吉林大学学报(医学版)  2017, Vol. 43 Issue (03): 572-576

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龙毅, 骆静方, 胡敏予, 陈继华, 朱柳凤, 魏巍, 乔楠, 杨丽娜
LONG Yi, LUO Jingfang, HU Minyu, CHEN Jihua, ZHU Liufeng, WEI Wei, QIAO Nan, YANG Lina
鞣花酸对CCl4诱导小鼠急性肝损伤的保护作用及其机制
Protective effect of ellagic acid on acute liver injury induced by CCl4 in mice and its mechanism
吉林大学学报(医学版), 2017, 43(03): 572-576
Journal of Jilin University (Medicine Edition), 2017, 43(03): 572-576
10.13481/j.1671-587x.20170321

文章历史

收稿日期: 2016-08-18
鞣花酸对CCl4诱导小鼠急性肝损伤的保护作用及其机制
龙毅1, 骆静方2, 胡敏予2, 陈继华2, 朱柳凤2, 魏巍2, 乔楠2, 杨丽娜2     
1. 湖南省人民医院儿童医学中心, 湖南 长沙 410005;
2. 中南大学湘雅公共卫生学院营养与食品卫生学教研室, 湖南 长沙 410078
[摘要]: 目的: 探讨鞣花酸对四氯化碳(CCl4)诱导的小鼠急性肝损伤的保护作用,阐明其可能的作用机制。 方法: 50只小鼠随机分为正常对照组,模型组,低、中和高剂量鞣花酸组;每组10只。正常对照组和模型组小鼠以1%羧甲基纤维素钠灌胃,低、中和高剂量鞣花酸组小鼠分别以160、320及480 mg·kg-1鞣花酸连续灌胃14d。模型组与各剂量鞣花酸组小鼠均腹腔注射0.1% CCl4,正常对照组小鼠腹腔注射等量植物油,16 h后观察各组小鼠体质量,计算肝脏指数;测定小鼠血清丙氨酸氨基转基转移(ALT)和天门冬氨酸氨基转移酸(AST)水平,检测肝组织匀浆中超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)和过氧化氢酶(CAT)水平。 结果: 实验前后各组小鼠体质量均无明显变化(P>0.05)。与正常对照组比较,模型组小鼠肝脏指数明显升高(P<0.05),模型组和各剂量鞣花酸组小鼠血清ALT、AST水平明显升高(P<0.05);与模型组比较,各剂量鞣花酸组小鼠肝脏指数均明显降低(P<0.05);高剂量鞣花酸组小鼠血清ALT和AST水平明显降低,肝组织匀浆中CAT水平明显升高(P<0.05);中和高剂量鞣花酸组小鼠肝组织匀浆中GSH-Px水平明显升高(P<0.05);各剂量鞣花酸组小鼠肝组织匀浆中SOD活性明显升高,MDA水平明显降低(P<0.05)。 结论: 鞣花酸对CC14诱导的小鼠急性肝损伤具有保护作用,以480 mg·kg-1剂量最为明显,其作用机制可能与抗氧化有关。
关键词: 鞣花酸    多酚    四氯化碳    肝损伤    抗氧化    
Protective effect of ellagic acid on acute liver injury induced by CCl4 in mice and its mechanism
LONG Yi1, LUO Jingfang2, HU Minyu2, CHEN Jihua2, ZHU Liufeng2, WEI Wei2, QIAO Nan2, YANG Lina2     
1. Children's Medical Center, People's Hospital, Hunan Province, Changsha 410005, China;
2. Department of Nutrition and Food Safety, XiangYa School of Public Health, Central South University, Changsha 410078, China
[Abstract]: Objective: To explore the protective effect of ellagic acid on the acute liver injury induced by carbon tetrachloride (CCl4) of the mice, and to explore its possible mechanism. Methods: A total of 50 mice were randomly divided into normal control group, model group, low, medium and high doses of ellagic acid groups (n=10). The mice in normal control group and model group were treated with 1% sodium carboxymethyl cellulose solvent by intragastic administration, and the mice in ellagic acid groups were treated with 160, 320, and 480 mg·kg-1 ellagic acid by intragastic administration, respectively. After 14 d, the mice in model group and different doses of ellagic acid groups were intraperitoneally injected with 10 mL·kg-1 0.1% CCl4, while the mice in normal control group were intraperitoneally injected with 10 mL·kg-1 plant oil. 16 h later, all the mice were sacrificed and the body weights and the liver indexes of the mice were detected; the serum levels of alanine transaminase(ALT), aspartate transaminase(AST) were detected; the activities of superoxide dismutase(SOD) and levels of GSH-Px, malonalde hyde(MDA) and catalase(CAT) in liver tissue of the mice were detected. Results: There were no significant differences of the body weights of the mice between each group before and after treatment(P > 0.05). Compared with normal control group, the liver indexes and the levels of serum ALT and AST of the mice in model group and different doses of ellagic acid groups were significantly increased (P < 0.05).Compared with model group, the liver indexes of the mice in different doses of ellagic acid groups were decreased(P < 0.05);the serum levels of ALT and AST of the mice in high dose of ellagic acid group were significantly decreased (P < 0.05), while the CAT level in liver homogenate was significantly increased (P < 0.05); the levels of GSH-Px in liver homogenate of the mice in medium and high doses of ellagic acid groups were significantly increased (P < 0.05); the activities of SOD in liver homogenate of the mice in different doses of ellagic acid groups were significantly increased (P < 0.05), and the MDA levels were significantly decreased (P < 0.05). Conclusion: The ellagic acid has the protective effect on acute chemical liver injury in the mice induced by CCl4 and the most effective dose is 480 mg·kg-1; its mechanism may be related to the anti-oxidation.
Key words: ellagic acid     polyphenol     carbon tetrachloride     liver injury     anti-oxidant    

