Study The Immunomodulatory Effect Of Ganoderma Atrum Polysaccharide On T Lymphocytes And Its Mechanism By Using RNA Sequencing

Ganoderma atrum is a valuable medicine and edible fungus,which is the best in Ganoderma,and has multifunctional bioactivity.In this study,we took polysaccharide from Ganoderma atrum?PSG-1?for research object,which was isolated and purified from the fruit bodies of Ganoderma atrum cultivated in Ganzhou,Jiangxi province,and investigated the immunomodulatory activity of PSG-1 on T lymphocytes as well as the underlying mechanism.The main research conclusions are concluded as follows:1.PSG-1 has immunomodulatory activity on T lymphocytes.In vitro studies,the results showed that PSG-1 could induce T lymphocytes proliferation,and increased the secretions of IL-2,IFN-?,and IL-12,but had an inhibitory action in Th17 cell and Treg cell differentiation.In vivo studies,the results demonstrated that PSG-1increased the Th17 cell-specific cytokines?IL-17A,IL-6?production,the transcription factors?STAT3,ROR?t?expressions and STAT3 phosphorylation.Meanwhile,the expressions of Treg cell-specific cytokines?TGF-?1,IL-10?and the transcription factor Foxp3 were also improved.Furthermore,PSG-1 increased the proportions of Th17 cells and Treg cells,and restored the ratio of Th17/Treg cells.The results above indicated that PSG-1 may play an important role in T lymphocytes immune regulation.2.By using the next generation sequencing technology?NGS?and based on Illumina NextSeq500 sequencing platform,the present study generated the transcriptome sequence information of T lymphocytes under the treatment of PSG-1.The raw data and clean reads of control groups were 6,383,864,400 bp,6,168,324,682bp,6,231,369,973 bp raw data and 6,176,198,712 bp,6,140,956,284 bp,6,204,194,525 bp clean reads.Meanwhile,the raw data and clean reads of PSG-1treatment groups were 6,486,661,500 bp,6,229,827,900 bp,6,479,696,700 bp and6,278,690,876 bp,6,128,994,770 bp,6,273,645,956 bp,respectively.The quality control of the sequence reads was estimated by various analysis methods,and the information indicated that the sequencing data could fully cover the expressed genes in both samples.3.Clean reads were aligned to mouse genome and unigenes with Ensembl databases.In totall,15629 genes were identified from control groups and PSG-1treated groups,of which,the up-regulated genes were 8156 and down-regulated genes were 7473.Among of them,394 differentially expressed genes?fold change>2,P-value<0.05?were screened,among them,283 genes were up-regulated and 111genes were down-regulated.These differentially expressed genes laid a good foundation for exploring the immunoregulatory mechanisms of PSG-1 on T lymphocytes.Pathway analysis identified that significant enrichment of differentially expressed genes involved in 152 canonical pathways,and 30 canonical pathways were significantly enriched.The analysis demonstrated 11 significant enriched canonical pathways associated with immune regulation,including Toll-like receptor,MAPK pathway,Calcium pathway,Jak-STAT,Cytokine-cytokine receptor interaction and so on.The results above suggested the relationship of the various genes involved in PSG-1-stimulated T lymphocytes in the signaling pathway.4.PSG-1 induces the proliferation and the cytokines production of T lymphocytes,and possesses immunomodulatory activity on T lymphocytes via stimulating Ca²⁺/CaN and MAPK pathways.