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Journal of Peking University (Health Sciences) ›› 2021, Vol. 53 ›› Issue (6): 1107-1114. doi: 10.19723/j.issn.1671-167X.2021.06.017

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Cytotoxicity and underlying mechanism of evodiamine in HepG2 cells

GAO Ya-dong1,ZHU An1,LI Lu-di1,ZHANG Tao1,WANG Shuo1,SHAN Dan-ping1,LI Ying-zi1,WANG Qi1,2,3,△()   

  1. 1. Department of Toxicology, Peking University School of Public Health, Beijing 100191, China
    2. Key Laboratory of State Administration of Traditional Chinese Medicine for Compatibility Toxicology, Beijing 100191, China
    3. Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, China
  • Received:2021-02-17 Online:2021-12-18 Published:2021-12-13
  • Contact: Qi WANG E-mail:wangqi@bjmu.edu.cn
  • Supported by:
    National Key Research and Development Program of China(2018YFC1704500);National Key Research and Development Program of China(2018YFC1704506)

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Abstract:

Objective: To investigate evodiamine (EVO)-induced hepatotoxicity and the underlying mechanism. Methods: HepG2 cells were treated with EVO (0.04-25 μmol/L) for different time intervals, and the cell survival rate was examined by cell counting kit-8 (CCK-8) method. After HepG2 cells were treated with EVO (0.2, 1 and 5 μmol/L) for 48 h, the alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) activities and total bilirubin (TBIL) content of supernatant were detected. A multifunctional microplate reader was used to detect the intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in HepG2 cells to evaluate the level of cell lipid peroxidation damage. The interactions between EVO and apoptosis, autophagy or ferroptosis-associated proteins were simulated by molecular docking. The HepG2 cells were stained by mitochondrial membrane potential (MMP) fluorescent probe (JC-10) and annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI), and MMP and apoptosis in HepG2 cells were detected by flow cytometry. The protein expression levels of caspase-9, caspase-3, bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were detected by Western blot. Results: The cell survival rate was significantly reduced after the HepG2 cells were exposed to EVO (0.04-25 μmol/L) in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) of the HepG2 cells treated with EVO for 24, 48 and 72 h were 85.3, 6.6 and 4.7 μmol/L, respectively. After exposure to EVO (0.2, 1 and 5 μmol/L) for 48 h, the ALT, AST, LDH, ALP activities and TBIL content in the HepG2 cell culture supernatant, and the MDA content in the cells were increased, and SOD enzyme activity was decreased. Molecular docking results showed that EVO interacted with apoptosis-associated proteins (caspase-9 and caspase-3) better. JC-10 and Annexin V-FITC/PI staining assays demonstrated that EVO could decrease MMP and promote apoptosis in the HepG2 cells. Western blot results indicated that the protein expressions of cleaved caspase-9 and cleaved caspase-3 were upregulated in the HepG2 cell treated with EVO for 48 h. In contrast, the protein expressions of pro-caspase-3, BSEP and MRP2 were downregulated. Conclusion: These results suggested that 0.2, 1 and 5 μmol/L EVO had the potential hepatotoxicity, and the possible mechanism involved lipid peroxidation damage, cell apoptosis, and cholestasis.

Key words: Evodiamine, Lipid peroxidation damage, Apoptosis, Cholestasis

CLC Number: 

  • R114

Figure 1

Effects of EVO on viability of HepG2 cells at different concentrations and time $\overline{x}$±s, n=3. # P<0.01, compared with control group at the same time. EVO, evodiamine."

Table 1

Effects of EVO on biochemical indicators in HepG2 cells for 48 h($\overline{x}±s$, n=3)"

Group ALT/(U/L) AST/(U/L) LDH/(U/L) ALP/(King’s unit/100 mL) TBIL/(μmol/L)
0 μmol/L 2.31±0.82 5.17±0.69 158.04±25.00 0.35±0.03 4.54±1.45
0.2 μmol/L 2.70±1.16 5.42±0.35 187.80±28.37* 0.34±0.02 6.09±1.66
1 μmol/L 2.61±1.17 5.43±0.84 231.92±19.92# 0.37±0.02 13.23±3.15#
5 μmol/L 5.82±1.26# 6.34±0.75# 242.11±19.72# 0.39±0.05# 16.06±3.72#

Figure 2

Effects of EVO on lipid peroxidation damage in HepG2 cells A, SOD activity; B, MDA content. $\overline{x}$±s. n=3. HepG2 cells were treated with EVO for 48h. # P<0.01, compared with control group. EVO, evodiamine; SOD, superoxide dismutase; MDA, malondialdehyde."

