Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 5: 3323 - 3332

DOI: https://doi.org/10.12418/CJAN2023.308

RESEARCH PAPER

Effects of Flavonoids from Ulmus pumila Fruits on Antioxidant Enzyme Activities and Related Gene Expression in Chronic Sleep Deprivation Mice

  • LI Xuehan , 1 ,
  • XU Xingjun , 1, 2, * ,
  • LI Haoyu 1 ,
  • ZHANG Zepeng 1 ,
  • ZHANG Weiwei 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, Qiqihar University, Qiqihaer 161006, China
*professor, E-mail:

Received date: 2022-07-27

  Online published: 2023-05-11

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Abstract

In order to explore the antioxidant effect of flavonoids from Ulmus pumila fruits on chronic sleep deprivation mice, forty-two 4-week-old specific pathogen free (SPF) male ICR mice were randomly divided into 7 groups according to body mass, they were blank control group, model control group, carboxymethylcellulose sodium (CMC-Na) control group, positive control group, and flavone high-, medium- and low-concentration groups. There was no significant difference in body mass among the groups (P>0.05). Except the blank control group, the other 6 groups of mice were used to establish chronic sleep deprivation model, and the chronic sleep deprivation model was established by using the modified multi water platform sleep deprivation method. After successful establishing the model, mice were gavaged for 4 weeks according to the following design: flavonoids high-, medium- and low- concentration groups were gavaged with 30, 60 and 120 mg/(kg BW·d) Ulmus pumila fruits flavone, blank control group was gavaged with the same amount of normal saline, positive control group was gavaged with 300 mg/(kg BW·d) melatonin, model control group was gavaged with the same amount of normal saline, CMC-Na control group was gavaged with the same amount of 0.5% CMC Na solution. The superoxide dismutase (SOD), glutathione peroxidase (GPX) activities, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content in liver, brain and duodenum of mice were measured by kits, and the mRNA relative expression levels of antioxidant genes SOD-1, SOD-2, GPX-1 and GPX-4 in liver, brain and duodenum of mice were measured by real-time fluorescent quantitative PCR method. The results showed that high-, medium- and low-concentrations of flavonoids from Ulmus pumila fruits could significantly increase the activities of SOD, GPX and T-AOC in brain, liver and duodenum of mice (P<0.05), significantly reduce the content of MDA in brain, liver and duodenum of mice (P<0.05), and significantly increase the mRNA relative expression levels of SOD-1, SOD-2, GPX-1 and GPX-4 in brain, liver and duodenum of mice (P<0.05). In conclusion, flavonoids from Ulmus pumila fruits can significantly improve the activities and related gene expression of antioxidant enzymes, and reduce the oxidative stress damage in chronic sleep deprivation mice.

Key words： flavonoids from Ulmus pumila fruits; sleep deprivation; antioxidant enzymes; gene expression

Cite this article

LI Xuehan , XU Xingjun , LI Haoyu , ZHANG Zepeng , ZHANG Weiwei . Effects of Flavonoids from Ulmus pumila Fruits on Antioxidant Enzyme Activities and Related Gene Expression in Chronic Sleep Deprivation Mice[J]. Chinese Journal of Animal Nutrition, 2023 , 35(5) : 3323 -3332 . DOI: 10.12418/CJAN2023.308

