# Anti-Aging Effects of Nanovesicles Derived from Human Tonsil-Derived Mesenchymal Stem Cells
## 3 Results
### Anti-Aging Effect of TMSC-NVs in the Passage-Associated Senescence Model  
To confirm the anti-aging properties, TMSC-NVs were added to the passage-associated senescence model and cell morphology and proliferation was observed (Figure 2a,b). As shown in Figure 2a,b, treatment with TMSC-NVs increased the cell proliferation of old HDFs. We performed a SA-\(\beta\)-galactosidase assay to confirm the anti-aging role of TMSC-NVs (Figure 2c). The results show that treatment with TMSC-NVs decreased \(\beta\)-galactosidase activity of old HDFs. Quantitative data from the SA-\(\beta\)-galactosidase assay showed that the ratio of senescent cells was reduced by treatment with TMSC-NVs, which supported that treatment with TMSC-NVs decreased the senescence level of HDFs (Figure 2d).  
Additionally, the protein expression of vinculin in focal adhesion and the morphological change in the actin cytoskeleton followed by treatment with TMSC-NVs was examined (Figure 2e,f). The results of the immunofluorescence analysis show increased commitment of vinculin in focal adhesion in old HDFs, which was decreased by treatment with TMSC-NVs. These results show that TMSC-NVs increase the proliferation of HDFs and decrease the senescence induced by passages.  
Figure 1: Production of nanovesicles derived from tonsil-derived mesenchymal stem cells (TMSC-NVs). (**a**) Morphology of human tonsil-derived mesenchymal stem cells, scale bar = 200 \(\upmu\)m. (**b**) Expression of surface markers of human tonsil-derived mesenchymal stem cells. (**c**) Protein expression and SEM image of TMSC-NVs. Protein level was normalized by \(\beta\)-actin. (**d**) Analysis of dynamic light scattering of TMSC-NVs.  
Regulation of Extracellular Matrix and Anti-Oxidant Gene by TMSC-NVs in the Passage-Associated Senescence Model  
To confirm the anti-aging properties of TMSC-NVs in terms of molecular biology, the gene expression of the extracellular matrix (ECM) production and senescence-related anti-oxidant gene followed by TMSC-NVs treatment was examined (Figure 3a). The mRNA level of collagen type 1 (_COL1_) and _ELASTIN_, which is decreased in old HDFs compared to young HDFs, showed that treatment with TMSC-NVs resulted in upregulated ECM production. Similarly, the mRNA expression of _SOD2_ and _HMOX1_, the anti-oxidant gene, was increased by treatment with TMSC-NV in old HDFs. In addition, the protein expression of COL1 examined by immunofluorescence showed that treatment with TMSC-NVs resulted in significantly increased ECM production in the passage-associated senescent HDFs (Figure 3b,c). According to these results, treatment with TMSC-NVs resulted in recovery of the ECM production and senescence-reducible anti-oxidant gene in senescent cells.  
Figure 2: Regulation of proliferation and senescence by treatment with TMSC-NVs in the passage-associated senescence model. (**a**) Morphological change in HDFs. Scale bar = 200 μm. (**b**) Proliferation test of passage-associated senescent HDFs after treatment with TMSC-NVs. (**c**) Senescence-associated (SA) \(\beta\)-galactosidase assay, scale bar = 200 μm. (**d**) Quantitative analysis of the SA-\(\beta\)-galactosidase assay. (**e**) Expression of vinculin in focal adhesion of HDFs. (**f**) Quantitative data of vinculin expression in focal adhesion. Significant differences among groups were determined by one-way ANOVA (ns > 0.05, * \(p\) < 0.05, ** \(p\) < 0.01, *** \(p\) < 0.001).  
Figure 3: Regulation of extracellular matrix and anti-oxidant gene by treatment with TMSC-NVs in the passage-associated senescence model. **(a)** mRNA expression of _COL1_, _ELASTIN_, _SOD2_, and _HMOX1_ in passage-associated senescent HDFs after treatment with TMSC-NVs. **(b)** Immunofluorescence analysis of collagen type 1 of passage-associated senescent HDFs after treatment with TMSC-NVs. **(c)** Quantitative data of immunofluorescence analysis. Significant differences among groups were determined by one-way ANOVA (* \(p<0.05\), *** \(p<0.01\), *** \(p<0.001\)).