Repairing Effect of Astragalin on Liver Oxidative Damage of Mice with Chronic Sleep Deprivation

ZHANG Wentian; ZHU Kaiye; LIANG Weiqi; GONG Zhian; MIAO Changjiu; LIN Yang; ZHANG Weiwei

doi:10.12418/CJAN2023.434

Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 7: 4678 - 4685

DOI: https://doi.org/10.12418/CJAN2023.434

RESEARCH PAPER

Repairing Effect of Astragalin on Liver Oxidative Damage of Mice with Chronic Sleep Deprivation

ZHANG Wentian ^,¹ ,
ZHU Kaiye ¹ ,
LIANG Weiqi ¹ ,
GONG Zhian ¹ ,
MIAO Changjiu ¹ ,
LIN Yang ¹ ,
ZHANG Weiwei ^,¹^,²^,^*

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¹ School of Life Science and Agriculture and Forestry, Qiqihar University, Qiqihar 161006, China
² Key Laboratory of Resistant Genetic Engineering and Cold Biodiversity Conservation of Heilongjiang Province, Qiqihar 161006, China

^* professor, E-mail: zww121@163.com

Received date: 2023-02-06

Online published: 2023-07-11

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Abstract

To investigate the repairing effect of astragalin on liver oxidative damage of mice with chronic sleep deprivation, thirty-six 4-week-old specific-pathogen-free (SPF) grade C57BL/6J male mice were randomly divided into three groups, each group consisted of three replicates of four animals each. They were normal sleep group (DMSO group), sleep deprivation group (SD group), and sleep deprivation+astragalin group (SD+AG group), the DMSO group did not undergo chronic sleep deprivation, and the remaining two groups underwent chronic sleep deprivation for 21 days using modified multi water platform sleep deprivation method. Before sleep deprivation was performed daily, mice in the DMSO group and SD group were intragastrically administered with 0.1% dimethylsulfoxide (DMSO) in saline configuration at the amount of 0.04 mL, and those in the SD+AG group were intragastrically administered with the same amount of astragalin dissolved in 0.1% DMSO at 20 mg/kg BW. The feed intake of mice in each group was measured daily at 13:00 to observe the status of mice. After the last administration, the mice were deprived of water and fasted, and then were sacrificed by cervical dislocation. The heart, liver, kidney and brain were weighed, and the total antioxidant capacity (T-AOC), the activities of alanine transaminase (ALT), alkaline phosphatase (AKP) and glutathione peroxidase (GSH-Px) and the content of reduced glutathione (GSH) in the liver of the mice were measured, as well as the changes of nuclear factor E2 related factor 2 (Nrf2) and downstream genes NAD(P)H:quinoneoxidoreductase 1 (NQO1), glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM) expression in the liver were detected. The results showed as follows: compared with the DMSO group, the liver AKP and ALT activities of mice in the SD group increased by 112.03% (P<0.05) and 32.96% (P<0.05), the liver T-AOC, GSH content and GSH-Px activity reduced by 8.33% (P<0.05), 39.79% (P<0.001) and 9.00%(P<0.01), and the mRNA relative expression levels of liver Nrf2, NQO1, GCLC and GCLM decreased by 19.00% (P>0.05), 8.91% (P>0.05), 13.00% (P>0.05) and 13.00% (P>0.05), respectively; the liver AKP activity of mice in SD+AG group decreased by 45.11% (P<0.001), the liver ALT activity increased by 14.88% (P<0.001), the liver T-AOC, GSH content and GSP-Px activity increased by 47.22% (P<0.05), 70.38% (P<0.01) and 36.07% (P<0.001), the mRNA relative expression levels of liver Nrf2, NQO1 and GCLC increased by 23.00% (P<0.001), 61.39% (P<0.01) and 85.00%(P<0.01), and the liver GCLM mRNA relative expression level decreased by 19.00% (P<0.05), respectively. Compared with the SD group, the brain coefficient of mice in the SD+AG group significantly increased (P<0.05), the liver coefficient significantly decreased (P<0.01), the liver AKP and ALT activities decreased by 74.11% (P<0.001) and 13.60% (P<0.05), the liver T-AOC, GSH content and GSP-Px activity increased by 60.61% (P<0.05), 182.99% (P<0.001) and 49.53% (P<0.001), the liver Nrf2, NQO1, GCLC mRNA relative expression levels increased by 51.85% (P<0.01), 77.17% (P<0.001) and 112.64% (P<0.001), and the liver GCLM mRNA relative expression level decreased by 7.03% (P>0.05), respectively. In summary, astragalin can up-regulate the expression of Nrf2 gene and its downstream genes NQO1, GCLC and GCLM, thereby repairing chronic sleep deprivation of oxidative damage to the liver of mice.

