动物营养学报 >

2023 , Vol. 35 >Issue 7: 4668 - 4677

DOI: https://doi.org/10.12418/CJAN2023.433

研究论文

罗汉果总黄酮对慢性睡眠剥夺小鼠抗氧化能力及炎症反应的影响

  • 李浩雨 , 1 ,
  • 徐兴军 , 1, 2, * ,
  • 李雪涵 1 ,
  • 张伟伟 1, 2 ,
  • 邵淑丽 1, 2
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  • 1 齐齐哈尔大学生命科学与农林学院,齐齐哈尔 161006
  • 2 抗性基因工程与寒地生物多样性保护黑龙江省重点实验室,齐齐哈尔 161006
* 徐兴军,教授,硕士生导师,E-mail:

李浩雨(1998—),女,黑龙江齐齐哈尔人,硕士研究生,从事动物生理生态学研究。E-mail:

Office editor: 菅景颖

收稿日期: 2023-01-19

  网络出版日期: 2023-07-11

基金资助

黑龙江省省属本科高校基本科研业务费面上项目(135509133)

收起

Effects of Total Flavonoids of Siraitia grosvenorii on Antioxidant Ability and Inflammatory Responses in Mice with Chronic Sleep Deprivation

  • LI Haoyu , 1 ,
  • XU Xingjun , 1, 2, * ,
  • LI Xuehan 1 ,
  • ZHANG Weiwei 1, 2 ,
  • SHAO Shuli 1, 2
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  • 1 College of Life Science and Agriculture and Forestry, Qiqihar University, Qiqihaer 161006, China
  • 2 Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas, Qiqihaer 161006, China
* professor, E-mail:

Received date: 2023-01-19

  Online published: 2023-07-11

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摘要

本试验旨在探究罗汉果总黄酮对慢性睡眠剥夺(CSD)小鼠抗氧化能力及炎症反应的影响。将42只4周龄无特定病原体(SPF)级雄性ICR小鼠按体质量随机分为7组,每组6只,组间体质量差异不显著(P>0.05)。空白对照组不进行慢性睡眠剥夺,其他6组使用改良多水平台睡眠剥夺法进行为期30 d的慢性睡眠剥夺,以构建CSD模型。造模成功后灌胃4周,其中低、中、高浓度罗汉果总黄酮组(分别简称低、中、高酮组)分别灌胃30、60、120 mg/(kg·d)的罗汉果总黄酮(溶于0.5 mL的1%DMSO溶液),空白对照组与模型对照组灌胃0.5 mL的生理盐水,阳性对照组灌胃30 mg/(kg·d)的褪黑素(溶于0.5 mL的1%DMSO溶液),二甲基亚砜(DMSO)对照组灌胃0.5 mL的1% DMSO溶液。利用相应试剂盒测定CSD小鼠血清和大脑中总抗氧化能力(T-AOC)、丙二醛(MDA)含量、血红素氧合酶-1(HO-1)活性、核因子-κB(NF-κB)和肿瘤坏死因子-α(TNF-α)含量,利用实时荧光定量PCR测定血清和大脑中核因子E2相关因子2(Nrf2)、HO-1、NF-κB、TNF-α mRNA相对表达量。结果显示:1)低、中、高酮组CSD小鼠大脑中T-AOC显著高于模型对照组(P<0.05),中、高酮组CSD小鼠大脑中HO-1活性显著高于模型对照组和DMSO对照组(P<0.05)。低、中、高酮组CSD小鼠血清中T-AOC、HO-1活性显著高于模型对照组和DMSO对照组(P<0.05),大脑和血清中MDA、NF-κB、TNF-α含量显著低于模型对照组和DMSO对照组(P<0.05)。2)低、中、高酮组CSD小鼠大脑和血清中HO-1 mRNA相对表达量显著高于模型对照组及DMSO对照组(P<0.05);中、高酮组CSD小鼠大脑和血清中Nrf2 mRNA相对表达量显著高于模型对照组及DMSO对照组(P<0.05);低、中、高酮组CSD小鼠大脑和血清中NF-κB、TNF-α mRNA相对表达量显著低于模型对照组及DMSO对照组(P<0.05)。综上所述,罗汉果总黄酮可提高CSD小鼠大脑、血清中抗氧化酶活性及相关基因mRNA相对表达量,降低大脑、血清中MDA与炎症因子含量及相关基因mRNA相对表达量,改善CSD小鼠的氧化应激及炎症反应。