睡眠剥夺指由于某些自身或外部环境因素导致机体睡眠时间减少或碎片化而导致严重缺乏有效睡眠[1]。研究表明,睡眠剥夺会引起氧化应激,降低身体抗氧化防御系统的能力[2]。睡眠剥夺不仅会导致白天嗜睡、注意力不集中、记忆力丧失、思维迟钝等问题,还与糖尿病、心脑血管疾病、肾病、情绪障碍等疾病有关[3]。Besedovsky等[4]研究表明,睡眠、免疫系统状态和防御系统强度是相互关联的。Rault等[5]研究发现,健康成年人在24 h睡眠剥夺后呼吸运动输出的减少可能是由大脑皮层呼吸命令的变化和吸气耐力的显著下降引起的。Cheung等[6]研究发现,睡眠不足与DNA损伤有关,DNA损伤可能导致基因相关疾病(如肿瘤)。总之,睡眠剥夺会导致人或动物的身体损害和代谢紊乱,导致各种疾病的发生,甚至死亡。
细胞中有许多重要的内源性抗氧化酶[7],如谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)、超氧化物歧化酶(superoxide dismutase,SOD)等,可清除自由基和超氧阴离子[8]。GPX主要存在于细胞质以及线粒体中,其中的过氧化氢和脂质过氧化物可以被去除以达到抗氧化作用[9-10]。生物膜中的大多数不饱和脂肪酸受到氧自由基的攻击,产生丙二醛(malondialdehyde,MDA)。脂质过氧化水平可通过MDA含量间接反映。当机体受到氧化损伤时,体内MDA含量会升高[11]。研究表明,抗氧化相关基因的表达可通过抗氧化酶活性调控[12],而SOD及GPX活性的升高可抑制脂质过氧化反应,从而降低MDA含量[13-14]。
榆树(Ulmus pumila L.)可作为一种食用植物,并具有一定的药用价值,其多种器官如果实、叶子等有镇静神经、利尿的作用,并且可以治疗神经衰弱、失眠等疾病[15]。榆荚仁(Ulmus pumila fruits)为榆树的果实或种子,已发现其提取物如黄酮等物质具有一定的抗氧化活性[16],但国内外关于榆荚仁的研究报道尚少。黄酮化合物中的酚羟基与自由基发生抽氢反应生成较稳定的半醌式自由基,从而终止自由基链式反应,起到自由基清除及抗氧化的作用[17]。本研究通过对慢性睡眠剥夺小鼠灌胃不同剂量的榆荚仁黄酮,在酶学及分子水平探讨榆荚仁黄酮的抗氧化作用。

1 材料与方法

1.1 试验材料

试验动物:42只4周龄无特定病原体(SPF)级雄性ICR小鼠[(22.00±2.00) g],购自吉林省长春市亿斯实验动物技术有限责任公司。
仪器设备:TECAN M1000酶标仪(帝肯上海贸易有限公司)、VORTEX-5旋涡混合器(德国IKA公司)、TS-DI-20L/H实验室超纯水设备(陶氏水处理设备工程有限公司)、恒温干燥箱(天津市南郊区东泥沽铁工厂)、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)、BS124S电子天平(德国赛多利斯股份公司)、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)、WD-9403D紫外仪(北京市六一仪器厂)、TH-86-340-LA(-80 ℃)超低温冰箱(北京天地精仪科技有限公司)、TGL-16C离心机(上海安亭科学仪器厂)、Applied Biosystems实时荧光定量PCR仪(美国应用生物系统公司)、2000/2000C Nanodrop(赛默飞世尔科技公司)、7220G可见分光光度计(上海精密科学仪器有限公司)。
主要试剂:纯度为90.55%的榆荚仁黄酮,溶解在0.5%羧甲基纤维素钠(CMC-Na)溶液中;反转录试剂盒、实时荧光定量PCR试剂盒、BCA蛋白定量试剂盒以及SOD、GPX、MDA、总抗氧化能力(T-AOC)试剂盒(试剂盒均购自哈尔滨卓瑞轩科技有限公司)。

1.2 试验方法

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、CMC-Na对照组、阳性对照组、榆荚仁黄酮低浓度组(简称低酮组)、榆荚仁黄酮中浓度组(简称中酮组)、榆荚仁黄酮高浓度组(简称高酮组),每组6只,组间体质量差异不显著(P>0.05)。除空白对照组外,其他6组小鼠采用改良多水平台睡眠剥夺法[18]进行为期30 d的慢性睡眠剥夺以构建睡眠剥夺模型,每日18 h(16:00至次日10:00)进行睡眠剥夺,睡眠剥夺过程中保证昼夜节律稳定,温度适宜。
慢性睡眠剥夺模型制备成功后,每天定时灌胃4周,各组小鼠自由饮食、饮水,低、中和高酮组分别灌胃30、60和120 mg/(kg BW·d)的榆荚仁黄酮(溶于0.5 mL的0.5% CMC-Na溶液);空白对照组灌胃0.5 mL的生理盐水;阳性对照组灌胃300 mg/(kg BW·d)的褪黑素(溶于0.5 mL生理盐水);模型对照组灌胃0.5 mL的生理盐水;CMC-Na对照组灌胃0.5 mL的0.5% CMC-Na溶液。
试验期间各组小鼠自由采食和饮水,末次灌胃后禁食24 h,小鼠采用断头法处死,冰上解剖并取出大脑、肝脏、十二指肠,用预冷生理盐水冲洗后滤纸吸干,置于-80 ℃冰箱备用。