Key words： chronic sleep deprivation; oxidative stress; liver damage; Nrf2; astragalin

Cite this article

ZHANG Wentian , ZHU Kaiye , LIANG Weiqi , GONG Zhian , MIAO Changjiu , LIN Yang , ZHANG Weiwei . Repairing Effect of Astragalin on Liver Oxidative Damage of Mice with Chronic Sleep Deprivation[J]. Chinese Journal of Animal Nutrition, 2023 , 35(7) : 4678 -4685 . DOI: 10.12418/CJAN2023.434

现代生活中各种因素影响着人们的睡眠质量,导致人们长期处于睡眠不足的状态。睡眠不足或睡眠剥夺(sleep deprivation,SD)会引起内皮功能、自主神经系统功能障碍以及炎症反应、内分泌失调和氧化应激反应等,导致体内活性氧(ROS)增多。过量的ROS会作用于膜脂、蛋白质、DNA等细胞组分,改变其结构和功能,进而对细胞产生损伤^[1]。

研究发现睡眠剥夺对小鼠的大脑有一定的损伤,导致小鼠的海马神经细胞自噬活性增强^[2-3]。张晓宁^[4]研究发现慢性睡眠剥夺会引起小鼠心肌氧化应激,同时还会破坏肝脏的抗氧化系统,早期睡眠剥夺会导致谷胱甘肽过氧化物酶(GSH-Px)活性降低,进而导致氧化应激;随着持续的慢性睡眠剥夺,肝脏的抗氧化系统进一步遭到破坏,还原型谷胱甘肽(GSH)含量及总抗氧化能力(T-AOC)也随之下降,而作为肝脏健康检测指标的谷丙转氨酶(ALT)、碱性磷酸酶(AKP)活性也会高于正常值^[5]。NAD(P)H:醌氧化还原酶1(NQO1)属于黄素酶,是一种重要的抗氧化酶,以NADPH为受体,催化ROS、辅酶Q在内的还原反应。NQO1表达受核转录相关因子2(Nrf2)-抗氧化反应元件(ARE)通路的调控,其表达量与Nrf2的表达有关,研究发现当发生氧化性肝损伤后,Nrf2及NQO1的表达显著降低^[6-7]。

紫云英苷(astragalin,AG)化学名为3,5,7,4'-四羟基黄酮-3-葡萄糖苷,是百蕊草^[8]、睡莲^[9]、荷叶^[10]、茶叶^[11]、月季^[12]等多种植物所含有的天然黄酮化合物之一,相对分子质量为448.38。常温状态下是黄色针晶状,不溶于水,溶于甲醇、乙醇等有机溶剂。研究发现紫云英苷具有抗炎、抗氧化、抗肿瘤、抗过敏、抗糖尿病、抗抑郁、抗真菌等多种作用,并且具有提高机体抵抗力、修复损伤的DNA、保护心肌和神经等作用^[13]。在本实验室的前期研究中发现,20 mg/kg的紫云英苷不会对肝细胞造成损伤,但紫云英苷对慢性睡眠剥夺导致的肝脏损伤的修复作用尚不清楚^[14]。因此,本试验以C57BL/6J雄性小鼠为研究对象,通过改良多平台睡眠剥夺法构建慢性睡眠剥夺模型,灌胃20 mg/kg的紫云英苷后检测小鼠肝脏组织氧自由基的清除能力、GSH含量和抗氧化酶的活性,同时检测肝脏抗氧化相关基因的表达情况,初步探索紫云英苷是否通过调节ALT、AKP、GSH-Px活性与GSH含量以及抗氧化相关基因Nrf2、NQO1、谷氨酸-半胱氨酸连接酶催化亚基(GCLC)和谷氨酸-半胱氨酸连接酶调节亚基(GCLM)的表达缓解慢性睡眠剥夺小鼠肝脏的氧化损伤,为治疗慢性睡眠剥夺导致的肝脏损伤提供新思路,同时为紫云英苷的开发利用提供试验依据。

