Psoriasis is characterized by chronic scaly papular plaque, skin damage that can be generalized throughout the body, and is associated with many comorbidities, such as cardiometabolic disorders, stroke, chronic kidney disease, inflammatory arthritis, depression, and lymphoma[4]. Long course and repeated attack of psoriasis can reduce the quality of life of patients, resulting in a heavy burden on society and individuals. As a common herb for clearing heat and cooling the blood, Paeonia veitchii Lynch is widely used in the treatment of psoriasis caused by blood-heat.[12–14] The results of network pharmacology indicate that Punica granatum Linn is the material basis for the main pharmacological effect of Paeonia veitchii Lynch, therefore, the present study verified the ability of Punica granatum Linn in the anti-psoriasis activity through the experiments of the animal model.
In this study, we started with the CIBERSORT algorithm to screen the key genes for psoriasis pathogenesis, to investigate the relationship between the key genes for psoriasis pathogenesis and immune infiltrating cells, and to screen traditional Chinese medicines with therapeutic potential for psoriasis. After further analyzing and screening the 348 DEGs, we obtained four key psoriasis genes: PCNA, CXCL8, S100A9 and CCNB1. PCNA is a metabolic protein related to DNA replication, repair and cell cycle progression, and it plays an essential role in cell proliferation[15], reflecting the abnormalities in the proliferation and differentiation of psoriasis epidermal cells, and it can be used as the most critical gene for assessing the progression of psoriasis. CXCL8, or IL-8, is an essential pro-inflammatory factor involved in the pathogenesis of psoriasis[16], making it a potential therapeutic target for psoriasis[17]. It is involved in regulating keratinocyte proliferation, neutrophil infiltration and angiogenesis in psoriasis. CXCL8 receptors are found in keratinocytes of psoriatic lesions, and CXCL8 activates keratinocytes through autocrine secretion, and produces and releases inflammatory mediators, which contribute to the migration of neutrophils to the lesion site[18, 19]. Some studies have shown that CXCL8 protein levels are positively correlated with PASI scores, further suggesting that CXCL8 may be closely related to the severity of psoriasis. CXCL8 may be involved in the development of psoriasis, and in the future, it may be used as an effective indicator to predict the severity of psoriasis, which may provide a useful reference value for psoriasis and clinical diagnosis. S100 calcium-binding protein A9 (S100A9) is mainly expressed in neutrophils, monocyte-macrophage cell lines, and diseased keratinocytes[20]. S100A9 and S100A8 form a dimer that binds target proteins, transmits calcium signals, and regulates the concentration of calcium ions in the cytoplasm[21], which is involved in the processes of cell proliferation and differentiation, inflammation, and apoptosis. The S100A8 and S100A9 genes have been identified near the psoriasis susceptibility locus PSORS4[22], and their expression has been found in the early stages of psoriasis[23]. Serum levels of S100A8/A9 in psoriasis patients correlate significantly with disease activity, suggesting that these S100 proteins are potential causative factors in psoriasis.[24]. The pathogenesis of psoriasis has a closely connection with the cell cycle of keratinocytes, and CCNB1 is a cell cycle-related gene. Some studies have shown that CCNB1 and CDC20 are highly expressed in psoriasis lesions, which further suggests that CCNB1 may be closely related to the development of psoriasis vulgaris[25]. In conclusion, these key target genes in psoriasis may play important roles in regulating keratinocyte proliferation and differentiation, participating in inflammatory response, maintaining normal skin barrier function, and regulating cytoplasmic calcium ion concentration.
