Hongguo Gao,1,2,* Te Liu,3,* Junfeng Liu,4,* Lian Yang,3,* Luxi Liu,5,* Zeyu Cui,3 Xiling Du,6 Yun Gu,1– 3 Peirong Huang7– 9 

1Department of Traditional Chinese Medicine, Graduate School, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330100, People’s Republic of China; 2Department of Geriatric Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200031, People’s Republic of China; 3Shanghai Geriatric Institute of Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200031, People’s Republic of China; 4Department of Nephrology, Shanghai Quyang Hospital, Shanghai, 200083, People’s Republic of China; 5Class 1, Grade 8, Shanghai Wenlai Middle School, Shanghai, 201101, People’s Republic of China; 6School of Life Science and Technology, Tongji University, Shanghai, 200092, People’s Republic of China; 7Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, People’s Republic of China; 8National Clinical Research Center for Eye Diseases, Shanghai, 200080, People’s Republic of China; 9Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai, 200080, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Peirong Huang, Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 haining Road, Hongkou District, Shanghai, 200031, People’s Republic of China, Tel +86-21-63240090, Fax +86-21-63240090, Email peirong_huang@126.com Yun Gu, Department of Traditional Chinese Medicine, Graduate School, Jiangxi University of Traditional Chinese Medicine, 1688 Meiling Avenue, Zhaoxian Town, Xinjian District, Nanchang, 330100, People’s Republic of China, Tel +86-791-87119529, Fax +86-791-87119529, Email birgitgu@163.com

Background and Aims: The incidence of and mortality due to atherosclerosis, a leading cause of cardiovascular disease, is rising annually. Oleanolic acid (OA), an active component of the traditional Chinese medicine Ligustrum lucidum, has been proven to have significant anti-inflammatory and lipid-lowering potential.
Methods: The fli1a::EGFP+ zebrafish fed with oxidized low-density lipoprotein (oxLDL) diet were used as Atherosclerosis model. The zebrafish Atherosclerosis model were fed with oxalic acid driven by superparamagnetic ferrite nanoparticles (OA@SPIONs). Isolation and enrichment of fli1a::EGFP+ zebrafish endothelial cells (zeECs) from each group and RNA-seq to analyze changes in gene transcription. The H&E, MASSION, Oil red O staining were used to identifying pathological phenotypes.
Results: Pathological staining and ultrastructural identification indicated that oxLDL-treated zebrafish exhibited significant lipid plaque deposition and signs of cellular senescence that were significantly alleviated by OA@SPIONs treatment. OA@SPIONs treatment notably improved the ultrastructural integrity of myocardial, liver, and intestinal tissues in oxLDL-treated zebrafish. The RNA-seq results showed that OA@SPIONs treatment significantly altered the expression levels of multiple gene transcripts in zeECs. The KEGG analysis revealed that in the OA@SPION-treated group zeECs, key genes in the JNK and MAPK signaling pathways, such as Cacna1c, Rab1ab (Ras), Map3k1 (MEKK1), Mapk8b (JNK), and JunD, had significantly lower sequencing signals than in the oxLDL+SPION-treated group zeECs. The qPCR results were highly consistent with the RNA-sequencing data.
Conclusion: Therefore, our results confirm that SPIONs can effectively deliver OA for stable release in zebrafish and provide strong evidence that OA@SPION-polyethyleneimine exerts protective effects against oxLDL-induced damage in zebrafish by downregulating the expression of the JNK and MAPK signaling pathways.

Keywords: atherosclerosis, super paramagnetic iron oxide nanoparticles (SPIONs), zebrafish, oleanolic acid (OA), JNK and MAPK signaling pathways