Xiao Ban Tong Mai Fang regulates autophagy via targeting miR-126-3p: bioinformatics analysis for prevention and treatment of atherosclerosis

Abstract

BACKGROUND: Studies have shown that miR-126-3p plays an important role in regulating autophagy and is closely related to the occurrence and development of atherosclerosis. Xiao Ban Tong Mai Fang can exert anti-atherosclerotic effects by exerting anti-inflammatory effects and inhibiting the proliferation of human aortic vascular smooth muscle cells. OBJECTIVE: To explore the mechanism of Xiao Ban Tong Mai Fang in the treatment of atherosclerosis by targeting microRNAs (miRNAs) to regulate autophagy using bioinformatics techniques. METHODS: Machine learning methods were used to screen the differentially expressed miRNAs in the atherosclerosis dataset GSE137580. The screened miRNAs were intersected with the key module genes screened by weighted gene co-expression network analysis. The potential regulatory genes of the intersected miRNAs were predicted based on the database, and then the predicted genes were intersected with the autophagy gene set. Protein-protein interaction analysis and enrichment analysis were performed on the intersection genes. Based on the enrichment analysis results, experimental validation was carried out. Human umbilical vein endothelial cells and RAW264.7 cells were cultured in vitro, and an atherosclerotic cell foam model was induced by oxidized low-density lipoprotein. qPCR was used to detect the differential expression of miR-126-3p. A miR-126-3p overexpression vector was constructed, and the transfection efficiency and the intervention effect of Xiao Ban Tong Mai Fang were detected by qPCR. Western blot was used to detect the regulation of Xiao Ban Tong Mai Fang on autophagy and mitogen-activated protein kinase pathway in human umbilical vein endothelial cells. RESULTS AND CONCLUSION: (1) Combining machine learning and weighted gene co-expression network analysis, 10 key miRNAs were identified. Based on literature review and previous foundation, miR-126-3p was selected for experimental validation. A total of 3,892 potential regulatory genes were predicted, and 257 autophagy-related genes were obtained by intersecting with the autophagy gene set. Enrichment analysis found that these genes were widely enriched in the mitogen-activated protein kinase signaling pathway, so this pathway was selected for experimental validation. (2) qPCR results showed that miR-126-3p was upregulated in oxidized low-density lipoprotein-induced foam cell models of human umbilical vein endothelial cells and RAW264.7 cells (P < 0.05). (3) After intervention with Xiao Ban Tong Mai Fang, the expression of miR-126-3p in the mimic group of human umbilical vein endothelial cells was significantly reduced (P < 0.05). (4) Western Blot results showed that Xiao Ban Tong Mai Fang intervention inhibited the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 lipidated/non-lipidated (LC3-II/LC3-I) and autophagy adaptor protein p62 in human umbilical vein endothelial cells (P < 0.05), and downregulated the expression of mitogen-activated protein kinase pathway proteins (P < 0.05), similar to the effect of autophagy inhibitor 3-methyladenine. These results indicate that miR-126-3p is upregulated in atherosclerosis models, and its abnormal expression may be involved in the pathogenesis of atherosclerosis. Xiao Ban Tong Mai Fang exerts its therapeutic effect on atherosclerosis by downregulating miR-126-3p, inhibiting the mitogen-activated protein kinase pathway, and inhibiting autophagy in human umbilical vein endothelial cells.

1. Introduction

Atherosclerosis is a vascular wall disease characterized by chronic inflammation, with a high incidence worldwide and serving as an important pathological basis for secondary cardiovascular and cerebrovascular diseases [1-2]. Modern medical treatments for atherosclerosis mainly focus on pharmacological and surgical interventions [3]. However, long-term use of drugs such as statins and fibrates increases the risk of liver and kidney damage and certain muscle pain [4-6]; surgical treatment is limited in clinical application due to its high cost, so finding safe and effective therapeutic drugs is urgent [7-8]. In traditional Chinese medicine, atherosclerosis can be classified under the categories of 'vein accumulation' and 'vein obstruction' [9-11]. Numerous studies have found that traditional Chinese medicine has advantages in the treatment of atherosclerosis, including multi-target synergy, holistic regulation [12], and low toxicity [13], and can act simultaneously on anti-inflammatory, lipid-regulating, and antioxidant mechanisms [14-16], which better matches the complex multifactorial pathogenesis of atherosclerosis. In-depth research on the prevention and treatment of atherosclerosis with traditional Chinese medicine will help expand treatment ideas and promote the modernization and scientific advancement of traditional Chinese medicine.

Xiao Ban Tong Mai Fang is composed of Radix Panacis Quinquefolii, Hirudo, steamed Radix Polygoni Multiflori, Salvia miltiorrhiza, Panax notoginseng, Trichosanthes kirilowii, and Gastrodia elata. It is an empirical formula created by Professor Cui Yingmin, a national-level renowned traditional Chinese medicine expert, with the effects of replenishing qi, activating blood, eliminating plaques, and unblocking meridians. It has shown good clinical efficacy in treating atherosclerosis [17]. Our previous research found that Xiao Ban Tong Mai Fang can exert anti-atherosclerotic effects by anti-inflammatory actions and inhibiting the proliferation of human aortic vascular smooth muscle cells [18]. However, whether Xiao Ban Tong Mai Fang can improve atherosclerosis by regulating miRNA-mediated autophagy remains unclear. miRNAs are newly discovered gene regulatory factors that play key roles in the epigenetic regulation of atherosclerosis, plaque formation, progression, and rupture [19-21]. Studies have shown that miR-126-3p plays an important role in regulating autophagy and is closely related to the occurrence and development of atherosclerosis.