1 材料与方法

1.1 试验材料

试验动物为36只4周龄无特定病原体(SPF)级C57BL/6J雄性小鼠,购自长春生物技术有限公司,试验程序遵循齐齐哈尔大学科技伦理委员会的《动物实验福利伦理审查指南》。

1.2 主要试剂

T-AOC测定试剂盒、ALT活性测定试剂盒、GSH含量测定试剂盒、AKP活性测定试剂盒购自南京建成生物工程研究所,超氧化歧化酶(SOD)活性测定试剂盒购自上海桥星贸易有限公司,蛋白质含量测定试剂盒、Trizol试剂购自美国赛默飞世尔科技有限公司,反转录试剂盒购自上海东洋纺生物科技有限公司,荧光定量PCR试剂盒购自宝生物工程(大连)有限公司。

1.3 试验仪器

Spark10M多功能酶标仪(Tecan,瑞士)、Z32HK高速台式冷冻离心机(Hermle,德国)、Thermo Electron (-80 ℃)超低温冰箱(Thermo,美国)、实时荧光定量PCR仪(Eppendorf,德国)、NanoDrop ND-2000C微量紫外分光光度计(Thermo,美国)、电子分析天平(北京赛多利斯仪器系统有限公司),Mastercycler nexus梯度PCR仪(Eppendorf,德国)。

1.4 试验方法

将36只4周龄C57BL/6J雄性小鼠实验室饲养1周,基础饲粮喂养,正常饮水,然后按照体质量将小鼠随机分成3组,即正常睡眠组(DMSO组)、睡眠剥夺组(SD组)、睡眠剥夺+紫云英苷组(SD+AG组),每组3个重复,每个重复4只,每个重复的4只放在1个笼子(27 cm×17 cm×13 cm)中。

利用改良多平台睡眠剥夺法对SD组和SD+AG组小鼠连续睡眠剥夺21 d^[15],每日睡眠剥夺18 h(17:00至次日11:00)制备慢性睡眠剥夺小鼠模型。每日睡眠剥夺前灌胃,其中DMSO组和SD组灌胃生理盐水配制的0.1% DMSO溶液,灌胃量为0.04 mL;SD+AG组灌胃20 mg/kg BW紫云英苷(将紫云英苷溶于0.04 mL的0.1%DMSO溶液中)。每日13:00测量各组小鼠的摄食量,观察小鼠状态。末次灌胃后禁水禁食,断颈法处死小鼠,取其脑、肝脏、肾脏和心脏,称其重量,将肝脏组织放入-80 ℃冰箱用于后续指标检测。

1.5 小鼠肝脏中抗氧化指标和肝脏功能指标的测定

在冰上制备肝脏组织的匀浆液,测定蛋白质浓度后,按照试剂盒说明书测定T-AOC以及SOD、AKP、ALT活性与GSH含量。

1.6 小鼠肝脏中抗氧化相关基因表达的检测

从-80 ℃冰箱取出肝脏组织,剪取一定量的肝脏组织于液氮中研磨成粉末,加入1 mL Trizol试剂提取总RNA,用NanoDrop ND-2000C微量紫外分光光度计进行定量分析。取1 μg RNA样品,按照ReverTra Ace qPCR RT Master Mix with gDNA Remover反转录试剂盒说明书进行反转录,获得cDNA。以cDNA为模板,β-肌动蛋白(β-actin)为内参基因,进行实时荧光定量PCR(qRT-PCR)。反应体系为10 μL:TB Green Premix Ex TaqⅡ 5 μL,cDNA 1.5 μL,上、下游引物各0.5 μL,ddH₂O 2.5 μL。反应条件:95 ℃预变性30 s,95 ℃ 1 min,60 ℃ 30 s,40个循环,得到Nrf2、GCLC、GCLM、β-actin mRNA的Ct值,用2^-△△Ct法计算各目的基因mRNA的相对表达量。所需引物均由上海生物工程有限公司合成,引物序列见表1^[10]。

