## 3 Results
### Iris-Exosome Improves Oxidative-Stress-Induced Impairment of Keratinocyte Barrier Function  
Keratinocytes play important roles in epidermal differentiation and skin regeneration. Oxidative stress induced by hydrogen peroxide causes premature keratinocyte senescence and abnormal terminal differentiation [25; 27]. According to our findings, _Iris_-exosomes protect nHEKs from oxidative stress and cellular damage. Therefore, we tested whether _Iris_-exosome exerts protective effects against H2O2-mediated cell dysfunction during differentiation and regeneration. First, to determine whether _Iris_-exosomes could restore  
Figure 5: Anti-senescence effect of _Iris_-exosome is associated with p38 and JNK phosphorylation. (**a**) Protein levels of senescence markers (p16, p21, p53, p-p53(S15), and Lamin B1) analyzed using Western blot. (**b**) The protein levels of MAPK Kinase (ERK, JNK, p38) were assessed using Western blot. \(\beta\)-actin was used as a loading control. The results are presented as mean \(\pm\) SD (\(\textit{t}=3\)). \(\mathbf{\texttt{{}^{***}}}\)\(\textit{p}\)\(<\) 0.001 compared with untreated cells; \(\#\)\(\textit{p}\)\(<\) 0.05, \(\#\)\(\textit{p}\)\(<\) 0.01 and \(\mathbf{\texttt{{}^{***}}}\)\(\textit{p}\)\(<\) 0.001 compared with H2O2-treated cells.  
the abnormal differentiation caused by H2O2 treatment in nHEKs, Western blotting was performed to target differentiation markers in nHEKs treated with CaCl2 (1.2 mM) for 12 days. In the presence of CaCl2, a significant increase in the expression of the intermediate differentiation markers K1, K10, and LOR was observed on days 8 and 12 after treatment with CaCl2 (Figure 6a). Interestingly, compared to the control cells (CaCl2-treated only), we found that the H2O2-treated group showed early signs of upregulated intermediate differentiation markers, K1, K10, and LOR proteins, on day 3 after treatment with CaCl2 (Figure 6a). _Iris_-exosomes, however, attenuated the early increase in the expression levels of the differentiation markers caused by H2O2 to levels similar to those of the control group (Figure 6a). Then, we tested the protective effects of exosomes against H2O2-induced cellular damage using a wound-healing assay. We treated nHEKs with _Iris_-exosomes for 4 h, treated them with 200 uM H2O2 for 4 h, and then scratched them with a sterile pipette tip. The change in the gap width after 48 h was used to determine wound-healing ability. The results show that groups treated with _Iris_-exosomes had higher scratch closure rates than those treated with H2O2 alone (Figure 6b). Altogether, these results indicate that _Iris_-exosome is a bioactive component found in _Iris germanica_ L. rhizome, displays limited cytotoxicity, and exhibits a protective effect on oxidative-stress-induced cellular dysfunction in nHEKs.  
Figure 6: Protective effects of _Iris_-exosome on H2O2-induced impairment of keratinocyte barrier function in nHEKs. (**) Protein levels of differentiation markers (K1, K10, and LOR) were assessed using Western blot. nHEKs were treated with _Iris_-exosomes for 4 h and with 200 uM H2O2 for 4 h. They were then incubated for 0, 4, 8, 12 days in the medium including 1.2 mM CaCl2. (**) Evaluation of scratch wound healing. Cells were treated using _Iris_-exosomes and/or 200 uM H2O2 for each 4 h and then scratched with a sterile pipette tip after cell confluence. The results were analyzed using a microscope and the ImageJ software. Data are expressed as the mean \(\pm\) SD (\(n=3\)). **\(p<0.01\) and ***\(p<0.001\) compared with untreated cells.