## 3 Results
### Iris-Exosome Is Capable of Attenuating Cellular Senescence Induced by H\({}_{2}\)O\({}_{2}\)  
In addition to assessing their radical scavenging activity in the intracellular environment, we investigated whether _Iris_-exosomes could protect nHEKs against oxidative-stress-induced cellular senescence. nHEKs were treated with various concentrations of _Iris_-exosomes and/or 200 uM H\({}_{2}\)O\({}_{2}\), and senescence-associated \(\beta\)-galactosidase (SA-\(\beta\)-gal) assay was conducted to examine the SA-\(\beta\)-gal-positive senescent cells. The results indicate that H\({}_{2}\)O\({}_{2}\) treatment only increased the number of senescent cells to 274.78% of the total reached in the untreated control group (100.00%); however, _Iris_-exosome-pretreated nHEKs reduced the number of senescent cells to 160.31%, 155.16%, and 138.37% (Figure 4a,b). It is well-known that _p16_ and _p21_ act as a cell cycle-dependent kinase inhibitor. RT-PCR was used to confirm these results via the analysis of the expression levels of senescent markers (_p16_ and _p21_). The results shown in Figure 4c,d indicate that the mRNA expression of _p21_ decreased concentration-dependently in the _Iris_-exosome-pretreated cells compared to the H\({}_{2}\)O\({}_{2}\)-treated cells. These results suggest that _Iris_-exosomes protect nHEKs against intracellular ROS production and cellular senescence induced by H\({}_{2}\)O\({}_{2}\). Iris-Exosome Inhibits H\({}_{2}\)O\({}_{2}\)-Induced Cellular Oxidative Stress via p38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway  
Regulation of p21, a senescence marker and cell cycle-dependent kinase inhibitor, is mediated by p53 in keratinocytes. Therefore, considering the above results, we examined the upstream regulators responsible for p21 regulation. Under the same conditions as the previous  
Figure 4: Anti-senescence effects of _Iris_-exosome against H\({}_{2}\)O\({}_{2}\)-induced cellular stress in nHEKs. (**a**) Representative images of nHEKs assayed for senescence-associated \(\beta\)-galactosidase (SA-\(\beta\)-gal; blue) (scale bar, 200 um). nHEKs were treated with _Iris_-exosomes for 4 h and oxidative stress caused via 4 h treatment of 200 uM H\({}_{2}\)O\({}_{2}\). The data were analyzed after 40 h incubation. (**b**) Senescent cells were counted within the same cell number at least three times independently. The level of senescent cells compared with total cells was visualized as a graph. (**c**) RT-PCR was performed as the previous condition to analyze senescence-related genes (_p16_, _p21_, and _p63_). (**d**) The mRNA levels of the senescence marker were analyzed via real-time qPCR. Each value was normalized to _GAPDH_ and _\(\beta\)-actin_ as a loading control. The result is expressed as the average of the values quantified by _GAPDH_ and _\(\beta\)-actin_. ***_p_ \(<\) 0.001 compared with untreated cells; ***_p_ \(<\) 0.01 and ***_p_ \(<\) 0.001 compared with H\({}_{2}\)O\({}_{2}\)-treated cells. experiments, immunoblotting, would be evaluated the expression of p53 and p-p53 (ser15). Similar to the previous RT-qPCR, the expression of p16 and Lamin B1, which are known to be cellular senescence markers, did not change (Figure 5a). Similar to p21, H2O2 treatment upregulated the expression of p53 and p-p53 (Figure 5a). Additionally, the expression of p21, p53, and p-p53 was downregulated by H2O2 after pretreatment with _Iris_-exosomes in a concentration-dependent manner (Figure 5a). According to these findings, the changes in p21 caused by H2O2 and the _Iris_-exosome treatment resulted from p53 action. Thus, we determined that the MAPK signaling pathway was involved in the nHEK treatment with H2O2 and _Iris_-exosomes. H2O2 significantly increased the phosphorylation levels of p38, ERK, and JNK in the nHEKs (Figure 5b). However, the _Iris_-exosomes markedly inhibited the H2O2-induced phosphorylation of Threonine180 and Tyrosine182 residues in p38 (Figure 5b). Similarly, the phosphorylation of JNK at threonine 183 and tyrosine 185 was also reduced by the _Iris_-exosomes (Figure 5b).