## 4 Discussion
In the current study, we indicated that an IL-22 treatment resulted in ROS generation at a low degree (increased approximately 10% of ROS-positive cells, compared with the normal control), hence, up-regulated ROS generation upon the stimulation of IL-22 might contribute to keratinocyte proliferation. Similarly, chronic low-grade inflammation enhances cell growth in proliferative diseases, however, high doses of pro-inflammatory cytokines can promote cell death [39; 40]. Interestingly, the pro-inflammatory capacity of IL-22 is weaker than that of IL-17, whereas IL-22 cannot only cause inflammation but also promote the proliferation of keratinocytes [15]. In the future, the relationship between low-grade inflammation and proliferation under IL-22 stimulation should be studied. IL-22 could induce keratinocyte proliferation, migration, and inhibit differentiation, leading to the histological features that are observed in psoriatic lesions such as epidermal hyperplasia and parakeratosis [26]. In this study, a cutaneous injection of IL-22 increased K16 expression and caused epidermal hyperplasia. Similarly, an IL-22 treatment increased cell proliferation and the proportion of cells entering the S phase. Indirubin could reduce the expression of IL-22-induced cell-proliferation markers K16 and Ki67 and suppress epidermal hyperplasia. Note that this concentration of indiribuin is comparable to that of common topical corticosteroid doses for psoriasis treatment [41]. The in vitro experiment was conducted at non-toxic concentrations and the in vivo experiment was performed by referring to the previous studies. The topical application of 0.1-10% indiribuin effectively suppressed skin inflammation and hyperkeraatosis in dermatitis animal models, and the 10% indigo naturalis ointment significantly improved the severity score in moderate psoriasis patients [22; 42]. Although indiribuin toxicity is low, further experiments should be performed to verify the optimal concentration of indiribuin [43]. The S phase of the cell cycle, which includes DNA replication, is critical for cell proliferation [44]. The progression is modulated tightly by cyclins and cyclin-dependent kinases (CDKs). Notably, cyclin D-CDK4/6 and cyclin E-CDK2 complexes contribute to the transition from the G1 to the S phase [45]. In psoriasis, the elevated proportion of cells entering the S phase was associated with epidermal hyper-proliferation [46]. The expressions of cyclin D1 and cyclin E-CDK2 complex were also up-regulated in psoriatic lesions [47; 48]. In this study, an IL-22 treatment increased the proportion of cells entering the S phase and increased the expression of cyclin D1 and cyclin E1. All of the components, i.e., indiribuin, indigo, and tryptophan, caused G0/G1 cell-cycle arrest, but only indiribuin lowered the proportion of cells entering the S phase and the expressions of cyclin D1 and cyclin E1. These results are comparable to those of a prior study reporting that indiribuin can regulate the cell cycle to inhibit keratinocyte proliferation and promote differentiation [49]. Previous results also showed that tryptophan treatment did not inhibit cyclin D or cyclin E expression, which play crucial roles in the G1/S phase transition in the cell cycle of vascular endothelial cells [24]. The signal transduction pathway of IL-22 in psoriasis pathogenesis has been reported. IL-22 binds to IL-22R, and activates tyrosine kinases Tyk2 and JAK1, resulting in the STAT3 and ERK phosphorylation [10; 50]. Despite the wide range of activation of MAPK signaling pathways (including p38, ERK, and JNK), ERK-mediated signaling might be considered the main pathway in IL-22 signaling [50; 51]. When activated by IL-22, STAT3 and ERK1/2 can aggravate psoriasis by promoting the expression of inflammatory and proliferation-promoting factors [2; 51]. In the current study, indirubin reduced ERK and STAT3 phosphorylation in IL-22-treated HaCaT keratinocytes.