关键词： 罗汉果总黄酮; 慢性睡眠剥夺; 抗氧化; 炎症因子

本文引用格式

李浩雨 , 徐兴军 , 李雪涵 , 张伟伟 , 邵淑丽 . 罗汉果总黄酮对慢性睡眠剥夺小鼠抗氧化能力及炎症反应的影响[J]. 动物营养学报, 2023 , 35(7) : 4668 -4677 . DOI: 10.12418/CJAN2023.433

Abstract

The aim of this experiment was to investigate the effects of total flavonoids of Siraitia grosvenorii on antioxidant ability and inflammatory responses in mice with chronic sleep deprivation (CSD). Forty-two 4-week-old specific-pathogen-free (SPF) grade male ICR mice were randomly divided into 7 groups of 6 mice each according to body mass, and the difference in body mass among groups was not significant (P>0.05). Blank control group did not undergo CSD, and the remaining 6 groups underwent CSD for 30 days to construct CSD model using a modified multilevel table sleep deprivation method. After successful establishing the model, mice were gavaged for 4 weeks. The low, medium and high concentrations of total flavonoids of Siraitia grosvenorii (hereinafter referred to as low-, medium- and high- flavonoids groups, respectively) were gavaged with 30, 60 and 120 mg/(kg·d) of total flavonoids of Siraitia grosvenorii (solved in 0.5 mL 1% DMSO solution), respectively, the blank control group and the model control group were both gavaged with 0.5 mL normal saline, the positive control group was gavaged with 30 mg/(kg·d) of melatonin (solved in 0.5 mL 1% DMSO solution), and the dimethyl sulfoxide (DMSO) control group was gavaged with 0.5 mL 1% DMSO solution. The total antioxidant capacity (T-AOC), malonic dialdehyde (MDA) content, heme oxygenase-1 (HO-1) activity, nuclear factor kappa-B (NF-κB) and tumour necrosis factor-α (TNF-α) contents in the serum and brain of CSD mice were measured by using the corresponding kits. The mRNA relative expression levels of nuclear factor E2 related factor 2 (Nrf2), HO-1, NF-κB and TNF-α in the serum and brain of CSD mice were determined by real-time fluorescence quantitative PCR. The results showed as follows: 1) the T-AOC in the brain of CSD mice in the low-, medium- and high- flavonoids groups was significantly higher than that in the model control group (P<0.05), the HO-1 activity in the brain of CSD mice in the medium- and high- flavonoids groups was significantly higher than that in the model control group and DMSO control group (P<0.05). The T-AOC and HO-1 activity in the serum of CSD mice in the low-, medium- and high- flavonoids groups were significantly higher than that in the model control group and DMSO control group (P<0.05), while the MDA, NF-κB and TNF-α contents in the serum and brain of CSD mice were significantly lower than that in the model control group and DMSO control group (P<0.05). 2) The mRNA relative expression level of HO-1 in the brain and serum of CSD mice in the low-, medium- and high- flavonoids groups were significantly higher than those in the model control group and DMSO control group (P<0.05); the mRNA relative expression level of Nrf2 in the brain and serum of CSD mice in the medium- and high- flavonoids groups was significantly higher than that in the model control group and DMSO control group (P<0.05); the mRNA relative expression levels of NF-κB and TNF-α in the brain and serum of CSD mice in the low-, medium- and high- flavonoids groups were significantly lower than those in the model control group and DMSO control group (P<0.05). In conclusion, the total flavonoids of Siraitia grosvenorii can increase the activities of antioxidant enzyme and the mRNA relative expression levels of related genes in the brain and serum of CSD mice, decrease the contents of MDA and inflammatory factors and the mRNA relative expression levels of related genes in the brain and serum, and then improve the oxidative stress and inflammatory responses in CSD mice.

