Species Differences of Bakuchiol-induced Liver Injury in Mice Based on Transcriptomics

Qian-jun KANG; Can TU; Zhao-juan GUO; Pin LI; Bing-qian JIANG; Ting WANG; Jing-xuan ZHANG

doi:10.13422/j.cnki.syfjx.20220429

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Species Differences of Bakuchiol-induced Liver Injury in Mice Based on Transcriptomics

更新时间：2022-02-07

- Species Differences of Bakuchiol-induced Liver Injury in Mice Based on Transcriptomics
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 5, Pages: 77-85(2022)
- 作者机构：
  
  北京中医药大学北京中医药研究院，国家药品监督管理局中医药研究与评价重点实验室，北京 100029
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220429
  CLC： R22;R242;R2-031;R285.5
- Received：13 October 2021，
  
  Published Online：13 December 2021，
  
  Published：05 March 2022
- 稿件说明：
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康倩君,涂灿,郭兆娟等.基于转录组学的补骨脂酚致小鼠肝损伤种属差异[J].中国实验方剂学杂志,2022,28(05):77-85.

KANG Qian-jun,TU Can,GUO Zhao-juan,et al.Species Differences of Bakuchiol-induced Liver Injury in Mice Based on Transcriptomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(05):77-85.
康倩君,涂灿,郭兆娟等.基于转录组学的补骨脂酚致小鼠肝损伤种属差异[J].中国实验方剂学杂志,2022,28(05):77-85. DOI： 10.13422/j.cnki.syfjx.20220429.

KANG Qian-jun,TU Can,GUO Zhao-juan,et al.Species Differences of Bakuchiol-induced Liver Injury in Mice Based on Transcriptomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(05):77-85. DOI： 10.13422/j.cnki.syfjx.20220429.

摘要

目的

探究补骨脂酚在Institute of Cancer Research（ICR）小鼠和昆明（KM）小鼠不同种属之间的肝毒性应答差异。

方法

通过急性毒性和亚急性毒性动物实验证实补骨脂酚致小鼠肝毒性的客观表现，运用转录组学比较正常ICR小鼠和KM小鼠2种不同种属小鼠间的差异基因富集通路，在此基础上采用实时荧光定量聚合酶链式反应（Real-time PCR）验证相关通路上关键基因的mRNA表达证实补骨脂酚致肝损伤的种属差异。

结果

补骨脂酚亚急性毒性实验，与正常组比较，ICR小鼠血清碱性磷酸酶（ALP），5′-核苷酸酶（5′-NT）含量均有不同程度升高，差异无统计学意义，KM小鼠无明显变化；病理结果显示ICR小鼠以肝细胞肥大为主要病理特征，KM小鼠主要表现为肝细胞脂肪变性。补骨脂酚急性毒性实验，给药3 d，KM小鼠出现竖毛，精神萎靡，濒死，ICR小鼠无明显毒性表现。与正常组比较，KM小鼠（400 mg·kg

-1

）血清丙氨酸氨基转移酶（ALT），天门冬氨酸氨基转移酶（AST）显著升高（

0.01），肝脏总活性氧（SOD）酶活力显著降低（

0.01）；ICR小鼠（400 mg·kg

-1

）血清5´-NT，胆碱酯酶（CHE）含量显著升高（

0.01）。ICR小鼠肝脑比增加20.34%，KM小鼠增加29.14%（

0.01）。ICR小鼠肝脏病理主要表现为肝细胞肥大，KM小鼠以局灶性炎症为主，伴肝细胞肥大、肝细胞脂肪变性。转录组学京都基因和基因组数据库（KEGG）通路和Reactome通路富集分析，发现ICR小鼠和KM小鼠不同种属间差异基因表达主要涉及氧化磷酸化、胆汁分泌、胆汁酸及胆汁酸盐合成和代谢通路。与正常组比较，细胞色素P450家族成员7A1（CYP7A1）在各给药组均显著上调（