1.3 检测指标

1.3.1 大脑、肝脏、十二指肠中抗氧化指标的测定

采用试剂盒检测大脑、肝脏、十二指肠中SOD、GPX活性与T-AOC及MDA含量。

1.3.2 大脑、肝脏、十二指肠中抗氧化酶相关基因表达的测定

Trizol法提取大脑、肝脏、十二指肠中总RNA,利用反转录试剂盒将Trizol提取后的总RNA反转录成cDNA。以cDNA为模板,用实时荧光定量PCR法检测SOD-1、SOD-2、GPX-1、GPX-4的mRNA相对表达量,以三磷酸甘油醛脱氢酶(GAPDH)作内参基因。引物信息见表1,所用引物均由上海生物工程有限公司设计并合成。实时荧光定量PCR(qRT-PCR)反应体系为15 μL,具体组成成分如下:7.5 μL 2×qPCR Mix,0.75 μL上游引物(2.5 μmol/L),0.75 μL下游引物(2.5 μmol/L),2 μL cDNA,4 μL ddH2O。反应程序如下: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。循环结束后检测其熔解曲线,采用2-△△Ct法计算目的基因mRNA相对表达量。
表1 引物信息

Table 1 Primer information

目的基因Target genes 引物序列Primer sequence (5'—3')
超氧化物歧化酶-1 F:CTTCCACCATGCCATGCCAGAG
SOD-1 R:AGCAACCGACCAACAAGCAAGG
超氧化物歧化酶-2 F:TGAGGAGAGCAGCGGTCGTG
SOD-2 R:TCGGTGGCGTTGAGATTGTTCAC
谷胱甘肽过氧化物酶-1 F:TGCGGAATGCCTTGCCAACAC
GPX-1 R:AGCCAGTAATCACCAAGCCAATGC
谷胱甘肽过氧化物酶-4 F:AGAAGAAGGTGTCTGGAGGTGAGG
GPX-4 R:AACCACGCAGCCAACCATGTC

1.4 数据统计与分析

采用SPSS 22.0软件进行单因素方差分析和Duncan氏法多重比较,试验结果以平均值±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。采用Graphpad Prism 8.0软件作图。

2 结果与分析

2.1 榆荚仁黄酮对慢性睡眠剥夺小鼠体内抗氧化应激水平的影响

2.1.1 榆荚仁黄酮对慢性睡眠剥夺小鼠大脑中SOD、GPX活性与T-AOC及MDA含量的影响

由表2可知,低、中、高酮组小鼠大脑中SOD、GPX活性与T-AOC显著高于模型对照组和CMC-Na对照组(P<0.05),表明榆荚仁黄酮对慢性睡眠剥夺小鼠大脑中SOD、GPX活性与T-AOC有显著的提高作用;模型对照组小鼠大脑中SOD、GPX活性与T-AOC和CMC-Na对照组差异不显著(P>0.05),表明CMC-Na对慢性睡眠剥夺小鼠大脑中抗氧化酶活性无显著影响;低、中、高酮组小鼠大脑中MDA含量均显著低于模型对照组和CMC-Na对照组(P<0.01),表明榆荚仁黄酮对小鼠大脑中MDA含量有显著降低作用。
表2 榆荚仁黄酮对慢性睡眠剥夺小鼠大脑中SOD、GPX活性与T-AOC及MDA含量的影响

Table 2 Effects of flavonoids from Ulmus pumila fruits on SOD, GPX activities, T-AOC and MDA content in brain of chronic sleep deprivation mice

项目
Items		 超氧化歧化酶
SOD/
(U/mg prot)		 谷胱甘肽过氧化物酶
GPX/
(U/mg prot)		 丙二醛
MDA/
(nmol/mg prot)		 总抗氧化能力
T-AOC/
(U/mg prot)
空白对照组Blank control group 40.64±0.52c 7.46±0.12a 1.26±0.02e 7.84±0.08c
模型对照组Model control group 28.56±0.66d 3.26±0.17e 2.32±0.04a 5.84±0.04d
CMC-Na对照组CMC-Na control group 29.19±0.29d 3.24±0.16e 2.81±0.16a 6.09±0.02d
阳性对照组Positive control group 49.43±0.58a 5.97±0.13d 1.57±0.04c 8.86±0.10b
低酮组Flavonoids low-concentration group 47.73±0.32b 6.61±0.19c 1.67±0.06b 10.66±0.09a
中酮组Flavonoids medium-concentration group 48.32±0.30b 7.14±0.09b 1.42±0.35d 10.71±0.11a
高酮组Flavonoids high-concentration group 41.36±0.16c 6.82±0.07c 1.72±0.06b 9.94±0.07b