Key words： total flavonoids of Siraitia grosvenorii; chronic sleep deprivation; antioxidant; inflammatory factors

人约有1/3的时间是在睡眠中度过的,在睡眠期间,身体的生理功能会发生许多变化,如细胞修复、激素分泌及免疫防御功能调节等[1-2],因此,保持充足的睡眠至关重要。而在现代社会,由于压力、睡前使用电子设备等因素导致褪黑激素分泌的变化,从而导致睡眠不足[3],即睡眠剥夺。睡眠剥夺分为短期的急性睡眠剥夺(acute sleep deprivation,ASD)和长期的慢性睡眠剥夺(chronic sleep deprivation,CSD),急性睡眠剥夺一般持续几小时至几十小时不等,而慢性睡眠剥夺则是指持续数天乃至数个月的连续剥夺[4]。慢性睡眠剥夺会导致氧化应激损伤以及一系列炎症反应的产生,最终引发各种疾病。Bellesi等[5]研究表明,慢性睡眠剥夺会使小胶质细胞处于持续激活的状态,促进其吞噬活动,可能使大脑受到进一步伤害。Xue等[6]研究表明,慢性睡眠剥夺激活神经胶质细胞,促进促炎细胞因子释放,抑制抗炎细胞因子释放。张晓宁[7]研究表明,慢性睡眠剥夺可诱导小鼠体内氧化应激反应产生,最终导致慢性炎症。以上研究表明,慢性睡眠剥夺严重影响动物生命健康。目前治疗睡眠障碍及缓解其损伤的常用药物为褪黑素,但褪黑素是一种内源性激素,虽可调节生物昼夜节律、保障睡眠,但长期使用的安全性并不明确,因此寻找安全有效的药物缓解或治疗慢性睡眠剥夺带来的损伤十分重要。
总抗氧化能力(total antioxidant capacity,T-AOC)是人体内各种抗氧化物质和抗氧化酶等共同构成的总的抗氧化水平,通常用来评价生物活性物质的抗氧化能力,可一定程度上反映机体清除活性氧(reactive oxygen species,ROS)的能力[8]。ROS积累过多会诱导脂质过氧化的产生,MDA是脂质过氧化的终产物,可作为反映脂质过氧化程度的指标[9]。核因子E2相关因子2(nuclear factor E2 related factor 2,Nrf2)被视为氧化应激反应的内源性主要调节因子,可协调下游抗氧化因子和Ⅱ相解毒酶的表达来对抗环境伤害和内源性应激源,以适应不同应激条件[10]。血红素氧合酶-1(heme oxygenase-1,HO-1)是一种重要的内源性抗氧化剂,可以在Nrf2的调控下通过清除羟自由基、超氧阴离子或单线态氧来抑制脂质或蛋白质的过氧化反应,发挥其抗氧化作用[11]。核因子-κB(nuclear factor κB,NF-κB)是一种多功能核转录因子,诱导细胞促炎因子[如肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α)等]及一氧化氮合酶(nitric oxide synthase,NOS)、环氧合酶-2(cyclooxygenase-2,COX-2)等的表达,导致炎症反应和细胞损伤的产生[12]。Nrf2可通过多种机制负性调控NF-κB信号通路,如Nrf2诱导细胞HO-1活性增加降低细胞内ROS水平来抑制氧化应激介导的NF-κB激活[13]。以上研究表明,Nrf2及其所调控的下游抗氧化因子在机体对抗氧化应激损伤及炎症反应产生的过程中发挥关键作用,同时Nrf2可受到节律基因和ROS的双向调节,使生物节律发生改变,进而影响睡眠[14]。因此,保证氧化还原系统内稳态对维持生物昼夜节律稳定及睡眠具有重要作用。
罗汉果(Siraitia grosvenorii)为葫芦科多年生藤本植物,主要产于我国广西壮族自治区,其果实是一种重要的药食两用材料,含多种营养元素及活性成分[15]。邵佩等[16]研究表明,罗汉果中提取的黄酮类物质具有良好的体外抗氧化作用。陈功等[17]研究表明,罗汉果黄酮增加运动大鼠供血能力,并提高氧运输能力。但应用罗汉果黄酮治疗慢性睡眠剥夺造成的氧化损伤及炎症反应的研究尚未见报道。因此,本试验使用不同浓度的罗汉果总黄酮对慢性睡眠剥夺小鼠进行灌胃,从基因及酶学等水平上探究罗汉果总黄酮能否通过调控慢性睡眠剥夺小鼠血清及大脑中T-AOC、HO-1等抗氧化因子与MDA、NF-κB、TNF-α等炎症因子及其相关基因的表达,缓解慢性睡眠剥夺对机体造成的氧化应激损伤及炎症反应。

