Rutin alleviates psoriasis‐related inflammation in keratinocytes by regulating the JAK2/STAT3 signaling

Abstract Background Psoriasis is a chronic inflammatory skin disease that can cause systemic inflammation in various organs. Rutin has been suggested to fight psoriasis, but the signaling pathways by which it works need to be explored. Materials and methods HaCaT cells co‐stimulated with interleukin (IL)‐17, IL‐22, tumor necrosis factor‐alpha (TNF‐α), IL‐1α, and oncostatin M (M5) were used as an in vitro cell model of psoriasis. The proliferation and viability of HaCaT cells were determined by 5‐ethynyl‐2′‐deoxyuridine and cell counting assays. Relative mRNA levels of IL‐6, TNF‐α, chemokines (CXCL1 and CXCL2), and anti‐microbial peptides (S100A7 and S100A8) were detected by reverse transcriptase‐quantitative PCR. Release of IL‐6 and TNF‐α from HaCaT cells was measured by enzyme‐linked immunosorbent assay. Keratin1, Keratin5, p‐JAK2, and p‐STAT3 protein levels were estimated with western blotting. Molecular docking predicted binding sites for Rutin and STAT3. Results Rutin treatment undercut M5‐urged viability increase and proliferation boost in HaCaT cells. Moreover, M5 stimulation mediated upregulation of IL‐6, TNF‐α, CXCL1, CXCL2, S100A7, and S100A8 was partially reversed after Rutin treatment. In addition, M5 stimulation induced downregulation of Keratin1 and Keratin5 proteins as well as upregulation of p‐JAK2 and p‐STAT3 proteins were attenuated in response to Rutin treatment, manifesting that Rutin treatment inhibited M5‐promoted aberrant differentiation and impaired M5‐mediated activation of the JAK2/STAT3 signaling in HaCaT cells. Molecular docking discovered that residues GLN326 and ASP334 in STAT3 might bind to Rutin. Conclusion Rutin treatment blocked the JAK2/STAT3 signaling, thus attenuating psoriasis‐related inflammation and anomalous differentiation in keratinocytes.

large amounts of pro-inflammatory cytokines [(such as interleukin (IL)-17, IL-22, IL-23, IL-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α], which can then trigger the onset of psoriasis. 6These cytokines compel hyperproliferation and aberrant differentiation of keratinocytes, causing the cells to produce a variety of cytokines, chemokines, and anti-microbial peptides, which promotes a sustained pro-inflammatory response. 7Existing therapies for psoriasis provide temporary symptomatic relief rather than a cure, focusing on the use of monoclonal antibodies (such as anti-IL-17 antibody) and immunosuppressive drugs (such as cyclosporine). 8Therefore, the development of new strategies for curing the disease or relieving its symptoms in the long-term is urgently needed.
Recently, bioflavonoids have been applied in the healthcare system, thanks to their wide range of biological activities, low cost, and high safety profile. 9Rutin (also called quercetin-3-rutinoside) is a polyphenolic bioflavonoid whose natural sources are fruits, herbs, and plants. 10Currently, several pharmacological properties of Rutin have been demonstrated, such as antihypertensive, hypolipidemic, antiallergic, cytoprotective, anti-inflammatory, and anti-viral. 11Peres et al.
uncovered that Rutin has a distinct anti-oxidant potential in dermocosmetic preparations. 12Specifically, Rutin-mediated anti-inflammatory activity has been reported in several chronic inflammatory diseases, including rheumatoid arthritis 13 and multiple sclerosis. 14As a potent anti-inflammatory agent, Rutin has a potentially protective role in psoriasis, 15 but the signaling pathways by which it works need to be validated and explored.
In the current research, we used HaCaT cells stimulated with a panel of pro-inflammatory cytokines as an in vitro cell model of psoriasis to characterize the role of Rutin and its associated pathways in psoriasis, providing evidence in support of Rutin as a potential therapeutic agent for psoriasis.
Following incubation with an appropriate secondary antibody, protein bands were visualized using a high-signal ECL western blotting substrate (Cat#E411-04, Vazyme, Nanjing, China), and gray-scale values of all bands were obtained using ImageJ (NIH, Bethesda, Maryland, USA), and relative protein expression levels were determined using β-actin as a standard.

Molecular docking
A molecular docking assessment was performed to determine the binding affinity of Rutin toward STAT3.The structure of Rutin was obtained by Pubchem (https://pubchem.ncbi.nlm.nih.gov/compound/5280805).
For the three-dimensional structure of STAT3, it was obtained from the Protein Data Bank database (https://www.uniprot.org/).By utilizing the AutoDock 4.2 software, the binding sites between Rutin and STAT3 were predicted.Visualization of docking results was performed using PyMol 2.2.

Data analysis
All experiments were performed in triplicate with at least three biological replicates.Data were shown as mean and standard error mean.
Statistical analysis was calculated by Prism 9 (GraphPad Software, USA).Tests for differences among multiple populations were conducted with analysis of variance (ANOVA) with Dunnet's post-hoc test.
A p-value of less than 0.05 was considered significant.

