Establish a Zebrafish Atherosclerosis Model Screening Platform to Evaluate the Therapeutic Effects of Qi-Regulating and Blood-Activating Prescriptions on Atherosclerosis

YIN Jiufeng, WANG Hui, WANG Haoyan, JIANG Tong, ZHANG Xinyi, LI Mingkai, ZHANG Dan, ZHAO Feng, YANG Wenqing

Abstract

Objective

To systematically identify traditional Chinese medicine (TCM) compound formulas with anti-atherosclerosis (AS) potential using a screening platform based on multi-strain apoeb gene knockout zebrafish models for anti-atherosclerosis TCM compounds

Methods

Screening was conducted using a formula dictionary database, and compound formulas such as Dan Shen Fang, Shang Ni San, Jin Lingzi San, and Li Qi Hua Liang Tang were selected from 933 blood-activating and stasis-resolving formulas. Five strains of apoeb－/－ zebrafish–AB, Tg(mpeg1:EGFP), Tg(lyzc:dsRed), Tg(gata1a:mCherry), and Tg(flk1:EGFP)—were fed a high-fat diet for 2 days, after which the AS model was established. DMSO (solvent control), rosuvastatin (western medicine positive control), compound Dan Shen Fang (TCM positive control), Shang Ni San, Jin Lingzi San, and Li Qi Hua Liang Tang were administered by bathing for 24 hours. Oil red O staining was used to detect lipid content, oxidative stress indicators, blood flow velocity, vascular endothelial cell damage, and the expression of inflammatory factors in zebrafish. The behavior patterns of inflammatory cells in zebrafish were observed, and the anti-AS efficacy of the Li Qi Hua Liang formulas was comprehensively evaluated.

Results 

 Compared with the AS model group, compound Dan Shen Fang and Shang Ni San improved lipid accumulation in the vascular lumen, reduced total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels, decreased oxidative stress response, inhibited the recruitment of neutrophils and macrophages, repaired vascular endothelial damage, improved blood flow velocity, and reduced the expression of inflammatory factors, thereby alleviating AS, with statistically significant differences. Jin Lingzi San and Li Qi Hua Liang Tang had limited effects on AS.

Conclusion

This platform provides an efficient and targeted tool for the screening and research evaluation of anti-atherosclerosis TCM compound formulas, accelerates the research progress of anti-atherosclerosis TCM compound formulas, and lays a foundation for subsequent drug development and clinical application.

 

Keywords: Atherosclerosis, Zebrafish, Drug screening, Chinese medicine compound formulas

 

Full Text:

PDF


References


SONG W, SHUAI Y Q, WANG K, et al. Clinical characteristics, syndrome type distribution and medication regularity of traditional Chinese medicine in treatment of patients with carotid atherosclerosis. Chinese Traditional Patent Medicine, 2025, 47(6): 2101-2108. doi: 10. 3969/j.issn.1001-1528.2025.06.051.

GETZ G S, REARDON C A. Apoprotein E and reverse cholesterol transport. Int J Mol Sci, 2018, 19(11): 3479. doi: 10.3390/ijms19113479.

GAO J. Meta analysis of the association between apolipoprotein E gene polymorphism and coronary heart disease in chinese elderly. Chongqing: Chongqing Medical University, 2020. doi: 10.27674/d.cnki.gcyku. 2020.001120.

HU Y X, YOU H M, ZHU R F, et al. Establishment of a lipid metabolism disorder model in ApoEb mutant zebrafish. Atherosclerosis, 2022, 361: 18-29. doi: 10.1016/j.atherosclerosis.2022.10.008.

KONDO H, MORINAGA K, MISAKI R, et al. Characterization of the pufferfish Takifugu rubripes apolipoprotein multigene family. Gene, 2005, 346: 257-266. doi: 10.1016/j.gene.2004.11.015.

KIM K Y, CHO Y S, BANG I C, et al. Isolation and characterization of the apolipoprotein multigene family in Hemibarbus mylodon (Teleostei: Cypriniformes). Comp Biochem Physiol B Biochem Mol Biol, 2009, 152(1): 38-46. doi: 10.1016/j.cbpb.2008.09.084.

OTIS J P, ZEITUNI E M, THIERER J H, et al. Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake. Dis Model Mech, 2015, 8(3): 295-309. doi: 10.1242/dmm.018754.