1 材料与方法

1.1 试验材料

试验材料:罗汉果总黄酮(纯度为90%),购自成都某生物科技有限公司。
试验动物:以购自长春市亿斯实验动物技术有限责任公司的42只4周龄无特定病原体(SPF)级雄性ICR小鼠为研究对象,体质量(26.00±2.00) g。
仪器设备:TECAN M1000酶标仪(帝肯上海贸易有限公司)、恒温干燥箱(天津市南郊区东泥沽铁工厂)、TH-86-340-LA(-80 ℃)超低温冰箱(北京天地精仪科技有限公司)、VORTEX-5旋涡混合器(德国IKA公司)、BS124S电子天平(德国赛多利斯股份公司)、大功率磁力加热搅拌器(江苏省金坛市荣华仪器制造有限公司)、Applied Biosystems实时荧光定量PCR仪(美国应用生物系统公司)、TGL-16M高速台式冷冻离心机(湖南湘仪实验室仪器开发有限公司)、DK-S26数显恒温水浴锅(上海三发科学仪器有限公司)、TGL-16C离心机(上海安亭科学仪器厂)、7220G可见分光光度计(上海精密科学仪器有限公司)。
主要试剂:BCA蛋白定量试剂盒、T-AOC检测试剂盒、MDA检测试剂盒、HO-1酶联免疫吸附测定(ELISA)检测试剂盒、小鼠NF-κB ELISA检测试剂盒、小鼠TNF-α ELISA 检测试剂盒、总RNA提取试剂盒、反转录试剂盒,以上试剂盒均购自百杰斯生物公司。

1.2 试验设计

将适应性饲养1周后的42只SPF级4周龄雄性ICR小鼠按体质量随机分为7组,分别为空白对照组、模型对照组、二甲基亚砜(DMSO)对照组、阳性对照组以及低、中和高浓度罗汉果总黄酮组(分别简称为低、中和高酮组),每组6只,各组间体质量差异不显著(P>0.05)。空白对照组不进行慢性睡眠剥夺,其他6组利用改良多水平台睡眠剥夺法[18]对小鼠进行为期30 d的慢性睡眠剥夺以构建慢性睡眠剥夺模型,每日18 h(14:00至次日08:00)。
慢性睡眠剥夺30 d后,各组每天定时灌胃,低、中、高酮组分别灌胃30、60、120 mg/(kg·d)的罗汉果总黄酮(溶于0.5 mL 1% DMSO溶液),空白对照组及模型对照组灌胃0.5 mL的生理盐水,阳性对照组灌胃30 mg/(kg·d)的褪黑素(溶于0.5 mL 1% DMSO溶液),DMSO对照组灌胃0.5 mL的1% DMSO溶液,各组小鼠自由饮食和饮水,室温下连续灌胃4周。

1.3 样品制备和检测指标

1.3.1 慢性睡眠剥夺小鼠大脑和血清样品制备

末次灌胃后禁食1 d,采用摘除眼球法取血后将小鼠用断颈法处死,于冰上解剖后取出大脑。血液经离心处理后制备成血清,大脑用预冷过的生理盐水冲洗后滤纸吸干,置于-80 ℃冰箱备用。

