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栀子苷对大鼠肝损伤及血清、肝脏、粪便中胆汁酸的影响
作者:
作者单位:

1.上海中医药大学 附属曙光医院,肝病研究所,肝肾疾病病证教育部重点实验室,上海市中医临床 重点实验室,上海 201203;2.上海中医药大学 中药研究所,交叉科学研究院,上海 201203;3.江西中医药大学 现代中药制剂教育部重点实验室,南昌 330004

作者简介:

刘毓,在读硕士,从事中医药防治慢性肝病的临床与基础研究,E-mail:doubing_xiangxiang@126.com

通讯作者:

刘伟,博士,副研究员,从事中药复方药效物质基础及体内过程研究,E-mail:lwhzayl@163.com

中图分类号:

R22;R242;R2-031;R285.5

基金项目:

国家自然科学基金项目(81703681);上海市科技创新行动计划项目(19401972700,20S21902600);上海市青年科技英才扬帆计划项目(17YF1419800);江西中医药大学现代中药制剂教育部重点实验室开放基金项目(TCM-202008)


Effect of Geniposide on Liver Injury and Bile Acid Profile in Serum, Liver and Excrement of Rats
Author:
Affiliation:

1.Shuguang Hospital, Institute of Liver Diseases, Key Laboratory of Liver and Kidney Disease (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine,Shanghai University of Traditional Chinese Medicine (TCM), Shanghai 201203, China;2.Institute of Chinese Materia Medica, Institute of Interdisciplinary Integrative Medicine, Shanghai University of TCM, Shanghai 201203, China;3.Key Laboratory of Modern Preparation of TCM Ministry of Education, Jiangxi University of TCM, Nanchang 330004, China

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    摘要:

    目的 探讨不同剂量栀子苷对大鼠肝脏的毒性,以及对其血清、肝组织及粪便中胆汁酸的影响。方法 60只SD大鼠,雌雄各半,按体质量随机分成空白组及栀子苷(50,100,200,400 mg·kg-1)组,共5组,每组12只,每天灌胃给药1次,连续7 d,于第8天收集大鼠血清、肝组织和盲肠内容物。检测各组大鼠血清丙氨酸氨基转移酶(ALT),门冬氨酸氨基转移酶(AST),碱性磷酸酶(ALP)活性,白蛋白(ALB),总胆红素(TBIL),总胆汁酸(TBA),肌酐(Crea),尿素(Urea)含量,肝脏组织切片分别作苏木素-伊红(HE)染色,免疫组化检测细胞角蛋白7(CK7),细胞角蛋白19(CK19)蛋白表达,蛋白免疫印迹法(Western blot)测定肝组织CK7,CK19蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测胆固醇7α-羟化酶(CYP7A1),胆固醇27α-羟化酶(CYP27A1),胆固醇12α-羟化酶(CYP8B1) mRNA表达情况,采用超高效液相色谱-串联质谱(UPLC-MS)测定血清、肝脏及盲肠内容物中18种胆汁酸含量。结果 与空白组比较,各剂量栀子苷组TBIL水平显著升高(P<0.01);400 mg·kg-1栀子苷组ALT,AST活性和TBA含量明显升高(P<0.05,P<0.01);HE染色表明,与空白组比较,栀子苷200,400 mg·kg-1组肝组织的汇管区有胆管反应,胆管周围有炎症细胞的浸润,尤以栀子苷400 mg·kg-1毒性明显。栀子苷400 mg·kg-1栀子苷组肝组织中CK7,CK19表达较空白组明显增加(P<0.05,P<0.01)。与空白组比较,400 mg·kg-1栀子苷组肝组织中甘氨熊脱氧胆酸(GUDCA),甘氨猪去氧胆酸(GHDCA)含量明显下降(P<0.05,P<0.01),牛磺鹅去氧胆酸(TCDCA),猪去氧胆酸(HDCA),胆酸(CA),鹅去氧胆酸(CDCA)含量显著升高(P<0.01),甘氨胆酸(GCA),甘氨鹅去氧胆酸(GCDCA),甘氨脱氧胆酸(GDCA),甘氨石胆酸(GLCA),牛磺熊去氧胆酸(TUDCA),GUDCA,GHDCA,熊去氧胆酸(UDCA),牛磺石胆酸(TLCA)的含量占比降低,TCDCA,HDCA,CA,CDCA,脱氧胆酸(DCA)的占比增加;血清中GHDCA,石胆酸(LCA)含量显著下降(P<0.01),牛磺猪去氧胆酸(THDCA),牛磺胆酸(TCA),GCA,TCDCA,UDCA,CA,CDCA,DCA含量明显增加(P<0.05,P<0.01),CA/DCA显著升高(P<0.01),且CA与CDCA含量占比分别上升19.60%和4.63%;与空白组比较,400 mg·kg-1栀子苷组盲肠内容物中所有的胆汁酸含量均下降,其中GCA,UDCA,HDCA,GCDCA,GDCA,TLCA,GLCA,CDCA,DCA,LCA含量明显下降(P<0.05,P<0.01)。Real-time PCR结果显示,与空白组比较,400 mg·kg-1栀子苷组肝组织中CYP7A1,CYP27A1 mRNA表达明显增加(P<0.05,P<0.01)。结论 400 mg·kg-1栀子苷灌胃,可导致大鼠明显的肝损伤,其肝脏、血清与粪便中胆汁酸发生显著改变,且同一种胆汁酸在肝脏、血清与粪便中的变化不尽相同。然而,由栀子苷所导致的肝损伤与体内胆汁酸变化间的因果关系尚不清楚,仍有待后续进一步研究给予阐明。