同列数据肩标不同字母表示差异显著(P<0.05),相同字母表示差异不显著(P>0.05)。下表同。

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

2.1.2 榆荚仁黄酮对慢性睡眠剥夺小鼠肝脏中SOD、GPX活性与T-AOC及MDA含量的影响

由表3可知,低、中、高酮组小鼠肝脏中SOD、GPX活性与T-AOC均显著高于模型对照组和CMC-Na对照组(P<0.05),表明榆荚仁黄酮对慢性睡眠剥夺小鼠肝脏中SOD、GPX活性与T-AOC有显著的提高作用;模型对照组和CMC-Na对照组小鼠肝脏中SOD、GPX活性与T-AOC差异不显著(P>0.05),表明CMC-Na对慢性睡眠剥夺小鼠肝脏中抗氧化酶活性无显著影响。低、中、高酮组小鼠肝脏中MDA含量均显著低于模型对照组和CMC-Na组(P<0.05),表明榆荚仁黄酮对慢性睡眠剥夺小鼠肝脏中MDA含量有显著降低作用。
表3 榆荚仁黄酮对慢性睡眠剥夺小鼠肝脏中SOD、GPX活性与T-AOC及MDA含量的影响

Table 3 Effects of flavonoids from Ulmus pumila fruits on SOD, GPX activities, T-AOC and MDA content in liver of chronic sleep deprivation mice

组别
Groups		 超氧化物歧化酶
SOD/
(U/mg prot)		 谷胱甘肽过氧化物酶
GPX/
(U/mg prot)		 丙二醛
MDA/
(nmol/mg prot)		 总抗氧化能力
T-AOC/
(U/mg prot)
空白对照组Blank control group 27.74±0.75e 8.01±0.03d 1.55±0.06e 3.84±0.07a
模型对照组Model control group 18.63±0.05f 5.95±0.06e 3.64±0.03a 1.93±0.06d
CMC-Na对照组CMC-Na control group 18.97±0.11f 6.03±0.07e 3.71±0.03a 2.03±0.02d
阳性对照组Positive control group 37.54±0.06c 9.92±0.07a 1.62±0.08e 3.63±0.04b
低酮组Flavonoids low-concentration group 35.28±0.33a 9.63±0.08b 1.97±0.05d 2.91±0.04a
中酮组Flavonoids medium-concentration group 39.86±0.12d 9.92±0.04a 2.07±0.03c 3.05±0.04c
高酮组Flavonoids high-concentration group 38.32±0.17b 8.60±0.45c 2.28±0.02b 3.01±0.04c

2.1.3 榆荚仁黄酮对慢性睡眠剥夺小鼠十二指肠中SOD、GPX活性与T-AOC及MDA含量的影响

由表4可知,低、中、高酮组小鼠十二指肠中SOD、GPX活性与T-AOC均显著高于模型对照组和CMC-Na对照组(P<0.05),表明榆荚仁黄酮对慢性睡眠剥夺小鼠十二指肠中SOD、GPX活性与T-AOC有显著的提高作用;模型对照组和CMC-Na对照组小鼠十二指肠中SOD、GPX活性与T-AOC差异不显著(P>0.05),表明CMC-Na对慢性睡眠剥夺小鼠十二指肠中抗氧化酶活性无显著影响。低、中、高酮组小鼠十二指肠中MDA含量均显著低于模型对照组和CMC-Na组(P<0.05),表明榆荚仁黄酮对慢性睡眠剥夺小鼠十二指肠中MDA含量有显著降低作用。
表4 榆荚仁黄酮对慢性睡眠剥夺小鼠十二指肠中SOD、GPX活性与T-AOC及MDA含量的影响

Table 4 Effects of flavonoids from Ulmus pumila fruits on SOD, GPX activities, T-AOC and MDA content in duodenum of chronic sleep deprivation mice

