATCT -
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