    Abstract:

    Objective To investigate the hepatotoxicity of different doses of geniposide on the liver of rats and the effects on bile acid profile in serum, liver tissue and feces.Method The 60 Sprague Dawley rats, half male and half female, were randomly divided into 5 groups according to body weight: blank group and four different doses (50, 100, 200, 400 mg·kg-1) geniposide groups, 12 rats in each group. The rats were treated by gavage once a day for 7 consecutive days, and the serum, liver and cecal contents were collected on the 8th day of treatment. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), the contents of albumin (ALB), total bilirubin (TBIL), total bile acid (TBA), creatinine (Crea) and carbamide (Urea) were detected in each group. The sections of liver tissue were stained with hematoxylin-eosin(HE), and the protein expressions of cytokeratin 7(CK7) and cytokeratin 19(CK19) were detected by immunohistochemistry. The protein expressions of CK7 and CK19 in the liver tissue were detected by Western blot. And the mRNA expressions of cholesterol 7α-hydroxylase (CYP7A1), cholesterol 27α-hydroxylase ( CYP27A1) and cholesterol 12α-hydroxylase (CYP8B1) were detected by real-time PCR. The contents of 18 kinds of bile acids in serum, liver and cecal contents were determined by ultra-performance liquid chromatography-mass spectrometry(UPLC-MS).Result Compared with the control group, TBIL level in each dose of geniposide group was increasesd significantly (P<0.01). ALT, AST activity and TBA content in 400 mg·kg-1 geniposide group were increased significantly (P<0.05, P<0.01). HE staining showed that, compared with control group, there was bile duct reaction in the portal area and inflammatory cells infiltrate around bile duct in 200 mg·kg-1 and 400 mg·kg-1 geniposide groups, especially 400 mg·kg-1. The expressions of CK7 and CK19 in liver tissue of 400 mg·kg-1 geniposide group were significantly higher than those in the control group (P<0.05, P<0.01). Compared with the control group, the contents of glycoursodeoxycholic acid (GUDCA) and glycohyodeoxycholic acid (GHDCA) in liver tissue of 400 mg·kg-1 geniposide group decreased significantly (P<0.05, P<0.01), the contents of sodium taurochenodeoxycholate (TCDCA), hyodeoxycholic acid (HDCA), cholic acid (CA) and chenodeoxycholic acid (CDCA) in liver tissue increased significantly (P<0.01), the contents of glycocholic acid hydrate (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid hydrate (GDCA), glycocholic acid (GLCA), tauroursodeoxycholic acid (TUDCA), GUDCA, GHDCA, ursodeoxycholic (UDCA) and taurolithocholic acid (TLCA) decreased, the proportions of TCDCA, HDCA, CA, CDCA and deoxycholic acid (DCA) in liver tissue increased, the contents of GHDCA and lithocholic acid (LCA) in serum decreased significantly (P<0.01), while sodium taurohyodeoxycholate hydrate (THDCA), taurocholic acid (TCA), GCA, TCDCA, UDCA, CA, CDCA, DCA in serum decreased significantly (P<0.05, P<0.01). The contents of CA, UDCA, CA, CDCA and DCA increased significantly (P<0.05), the ratio of CA/DCA increased significantly (P<0.05), and the ratio of CA and CDCA increased by 19.60% and 4.63%, respectively; Compared with the control group, the contents of all bile acids in cecal contents of 400 mg·kg-1 were decreased, and the contents of GCA, UDCA, HDCA, GCDCA, GDCA, TLCA, GLCA, CDCA, DCA and LCA were decreased significantly (P<0.05, P<0.01). In addition, real-time PCR results showed that the mRNA expressions of CYP7A1, CYP27A1 in the 400 mg·kg-1 geniposide group were significantly higher than those in the control group (P<0.05, P<0.01).Conclusion The 400 mg·kg-1 geniposide can cause obvious hepatotoxicity in rats, and the bile acid profile in liver, serum and excrement changes significantly, and the changes of the each bile acid in liver, serum and feces are different. However, the causal relationship between the gardenoside-induced liver injury and the changes in bile acid profile are not clear. It needs to be further studied.

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刘毓,张聪聪,胡永红,陈高峰,杨海琳,张定棋,陈佳美,刘平,刘伟,张华.栀子苷对大鼠肝损伤及血清、肝脏、粪便中胆汁酸的影响[J].中国实验方剂学杂志,2021,27(11):63~75

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  • 收稿日期:2020-11-03
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  • 在线发布日期: 2021-05-06
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