Rutin restrained the abnormal proliferation but not the survival of M5-treated HaCaT cells
Rutin is a natural flavonoid glycoside with anti-inflammatory properties, whose chemical structure (molecular formula: These results manifested that Rutin could suppress the proliferation but not the survival of M5-treated HaCaT cells.

Rutin contributed to the differentiation of M5-stimulated HaCaT cells
Considering that the decreased terminal differentiation capacity of keratinocytes is a phenotypic feature of psoriatic lesions, 18 we further characterized the effect of Rutin on the differentiation capacity of M5-stimulated HaCaT cells by detecting the keratinocyte differentiation markers Keratin1 and Keratin5.The results showed that Keratin1 and Keratin5 protein levels were decreased in M5-stimulated HaCaT cells compared to control cells, but these decreased protein levels were partially reversed upon Rutin treatment (Figure 2A-C).These outcomes suggested that Rutin could ameliorate M5-induced aberrant differentiation of HaCaT cells.

Rutin repressed the inflammatory response of M5-treated HaCaT cells
Because psoriasis is a chronic inflammatory skin disease, we further analyzed the influence of Rutin on psoriasis-associated inflammation.
Consistently, M5-induced release of IL-6 and TNF-α was also attenuated by Rutin treatment (Figure 3C, D).As for the chemokines C-X-C motif chemokine ligand (CXCL) 1 and CXCL2, their mRNA levels were also upregulated after M5 stimulation, whereas Rutin treatment reversed M5 stimulation-mediated effects on CXCL1 and CXCL2 mRNA levels (Figure 3E, F).Studies have shown that anti-microbial peptides secreted by keratinocytes are associated with the severity of psoriatic lesions. 19Therefore, we explored the effect of Rutin on the anti-microbial peptides S100A7 and S100A8.Data showed that M5 stimulation significantly enhanced S100A7 and S100A8 mRNA levels, whereas M5-mediated upregulation of S100A7 and S100A8 mRNA levels was diminished post-treatment with Rutin (Figure 3G, H).Collectively, these results suggested that Rutin treatment decreased the inflammatory response of M5-treated HaCaT Cells.

Rutin suppressed the JAK2/STAT3 signaling in M5-treated HaCaT cells
Available evidence suggests that cytokines in the JAK/STAT pathway play a crucial role in common skin diseases, including psoriasis. 20 stimulation with M5 significantly elevated the protein levels of p-JAK2 and p-STAT3 in HaCaT cells, but the addition of Rutin weakened M5induced upregulation of p-JAK2 and p-STAT3 protein levels (Figure 4A-C).Application of computer-simulated molecular docking revealed that residues GLN326 and ASP334 in STAT3 might bind to Rutin, suggesting that Rutin may exert its effects by targeting STAT3 (Figure 4D).These data manifested that Rutin might target STAT3 to suppress the JAK2/STAT3 signaling in M5-treated HaCaT cells.
Therefore, exploration of novel botanical agents to alleviate the skin barrier disruption associated with psoriasis is necessary.Evidence to date has demonstrated that the prevention of inflammation-related damage is a definitive and attainable therapeutic goal for psoriasis. 4 is well established that Rutin is effective against multiple inflam-matory diseases, including psoriasis. 25,26Although Rutin attenuates inflammation associated with psoriasis, 26 the mechanisms mediated by which Rutin exerts its effects are unclear.Here, we demonstrated that Rutin mitigated psoriasis-associated inflammation by inactivating the JAK2/STAT3 signaling.
A mixture of M5 cytokines was able to induce keratinocytes to exhibit several features of psoriatic keratinocytes, 27 and many studies have used M5 cytokine mixtures to establish an in vitro model of psoriatic keratinocytes. 28,29The present study established an in vitro cell model of psoriasis by stimulating HaCaT cells with 10 ng/mL of M5 to investigate the role of Rutin and its associated signaling pathways.
Keratinocytes are not only an important component in maintaining skin structure but also a major player in the skin's immune sys- HaCaT cells through the Nrf2 signaling. 26Wang et al. exposed that the inflammatory response in psoriasis was lessened by Rutin through inactivating the AGE/RAGE signaling. 30Here, our results exhibited that Rutin treatment relieved M5-induced viability elevation and proliferation promotion for HaCaT cells.Moreover, M5-induced elevation of chemokines (CXCL1 and CXCL2), pro-inflammatory cytokines (IL-6 and TNF-α), and anti-microbial peptides (S100A7 and S100A8) were partly reversed following Rutin treatment, suggesting that Rutin treatment could mitigate psoriasis-related inflammation.
Keratin is the major structural protein of keratinocytes, maintaining the structural stability and integrity of keratinocytes. 31Abnormal expression of keratins is associated with a variety of skin diseases, and changes in keratin expression may cause incomplete differentiation of epidermal keratinocytes, leading to skin hyperplasia. 32Roth et al. reported that Keratin1 has an important role in the maintenance of skin integrity, and it is involved in the skin inflammatory network by mediating IL-18. 33Abnormal keratin5 expression impairs the structure and function of the basal cell keratin cytoskeleton, leading to skin fragility. 34Here, we observed that the inhibitory effects of M5 cytokines on Keratin1 and Keratin5 were impaired upon Rutin treatment, manifesting that Rutin treatment eased the abnormal differentiation of keratinocytes in psoriasis.
The JAK/STAT signaling is affected by a variety of growth factors and cytokines, participating in many important biological processes, such as cell apoptosis, proliferation, differentiation, and immunomodulation. 35Studies have shown that various herbal monomers or active ingredients may be involved in psoriasis by modulating the JAK/STAT signaling. 36For instance, kaempferol improved psoriasis by modulating IFN-γ-induced the JAK/STAT signaling. 37Total glucosides of paeony repressed the phosphorylation of STAT1 and STAT3, thus attenuating animal psoriasis. 38Centella asiatica blocked the JAK/STAT3 signaling, resulting in alleviating psoriasis. 39STAT3 is a key player in psoriasis pathogenesis and psoriasiform inflammation. 40AT3 activation in psoriatic keratinocytes is caused by phosphorylation of Tyr705 in STAT3 by stimulation of pro-inflammatory factors.41 A study showed that a strong increase in STAT3 transcriptional activity resulted from JAK2-dependent phosphorylation of Tyr705 mediated by IL-20 and IL-6.42 Here, the upregulation of p-JAK2 and p-STAT3 mediated by M5 stimulation in HaCaT Cells was undercut following Rutin treatment.Notably, computer-simulated molecular docking displayed that residues GLN326 and ASP334 in the DNA-binding domain (DBD) structural domain of STAT3 might bind to Rutin.The DBD domain of STAT3 helps to recognize and bind to its target DNA-GAS sequence.43 Also, the DBD domain is a "druggable" domain in STAT3, and interfering with DBD activity can directly inhibit the interaction of STAT3 with its target double-stranded DNA.44 Therefore, we inferred that Rutin may work by binding to residues GLN326 and ASP334 in STAT3 to inhibit the interaction of STAT3 with its target doublestranded DNA. Alutcomes manifested that Rutin might exert its role in M5-stimulated HaCaT Cells through controlling the JAK2/STAT3 signaling.The limitations of this study are as follows: (1) it was not verified whether STAT3 is a target of Rutin; (2) it was not verified that Rutin is effective against psoriasis by targeting STAT3 through animal models with psoriatic lesions.The above-mentioned limitations are the direction of our further research.

