## 4 Discussion  
This study shows that exosomes from the rhizomes of _Iris germanica_ L. suppress oxidative stress in keratinocytes. Interestingly, the exosomes were not very effective at scavenging radicals (Figure 3). Nevertheless, cell viability loss caused by H\({}_{2}\)O\({}_{2}\) treatment was suppressed via treatment with exosomes, suggesting that they are responsible for removing ROS from keratinocytes. The results of the WST-1 and DCF-DA assays show that exosome treatment significantly reduced intracellular ROS generation caused by H\({}_{2}\)O\({}_{2}\) (Figures 2 and 3). Thus, _Iris_-exosomes are considered to indirectly improve intracellular antioxidant function rather than provide benefits directly. It is possible to analyze the purity of human-derived exosomes by comparing membrane protein content before and after separation using membrane proteins such as CD81, CD63, and CD9. However, in the case of plant exosomes, no clearly identifiable membrane proteins have yet been identified; even in the case of some membrane proteins that have been identified, significant differences are expected among species. Our group, therefore, conducted additional follow-up studies to overcome this limitation (phospholipid material analysis, unique marker membrane protein analysis, etc.). As a quantitative method, NTA was used to verify plant exosomes, while oil red O and cryo-TEM were used as qualitative methods. In the future, more sophisticated analytical methods should be developed to identify the characteristics of plant-based exosomes in detail. The results of our study indicate that p21-p53 molecules play a more important role in protecting nHEK cells from H\({}_{2}\)O\({}_{2}\)-induced cellular senescence than p16 molecules. ROS production is closely associated with cellular senescence. Antioxidant enzymes, such as CAT and SOD, can alleviate cellular senescence by regulating intracellular ROS levels [67]. Because _Iris_-exosomes upregulate antioxidant enzymes, we examined whether _Iris_-exosomes can counteract cellular senescence in H\({}_{2}\)O\({}_{2}\)-induced nHEKs. Treatment with _Iris_-exosomes reduced the activity of SA-\(\beta\)-gal, a marker of senescence (Figure 4). Furthermore, the expression of _p21_ mRNA decreased in nHEKs treated with _Iris_-exosomes rather than just H\({}_{2}\)O\({}_{2}\) (Figure 4). Moreover, H\({}_{2}\)O\({}_{2}\) treatment increased the protein levels of p21, p53, and p-p53, whereas _Iris_-exosome treatment decreased their levels in a concentration-dependent manner (Figure 5). These results indicate that _Iris_-exosome inhibits cellular senescence caused by H\({}_{2}\)O\({}_{2}\) via the activation of the p53-p21 pathway. Another interesting finding is that the expression of p16 and Lamin B1, other known senescence markers, did not change. The relationship between p16 expression and cellular senescence has been previously reported in many research papers. With age, the epidermis and dermis accumulate p16- and SA-\(\beta\)-gal-positive cells [68; 69; 70; 71; 72; 73; 74]. [PERSON] et al. showed that ROS-induced senescence may be triggered by the demethylation of the promoter region of p16 in normal epidermal keratinocytes [75]. These findings indicate that p16 is a marker of cellular senescence in keratinocytes. However, other studies have suggested that p16 is not a reliable senescence marker in keratinocytes. From the in vitro study of skin equivalents, different investigators have reported different results for p16 expression. Some have observed an inverse correlation with the expression of Ki-67, a proliferation marker in skin biopsy, whereas others have reported no change or an increase [76; 77]. In addition, nHEKs treated with H\({}_{2}\)O\({}_{2}\) and/or _Iris_-exosomes did not exhibit any changes in p16 mRNA or protein levels.