项目
Items		 超氧化歧化酶
SOD/
(U/mg prot)		 谷胱甘肽过氧化物酶
GPX/
(U/mg prot)		 丙二醛
MDA/
(nmol/mg prot)		 总抗氧化能力
T-AOC/
(U/mg prot)
空白对照组Blank control group 66.19±0.17b 5.09±0.06b 3.41±0.03c 2.67±0.03d
模型对照组Model control group 38.70±0.13d 3.92±0.07d 6.98±0.01a 0.97±0.02f
CMC-Na对照组CMC-Na control group 39.18±0.14d 4.01±0.02d 7.00±0.01a 0.94±0.01f
阳性对照组Positive control group 67.32±0.21a 4.93±0.06c 3.63±0.02b 2.95±0.01b
低酮组Flavonoids low-concentration group 65.10±0.59c 5.23±0.09b 2.61±0.02e 2.09±0.02e
中酮组Flavonoids medium-concentration group 67.74±0.23a 5.56±0.05b 2.48±0.02f 3.05±0.02a
高酮组Flavonoids high-concentration group 66.16±0.04b 5.17±0.09a 3.20±0.04d 2.80±0.01c

2.2 榆荚仁黄酮对慢性睡眠剥夺小鼠体内抗氧化酶相关基因表达的影响

2.2.1 榆荚仁黄酮对慢性睡眠剥夺小鼠大脑中抗氧化酶相关基因表达的影响

由图1可知,阳性对照组以及低、中、高酮组小鼠大脑中SOD-1、SOD-2、GPX-1和GPX-4 mRNA相对表达量均显著高于空白对照组、模型对照组和CMC-Na对照组(P<0.05)。
图1 榆荚仁黄酮对慢性睡眠剥夺小鼠大脑中SOD-1、SOD-2、GPX-1和GPX-4 mRNA相对表达量的影响

空白对照组:blank control group;模型对照组:model control group;CMC-Na对照组:CMC-Na control group;阳性对照组:positive control group;低酮组:flavonoids low-concentration group;中酮组:flavonoids medium-concentration group;高酮组:flavonoids high-concentration group。下图同 the same as below。
数据柱标注不同字母表示差异显著(P<0.05),相同字母表示差异不显著(P>0.05)。下图同。

Fig.1 Effects of flavonoids from Ulmus pumila fruits on mRNA relative expression levels of SOD-1, SOD-2, GPX-1 and GPX-4 in brain of chronic sleep deprivation mice

Data columns with different letters indicated significant difference (P<0.05), while with the same letters indicated no significant difference (P>0.05). The same as below.

2.2.2 榆荚仁黄酮对慢性睡眠剥夺小鼠肝脏中抗氧化酶相关基因表达的影响

由图2可知,阳性对照组以及低、中、高酮组小鼠肝脏中SOD-1、SOD-2、GPX-1和GPX-4 mRNA相对表达量均显著高于空白对照组、模型对照组和CMC-Na对照组(P<0.05)。
图2 榆荚仁黄酮对慢性睡眠剥夺小鼠肝脏中SOD-1、SOD-2、GPX-1和GPX-4 mRNA相对表达量的影响

Fig.2 Effects of flavonoids from Ulmus pumila fruits on mRNA relative expression levels of SOD-1, SOD-2, GPX-1 and GPX-4 in liver of chronic sleep deprivation mice

2.2.3 榆荚仁黄酮对慢性睡眠剥夺小鼠十二指肠中抗氧化酶相关基因表达的影响

由图3可知,阳性对照组以及低、中、高酮组小鼠十二指肠中SOD-1、SOD-2、GPX-1和GPX-4 mRNA相对表达量均显著高于模型对照组和CMC-Na对照组(P<0.05)。
图3 榆荚仁黄酮对慢性睡眠剥夺小鼠十二指肠中SOD-1、SOD-2、GPX-1和GPX-4 mRNA相对表达量的影响

Fig.3 Effects of flavonoids from Ulmus pumila fruits on mRNA relative expression levels of SOD-1, SOD-2, GPX-1 and GPX-4 in duodenum of chronic sleep deprivation mice

