关键词: 原儿茶醛, 动脉粥样硬化, 转录组学, 靶点验证, 差异表达基因

Abstract: BACKGROUND: Protocatechualdehyde has the potential to delay the progression of atherosclerosis. Nevertheless, its specific mechanisms of action within multi-target regulatory networks remain unclear and require further investigation.
OBJECTIVE: To investigate the potential targets of protocatechualdehyde in intervening atherosclerosis based on transcriptomics.
METHODS: (1) Thirty ApoE-/- mice were randomly divided into a model group (n=10), a rosuvastatin group (n=10), and a protocatechualdehyde group (n=10). An atherosclerosis model was induced by feeding the mice with a high-fat diet for 12 weeks. Seven C57BL/6J mice were selected as a control group (without modeling). After successful modeling, the control group and model group were given physiological saline by gavage; the rosuvastatin group was given rosuvastatin by gavage, and the protocatechualdehyde group was given protocatechualdehyde by gavage, once a day for 12 consecutive weeks. After the last administration, samples were collected. An automatic biochemical analyzer was used to detect serum lipid levels in mice. Gross Oil Red O staining and hematoxylin-eosin and Masson staining of paraffin sections of the aortic root were used to detect the pathological conditions of arterial plaques. (2) Aortic sample transcriptome expression profile from the control group, model group, and protocatechualdehyde group mice were analyzed using high-throughput sequencing technology. Differential gene screening FC > 2, q < 0.05, gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, weighted gene co-expression network analysis, and short time series expression miner analysis were performed based on the Ouyi Cloud platform. A protein-protein interaction network was constructed by combining the string database, and core genes were screened using Cytoscape. (3) The mRNA expression of Calm4 (calmodulin pseudogene 4), Kprp (keratinocyte proline-rich protein), Hrnr (hornerin 2), and Lor (loricrin) in aortic samples of mice from the control group, model group, and protocatechualdehyde group was detected by RT-PCR to verify candidate targets.
RESULTS AND CONCLUSION: (1) Protocatechualdehyde significantly reduced serum levels of total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in atherosclerosis mice. Gross Oil Red O staining and hematoxylin-eosin and Masson staining of paraffin sections of the aortic root showed that protocatechualdehyde decreased plaque formation, suppressed intimal thickening, increased collagen fiber content, thereby promoting plaque stabilization. (2) Transcriptome analysis identified 191 differentially expressed genes. Through Cytoscape analysis, Kprp, Calm4, Hrnr, and Lor were preliminarily identified as key candidate targets. (3) RT-PCR analysis showed that the expression levels of Kprp, Calm4, Hrnr, and Lor mRNA in the model group were higher than those in the control group (P < 0.05), while the expression levels of Kprp, Calm4, and Lor mRNA in the protocatechualdehyde group were lower than those in the model group (P < 0.05). These results indicate that protocatechualdehyde intervention can significantly improve atherosclerotic plaques, and the three genes Kprp, Calm4, and Lor may be potential targets for protocatechualdehyde treatment of atherosclerosis.

Key words: protocatechualdehyde, atherosclerosis, transcriptomics, target validation, differentially expressed genes