Study on the regulatory effect of leech peptide HE-D on macrophages in atherosclerosis by transcriptome sequencing

Abstract

Ethnopharmacological relevance: The incidence of atherosclerotic cardiovascular disease is a serious threat to human health. Leeches are used in traditional Chinese medicine to treat cardiovascular diseases. HE-D is an active peptide extracted and isolated from leeches, which can inhibit the migration of RAW264.7 macrophages.

Aim: This study shows the effects of HE-D on macrophages in atherosclerosis and the mechanism of inhibition on the migration of macrophages based on transcriptome sequencing (RNA-Seq).

Materials and methods: The transwell method was used to detect the activity of HE-D in inhibiting the migration of macrophages. Macrophages were divided into control group, lipopolysaccharide group, and HE-D group. Samples were collected and RNA-Seq performed. The DEseq2 method detected significantly differentially expressed genes (DEGs), GO and KEGG Pathway databases were used to analyze the functions and pathway enrichment of DEGs. Finally, qRT-PCR and Western blotting were used to verify the genes screened by RNA-Seq analyses.

Results: Cell experiments showed that HE-D can inhibit the migration of RAW264.7 macrophages induced by LPS. DEseq2 analyses showed that there were 363 DEGs after HE-D administration in the result of RNA-Seq. The GO function of DEGs was significantly enriched in cell migration and inflammation, and the DEGs related to cell migration were significantly enriched in the NF-κB signaling pathway. qRT-PCR and Western blot analyses, showed that when compared with the LPS group, the related genes IKKα, IKKγ, TRAF6, TLR4, and TRAF5 in the NF-κB pathway were significantly down-regulated in the HE-D group. In addition, it was found that the inflammatory factors iNOS and TNF-α were significantly down-regulated, and Arg-1 and IL-10 were up-regulated.

Conclusion: HE-D can inhibit the migration of macrophages by inhibiting IKKα and IKKγ in the NF-κB signaling pathway, and promote the transformation of macrophages from M1to M2 subtypes. Therefore, HE-D can potentially be used as a drug for the treatment of atherosclerosis.

Keywords: Atherosclerosis; Leech peptide HE-D; Macrophage migration; Macrophage polarization; Transcriptome sequencing.