Abstract: BACKGROUND: Stachydrine has anti-inflammatory, antioxidant, and antiplatelet properties that promote angiogenesis and has potential benefits on the cardiovascular system and central nervous system. Recently, it has been found that stachydrine effectively reverses homocysteine-induced endothelial dysfunction and ameliorates endothelial dysfunction by increasing the expression of guanosine triphosphate cyclase hydrolase and dihydrofolate reductase, but the role of stachydrine in atherosclerosis is yet unclear.
OBJECTIVE: To explore the effect and molecular mechanism of stachydrine on atherosclerosis induced by a high-fat diet in ApoE-/- mice.
METHODS: A total of 48 ApoE-/- mice were randomly divided into blank control group, model group, stachydrine group and atorvastatin group, with 12 mice in each group. Mice in the latter three groups were fed with high-fat diet for 12 weeks to establish animal models of atherosclerosis. After successful modeling, the stachydrine group was treated with stachydrine (30 mg/kg) by gavage, the atorvastatin group was treated with atorvastatin (2.6 mg/kg) by gavage, and the blank control group and the model group were treated with the same volume of sodium carboxymethyl cellulose by gavage once a day for 30 days. After administration, hematoxylin-eosin staining was used to observe the pathological changes of the aortic root. Oil red O staining was used to detect lipid deposition in aortic plaques and the aortic root. Real-time fluorescent quantitative PCR was used to detect mRNA expression of adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and selectin E) and chemokines (CXCL1, CXCL4, and monocyte chemotactic protein 1) in the aorta. RNA sequencing was used to analyze differential expression of genes between groups of aortic tissues and enrich for significantly upregulated signaling pathways. Western blot was used to detect the expression levels of autophagy marker proteins, autophagy microtubule-associated protein light chain β3 antibody (LC3BII/LC3BI), SQSTM1, phosphorylated AMp-activated protein kinase α and silent information regulator. Autophagy-lysosome changes were observed under transmission electron microscope.
RESULTS AND CONCLUSION: Compared with the blank control group, the model group had increased aortic plaques and lipid deposition, and increased mRNA expression of adhesion molecules and chemokines (P < 0.05). Compared with the model group, the stachydrine group or atorvastatin group had reduced aortic plaques and lipid deposition, and decreased mRNA expression of adhesion molecules and chemokines (P < 0.05). RNA sequencing analysis showed that 972 genes were up-regulated and 781 genes were down-regulated in the stachydrine group compared with the model group. KEGG enrichment analysis of the up-regulated genes showed that autophagy signaling pathway and AMPK signaling pathway were significantly up-regulated. Western blot results showed that compared with the model group, the stachydrine group had a significantly increased LC3BII/LC3BI ratio and protein expression of phosphorylated AMp-activated protein kinase α and silent information regulator (P < 0.05), and a significantly decreased protein level of SQSTM1. Transmission electron microscope analysis of mouse aorta showed that the stachydrine group had a significantly increased number of autophagolysosomes compared with the model group. To conclude, stachydrine may activate autophagy by up-regulating AMp-activated protein kinase/silent information regulator signaling pathway, thereby alleviating vascular endothelial inflammation and plaque deposition in atherosclerosis mice. 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: stachydrine, atherosclerosis, autophagy, AMPK/SIRT1, inflammatory response, engineered tissue construction