肝脏是人体消化、吸收、代谢和生成胆汁的重要器官,易被各种毒物、药物、化合物及其代谢产物损伤,导致急性肝损伤,这是许多肝脏疾病的初始环节和共同途径[1]。目前对肝损伤的防治仍是一个全球性的严峻课题,因而从天然植物化学物中筛选抗氧化损伤的物质或成分并对其作用机制进行探讨对预防急性肝损伤具有重要意义。

鞣花酸(C14H6O8)又名并没食子酸、胡颓子酸,是一种天然多酚类植物化学物的衍生物,广泛存在于各种软果和坚果等植物组织(尤其是果皮)中[2]。研究[3-9]显示:鞣花酸具有抗氧化、抗癌、抗动脉粥样硬化、抗炎、抗菌和抗毒性等作用。研究[10-12]表明:鞣花酸所含的羟基能明显增强其抗氧化损伤的能力,在口服吸收后0.5~1.0 h内血清中鞣花酸浓度达到峰值,与其他器官比较,其在肾和肝组织中浓度最高。四氯化碳(carbon tetrachloride, CCl4)是一种亲肝毒物,广泛应用于化学性肝损伤模型的建立[13-15]。目前鞣花酸保肝护肝机制研究国内外甚少。本研究采用CCl4诱导并建立小鼠急性肝损伤模型,观察不同剂量鞣花酸对急性肝损伤的保护作用,为其应用于急性肝脏损伤的防治提供实验依据。

1 材料与方法 1.1 动物、主要试剂和仪器

50只SPF级雄性昆明小鼠,体质量18~22g,购自中南大学湘雅医学院动物实验中心,动物许可证号:SCXK(湘)2009-0012。鞣花酸(纯度为90%)为陕西锦泰生物工程有限公司产品,CCl4为河南金海化工产品,超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)测定试剂盒为南京建成生物工程研究所产品。全自动生化分析仪(AU2700) 为日本奥林巴斯公司产品,酶标仪(Power Waves XSC2) 为美国MD公司产品。

