# Transcriptome Analysis Reveals the Immunoregulatory Effect of Two Polysaccharides from Rhodomyrtus tomentosa

## TL;DR

This study shows that two polysaccharides from Rhodomyrtus tomentosa berries can boost immune responses in macrophages, with one being more effective.

## Contribution

The study identifies RTP-2 as a more potent immunostimulatory polysaccharide through transcriptome and functional analyses in macrophages.

## Key findings

- Both RTP-1 and RTP-2 enhanced phagocytosis, NO/ROS production, and cytokine secretion in macrophages.
- RTP-2 activated a co-expression module linked to mitophagy, proteostasis, and innate immunity.
- RT-qPCR confirmed significant gene expression changes in the RTP-2 group, including upregulation of Dram1 and Bmf1.

## Abstract

The Rhodomyrtus tomentosa (Aiton.) Hassk. berry is rich in structurally diverse polysaccharides with potential biological activity. However, its immunomodulatory properties remain understudied, limiting our current understanding of its functional significance. Two structurally distinct polysaccharides from Rhodomyrtus tomentosa (RTP-1 and RTP-2) were evaluated for immunostimulatory activity in RAW264.7 macrophages. Phagocytic function was assessed by neutral red assay, nitric oxide (NO) and reactive oxygen species were measured using the Griess assay and fluorescent probes, and cytokines (TNF-α, IL-6 and IL-1β) were quantified by enzyme-linked immunosorbent assay. Analysis of RNA-seq data using weighted gene co-expression network analysis revealed co-expression modules. The selected transcripts were independently validated by quantitative real-time PCR (RT-qPCR). The results showed that both polysaccharides enhanced phagocytosis, increased NO/ROS levels, and promoted cytokine secretion. Transcriptome results indicated that RTP-2 activated the MEturquoise co-expression module containing 222 hub genes, whereas RTP-1 was mainly associated with the MECyan module containing 49 hub genes. Module enrichment for RTP-2 revealed links with mitophagy–immune regulation, proteostasis/stress, and innate immune signaling. RT-qPCR further confirmed that in the RTP-2 group, Dram1 expression was upregulated approximately 121 times, Bmf1 expression was upregulated approximately 18 times, and Bnip3 was significantly downregulated, whereas Bnip3l expression remained unchanged. Overall, RTP-2 exhibited a more pronounced and coherent macrophage-stimulating profile in vitro, supporting its potential as a macrophage-targeted immunostimulatory ingredient.