1.3.2 慢性睡眠剥夺小鼠大脑和血清中抗氧化指标及炎症因子含量的测定

采用试剂盒法检测大脑、血清中T-AOC、HO-1活性以及MDA、NF-κB、TNF-α含量。

1.3.3 慢性睡眠剥夺小鼠大脑和血清中抗氧化酶及炎症因子相关基因表达的测定

采用相关试剂盒提取大脑和血清中总RNA,利用反转录试剂盒将总RNA反转录成cDNA。以cDNA为模板,采用实时荧光定量PCR法检测目的基因的mRNA相对表达量。PCR反应体系为15.0 μL,包括7.5 μL 2×qPCR Mix,1.5 μL 2.5 μmol/L引物,2.0 μL cDNA,4.0 μL ddH2O。反应条件:95 ℃预变性5 min,95 ℃变性30 s,60 ℃退火10 s,72 ℃延伸30 s,循环40次。以甘油醛-3-磷酸脱氢酶(GAPDH)作内参基因,采用2-△△Ct法计算相关目的基因的mRNA相对表达量。引物均由上海生物工程有限公司设计并合成,引物信息见表1。
表1 引物信息

Table 1 Primer information

基因Genes 引物序列Primer sequences (5'—3')
血红素氧合酶-1
HO-1		 F:ACCGCCTTCCTGCTCAACATTG
R:CTCTGACGAAGTGACGCCATCTG
核因子E2相关因子2
Nrf2		 F:AAGCACAGCCAGCACATTCTCC
R:TGACCAGGACTCACGGGAACTTC
核因子-κB
NF-κB		 F:TCGAGTCTCCATGCAGCTACGG
R:CGGTGGCGATCATCTGTGTCTG
肿瘤坏死因子-α
TNF-α		 F:GCGACGTGGAACTGGCAGAAG
R:GCCACAAGCAGGAATGAGAAGAGG
甘油醛-3-磷酸脱氢酶
GAPDH		 F:GGGCTCTCTGCTCCTCCCTGT
R:ACGGCCAAATCCGTTCACACC

1.4 数据统计与分析

采用Graphpad Prism 8.0及SPSS 22.0软件进行作图及统计学分析。试验结果进行单因素方差分析,采用LSD法进行组间多重比较,以平均值±标准差(mean±SD)表示。P<0.05表示差异显著,P>0.05表示差异不显著。

2 结果与分析

2.1 罗汉果总黄酮对慢性睡眠剥夺小鼠大脑中T-AOC、HO-1活性及MDA、NF-κB、TNF-α含量的影响

由表2可知,低、中、高酮组大脑中T-AOC显著低于空白对照组(P<0.05),显著高于模型对照组(P<0.05);中、高酮组大脑中HO-1活性显著低于空白对照组和阳性对照组(P<0.05),显著高于模型对照组和DMSO对照组(P<0.05);低、中、高酮组大脑中MDA、TNF-α含量显著高于空白对照组和阳性对照组(P<0.05),显著低于模型对照组和DMSO对照组(P<0.05);低、中、高酮组大脑中NF-κB含量显著高于阳性对照组(P<0.05),显著低于模型对照组和DMSO对照组(P<0.05)。上述结果说明罗汉果总黄酮能显著提高慢性睡眠剥夺小鼠大脑中T-AOC、HO-1活性,显著降低大脑中MDA、NF-κB、TNF-α含量。上述指标模型对照组与DMSO对照组均差异不显著(P>0.05),说明DMSO对慢性睡眠剥夺小鼠无毒害作用,对其大脑中T-AOC、HO-1活性及MDA、NF-κB、TNF-α含量无显著影响。
表2 罗汉果总黄酮对慢性睡眠剥夺小鼠大脑中T-AOC、HO-1活性及MDA、NF-κB、TNF-α含量的影响

Table 2 Effects of total flavonoids of Momordica grosvenori on T-AOC, HO-1 activity and MDA, NF-κB and TNF-α contents in brain of mice with chronic sleep deprivation

