Transcriptomic analysis of potential targets of protocatechualdehyde in treatment of atherosclerosis

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. Serum lipid levels were measured using an automatic biochemical analyzer. Aortic plaque pathology was assessed by gross oil red O staining, hematoxylin-eosin staining, and Masson staining of aortic root paraffin sections. (2) High-throughput sequencing was used to analyze the transcriptome expression profiles of aortic samples from the control, model, and protocatechualdehyde groups. Differential gene screening (FC > 2, q < 0.05), GO and KEGG enrichment analyses, 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 using the STRING database, and core genes were screened using Cytoscape. (3) RT-PCR was used to detect the mRNA expression of Calm4 (calmodulin pseudogene 4), Kprp (keratinocyte proline-rich protein), Hrnr (filaggrin 2), and Lor (loricrin) in aortic samples from the control, model, and protocatechualdehyde groups to validate candidate targets. RESULTS AND CONCLUSION: (1) Protocatechualdehyde significantly reduced serum total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels in atherosclerotic mice. Gross oil red O staining and hematoxylin-eosin and Masson staining of aortic root paraffin sections showed that protocatechualdehyde reduced plaque formation, inhibited intimal thickening, increased collagen fiber content in plaques, and stabilized plaques. (2) Transcriptome analysis identified 191 differentially expressed genes, and Cytoscape analysis preliminarily identified Kprp, Calm4, Hrnr, and Lor as key candidate targets. (3) RT-PCR showed that the mRNA expression of Kprp, Calm4, Hrnr, and Lor in the model group was higher than that in the control group (P < 0.05), while the mRNA expression of Kprp, Calm4, and Lor in the protocatechualdehyde group was lower than that in the model group (P < 0.05). These results indicate that protocatechualdehyde intervention can significantly improve atherosclerotic plaques, and Kprp, Calm4, and Lor may be potential targets for protocatechualdehyde in the treatment of atherosclerosis.

1. Introduction

Atherosclerosis, a chronic inflammatory disease initiated by lipid deposition, is a major pathological basis for various cardiovascular diseases, including peripheral vascular disease, cerebral infarction, and coronary atherosclerotic heart disease [1-7]. Its typical pathological features include intimal injury of large and medium arteries, lipid metabolism disorders, inflammatory cell infiltration, and abnormal proliferation of vascular smooth muscle cells [8-9].

Currently, statins are widely used in the treatment of atherosclerosis, but they may cause adverse reactions such as rhabdomyolysis, liver damage, and elevated blood glucose, limiting their long-term safety [10-12]. Therefore, medicinal and edible traditional Chinese medicines have attracted increasing attention in the treatment of atherosclerosis. Salvia miltiorrhiza (Danshen) has the effects of promoting blood circulation, removing blood stasis, calming the mind, and relieving pain, and can be used for the treatment of atherosclerosis [13-14]. Modern pharmacology has further confirmed that Danshen can improve circulation, exert anti-inflammatory and anti-edema effects, and inhibit thrombosis [15].

Protocatechualdehyde is a phenolic compound extracted from the root of Danshen, with anti-proliferative and antioxidant effects [16-17]. Numerous studies have shown that protocatechualdehyde can delay the progression of atherosclerosis by reducing pericyte injury, inhibiting apoptosis, and inhibiting vascular smooth muscle migration [18-20]. Studies have confirmed that the combination of protocatechualdehyde with ginsenoside and notoginsenoside can inhibit the progression of atherosclerosis by inhibiting cellular senescence [21]; in addition, the combination of protocatechualdehyde and notoginsenoside can significantly alleviate vascular inflammation and calcification, exerting a synergistic anti-atherosclerotic effect [22].

Atherosclerosis involves a multi-target regulatory network with complex mechanisms [23], and the specific targets of protocatechualdehyde have not yet been clarified. Therefore, this study combined transcriptome sequencing and bioinformatics analysis to systematically screen key targets of protocatechualdehyde in intervening atherosclerosis, aiming to provide a theoretical basis for elucidating its molecular mechanism.