Panax notoginseng Saponins Alleviate Coronary Artery Disease Through Hypermethylation of the miR-194-MAPK Pathway

Lian Duan, Yongmei Liu, Jun Li, Yun Zhang, Yan Dong, Chao Liu, Jie Wang, Lian Duan, Yongmei Liu, Jun Li, Yun Zhang, Yan Dong, Chao Liu, Jie Wang

Abstract

Background: Panax notoginseng saponins (PNS) may have an inhibitory effect against coronary artery disease (CAD); however, the mechanism is unclear. Recent research has begun to evaluate the role of epigenetics in CAD. Our team found that hypomethylation of miR-194 could be an important mechanism of CAD. Purpose: The aim of this study was to investigate the effect of PNS against CAD and evaluate whether the mechanism is related to methylation of mi-R194. Methods: We conducted a randomized controlled trial with a double-blind placebo design on 84 patients with CAD. Treatment was continued for 4 weeks, and the clinical effect of PNS on CAD was observed. Methylation of miR-194, its promoter, and the key nodes of the MAPK pathway were measured by pyrosequencing and qRT-PCR. We then conducted a pharmacological analysis of the active components of PNS. The effects of PNS on oxidized human umbilical vein endothelial cells and the methylation of miR-194, its promoter, and the key nodes of the MAPK pathway were measured in vitro through methylation-specific PCR (MSPCR), qRT-PCR, Western blot analysis, and annexin V/propidium iodide apoptosis assay. Results: PNS improved symptoms of CAD. High-density lipoprotein and white blood cell count demonstrated significant changes after treatment in the PNS group. No significant difference was observed between miR-194 and mRNA MAPK, FAS, RAS, and FOS in the PNS group after treatment. However, some notable trends were observed in these genes. The targets of PNS were predicted by the pharmacological components. Some targets were found to be differentially expressed genes in CAD sequencing. Six genes, including MAPK1, RAS, and FASL, were common targets of PNS in CAD sequencing. Correlations were observed between genes in the interaction network and clinical parameters. In vitro experiments confirmed that PNS could change the methylation of miR-194, its promoter, and MAPK, FAS, RAS, and FOS. Intervention with PNS is likely to improve apoptosis. Conclusion: We reported the regulation of miR-194 promoter, miR-194, and MAPK methylation by PNS through cell experiments and a randomized controlled trial. PNS can be used for intervention in CAD by targeting the miR-194 promoter-miR-194-MAPK signaling pathway. Clinical Trial Registration: https://www.clinicaltrials.gov/, NCT03083119.

Keywords: DNA methylation; MAPK; Panax notoginseng saponins; coronary artery disease; miRNA.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Duan, Liu, Li, Zhang, Dong, Liu and Wang.

Figures

FIGURE 1
FIGURE 1
Trial protocol timeline as described in detail in the Materials and Methods section.
FIGURE 2
FIGURE 2
Cell experimental protocol as described in detail in the Materials and Methods section.
FIGURE 3
FIGURE 3
Study participant flow diagram.
FIGURE 4
FIGURE 4
Blood tests before and after treatment in the PNS and placebo groups. p < 0.05 and **p < 0.01, significantly different from the PNS group before.
FIGURE 5
FIGURE 5
Prediction of PNS targets by pharmacological analysis. (A) Top 20 potential targets of PNS. The relationship of the top 20 best matching genes of PNS was exhibited by Cytoscape. Yellow squares indicate the main ingredients of PNS. The circles indicate the potential targets. The nodes with a higher degree appear redder in color. The nodes with a higher fit value have thicker edges. (B) Common targets of the main ingredients of PNS. (C) Common targets of PNS potential targets and CAD-related genes.
FIGURE 6
FIGURE 6
Expression of key genes in the miR-194 promoter-miR914-MAPK pathway according to pyrosequencing and PCR.
FIGURE 7
FIGURE 7
Expression of key genes in the miR-194 promoter-miR-194-MAPK pathway by MSPCR, qRT-PCR, and Western blotting in vitro(A) DNA methylation level of the miR-194 promoter by MSPCR; (B) miR-194 expression; (C)RAS expression; (D) MAPK1 expression; (E) FOS expression; (F) FAS expression; (G) FASL expression; (H) c-Jun expression; (I) MAPK1 protein level; and (J) MAPK1 protein.
FIGURE 8
FIGURE 8
Annexin V/PI apoptosis assay results. (A) Level of apoptosis in HUVECs; (B) control group; (C) H2O2 group; and (D) PNS group. *p < 0.05 and **p < 0.01, significantly different from the control group. #p < 0.05, significantly different from the H2O2 group.

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