# Anti-Aging Effects of Nanovesicles Derived from Human Tonsil-Derived Mesenchymal Stem Cells
## 3 Results
### Anti-Aging Effect of TMSC-NV in the UV-Induced Senescence Model  
Based on the results of the intrinsic senescence model, TMSC-NVs were added to the extrinsic senescence model, the UV-induced senescence model. As a result, proliferation of HDFs was decreased after UV irradiation and increased by treatment with TMSC-NVs, compared with UV-induced senescent HDFs (Figure 4a,b). In addition, the result of the SA-\(\beta\)-galactosidase assay shows that TMSC-NVs decreased the \(\beta\)-galactosidase activity of UV-induced senescent cells (Figure 4c) and the quantitative data from the SA-\(\beta\)-galactosidase assay support that UV-induced senescence was significantly decreased after treatment with TMSC-NVs, similar to the passage-associated senescence model (Figure 4d).  
Additionally, vinculin expression in the focal adhesion and arrangement of the cytoskeleton of HDFs was examined through treatment with TMSC-NVs (Figure 4e,f). The results of the immunofluorescence analysis show increased expression of vinculin in the UV-induced senescent HDFs and treatment with TMSC-NVs resulted in decreased expression of vinculin in focal adhesion. These results show that TMSC-NVs increase the proliferation of HDFs and decrease the cellular senescence in the UV-induced senescence model.  
Figure 4: Regulation of proliferation and senescence by treatment with TMSC-NVs in the UV-induced senescence model. (**a**) Morphological change in HDFs by treatment. Scale bar = 200 μm. (**b**) Proliferation test of UV-induced senescent HDFs after treatment with TMSC-NVs. (**c**) SA-\(\beta\)-galactosidase assay of UV-induced senescent HDFs after treatment with TMSC-NVs, 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 group were determined by one-way ANOVA (ns; not significant, * \(p<\) 0.05, ** \(p<\) 0.01, *** \(p<\) 0.001).  
Regulation of Extracellular Matrix and Anti-Oxidant Gene by Treatment with TMSC-NV in the UV-Induced Senescence Model  
To confirm the anti-aging properties of TMSC-NVs in a UV-induced senescence model in terms of molecular biology, mRNA expression of senescence-related ECM production and anti-oxidant gene was examined by qPCR. The results of qPCR show that _COL1_ and _ELASTIN_ were decreased after UV irradiation and treatment with TMSC-NVs resulted in significantly increased _COL1_; however, _ELASTIN_ was not increased. The anti-oxidant genes, _SOD2_ and _HMOX1_, were decreased by UV irradiation and increased by treatment with TMSC-NVs (Figure 5a). In addition, the results of immunofluorescence analysis show decreased expression of collagen type 1 in UV-induced senescent HDFs, which was increased by treatment with TMSC-NVs (Figure 5b,c). These results show that TMSC-NVs increase the ECM production and senescence-reducible anti-oxidant gene.  
Figure 5: Regulation of extracellular matrix and anti-oxidant gene by treatment with TMSC-NVs in the UV-induced senescence model. (**a**) mRNA expression of _COL1_, _ELASTIN_, _SOD2_, and _HMOX1_ in UV-induced senescent HDFs after treatment with TMSC-NVs. (**b**) Immunofluorescence analysis of collagen type 1 of UV-induced 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).