Immune infiltration analysis of 22 immune cells in the skin lesion tissue samples was performed by the CIBERSORT reverse convolution algorithm. From the results, it can be seen that helper T cells, unactivated dendritic cells, and CD8 + T cells had the highest percentage, and helper T cells and unactivated dendritic cells were also significantly elevated in the case samples. Multiple immune cells were elevated in psoriasis case samples, suggesting their importance in psoriasis. Immature DCs are very few in inflammatory diseases and the ability of migration is great; mature DCs can activate the initial T cells effectively, which is the central link to start, regulate and maintain the immune response[26, 27]. Immunological and inflammatory stimuli are essential for the development of psoriasis, and an imbalance in peripheral blood Th1/Th2 balance and activation of related signaling pathways are the main drivers of psoriasis in both the initiation and amplification phases of psoriasis development[28]. Among them, Th1 mainly secretes IL-2 and IFN-γ, which participate in cellular immunity and inflammation[29], while Th2 secretes IL-4 and IL-10, which participate in humoral immunity and inhibit inflammation[30]. Under normal circumstances, the relative balance of Th1/Th2 in the peripheral blood plays an immunoprotective role, while its imbalance will cause excessive inflammatory reactions[31]. Immune cell correlation analysis showed a significant positive correlation between γδ T cells and activated CD4 memory T cells, suggesting that the synergistic effect of these two immune cells has an important impact on psoriasis pathogenesis. An important feature that distinguishes memory T cells from initial T cells is the ability to produce cytokines efficiently and rapidly, and is a key mechanism by which CD4 + memory T cells exert their anti-infective effects. Studies on human psoriasis have shown that plasmacytoid dendritic cells contribute to psoriasis through the secretion of IFNα, and that IFNα induces the secretion of IFNγ and the psoriasis-associated growth factor IGF-1 by γδ T cells. In addition, γδ T cells also secrete TNF-α, IL- 17A and other cytokines related to the development of psoriasis, and IL-8, CCL3, CCL4, CCL5 and other inflammatory factors that chemotaxis inflammatory cells to the local area of the skin, and γδ T cells may play a key role in the disease progression of psoriasis. It can be hypothesized that the synergistic effect of these two types of immune cells may have an important influence on the pathogenesis of psoriasis, which can be verified by relevant experimental studies. The key psoriasis genes PCNA and CCNB1 were positively correlated with neutrophils and activated dendritic cells, and S100A9 and CXCL8 were negatively correlated with monocytes. It is suggested that the key psoriasis genes are related to the immune cell infiltration mechanism of psoriasis.
The mechanism of action of Paeonia veitchii Lynch on psoriasis was analyzed by using network pharmacology methods. The analysis and screening yielded 23 active ingredients, including catechin, baicalein, β-sitosterol, Punica granatum Linn, paeoniflorin, and salvianolic acid, and 150 targets of action, including CCNB1, CXCL8, PCNA, and S100A9, in this drug. The molecular docking results confirmed that Punica granatum Linn had the strongest binding ability and the most stable binding effect. KEGG showed that the highly relevant DEGs for psoriasis were enriched in NF- κB pathway and TLR4 pathway. Therefore, we constructed an IMQ-induced psoriasis model to validate the efficacy of the active ingredient. TLR4 plays an essential role in various inflammation-related diseases. Meanwhile, IMQ, as a TLR agonist, over-activates the TLR4 pathway, resulting in a prolonged and excessive inflammatory response. NF-κB, as a major pathway downstream of TLR4, was also over-activated in IMQ-stimulated and psoriasis patients. Western blotting results showed that NF-κB pathway was included in IMQ-induced inflammatory skin injury in mice, and Punica granatum Linn was able to alleviate IMQ-induced skin injury in mice by inhibiting the over-activation of TLR4/NF-κB signaling pathway. Mouse skin damage by restraining the over-activation of TLR4/NF-κB signaling pathway.
NF-κB p65 plays a key role in mediating a positive feedback loop in psoriasis, where its activation moves to the nucleus and stimulates the transcription of proliferative and inflammatory regulatory genes[32]. As a protein transcription factor, NF-ΚB is involved in regulating inflammation and some complicated biological processes. It is a key regulator of various immune and inflammatory responses, cell proliferation and differentiation. It promotes the expression of cytokines involved in psoriasis, involving IL-6, IL-1β, and TNF-α, among others. The results confirm Punica granatum Linn ameliorates histopathological changes and reduces the production of TNF-α and IL-6 by inhibiting the expression of TLR4 and the activation of NF-κB.