## 4 Discussion
A further investigation of the putative mechanism underlying _Iris_-exosome's protective effect against oxidative stress revealed that the exosome inhibits p53 activation by inhibiting the expression of p-p38 and p-JNK proteins, which are increased by H\({}_{2}\)O\({}_{2}\), thus reducing the expression of p21. MAPKs (ERK1/2, JNK, and p38) are activated by a variety of stimuli, such as heat shock and hydrogen peroxide [78; 79; 80; 81]. While these MAPK proteins are activated by ROS produced via UV irradiation or H\({}_{2}\)O\({}_{2}\) addition, JNK and p38 appear to be more sensitive to ROS than ERK1/2. It has been reported that excessive ROS production stimulates JNK activity, resulting in pathological conditions such as arthritis and cancer [79]. Additionally, various human cell lines have shown that ROS activates c-Jun N-terminal kinase (JNK) and/or p38 [82; 83; 84; 85]. In human keratinocytes, the same cell lines act independently in response to ROS production. These findings indicate that ROSmediated MAPKs are important mediators of ROS in the regulation of cellular function and the fate of keratinocytes. Additionally, MAPKs have been reported to regulate p53-p21 pathways upstream. According to one study, oxidative stress induced by H\({}_{2}\)O\({}_{2}\) activates both the p53/p21/Rb and p38 MAPK/MAPKAPK-2 pathways involved in the premature senescence of human endometrium-derived mesenchymal stem cells (hMESC) [86]. In other studies, p53 was shown to interact with MAPKs (ERK1/2, JNK, and p38) and phosphorylate and activate p53 in response to stress [87; 88; 89; 90]. According to these results, the MAPK-p53 signaling pathway contributes to the oxidative stress response of keratinocytes. Therefore, we examined how exosomes downregulate these senescence marker proteins. H\({}_{2}\)O\({}_{2}\) treatment increased the phosphorylation of p38 and JNK, whereas _Iris_-exosomes decreased this phosphorylation in a dose-dependent manner. In line with previous studies, oxidative stress was found to be associated with p38 and JNK mitogen-activated protein kinases (MAPKs). Consequently, it can be stated that _Iris_-exosome inhibits p38 and JNK protein expression, thus preventing p53 activation. As keratinocytes differentiate, the following four layers of structure form in the epidermis: the basal layer, spinous layer, granular layer, and stratum corneum. They play crucial roles in maintaining the skin barrier [24; 25]. Therefore, it is important to maintain a normal level of keratinocyte differentiation if one is to sustain a healthy epidermal layer. Studies have shown that ROS abnormally promotes keratinocyte differentiation, resulting in an incomplete epidermal layer [25]. Additionally, UV irradiation accumulates ROS levels in nHEKs and improves SA-\(\beta\)-gal activity, and 3D-reconstructed epidermis from UV-irradiated nHEKs shows thinner cross-sectional structures and more extensively differentiated cells [76]. These results indicate that oxidative stress disrupts the balance between the proliferation and differentiation of keratinocytes, resulting in the degeneration of most cells into aged and differentiated cells, as well as the breakdown of the skin barrier and a thinner epidermis. In this study, we found that the _Iris_-exosome offers recovery from intracellular ROS and investigated whether it protects keratinocytes from oxidative-stress-induced degeneration (differentiation). In our study, early-stage H\({}_{2}\)O\({}_{2}\) treatment resulted in significantly increased levels of intermediate differentiation markers, K1, K10, and LOR (3 days after CaCl\({}_{2}\) treatment). However, the changes in the levels of the differentiation markers were close to the control levels in the nHEKs that had undergone pretreatment with _Iris_-exosomes. These findings suggest that _Iris_-exosome may exhibit a potential protective effect against H\({}_{2}\)O\({}_{2}\)-induced degeneration of nHEKs.