1.2 动物分组和模型制备

50只小鼠按体质量分成正常对照组,模型组,低、中、高剂量鞣花酸组,每组10只。各组小鼠均灌胃给药,正常对照组和模型组小鼠给予羧甲基纤维素钠溶液,低、中和高剂量鞣花酸组小鼠分别给予160、320和480 mg·kg-1鞣花酸,每日1次,连续14d。末次灌胃后1h,除正常对照组外,其余各组小鼠均腹腔注射0.1% CC14 (10 mL·kg-1),正常对照组小鼠腹腔注射等量1%植物油,禁食不禁水,16 h后摘除眼球取血,颈椎脱臼法处死动物,解剖,取小鼠肝组织,称质量。实验过程中,小鼠定期称质量,观察其质量改变情况,并记录其进食量和饮水量,计算肝脏指数,肝脏指数=肝质量/体质量。

1.3 各组小鼠肝组织匀浆中SOD活性、MDA、GSH-Px和CAT水平及血清ALT和AST水平的测定

各组小鼠摘除眼球法取血,采集血样,3 000 r·min-1离心10 min,将血清分离,采用全自动生化分析仪测定血清ALT和AST水平。取相应各组小鼠肝脏称质量,以脏器质量:生理盐水=1:9的体积比取冰冷的生理盐水,充分研磨得到10%组织匀浆,3 000 r·min-1离心10 min,用移液器取离心后的上清液,按试剂盒说明书测定血清ALT和AST水平、肝组织匀浆中SOD活性、GSH-Px、MDA和CAT水平。

1.4 统计学分析

采用SPSS 18.0统计软件进行统计学分析。各组小鼠体质量、肝脏指数、血清ALT和AST水平、肝组织匀浆中SOD活性和MDA、GSH-Px及CAT水平均以x±s表示,组间均数比较采用单因素方差分析。以α=0.05为检验水准。

2 结果 2.1 各组小鼠体质量和肝脏指数

实验前,各组小鼠饮食量和饮水量基本一致,无明显差异。与实验前比较,实验后各组小鼠体质量均无明显变化(P>0.05)。见表 1。与正常对照组比较,模型组小鼠肝脏指数明显升高(P<0.05);与模型组比较,各剂量鞣花酸组小鼠肝脏指数均明显降低(P<0.05)。见表 1

表 1 各组小鼠体质量和肝脏指数 Table 1 Body weights and liver indexes of mice in various groups
(n=10,x±s)
Group Body weight(m/g) Liver index
Before treatment After treatment
Normal control 23.49±1.44 27.48±4.00 4.685±0.416
Model 23.93±1.88 29.64±4.46 5.214±0.455*
Ellagic acid
  Low dose 23.55±1.07 29.19±2.37 4.658±0.30
  Medium dose 24.27±1.76 28.58±2.78 4.572±0.441
  High dose 24.44±0.87 28.10±3.44 4.363±0.542
* P < 0.05 compared with normal control group; P < 0.05 compared with model group.
2.2 各组小鼠血清ALT和AST水平

与正常对照组比较,模型组和低、中及高剂量鞣花酸组小鼠血清ALT和AST水平明显升高(P<0.05), 说明造模成功。高剂量鞣花酸组小鼠血清ALT和AST水平均明显低于模型组(P<0.05)。见表 2

表 2 各组小鼠血清ALT和AST水平 Table 2 Levels of serum ALT and AST of mice in various groups
[n=10,x±sB/(U·L-1)]
Group ALT AST
Normal control 42.57±13.59 57.66±26.68
Model 250.39±65.66* 316.81±88.97*
Ellagic acid
  Low dose 251.16±65.40* 252.61±76.60*
  Medium dose 208.30±84.42* 196.34±87.43*
  High dose 155.86±53.94*△ 175.34±81.10*△
* P < 0.05 compared with normal control group; P < 0.05 compared with model group.
2.3 各组小鼠肝组织匀浆中SOD活性和MDA、GSH-Px及CAT水平