组别
Groups		 总抗氧化能力
T-AOC/
(nmol/g prot)		 血红素氧合酶-1
HO-1/
(U/g prot)		 丙二醛
MDA/
(nmol/mg prot)		 肿瘤坏死因子-α
TNF-α/
(pg/mg prot)		 核因子-κB
NF-κB/
(pg/mg prot)
空白对照组Blank control group 0.36±0.01a 66.56±2.09a 2.91±0.03f 661.65±4.13e 227.43±2.05c
模型对照组Model control group 0.23±0.02c 33.80±0.61ef 5.51±0.15a 1 164.40±5.78a 405.58±4.56a
阳性对照组Positive control group 0.32±0.01b 53.08±1.06b 3.35±0.07e 705.53±3.51d 193.55±5.07d
DMSO对照组DMSO control group 0.27±0.09bc 31.98±0.96ef 5.66±0.08a 1 159.48±4.61a 415.58±3.43a
低酮组Low-flavonoids group 0.34±0.04b 36.32±0.31de 4.56±0.11b 822.50±2.37b 367.96±1.67b
中酮组Medium-flavonoids group 0.35±0.02b 38.70±0.72d 3.94±0.10c 783.76±5.26c 367.56±4.35b
高酮组High-flavonoids group 0.35±0.01b 46.43±0.41c 3.62±0.09d 816.70±3.06b 231.03±3.80c

同列数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。下表同。

In the same column, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as below.

2.2 罗汉果总黄酮对慢性睡眠剥夺小鼠血清中T-AOC、HO-1活性及MDA、NF-κB、TNF-α含量的影响

由表3可知,低、中、高酮组及阳性对照组血清中T-AOC、HO-1活性显著高于模型对照组和DMSO对照组(P<0.05),MDA、NF-κB、TNF-α含量显著低于模型对照组和DMSO对照组(P<0.05),说明罗汉果总黄酮能显著提高慢性睡眠剥夺小鼠血清中T-AOC、HO-1活性,显著降低血清中MDA、NF-κB、TNF-α含量。模型对照组与DMSO对照组除血清中T-AOC差异显著(P<0.05)外,血清HO-1活性及MDA、NF-κB、TNF-α含量均差异不显著(P>0.05),说明DMSO对慢性睡眠剥夺小鼠无毒害作用。
表3 罗汉果总黄酮对慢性睡眠剥夺小鼠血清中T-AOC、HO-1活性及MDA、NF-κB、TNF-α含量的影响

Table 3 Effects of total flavonoids of Momordica grosvenori on T-AOC, HO-1 activity and MDA, NF-κB and TNF-α contents in serum of mice with chronic sleep deprivation

组别
Groups		 总抗氧化能力
T-AOC/
(nmol/mL)		 血红素氧合酶-1
HO-1/
(U/mL)		 丙二醛
MDA/
(nmol/mL)		 肿瘤坏死因子-α
TNF-α/
(pg/mL)		 核因子-κB
NF-κB/
(pg/mL)
空白对照组Blank control group 0.52±0.02a 69.70±2.18a 1.56±0.01f 703.15±5.91e 203.45±3.57d
模型对照组Model control group 0.43±0.01f 32.07±1.12e 5.46±0.14a 1 254.38±4.33a 476.68±4.96a
阳性对照组Positive control group 0.49±0.01b 50.48±1.06b 2.02±0.09e 826.38±5.59d 284.80±5.09c
DMSO对照组DMSO control group 0.42±0.01g 30.68±0.54e 5.49±0.21a 1 242.76±5.47a 482.50±6.45a
低酮组Low-flavonoids group 0.45±0.01e 40.07±0.50d 4.20±0.13b 990.28±4.79b 367.68±2.31b
中酮组Medium-flavonoids group 0.46±0.01d 42.10±0.80cd 3.34±0.06c 971.86±4.20b 354.61±4.49b
高酮组High-flavonoids group 0.48±0.01c 45.45±0.54c 2.73±0.16d 930.01±5.65c 353.46±6.18b

2.3 罗汉果总黄酮对慢性睡眠剥夺小鼠抗氧化酶及炎症因子相关基因表达的影响

2.3.1 罗汉果总黄酮对慢性睡眠剥夺小鼠大脑中HO-1、Nrf2、NF-κB、TNF-α mRNA相对表达量的影响

由图1可知,低、中、高酮组及阳性对照组大脑中HO-1 mRNA相对表达量显著高于模型对照组和DMSO对照组(P<0.05),NF-κB、TNF-α mRNA相对表达量显著低于模型对照组和DMSO对照组(P<0.05);中、高酮组及阳性对照组大脑中Nrf2 mRNA相对表达量显著高于模型对照组和DMSO对照组(P<0.05),低酮组大脑中Nrf2 mRNA相对表达量与模型对照组和DMSO对照组相比差异不显著(P>0.05)。而模型对照组与DMSO对照组之间上述指标均不存在显著差异(P>0.05)。
图1 罗汉果总黄酮对慢性睡眠剥夺小鼠大脑中HO-1、Nrf2、NF-κB、TNF-α mRNA相对表达量的影响

