## 4 Discussion  
Several different mechanisms have been suggested for the effectiveness of indigo naturalis on psoriasis. Indigo naturalis and its constituent tryptanthrin showed anti-angiogenic activity by inhibiting cell-cycle progression in endothelial cells [24]. Tryptanthrin was also reported to inhibit IL-17-induced inflammatory responses in human keratinocytes [22]. The other components of indigo naturals may have therapeutic potential through different mechanisms. Hence, this study examined their IL-22 targeting mechanism. Indeed, IL-22 stimulation increased ROS production and inflammatory mediator levels in keratinocytes, which were reversed by a pre-treatment with indiirubin. This study also identified the potent anti-proliferation and cell-cycle-modulating activities of indiribin in IL-22 treated keratinocytes. For the first time, the present study indicated that indiribin, the active compound of indigo naturalis, has anti-posoriatic effects through the IL-22 targeting mechanism. Our findings may provide new insights into the anti-posoriatic efficacy of indigo naturals and help to set a more accurate target in the further development of indigo naturalis components as effective therapeutic agents. IL-22 is a Th17-derived cytokine that plays a crucial role in psoriasis pathogenesis [25]. Keratinocytes, which are located in the outermost skin layer, are the main downstream targets of IL-22 [26]. IL-22 elicits profound effects on cellular processes in keratinocytes, such as proliferation and secretion of inflammatory mediators [15; 27]. The administration of IL-22 to human skin xenografts resulted in psoriasis-like symptoms, including epidermal hyperplasia and increased vascularization [28]. Similarly, the topical application of imiquimod on the skin failed to induce epidermal hyperplasia or inflammation in IL-22-deficient mice [29]. These results suggest that targeting IL-22 production and the related signaling pathways might be potential therapeutic approaches for psoriasis treatment. Figure 5: Effects of indiribin on epidermal hyperplasia and keratinocyte proliferation in IL-22-treated BALB/c mice. (**A**) Ear thicknesses were measured using a digital thickness gauge. (**B**) H&E staining and immunohistochemical analysis using anti-K16 in mouse ear tissues. Scale bar = 50 \(\upmu\)m. (**C**) Protein levels of K16 in ear tissue lysates. Data represent the means \(\pm\) SDs (n = 5 per group). # \(p\) \(<\) 0.05 vs. the normal control (NC) group, * \(p\) \(<\) 0.05 vs. the IL-22-treated group. ROS can induce angiogenesis and an inflammatory response to trigger the development of psoriasis [30]. Notably, IL-22 can induce an inflammatory response via ROS generation in keratinocytes [15]. This study indicated that IL-22 increased ROS production and TNF-\(\alpha\), IL-6, ICAM-1 secretion in HaCaT cells. These inflammatory mediators could regulate the development of Th17 or induce neutrophil infiltration in psoriatic lesions [31; 32; 33]. In comparison to IL-17, IL-22 has a minor influence on the inflammatory responses in keratinocytes, but it can amplify the effects of the pro-inflammatory cytokines in psoriatic lesions [27; 34]. In the present study, IL-22 significantly, but weakly, induced ICAM-1 production and failed to stimulate the production of IL-8 in HaCaT cells. Among the three components, only indiribuin inhibited ROS production and TNF-\(\alpha\), IL-6, and ICAM-1 secretion in IL-22-treated keratinocytes. This is consistent with previous research results that reported the antioxidant and anti-inflammatory potential of indiribuin [35; 36; 37]. Oxidative stress and inflammatory response play a crucial role in the modulation of cell proliferation. Although higher levels of ROS can induce signal transductions leading to apoptosis and necrosis, lower doses of ROS, in contrast, trigger cell survival and proliferation, which is an important hallmark of psoriasis [38].