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Study on the regulatory effect of leech peptide HE-D on macrophages in atherosclerosis by transcriptome sequencing
Ke Wang1, Qi Cao2, Qiong Yang2
1Marine College, Shandong University, Weihai, 264209, China; Heping Hospital Affiliated to Changzhi Medical College, Changzhi, 046000, China.
Insights
HE-D, a peptide from leeches, inhibits macrophage migration by targeting the NF-κB pathway, offering potential for atherosclerosis treatment. This study used transcriptome sequencing to elucidate its mechanism.
Area of Science:
- Cardiovascular Research
- Immunology
- Pharmacology
Background:
- Atherosclerotic cardiovascular disease poses a significant health risk.
- Leeches have a history in traditional Chinese medicine for cardiovascular conditions.
- HE-D, a leech peptide, demonstrates inhibitory effects on macrophage migration.
Purpose of the Study:
- To investigate the effects of HE-D on macrophages in atherosclerosis.
- To elucidate the mechanism of HE-D's inhibition of macrophage migration using transcriptome sequencing (RNA-Seq).
Main Methods:
- Transwell assays to assess HE-D's inhibition of macrophage migration.
- RNA-Seq to identify differentially expressed genes (DEGs) after HE-D treatment.
- Gene Ontology (GO) and KEGG pathway analyses for DEG functional enrichment.
- Quantitative reverse transcription PCR (qRT-PCR) and Western blotting for gene validation.
Main Results:
- HE-D significantly inhibited lipopolysaccharide (LPS)-induced RAW264.7 macrophage migration.
- RNA-Seq identified 363 DEGs, enriched in cell migration and inflammation pathways, notably the NF-κB signaling pathway.
- HE-D down-regulated key NF-κB pathway genes (IKKα, IKKγ, TRAF6, TLR4, TRAF5) and inflammatory factors (iNOS, TNF-α), while up-regulating Arg-1 and IL-10.
Conclusions:
- HE-D inhibits macrophage migration by suppressing IKKα and IKKγ within the NF-κB pathway.
- HE-D promotes the M1 to M2 macrophage subtype transition.
- HE-D shows potential as a therapeutic agent for atherosclerosis.
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.
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