Table 2

Molecular interactions between EVO and apoptosis, autophagy or ferroptosis-associated proteins"

Protein PDB ID Total score H-bond number Residues involved in H-bond formation
Caspase-9 1JXQ 6.31 1 A/Lys396
Caspase-3 3H0E 5.13 1 A/Thr62
p62 6MJ7 3.01 0
Beclin-1 5EFM 2.61 0
LC3 3WAM 3.99 0
Gpx4 6HKQ 4.03 1 A/Lys48

Figure 3

Molecular interaction between EVO and human apoptosis-associated proteins A, the 3D model of caspase-9; B, the 2D model of caspase-9; C, the 3D model of caspase-3; D, the 2D model of caspase-3. EVO, evodiamine."

Figure 4

Effects of EVO on mitochondrial membrane potential in HepG2 cells $\overline{x}$±s. n=3. HepG2 cells were treated with EVO for 48 h. # P<0.01, compared with control group. EVO, evodiamine."

Figure 5

Effects of EVO on apoptosis rate in HepG2 cells A, apoptosis was evaluated by Annexin V-FITC and PI staining; B, quantitative statistical results of the proportion of apoptotic cells. $\overline{x}$±s. n=3. HepG2 cells were treated with EVO for 48h. # P<0.01 compared with control group. FITC, fluorescein isothiocyanate; PI, propidium iodide; EVO, evodiamine."

Figure 6

Effects of EVO on the protein expressions of caspase-9 and caspase-3 in HepG2 cells A, relative protein expression levels by Western blot; B, semi-quantitative analysis. $\overline{x}$±s. n=3. HepG2 cells were treated with EVO for 48 h. * P<0.05, # P<0.01, compared with control group. EVO, evodiamine."

Figure 7

Effects of EVO on the protein expressions of BSEP and MRP2 in HepG2 cells A, relative protein expression levels by Western blot; B, semi-quantitative analysis. $\overline{x}$±s. n=3. HepG2 cells were treated with EVO for 48 h. * P<0.05, # P<0.01, compared with control group. EVO, evodiamine; BSEP, bile salt export pump; MRP2, multidrug resistance-associated protein 2."