表1 实时荧光定量PCR引物序列

Table 1 Primer sequences for qRT-PCR

基因 Genes	上游引物 Forward primers (5'—3')	下游引物 Reverse primers (5'—3')
谷氨酸-半胱氨酸连接酶调节亚基GCLM	AGTTGGAGCAGCTGTATCAGTGG	TTTAGCAAAGGCAGTCAAATCTGG
谷氨酸-半胱氨酸连接酶催化亚基GCLC	ATCTGCAAAGGCGGCAAC	ACTCCTCTGCAGCTGGCTC
NAD(P)H:醌氧化还原酶1 NQO1	GAAGAGCACTGATCGTACTGGC	GGATACTGAAAGTTCGCAGGG
核转录因子E2相关因子2 Nrf2	CTTTAGTCAGCGACAGAAGGAC	AGGCATCTTGTTTGGGAATGTG
β-肌动蛋白β-actin	ACCGTGAAAAGATGACCCAG	AGCCTGGATGGCTACGTACA

1.7 数据处理与分析

所有数据采用SPSS 22.0统计学软件进行处理,计量资料数据以平均值±标准差表示,进行t检验或方差分析,P<0.05表示差异显著,P<0.01表示差异极显著。数据通过GraphPad Prism 9.4.0软件进行作图。

2 结果与分析

2.1 紫云英苷对慢性睡眠剥夺小鼠体重及脏器系数的影响

睡眠剥夺3周后,SD组小鼠体重增加了2.04 g,DMSO组小鼠体重增加了2.30 g,SD+AG组小鼠体重增加了4.57 g。由表2可知,与DMSO组相比,SD组小鼠脑、心脏和肾脏系数下降,但差异不显著(P>0.05),肝脏系数显著升高(P<0.05);与SD组相比,SD+AG组小鼠脑(P<0.05)、心脏(P>0.05)、肾脏系数(P>0.05)有不同程度升高,而肝脏系数显著下降(P<0.05),脑、心脏、肝脏和肾脏系数均恢复到了DMSO组水平(P>0.05)。

表2 慢性睡眠剥夺小鼠脏器系数的变化

Table 2 Changes of organ coefficients of chronic sleep-deprived mice %

项目 Items	组别Groups
项目 Items	DMSO	SD	SD+AG
脑Brain	5.43±0.23	4.96±0.11	5.38±0.07^#
心脏Heart	5.38±0.12	5.14±0.13	5.32±0.21
肝脏Liver	7.32±0.08	8.72±0.02^*	7.48±0.09^#
肾脏Kidney	6.14±0.15	5.88±0.42	6.02±0.34

	“”:与DMSO组相比;“#”:与SD组相比。“、#”表示差异显著(P<0.05),“、##”表示差异极显著(P<0.01),“*、###”表示差异极显著(P<0.001)。下表同。
	“”: compared with DMSO group; “#”: compared with SD group. “, #” indicated significant difference (P<0.05), “, ##” indicated extremely significant difference (P<0.01), and “*, ###” indicated extremely significant difference (P<0.001). The same as below.

2.2 紫云英苷对慢性睡眠剥夺小鼠肝脏功能的影响

由表4可知,与DMSO组相比,SD组小鼠的肝脏AKP和ALT活性分别升高了112.03%(P<0.05)和32.96%(P<0.05);SD+AG组小鼠的肝脏AKP活性降低了45.11%(P<0.001),肝脏ALT活性升高了14.88%(P<0.001)。与SD组相比,SD+AG组小鼠肝脏AKP和ALT活性分别降低了74.11%(P<0.001)和13.60%(P<0.05)。