MACRAE C A, PETERSON R T. Zebrafish as tools for drug discovery. Nat Rev Drug Discov, 2015, 14(10): 721-731. doi: 10.1038/nrd4627.

Van WIJK R C, KREKELS E H J, KANTAE V, et al. Mechanistic and quantitative understanding of pharmacokinetics in zebrafish larvae through nanoscale blood sampling and metabolite modeling of paracetamol. J Pharmacol Exp Ther, 2019, 371(1): 15-24. doi: 10.1124/jpet.119.260299.

GAO Y X, XIE X P, WANG H, et al. Viewing the Spring-Autumn Normal Pulse from 'Suwen · Maiyao Jingwei Lun'. Journal of Chinese Medicine, 2024, 39(6): 1166-1170. doi: 10.13424/j.cnki.mtcm.1983.01.002.

LI S S, YU H H, TIAN W Y. Research progress of effect of Traditional Chinese Medicine in prevention and treatment of atherosclerosis based on signal pathway related to inflammatory response. Chinese Journal of Experimental Traditional Medical Formulae, 2020, 26(23): 180-186. doi: 10.13422/j.cnki.syfjx.20201868.

RAGGI P, GENEST J, GILES J T, et al. Role of inflammation in the pathogenesis of atherosclerosis and therapeutic interventions. Atherosclerosis, 2018, 276: 98-108. doi: 10.1016/j.atherosclerosis.2018.07.014.

LUO Y Y. Experimental study of intervenient effects the recipes for promoting blood circulation and removing blood stasis on heart blood stasis syndrome in atheriosclerosis. Changsha: Hunan University of Traditional Chinese Medicine, 2004.

FAN H. Intervention effect and mechanism of scutellarin from Erigeron breviscapus on atherosclerosis of qi stagnation and blood stasis type. Shenyang: Liaoning University of Traditional Chinese Medicine, 2018.

ZHANG L F. Observation on the therapeutic effect of the method of Regulating Qi And Activating Blood circulation on angina pectoris with tiredness and stability of Qi Stagnation And Blood Stasis. Shanghai: Shanghai University of Traditional Chinese Medicine, 2020. doi: 10.27320/d.cnki.gszyu.2020.000333.

MONTASSER M E, O'HARE E A, WANG X, et al. An APOO pseudogene on chromosome 5q is associated with low-density lipoprotein cholesterol levels. Circulation, 2018, 138(13): 1343-1355. doi: 10.1161/CIRCULATIONAHA.118.034016.

HE F. Mustard seeds prevent atherosclerosis in hypercholesterolemic zebrafish. Guangzhou: Southern Medical University, 2013.

SHUI D, WANG Y Q, LI S J, et al. Comparative study on anti-atherosclerosis effect of Xiexin decoction and its compositions of effective components. Chinese Journal of Hospital Pharmacy, 2021, 41(18): 1836-1841. doi: 10.13286/j.1001-5213.2021.18.04.

SHENG J. Research on drug screening based on transgenic zebrafish disease models of atherosclerosis and thrombosis. Hangzhou: Zhejiang University, 2021. doi: 10.27461/d.cnki.gzjdx.2021.004027.

HU Y X. Establishment of a lipid-lowering drug screening platform in zebrafish by ApoEb knockout. Shanghai: Naval Medical University of the Chinese People's Liberation Army, 2020. doi: 10.26998/d.cnki.gjuyu.2020. 000301.

LI X, WU L H, FAN X H, et al. Network pharmacology study on major active compounds of Fufang Danshen formula. China Journal of Chinese Materia Medica, 2011, 36(21): 2911-2915.

WANG Y Z, LIANG L Y, CAO J L, et al. Key information research and contemporary research progress of classical formula Jinlingzi San. Chinese Journal of Experimental Traditional Medical Formulae, 2024, 30(24): 215-223. doi: 10.13422/j.cnki.syfjx.20240817.

DU Q, SHAO R, WANG W, et al. Vitamin D3 regulates energy homeostasis under short-term fasting condition in zebrafish (Danio Rerio). Nutrients, 2024, 16(9): 1271. doi: 10.3390/nu16091271.


Refbacks

  • There are currently no refbacks.