CONCLUSION
Rutin could lighten M5-induced inflammation and abnormal differentiation in HaCaT cells by mediating the JAK2/STAT3 signaling, which offers evidence to bolster the potential of Rutin as a drug for managing psoriasis.
C 27 H 30 O 16 ) is shown in Figure 1A.Inflammatory responses associated with psoriasis have been shown to be mitigated by Rutin, but the role of Rutin in psoriasis still needs to be further investigated.Here, we first estimated the cytotoxicity of Rutin on normal keratinocytes.CCK-8 assays showed that Rutin exhibited a suppressive influence on HaCaT cell viability at concentrations up to 50 µM, whereas 100 µM was close to the semiinhibitory concentration (Figure 1B).Based on safety considerations, we chose 10 and 30 µM for subsequent experiments.Subsequently, HaCaT cells were then co-stimulated with M5 to mimic psoriasis in vitro.As expected, M5 stimulation elevated cell viability and facilitated cell proliferation in HaCaT cells, but Rutin treatment restrained HaCaT cell viability and proliferation under M5 stimulation (Figure 1C, D).
tem.Multiple inflammatory mediators in psoriasis-affected lesions act directly or indirectly on keratinocytes, leading to hyperproliferation and activation of keratinocytes.Moreover, keratinocytes recruit T cells and neutrophils by secreting IL-6 and TNF-α, chemokines, and antimicrobial peptides, thereby contributing to dermal infiltration.The neutrophil chemokines CXCL1 and CXCL2 released by keratinocytes can further exacerbate inflammation by acting on vascular endothelial cells and mast cells to reactivate the central granulocyte axis.Thus, targeting the inflammation of keratinocytes is crucial for psoriasis.RecentF I G U R E 4 Rutin repressed the JAK2/STAT3 signaling in M5-treated HaCaT Cells.(A-C) Relative protein levels of p-JAK2 and p-STAT3 in M5-stimulated HaCaT cells and Rutin-treated M5-stimulated HaCaT cells were estimated by western blotting.***p < 0.001 vs. control; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. M5; ns: not significant (p > 0.05).Statistical differences were estimated by ANOVA.(D) Structural demonstration of the predicted binding mode of Rutin to STAT3.studies found that Rutin can alleviate psoriasis.Lang et al. uncovered that Rutin could lessen oxidative stress injury induced by H 2 O 2 in