与正常对照组比较,模型组和低剂量鞣花酸组小鼠肝组织匀浆中SOD活性明显降低,MDA水平明显升高(P<0.05);低、中和高剂量鞣花酸组小鼠肝组织匀浆中SOD活性均明显高于模型组,MDA水平明显低于模型组(P<0.05)。与正常对照组比较,模型组小鼠肝组织匀浆中GSH-Px和CAT水平明显降低(P<0.01);与模型组比较,中和高剂量鞣花酸组小鼠肝组织匀浆中GSH-Px水平明显升高(P<0.01);高剂量鞣花酸组小鼠肝组织匀浆中CAT水平明显升高(P<0.05)。见表 3

表 3 各组小鼠肝组织匀浆中SOD活性和MDA、GSH-Px及CAT水平 Table 3 Activities of SOD and levels of MDA, GSH-Px, CAT in liver homogenate of mice in various groups
[n=10,x±sB/(U·mg-1)]
Group SOD MDA GSH-Px CAT
Normal control 145.60±20.01 8.52±1.18 796.80±28.71 127.30±10.34
Model 108.01±13.54* 11.70±0.91* 581.30±48.37* 106.10±8.74*
Ellagic acid
  Low dose 112.87±18.65*△ 0.06±1.35*△ 618.00±28.33* 105.00±9.55*
  Medium dose 126.30±9.33 9.14±1.23 650.80±61.62**△△ 112.60±8.25*
  High dose 136.95±14.96 8.92±0.94 679.50±49.22**△△ 117.40±12.26*△
* P < 0.05,** P < 0.01 compared with normal control group; P < 0.05,△△ P < 0.01 compared with model group.
3 讨论

肝脏是各种外来物质和有毒代谢产物转化的最终场所,一些有毒物质及其代谢产物可引起肝脏各种急性或慢性损伤,导致肝细胞坏死、凋亡和肝组织纤维化等病理改变[5]。CCl4是一种能通过氧化损伤、炎症反应、脂肪变性和纤维化等作用引起急性肝损伤的化合物,其可在动物体内部分重现人类肝病模式。肝细胞膜结构和功能损伤会引起一些肝细胞酶类(如AST和ALT等)进入血液。血清转氨酶(主要是ALT和AST)水平是检验肝损伤程度的敏感和特异性指标。本研究结果显示:与正常对照组比较,模型组小鼠血清ALT和AST水平升高,说明CCl4诱导的肝损伤模型制备成功。

目前认为CCl4致肝损伤的作用机制是肝细胞膜的脂质过氧化以及细胞炎症反应,而这些导致的肝损伤与自由基的产生增加或清除率降低有关[17]。体内有一系列酶性抗氧化物,主要包括SOD、GSH-Px和CAT等。SOD是人体重要的抗氧化酶和氧自由基清除剂,存在于有氧代谢的细胞中,构成体内第一道抗氧化酶防御体系,平衡了机体的氧化和抗氧化反应,其主要功能是清除O2-[18]。临床研究[19]表明:肝损伤程度高的患者,体内的SOD活性明显降低。GSH-Px和CAT能催化H2O2分解为水和氧,能阻断脂质过氧化的过程,从而达到抗氧化的作用。研究[20]表明:在氧化应激的作用下,GSH大量消耗,体内GSH-Px的活性明显下降。SOD、GSH-Px和CAT水平间接反映机体消除氧自由基的能力。MDA为脂质过氧化反应中分解的终产物之一,可间接反映体内脂质过氧化作用的情况和肝损伤程度[21-23]。本研究结果表明:鞣花酸能明显降低CC14所致急性肝损伤小鼠肝组织匀浆中MDA水平,提高SOD活性和GSH-Px及CAT水平,提示鞣花酸对急性化学性肝损伤的保护作用可能是通过抗自由基和抑制脂质过氧化机制发挥作用的,但其具体机制有待继续探讨。

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