Abstract

Prolactin-releasing Peptide (PrRP) is a neuropeptide whose receptor is GPR10. Recently, the regulatory role of PrRP in the neuroendocrine field has attracted increasing attention. However, the influence of PrRP on macrophages, the critical housekeeper in the neuroendocrine field, has not yet been fully elucidated. Here, we investigated the effect of PrRP on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) with RNA sequencing, bioinformatics, and molecular simulation. BMDMs were exposed to PrRP (18 h) and were subjected to RNA sequencing. Differentially expressed genes (DEGs) were acquired, followed by GO, KEGG, and PPI analysis. Eight qPCR-validated DEGs were chosen as hub genes. Next, the three-dimensional structures of the proteins encoded by these hub genes were modeled by Rosetta and Modeller, followed by molecular dynamics simulation by the Gromacs program. Finally, the binding modes between PrRP and hub proteins were investigated with the Rosetta program. PrRP showed no noticeable effect on the morphology of macrophages. A total of 410 DEGs were acquired, and PrRP regulated multiple BMDM-mediated functional pathways. Besides, the possible docking modes between PrRP and hub proteins were investigated. Moreover, GPR10 was expressed on the cell membrane of BMDMs, which increased after PrRP exposure. Collectively, PrRP significantly changed the transcriptome profile of BMDMs, implying that PrRP may be involved in various physiological activities mastered by macrophages.

Highlights

  • GPR10 was expressed on the cell membrane of bone marrow-derived macrophages (BMDMs), which increased after Prolactin-releasing peptide (PrRP) exposure

  • PrRP significantly changed the transcriptome profile of BMDMs, implying that PrRP may be involved in various physiological activities mastered by macrophages

  • In 2012, the Romeroa team discoveries that PrRP promotes the production of proinflammatory cytokines (IL-8, IL-12, IL-1β, and IL-6) in Salmon salar, indicating that PrRP is a local transmitter of the innate immune pathway in leukocytes [13]

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Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. (RNA-sequencing)isisaauseful usefulmethod methodtoto explore effect of the peptide the gene expression profile of macrophages from the transcriptome level [14,15,16]. It on the gene expression profile of macrophages from the transcriptome level [14,15,16]. Would useful be to investigate the PrRP-induced gene expression profile ofprofile macrophages. Be it would useful to investigate the PrRP-induced gene expression of macby using RNA-seq. (a) PrRP31-triggered (DEGs) were were acquired acquired from from murine murine bone marrow-derived macrophages (BMDMs). Genes (c) The three-dimensional structures of hub proteins were studied.

PrRP Demonstrated No Significant Effect on the Shape of BMDMs
Functional and Pathway Enrichment Analysis of DEGs
Functional
Common Transcription Factors Tied to Genes Down-Regulated by PrRP
Verification of Hub Genes with qPCR and Western Blot
Protein Modeling of Hub Proteins
10. Ramachandran of the homology modeled-models of hub proteins
Molecular Dynamics Simulation of Hub Proteins
Peptide-Hub
Peptide-Hub Protein Docking
15. The docking ofand
2.10. Expression of GPR10 on BMDMs
PrRP Modulated Different Functional Enrichment Pathways of BMDMs
Common Transcription Factors Tied to PrRP-Regulated DEGs in BMDMs
Expression of PrRP-GPR10 on BMDMs
Prolactin and Inflammatory Processes
Prolactin and Macrophages
PrRP and Microglia
Non-Specific Fluorescent Signal Caused by Dead Cells
Non-Specific Fluorescent Signal from Cell Debris or Tiny Tissue Pieces
Non-Specific Fluorescence Signals Caused by BMDMs
The Limitations of Our Study
Animals
Reagents
BMDM Preparation
RNA-seq Sample Preparation and Microscope Detection
Flow Cytometry Detection
RNA Quantification and Qualification
Sequencing and Clustering
RNA-seq Data Interpretation
Differential Expression Interpretation
GO and KEGG Enrichment Interpretation
4.10. Western Blot Detection
4.11. Immunofluorescence Stain
4.12. Homology Modeling of Proteins
4.13.1. Molecular Dynamic Simulation
4.13.2. Molecular Dynamic Simulation
Conclusions
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