数据柱标注相同小写字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。下图同。

Fig.1 Effects of total flavonoids of Siraitia grosvenorii on relative expression levels of HO-1, Nrf2, NF-κB and TNF-α mRNA in brain of mice with chronic sleep deprivation

Value columns with the same small letter mean no significant difference (P>0.05), while with different small letters mean significant difference (P<0.05). The same as below.

2.3.2 罗汉果总黄酮对慢性睡眠剥夺小鼠血清中HO-1、Nrf2、NF-κB、TNF-α mRNA相对表达量的影响

由图2可知,低、中、高酮组及阳性对照组血清中HO-1、Nrf2 mRNA相对表达量显著高于模型对照组和DMSO对照组(P<0.05),NF-κB、TNF-α mRNA相对表达量显著低于模型对照组和DMSO对照组(P<0.05)。模型对照组与DMSO对照组之间上述指标均差异不显著(P>0.05)。
图2 罗汉果总黄酮对慢性睡眠剥夺小鼠血清中HO-1、Nrf2、NF-κB、TNF-α mRNA相对表达量的影响

Fig.2 Effects of total flavonoids of Siraitia grosvenorii on relative expression levels of HO-1, Nrf2, NF-κB and TNF-α mRNA in serum of mice with chronic sleep deprivation

3 讨论

氧化应激是ROS与抗氧化反应的失衡,而睡眠则能够促进抗氧化,抵御氧化应激产生[19]。Vaccaro等[20]研究表明,睡眠剥夺会导致ROS的积累和极大的氧化压力,造成机体的氧化应激损伤。而植物中的一些活性成分,如黄酮类物质,具有良好的抗氧化作用和抗菌、抗炎等特性[21]。Gou等[22]研究表明,甘草总黄酮可以通过缓解炎症、提高抗氧化酶的活性和减轻肝脏损伤缓解小鼠肝脏组织的氧化应激损伤。Chen等[23]研究表明,松香素通过Nrf2/HO-1通路上调相关抗氧化酶活性,从而清除ROS并改善心脏功能恶化。本试验测定了慢性睡眠剥夺小鼠大脑和血清中T-AOC、HO-1活性,结果表明罗汉果总黄酮能够显著提高慢性睡眠剥夺小鼠大脑和血清中T-AOC、HO-1活性,与模型对照组及DMSO对照组相比,阳性对照组效果最好,高酮组次之,与上述研究结果相符。HO-1作为限速保护酶,可催化血红素氧化形成一氧化碳(CO)、亚铁离子(Fe2+)以及胆绿素,并将这些物质转化为胆红素,从而在抗氧化防御系统中发挥重要作用[24]。T-AOC代表机体抗氧化物质的总抗氧化水平,它的提高代表着机体清除ROS能力的增强,可有效减轻机体氧化应激损伤[8]。罗汉果总黄酮通过提高相关抗氧化酶的活性,增强其抗氧化能力,从而减轻慢性睡眠剥夺小鼠的氧化应激损伤。