[1] 国家药典委员会. 中华人民共和国药典一部[M]. 11版. 北京: 中国医药科技出版社, 2020: 178.
[2] 刘颖, 杨润芳, 夏祺悦, 等. 吴茱萸醇提物重复给药的靶器官毒性研究[J]. 现代预防医学, 2015, 42(14):2600-2603.
[3] 黄伟, 李晓骄阳, 孙蓉. 吴茱萸水提组分多次给药对小鼠肝毒性的“量-时-毒”关系研究[J]. 中国中药杂志, 2012, 37(15):2223-2227.
[4] Wang L, Fang K, Cheng J, et al. Scaffold hopping of natural product evodiamine: discovery of a novel antitumor scaffold with excellent potency against colon cancer[J]. J Med Chem, 2020, 63(2):696-713.
doi: 10.1021/acs.jmedchem.9b01626 pmid: 31880942
[5] Meng T, Fu S, He D, et al. Evodiamine inhibits lipopolysaccharide (LPS)-induced inflammation in BV-2 cells via regulating AKT/Nrf2-HO-1/NF-κB signaling axis[J]. Cell Mol Neurobiol, 2020, 41(1):115-127.
doi: 10.1007/s10571-020-00839-w
[6] Wu JY, Chang MC, Chen CS, et al. Topoisomerase Ⅰ inhibitor evodiamine acts as an antibacterial agent against drug-resistant Klebsiella pneumoniae[J]. Planta Med, 2013, 79(1):27-29.
doi: 10.1055/s-00000058
[7] Lin J, Zhang X, Li C, et al. Evodiamine via targeting nNOS and AMPA receptor GluA1 inhibits nitroglycerin-induced migraine-like response[J]. J Ethnopharmacol, 2020, 254:112727.
doi: 10.1016/j.jep.2020.112727
[8] Zhang Y, Wang J, Wang C, et al. Pharmacological basis for the use of evodiamine in Alzheimer’s disease: antioxidation and antiapoptosis[J]. Int J Mol Sci, 2018, 19(5):1527.
doi: 10.3390/ijms19051527
[9] Li F, Dong YZ, Zhang D, et al. Molecular mechanisms involved in drug-induced liver injury caused by urate-lowering Chinese herbs: a network pharmacology study and biology experiments[J]. PLoS One, 2019, 14(5):e216948.
[10] 黄伟, 孙蓉. 吴茱萸水提组分多次给药致小鼠肝毒性氧化损伤机制研究[J]. 中药药理与临床, 2012, 28(5):114-116.
[11] 蔡卿嫣. 吴茱萸水提物的大鼠肝毒性及其线粒体损伤机制研究[D]. 广州: 广州中医药大学, 2014.
[12] Tolosa L, Gómez-Lechón MJ, Pérez-Cataldo G, et al. HepG2 cells simultaneously expressing five P450 enzymes for the screening of hepatotoxicity: identification of bioactivable drugs and the potential mechanism of toxicity involved[J]. Arch Toxicol, 2013, 87(6):1115-1127.
doi: 10.1007/s00204-013-1012-x pmid: 23397584
[13] Jain AN. Surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine[J]. J Med Chem, 2003, 46(4):499-511.
doi: 10.1021/jm020406h
[14] 李文兰, 孙向明, 陈晨, 等. 基于UPLC-Q-TOF MS的吴茱萸致肝毒性部位及入血成分分析[J]. 质谱学报, 2017, 38(3):282-293.
[15] 高绪聪, 柴振海, 张宗鹏. 药物性肝损伤的生物标志物及其评价的研究进展[J]. 中国药理学与毒理学杂志, 2012, 5(26):692-696.
[16] Huang P, Feng L, Oldham EA, et al. Superoxide dismutase as a target for the selective killing of cancer cells[J]. Nature, 2000, 407(6802):390-395.
doi: 10.1038/35030140
[17] Ho E, Karimi Galougahi K, Liu C, et al. Biological markers of oxidative stress: applications to cardiovascular research and practice[J]. Redox Biol, 2013, 1(1):483-491.
doi: 10.1016/j.redox.2013.07.006
[18] 刘晓婷, 王延让, 张明. 线粒体介导细胞凋亡的研究进展[J]. 环境与健康杂志, 2013, 30(2):182-185.
[19] Perez MJ, Briz O. Bile-acid-induced cell injury and protection[J]. World J Gastroenterol, 2009, 15(14):1677-1689.
doi: 10.3748/wjg.15.1677
[20] Pauli-Magnus C, Stieger B, Meier Y, et al. Enterohepatic transport of bile salts and genetics of cholestasis[J]. J Hepatol, 2005, 43(2):342-357.
pmid: 15975683
[21] Yang K, Woodhead JL, Watkins PB, et al. Systems pharmacology modeling predicts delayed presentation and species differences in bile acid-mediated Troglitazone hepatotoxicity[J]. Clin Pharmacol Ther, 2014, 96(5):589-598.
doi: 10.1038/clpt.2014.158 pmid: 25068506
[22] Fattinger K. The endothelin antagonist bosentan inhibits the cana-licular bile salt export pump: a potential mechanism for hepatic adverse reactions[J]. Clin Pharmacol Ther, 2001, 69(4):223-231.
pmid: 11309550
[23] Kenna JG. Current concepts in drug-induced bile salt export pump (BSEP) interference[J]. Curr Protoc Toxicol, 2014, 61(1):1-15.
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