Endocrine Abstracts

Mesenchymal stem cell (MSC), as a significant part of regenerative medicine, has been considered as a promising therapy in several diseases. Recently, the secretion effect of MSCs has been found to play a key role in the regulation of inflammatory. Here, we screened different peptides with high expression abundance by mass spectrometry from the supernatant of human umbilical cord MSCs. Also, we identified a peptide (AQTKRLVGDVLLATAFLSYSGP) named MSC-P2 derived from dynein axonemal heavy chain 5. Thus, we explored the role and potential mechanism of MSC-P2 in regulating inflammation of RAW 264.7 cells. ProtParam tool was used to analyze the physicochemical properties of MSC-P2. The instability index (II) of MSC-P2 is computed to be 20.50, classifying itself as a stable peptide. The aliphatic index is 110.91 and grand average of hydropathicity (GRAVY) is 0.441, proving its hydrophobicity. MSC-P2 entered into the cytoplasm of RAW 264.7 cells in 24 hours by fluorescence microscope. Cell Counting Kit-8 detection showed that MSC-P2 exerted no significant cytotoxicity at the concentration from 1uM to 100 uM. Besides, Real-time PCR showed that the mRNA expression levels of iNOS, TNF-α, and IL-6 were significantly down-regulated in the MSC-P2 group, compared with the control group (P < 0.05). Consistently, the protein expression levels of iNOS and TNF-α were significantly reduced in the MSC-P2 group, compared with the control group (P < 0.05) by Western blot and immunofluorescence. RNA transcriptome sequencing identified 2028 up-regulated genes and 1819 down-regulated genes in the MSC-P2 group (| log₂FC | > 1 and FDR < 0.05). KEEG analysis revealed that the down-regulated differentially expressed genes (DEGs) were mainly enriched in the pro-inflammation pathways including cytokine−cytokine receptor interaction, NF−kappa B signaling pathway, and NOD−like receptor signaling pathways. Besides, the up-regulated DEGs were mainly concentrated in metabolic pathways associated with anti-inflammation roles including fatty acid metabolism, biosynthesis of unsaturated fatty acids and PPAR signaling pathway. PPI network of top 30 DEGs was built by using the STRING database and it revealed 40 nodes and 92 edges. Most of these genes involving the network, such as Isg15, Fasn, Psmd11 and Tlr4, have been reported with the roles in the regulation of inflammation. This study will help us better understand the protective functions of the secretome from MSCs. In addition, it will provide an anti-inflammatory peptide, which will become a potential drug for prevention and treatment of inflammatory-related metabolic diseases.