表3 紫云英苷对慢性睡眠剥夺小鼠肝脏AKP和ALT活性的影响

Table 3 Effects of astragalin on liver AKP and ALT activities of chronic sleep-deprived mice

项目 Items	组别Groups
项目 Items	DMSO	SD	SD+AG
碱性磷酸酶AKP/(金氏单位/g prot)	1.33±0.18	2.82±0.45^*	0.73±0.15^***,###
谷丙转氨酶ALT/(U/g prot)	21.24±0.76	28.24±1.90^*	24.40±1.10^*,#

表4 紫云英苷对慢性睡眠剥夺小鼠肝脏T-AOC、GSH-Px活性和GSH含量的影响

Table 4 Effects of astragalin on liver T-AOC, GSH-Px activity and GSH content of chronic sleep-deprived mice

项目 Items	组别Groups
项目 Items	DMSO	SD	SD+AG
总抗氧化能力T-AOC/(U/mg prot)	1.44±0.14	1.32±0.17^*	2.12±0.23^*,#
谷胱甘肽GSH/(μmol/g prot)	2 254.19±235.21	1 357.17±69.66^***	3 840.66±248.00^**,###
谷胱甘肽过氧化物酶GSH-Px/(U/mg prot)	26.67±1.07	24.27±1.17^**	36.29±2.79^***,###

2.3 紫云英苷对慢性睡眠剥夺小鼠肝脏中抗氧化指标的影响

由表4可知,与DMSO组相比,SD组肝脏T-AOC、GSH含量和GSH-Px活性分别降低了8.33%(P<0.05)、39.79%(P<0.001)和9.00%(P<0.01);SD+AG组肝脏T-AOC、GSH含量和GSH-Px活性分别升高了47.22%(P<0.05)、70.38%(P<0.01)和36.07%(P<0.001)。与SD组相比,SD+AG组肝脏T-AOC、GSH含量和GSH-Px活性分别升高了60.61%(P<0.05)、182.99%(P<0.001)和49.53%(P<0.001)。

2.4 紫云英苷对慢性睡眠剥夺小鼠肝脏中抗氧化相关酶基因表达的影响

由表5可知,与DMSO组相比,SD组肝脏中Nrf2、NQO1、GCLC和GCLM的mRNA相对表达量分别降低了19.00%(P>0.05)、8.91%(P>0.05)、13.00%(P>0.05)和13.00%(P>0.05);SD+AG组肝脏中Nrf2、NQO1和GCLC mRNA相对表达量分别升高了23.00%(P<0.001)、61.39%(P<0.01)、85.00%(P<0.01),GCLM mRNA相对表达量降低了19.00%(P<0.05)。与SD组相比,SD+AG组小鼠肝脏中Nrf2、NQO1、GCLC mRNA相对表达量分别升高了51.85%(P<0.01)、77.17%(P<0.001)、112.64%(P<0.001),而GCLM mRNA相对表达量下降了7.03%(P>0.05)。

**表5 紫云英苷对慢性睡眠剥夺小鼠肝脏Nrf2、NQO1、GCLC和GCLM mRNA相对表达量的影响**

Table 5 Effects of astragalin on mRNA relative expression levels of liver Nrf2, NQO1, GCLC and GCLM of chronic sleep-deprived mice

项目 Items	组别Groups
项目 Items	DMSO	SD	SD+AG
核转录因子E2相关因子2 Nrf2	1.00±0.01	0.81±0.08	1.23±0.02^***##
NAD(P)H:醌氧化还原酶1 NQO1	1.01±0.03	0.92±0.03	1.63±0.05^**###
谷氨酸-半胱氨酸连接酶催化亚基GCLC	1.00±0.03	0.87±0.03	1.85±0.09^**###
谷氨酸-半胱氨酸连接酶调节亚基GCLM	1.00±0.01	0.87±0.05	0.81±0.02^*

