中国中药杂志

2020, v.45(12) 2916-2923

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补骨脂素通过延缓肝再生加重四氯化碳所致肝毒性的机制研究
Mechanism of psoralen in aggravating hepatotoxicity induced by CCl_4 by delaying liver regeneration

梁佩诗;周旺;江振洲;张陆勇;
LIANG Pei-shi;ZHOU Wang;JIANG Zhen-zhou;ZHANG Lu-yong;School of Traditional Chinese Medicine, Guangdong Pharmaceutical University;Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University;

摘要(Abstract):

研究补骨脂素(psoralen,PSO)是否通过诱导肝细胞周期阻滞而延缓肝再生,从而加重四氯化碳(CCl_4)所致的肝毒性。将6~8周龄雌性C57BL/6小鼠随机分成对照组(control组)、模型组(CCl_4组)、联用组(CCl_4+PSO组)和补骨脂素组(PSO组)。给予CCl_4组和CCl_4+PSO组小鼠单次腹腔注射100μL·kg~(-1)的CCl_4建立小鼠急性肝损伤模型;CCl_4注射后12,36,60 h,给予CCl_4+PSO组和PSO组小鼠灌胃200 mg·kg~(-1)的PSO。检测CCl_4注射后36,60,84 h的血清ALT和AST水平;使用HE染色法观察肝组织病理变化;RT-qPCR法检测肝脏HGF,TGF-β,TNF-α,p53和p21的mRNA水平;Western blot法检测肝脏中细胞周期相关蛋白的表达。结果显示,与control组相比,CCl_4单次腹腔注射36 h后,CCl_4组的ALT和AST水平显著升高,在84 h出现明显回落;与CCl_4组相比,CCl_4+PSO组在36 h的ALT和AST水平与CCl_4组相当,而在60,84 h时,ALT和AST水平均显著高于CCl_4组。HE染色结果显示,CCl_4组呈现弥散性分布的炎细胞浸润,未观察到CCl_4模型经典肝损伤表现;CCl_4联用PSO可致小叶中央型坏死和大量的炎细胞浸润;PSO组仅出现弥漫性水肿,未见肝细胞坏死或炎细胞浸润。RT-qPCR结果显示,PSO显著抑制CCl_4造模后HGF和TNF-α的mRNA表达,并且上调TGF-β,p53,p21的mRNA水平。Western blot结果显示,CCl_4组和CCl_4+PSO组的PCNA表达显著升高,CCl_4+PSO组的升高幅度小于CCl_4组;CCl_4与PSO共同作用可致p27的表达显著升高。初步的机制研究表明,PSO可延缓CCl_4损伤后肝脏修复过程,这一作用主要与PSO抑制肝脏发生急性损伤后PCNA的上调,以及对一些细胞周期相关蛋白的调节有关,其中p27,p53和p21可能在其中发挥重要的作用。
This study aimed to investigate whether psoralen can aggravate hepatotoxicity induced by carbon tetrachloride(CCl_4) by inducing hepatocyte cycle arrest and delaying liver regeneration. Female C57 BL/6 mice aged 6-8 weeks were randomly divided into control group, model group(CCl_4 group), combined group(CCl_4+PSO group) and psoralen group(PSO group). CCl_4 group and CCl_4+PSO group were given CCl_4 intraperitoneally at a dose of 100 μL·kg~(-1) once; olive oil of the same volume was given to control group and PSO group intraperitoneally; 12 h, 36 h and 60 h after CCl_4 injection, PSO group and CCl_4+PSO group were administrated with PSO intragastrically at a dose of 200 mg·kg~(-1); 0.5% CMC-Na of the same volume was administrated to control group and PSO group intragastrically. The weight of mice was recorded every day. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were measured at 36 h, 60 h and 84 h after CCl_4 injection. Mice were sacrificed after collection of the last serum samples. Liver samples were collected, and liver weight was recorded. Histopathological and morphological changes of liver were observed by haematoxylin and eosin staining. The mRNA levels of HGF, TGF-β, TNF-α, p53 and p21 in liver were detected by RT-qPCR. Western blot was used to detect the levels of cell cycle-related proteins. According to the results, significant increase of serum ALT and AST and centrilobular necrosis with massive inflammatory cell infiltration were observed in CCl_4+PSO group. After PSO administration in CCl_4 model, the mRNA levels of HGF(hepatocyte growth factor) and TNF-α were reduced, while the mRNA expressions of TGF-β, p53 and p21 was up-regulated. The expression of PCNA(proliferating cell nuclear antigen) was significantly increased in CCl_4 and CCl_4+PSO group, while the relative protein level in CCl_4+PSO group was slightly lower than that in CCl_4 group. Compared with control and CCl_4 group, the expression of p27(cyclic dependent kinase inhibitor protein p27) was prominently increased in CCl_4+PSO group. These results indicated that hepatotoxicity induced by CCl_4 could be aggravated by intraperitoneal administration with PSO, and the repair process of liver could be delayed. The preliminary mechanism may be related to the inhibition of PCNA and regulation of some cell cycle-associated protein by psoralen, in which the significant up-regulation of p27, p53 and p21 may play important roles.

关键词(KeyWords): 补骨脂素;肝毒性;细胞周期阻滞;四氯化碳
psoralen;hepatotoxicity;cycle arrest;carbon tetrachloride

Abstract:

Keywords:

基金项目(Foundation): 国家“重大新药创制”科技重大专项(2015ZX09501004-002-004);; 公益性行业科研专项(201507004-002);; 新疆维吾尔自治区重点研发计划项目(2016B03044-3)

作者(Author): 梁佩诗;周旺;江振洲;张陆勇;
LIANG Pei-shi;ZHOU Wang;JIANG Zhen-zhou;ZHANG Lu-yong;School of Traditional Chinese Medicine, Guangdong Pharmaceutical University;Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University;

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