3 讨论

当机体受到氧化损伤时,会产生超氧阴离子自由基、过氧化氢等活性氧自由基,这些活性氧自由基可引起细胞DNA损伤和细胞毒性,从而产生氧化应激损伤[19]。自由基是正常代谢的产物,当自由基在体内的动态平衡被破坏,就会对身体造成损害并引发疾病[20]。高珍琦等[21]发现马齿苋总黄酮提取液对1,1-二苯基-2-三硝基苯肼(DPPH)自由基具有较好的清除效果。榆荚仁黄酮因酚羟基上的氢原子与过氧自由基结合生成黄酮自由基,进而与其他自由基反应,从而终止自由基链式反应[22],因此榆荚仁黄酮能够提高机体的抗氧化及清除自由基能力[23]。MDA是体内重要的脂质过氧化产物之一,故MDA的含量可反映组织的脂质过氧化状态,间接反映组织细胞受损伤的程度[24]。睡眠剥夺小鼠经榆荚仁黄酮灌胃4周后,体内MDA含量显著降低,说明其氧化损伤有所改善。Pulido-Hornedo等[25]研究发现,黄芪黄酮可以通过增加肝纤维化模型大鼠体内GPX活性,降低MDA含量,从而改善大鼠肝纤维化程度。组织细胞中SOD活性可以维持机体氧化与抗氧化系统的平衡,并清除超氧阴离子,是中枢神经系统中重要的抗氧化酶,保护机体组织细胞免受损伤[26]。Li等[27]研究发现,黄芩苷可以通过调节NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体通路改善细胞凋亡水平,显著提高SOD等抗氧化酶活性,并降低MDA含量。在本试验中,睡眠剥夺小鼠大脑、肝脏和十二指肠中SOD、GPX活性和T-AOC均显著降低且MDA含量升高,灌胃榆荚仁黄酮后,相关酶的活性均显著升高,其中中酮组的效果最明显,与上述研究结果相符。Li等[28]研究表明,何首乌多酚可显著调节体内抗氧化反应和脂质积累,表现为SOD活性增加、MDA含量降低以及相关基因mRNA表达水平的变化。Beiranvand等[29]研究表明,牛叶水醇提取物可通过降低ROS、MDA含量减轻机体氧化应激损伤。这些结果均与本试验结果一致。
da Purificação等[30]研究发现,槲皮素能够改善高血糖诱导的氧化应激引起的细胞损伤,上调SOD-1和GPX-1基因的表达。Zuo等[31]研究发现,淫羊藿苷可通过激活NF-E2相关因子2/抗氧化反应元件(Nrf2/ARE)通路,从而上调GPX和SOD的生成,提高抗氧化相关基因的表达以起到对软骨细胞的保护作用。本试验使用浓度为30、60和120 mg/(kg BW·d)的榆荚仁黄酮灌胃慢性睡眠剥夺小鼠,结果表明,慢性睡眠剥夺小鼠大脑、肝脏和十二指肠中SOD-1、SOD-2、GPX-1和GPX-4的mRNA相对表达量均显著下调,灌胃榆荚仁黄酮后,上述抗氧化酶相关基因的mRNA相对表达量均显著上升。给睡眠剥夺小鼠灌胃4周榆荚仁黄酮后,其体内SOD-1、SOD-2、GPX-1及GPX-4这4种抗氧化基因的表达上调可能是因为睡眠剥夺小鼠的氧化应激相对严重,机体自由基的攻击破坏抗氧化系统,损伤细胞,而机体的内源性抗氧化系统不足以清除自由基,榆荚仁黄酮可以清除体内的自由基,也可以与金属离子络合,减少自由基和金属离子对细胞的氧化损伤,提高SOD和GPX活性,进而提高睡眠剥夺小鼠体内SOD-1、SOD-2、GPX-1及GPX-4基因的表达,另一个原因可能是由于睡眠剥夺小鼠体内SOD、GPX活性降低,体内自由基数量增加,损伤细胞,导致细胞供能不足以及DNA、RNA转录受阻,显著降低患病小鼠体内抗氧化基因的表达量,而榆荚仁黄酮有清除体内自由基的作用,灌胃榆荚仁黄酮小鼠细胞DNA、RNA转录得到改善,从而提高睡眠剥夺小鼠体内SOD-1、SOD-2、GPX-1及GPX-4基因的表达。但睡眠剥夺小鼠体内SOD-1和SOD-2的mRNA相对表达量无明显剂量依赖规律,原因可能是SOD-1基因的表达受多种因子调控[32],但榆荚仁黄酮对相关因子的作用机制尚不明确;而GPX-1及GPX-4基因在不同器官中的表达有一定规律,其在大脑中的表达量以低酮组最高,其在肝脏中的表达量以中酮组最高,在十二指肠中则以高酮组最高,其原因可能是榆荚仁黄酮对不同器官作用效应不同。

4 结论

综上所述,榆荚仁黄酮能显著上调SOD-1、SOD-2、GPX-1及GPX-4基因在慢性睡眠剥夺小鼠大脑、肝脏、十二指肠中的表达,从而提高慢性睡眠剥夺小鼠大脑、肝脏、十二指肠中SOD、GPX活性以及T-AOC,并降低MDA含量,进而改善慢性睡眠剥夺小鼠的氧化应激状态。

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