慢性睡眠剥夺会导致体内抗氧化系统失衡,造成氧化应激损伤,而Nrf2信号通路则能够在受到氧化应激损伤刺激后被激活,增强Nrf2的核转位,促使其与抗氧化原件结合,上调相关位点的抗氧化基因的表达,进而增强机体的抗氧化能力[25⇓-27]。Nrf2也被认为是调节Ⅱ相解毒酶编码基因表达的细胞保护因子,能够调节解毒酶如相关谷氨酸-半胱氨酸连接酶(GCL)、硫氧还蛋白还原酶1(Txnrd1)、NAD(P)H-苯二酚氧化还原酶1(NQO1)和HO-1等的表达,最终影响细胞的氧化状态,并针对氧化应激提供保护。植物中的黄酮类物质能够通过激活Nrf2信号通路发挥抗氧化作用。本试验结果显示,慢性睡眠剥夺小鼠大脑、血清中Nrf2、HO-1 mRNA相对表达量显著下调,使用不同浓度的罗汉果总黄酮灌胃后,慢性睡眠剥夺小鼠大脑、血清中Nrf2、HO-1 mRNA相对表达量显著上调,与模型对照组及DMSO对照组相比,阳性对照组效果最好,高酮组次之。其原因可能是罗汉果总黄酮激活了Nrf2信号通路,使Nrf2通过磷酸化和Keap1解离进入细胞核,与相关基因启动子结合并诱导下游抗氧化基因表达的调节,从而上调HO-1的表达,进而提高HO-1的活性,减轻慢性睡眠剥夺小鼠的氧化应激损伤。
Ren等[28]研究表明,漆树黄酮通过抑制Src介导的NF-κB p65和丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路减轻内毒素诱导的脓毒症小鼠肾脏炎症和细胞凋亡。Ren等[29]研究表明,金鱼草素通过阻断NF-κB信号通路和激活MAPK和Nrf2/HO-1信号通路发挥抗炎活性。NF-κB是一种快速反应因子,通过与其抑制因子核因子-κB抑制蛋白(inhibitor of nuclear factor-κB,IκB)结合,使其处于静息状态。慢性睡眠剥夺使机体产生过量的ROS,诱导脂质过氧化并产生氧化应激。ROS的过量积累会触发IκB蛋白复合体的激活,并使IκB蛋白磷酸化、泛素化、降解,导致NF-κB二聚体转位到细胞核,促进靶基因如TNF-α等的表达[12,30-31]。而Nrf2与在炎症中起关键作用的NF-κB相互作用,Nrf2可以阻止IκB蛋白复合体降解,并抑制NF-κB信号通路激活[32]。以上研究表明,植物中的黄酮类物质可能通过激活Nrf2信号通路来抑制NF-κB信号通路发挥抗炎作用。本试验结果表明,慢性睡眠剥夺小鼠大脑、血清中NF-κB、TNF-α mRNA相对表达量显著上调,MDA、NF-κB、TNF-α含量也显著提高,使用不同浓度的罗汉果总黄酮灌胃后,慢性睡眠剥夺小鼠大脑、血清中NF-κB、TNF-α mRNA相对表达量显著下调,MDA、NF-κB、TNF-α含量也显著降低,其中高酮组效果最明显,与上述研究结果一致。其原因可能是慢性睡眠剥夺使机体产生过量的ROS,导致脂质过氧化产生,使得慢性睡眠剥夺小鼠大脑和血清中MDA含量的提高;同时,ROS的过量积累促进了NF-κB核转位,导致NF-κB及其下游炎症因子TNF-α mRNA相对表达量显著上调,进而提高NF-κB、TNF-α在慢性睡眠剥夺小鼠大脑和血清中的含量;而罗汉果总黄酮通过上调抗氧化酶相关基因表达提高抗氧化酶活性,从而提高机体抗氧化能力,降低ROS含量,减轻脂质过氧化程度,降低慢性睡眠剥夺小鼠大脑及血清中MDA含量;同时,ROS含量降低抑制了IκB蛋白复合体降解,阻止了NF-κB核转位,导致慢性睡眠剥夺小鼠大脑和血清中NF-κB、TNF-α mRNA相对表达量显著下调,进而降低NF-κB、TNF-α在慢性睡眠剥夺小鼠大脑和血清中的含量。
综上所述,罗汉果总黄酮能够通过提高慢性睡眠剥夺小鼠大脑和血清中T-AOC、HO-1活性及相关基因表达,降低MDA、NF-κB、TNF-α含量及相关基因表达,缓解慢性睡眠剥夺小鼠的氧化应激损伤及炎症反应。

4 结论

罗汉果总黄酮可通过调控慢性睡眠剥夺小鼠血清及大脑中T-AOC、HO-1等抗氧化因子以及MDA和炎症因子及其相关基因表达,缓解慢性睡眠剥夺造成的氧化应激损伤及炎症反应。

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