3 讨论

研究表明,充足的睡眠可以使人精力充沛,同时睡眠期间也是机体进行自我修复的一个过程,当睡眠剥夺时,机体修复有限,持续睡眠不足会导致机体的多个器官产生炎症反应及氧化应激反应^[16]。肝脏作为人体最大的解毒器官,其细胞内含有大量的线粒体,线粒体是呼吸的主要场所,也是产生ROS和氧化应激的主要场所^[17]。本试验通过改良多平台睡眠剥夺法构建小鼠慢性睡眠剥夺模型,结果表明,与DMSO组相比,SD组小鼠的心脏、肾脏系数变化不大,但肝脏系数显著升高,而SD+AG组小鼠的肝脏系数与SD组相比显著下降,且与DMSO组接近,因此本试验选择肝脏组织进行后续研究。

SD会导致小鼠肝脏的抗氧化能力下降和氧化损伤,当有1%的肝细胞坏死后,ALT的活性会显著升高,所以ALT被推荐为检测肝脏损伤的敏感指标^[18],而AKP是反映肝脏损伤的经典指标,可以根据ALT和AKP活性的变化来检测肝脏的健康状态^[19]。本试验结果发现SD组小鼠的肝脏ALT、AKP活性较DMSO组升高,表明慢性睡眠剥夺已对小鼠肝脏造成损伤。

在本实验室的前期研究中,检测了不同浓度紫云英苷灌胃后C57BL/6J雄性小鼠血清中ALT活性的变化,结果表明20 mg/kg BW的紫云英苷不会对小鼠肝脏组织造成损伤^[14]。紫云英苷是黄酮类化合物的一种,可以有效清除ROS,减少自由基对细胞的攻击^[20]。本试验结果表明,与SD组相比,在睡眠剥夺过程中灌胃20 mg/kg BW紫云英苷的小鼠肝脏ALT、AKP活性均显著下降。这一结果说明20 mg/kg BW的紫云英苷对小鼠因为睡眠剥夺而造成的肝脏损伤有一定的修复作用。

T-AOC是反映机体清除ROS的总能力,主要检测肝脏中酶促体系和非酶促体系抗氧化物质抗氧化能力的总和,能够清楚地显示机体的抗氧化能力,比单个指标的抗氧化能力测定能更好地反映抗氧化状态^[21]。GSH是一种可溶性硫醇抗氧化剂,在细胞中含量丰富,主要在肝脏中合成,是细胞内抗氧化系统中重要的一员,能够持续清除自由基,让细胞免受氧化损伤,当发生氧化应激导致肝脏损伤后,大鼠肝脏T-AOC下降^[22]。本试验结果显示,SD组小鼠的肝脏T-AOC相比于DMSO组显著下降,肝脏GSH含量和GSH-Px活性显著降低,而与SD组相比,SD+AG组小鼠的肝脏T-AOC上升,肝脏GSH含量和GSH-Px活性均显著升高,说明紫云英苷可以在一定程度上修复肝脏的氧化损伤。

Nrf2是机体抗氧化应激的主要调节因子,可以预防氧化应激导致的细胞损伤。研究发现,紫云英苷可以通过调节Nrf2进而调控NQO1的表达^[23];紫云英苷可以通过Nrf2通路调控其下游基因谷氨酸-半胱氨酸连接酶(GCL)的表达^[24]。当发生氧化应激时,Nrf2的表达降低会影响GCLC和GCLM的表达,进而影响GSH和GSH-Px的表达,从而影响机体清除自由基的合成速度,导致机体产生氧化损伤^[25]。本试验结果表明,与DMSO组相比,SD组小鼠的肝脏Nrf2、NQO1、GCLC和GCLM mRNA相对表达量略有降低,与SD组相比,SD+AG组小鼠的肝脏Nrf2、NQO1和GCLC mRNA相对表达量显著升高,GCLC mRNA相对表达量的变化不明显。

综上所述,紫云英苷可上调Nrf2及其下游基因NQO1、GCLC和GCLM的表达,增强小鼠的抗氧化能力,一定程度上可修复慢性睡眠剥夺导致的小鼠肝脏氧化损伤。

4 结论

紫云英苷可通过调节肝脏中ALT、AKP、GSH-Px活性与GSH含量以及抗氧化相关基因Nrf2、NQO-1、GCLC和GCLM的表达,在一定程度上修复慢性睡眠剥夺引起的小鼠肝脏氧化损伤。

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