0.01），多耐药相关蛋白（MRP2）在KM小鼠各给药组均显著降低（

0.01），在ICR小鼠各给药组显著升高（

0.01）；胆汁酸盐输出泵（BSEP）在ICR小鼠急性肝损伤（400 mg·kg

-1

）组明显降低（

0.05）；SHP1在KM小鼠急性肝损伤（400 mg·kg

-1

）组高表达，法尼醇X受体（FXR）在ICR小鼠亚急性肝损伤（200 mg·kg

-1

）组显著降低（

0.01）；SOD1，过氧化氢酶（CAT），核转录因子E

相关因子2（NFR2）在KM小鼠急性肝损伤（400 mg·kg

-1

）组显著降低（

0.01），CAT在KM小鼠亚急性肝损伤（200 mg·kg

-1

）组显著降低（

0.01）；谷胱甘肽巯基转移酶1（GSTA1），谷胱甘肽过氧化物酶1（GPX1）在KM小鼠急性肝损伤（400 mg·kg

-1

）组显著增强（

0.01），SOD1，CAT，NRF2，GSTA1在ICR小鼠亚急性肝损伤（200 mg·kg

-1

）组显著升高（

0.01），CAT，NRF2在ICR小鼠急性肝损伤（400 mg·kg

-1

）组显著升高（

0.01）。

结论

随着补骨脂酚给药剂量增加，KM小鼠氧化应激所致肝损伤逐渐加重；ICR小鼠表现出更强的抗氧化能力。通过比较2种小鼠对补骨脂酚的毒性应答反应，关于研究补骨脂酚所致小鼠肝脏肝毒性机制，ICR小鼠更适合研究胆汁分泌及胆汁酸代谢相关的机制，KM小鼠更易受氧化应激的影响而发生肝损伤。

Abstract

Objective

To explore the differences in response to bakuchiol-induced hepatotoxicity between Institute of Cancer Research （ICR） mice and Kunming （KM） mice.

Method

The objective manifestations of bakuchiol-induced hepatotoxicity in mice were confirmed by acute and subacute toxicity animal experiments， and enrichment pathways of differential genes between normal ICR mice and KM mice were compared by transcriptomics. The real-time quantitative polymerase chain reaction （real-time qPCR） assay was used to verify the mRNA expression of key genes in the related pathways to confirm the species differences of bakuchiol-induced liver injury.

Result

In the subacute toxicity experiment， compared with the normal mice， the ICR mice showed increased serum content of alkaline phosphatase （ALP）， and 5′-nucleotidase （5′-NT）， without significant difference， and no manifest change was observed in KM mice. Pathological results showed that hepatocyte hypertrophy was the main pathological feature in ICR mice and hepatocyte steatosis in KM mice. In the acute toxicity experiment， KM mice showed erect hair， mental malaise， and near-death 3 days after administration. The levels of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in KM mice （400 mg·kg

-1

） significantly increased（

0.01）， and the activity of total reactive oxygen species （SOD） in liver significantly decreased（

0.01）compared with those in normal mice， while the serum content of 5′-NT and cholinesterase （CHE） in ICR mice （400 mg·kg

-1

） were significantly elevated （

0.01）. The liver/brain ratio in ICR mice increased by 20.34% and that in KM mice increased by 29.14% （

0.01）. The main pathological manifestation of the liver in ICR mice was hepatocyte hypertrophy， while those in KM mice were focal inflammation， hepatocyte hypertrophy， and hepatocyte steatosis. Kyoto Encyclopedia of Genes and Genomes（KEGG）and Reactome pathway enrichment analyses showed that the differential gene expression between ICR mice and KM mice was mainly involved in oxidative phosphorylation， bile secretion， bile acid and bile salts synthesis， and metabolism pathway. CYP7A1 was up-regulated in all groups with drug intervention （

0.01） and MRP2 was reduced in all groups with drug intervention of KM mice （

0.01） and elevated in all groups with drug intervention of ICR mice （

0.01） compared with those in the normal group. The expression of BSEP was lowered in ICR mice with acute liver injury （400 mg·kg

-1

）（

0.05）. SHP1 was highly expressed in KM mice with acute liver injury （400 mg·kg

-1

）. The expression of FXR was diminished in ICR mice with subacute liver injury （200 mg·kg

-1

）（

0.01）. SOD1， CAT， and NFR2 significantly decreased in KM mice with acute liver injury （400 mg·kg

-1

）， and CAT dwindled in KM mice with subacute liver injury （200 mg·kg

-1

）（

0.01）. GSTA1 and GPX1 significantly increased in KM mice with acute liver injury （400 mg·kg

-1

）（

0.01） and SOD1， CAT， NRF2， and GSTA1 significantly increased in ICR mice with subacute liver injury （200 mg·kg

-1

）（

0.01）. CAT and NRF2 significantly increased in ICR mice with acute liver injury （400 mg·kg

-1

）（

0.01）.

Conclusion

With the increase in the dosage of bakuchiol， the liver injury induced by oxidative stress in KM mice was gradually aggravated， and ICR mice showed stronger antioxidant capacity. The comparison of responses to bakuchiol-induced hepatotoxicity between ICR mice and KM mice reveals that ICR mice are more suitable for the investigation of the mechanisms related to bile secretion and bile acid metabolism in the research on bakuchiol-induced hepatotoxicity in mice. KM mice are more prone to liver injury caused by oxidative stress.

关键词

Keywords

references

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