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The use of adipose-derived stem cells (ADSCs) to enhance wound healing and tissue regeneration is progressively being accepted. Proteomic profiling of cultured ADSCs by mass spectrometry (MS) is a valuable tool to determine the identity of the proteins involved in multiple pathways, which make these ADSCs unique. In the current study, Nano-LC-MS/MS analysis was implemented on the membrane-free stem cell component (MFSCC), and the MS analysis revealed the presence of 252 proteins, that are involved in several biological functions, like metabolic process, biological regulation, developmental process, cell proliferation, and many more. Furthermore, bioinformatic analyses of the identified proteins in MFSCC found them to be involved in versatile pathways, like integrin pathway and wound healing response-related pathways. In addition, we also investigated the anti-inflammatory effects of MFSCC on lipopolysaccharide (LPS) stimulated mouse macrophage (RAW264.7) cells. The cell cytotoxicity of MFSCC was measured using MTT and LDH assays, the production of nitric oxide (NO) was measured by the Griess assay, and the protein expression levels of inducible nitric oxide (iNOS) and cyclooxygenase (COX-2) were examined by western blot analysis. The results showed that MFSCC concentrations ranging from 0.1 to 3  μ g/mL did not show any significant cytotoxicity in LPS-induced RAW264.7 cells. Treatment with MFSCC of LPS-stimulated RAW264.7 cells significantly suppressed the production of NO and the expression of iNOS and COX-2 proteins related to inflammation. The present findings lead to a better understanding of the therapeutic potential of MFSCC and strongly promote it for the future clinical development of novel non-cell-based stem cell therapeutics.

Proteome Profiling of Membrane-Free Stem Cell Components by Nano-LS/MS Analysis and Its Anti-Inflammatory Activity