Etanercept ameliorates psoriasis progression through regulating high mobility group box 1 pathway

Abstract Background As a common skin disease, psoriasis is related to inflammation and immune response. Due to the frequent recurrence of psoriasis, the treatment of psoriasis remains a clinical challenge. As an effective tumor necrosis factor‐alpha (TNF‐α) inhibitor, etanercept has been used for the treatment of psoriasis. However, some patients with psoriasis have no response to etanercept or discontinue treatment. To improve the therapeutic effect of etanercept, searching the potential biomarkers and investigating the related mechanisms of etanercept in the treatment of psoriasis are vital. Materials and methods We stimulated HaCaT cells with lipopolysaccharide (LPS) to generate cellular psoriatic changes and established an imiquimod (IMQ)‐induced psoriasis‐like mouse model, and then used an etanercept to treat cell and mouse model. Results Etanercept alleviated IMQ‐induced pathological changes and inflammation, and it also decreased the protein expression of high mobility group box 1 (HMGB1), receptor for advanced glycation end‐products, and toll‐like receptor 4. Moreover, the results of in vitro experiments showed that etanercept inhibited proliferation and inflammation, and promoted cell cycle arrest and apoptosis in LPS‐treated HaCaT cells. Knockdown of HMGB1 further enhanced the inhibitory effects of etanercept on LPS‐treated HaCaT cell viability and inflammation, while overexpression of HMGB1 notably reversed the inhibitory effects of etanercept on LPS‐induced HaCaT cell viability and inflammation. Conclusion Etanercept inhibited proliferation and inflammation and promoted cell cycle arrest and apoptosis in LPS‐induced HaCaT cells, and etanercept ameliorated inflammation in a psoriasis‐like mouse model.


INTRODUCTION
Psoriasis is a chronic autoimmune disease, which affects 1%-3% of the population worldwide. 1 Psoriasis is clinically manifested as thickening and scaling of skin lesions, which are characterized by epidermal hyperplasia, aberrant differentiation of keratinocytes, and immune cell infiltration. 2 Psoriasis has a high incidence rate and is prone to relapse, and is usually accompanied by complications, including type 2 diabetes, cardiovascular disease, and depression. 3,4 Notably, psoriasis has a substantial negative impact on the daily quality of life of the patients, such as limitations on clothing, relationship problems, and fertility problems. 5 However, the exact pathogenesis of psoriasis has not been fully understood, and there is often no complete cure without a complete treatment plan.
The pathogenesis of psoriasis is complex and uncertain and involves heredity, immune regulation disorders, environmental factors, and so on. 6 According to the evidence from molecule and clinic, neutrophils, T cells, and inflammatory cytokines (such as tumor necrosis factoralpha [TNF-α], interleukin [IL]-23, IL-6, and IL-8) are the key mediators for psoriasis progression. [7][8][9] Compared with the uninvolved skin of patients with psoriasis and healthy individuals, TNF levels in psoriasis tissue are elevated, suggesting that blocking TNF activity contributes to treating psoriasis. 10 At present, some patients with psoriasis are incurable, but it can be controlled with medication. 11 Etanercept, a dimeric fusion protein, is considered to be an effective TNF-α inhibitor. 12,13 Clinically, etanercept can be used to treat rheumatoid arthritis, ankylosing spondylitis, and psoriasis. [14][15][16][17][18][19] For patients with moderate to severe plaque psoriasis, etanercept is an important therapeutic option. 19 In the USA, 50 mg twice weekly (BIW) for 3 months followed by a maintenance dosage of 50 mg/week is the recommended dosage of etanercept for patients with psoriasis. 18 Nestorov 20 High mobility group box 1 (HMGB1), also known as amphoterin, is a ubiquitous nuclear protein in all cell types. 21,22 After cell damage, HMGB1 can be released into the extracellular milieu, where it functions as a damage-associated molecular pattern molecule. 23

HMGB1
can activate the nuclear factor kappa B (NF-κB) pathway and subsequently induce pro-inflammatory cytokines, such as IL-1 and TNF-α. 24 HMGB1 is reported to modulate cytokine-like activity by interacting with cellular receptors, such as receptor for advanced glycation end products (RAGE) and toll-like receptor 2/4 (TLR2/4). 25 Moreover, extracellular HMGB1 enhances the inflammatory response by binding to other pro-inflammatory mediators, such as nucleic acids, lipopolysaccharide (LPS), IL-1α, and β. 26 Recently, a large number of studies have revealed that HMGB1 plays important role in many types of inflammatory and autoimmune diseases. [27][28][29] It has been found that the level of HMGB1 in the serum of patients with psoriasis was increased, and HMGB1 expression was detected in the cytoplasm of psoriatic lesions. 21,30 These studies suggest that HMGB1 may play a key role in the pathogenesis of psoriasis. However, detailed studies investigating the role of etanercept and HMGB1 in psoriasis are lacking.
In this research, we hypothesized that etanercept regulated the HMGB1 pathway to influence psoriasis progression. Psoriasis-like lesions induced by imiquimod (IMQ) are similar to human psoriasis in phenotype and histological characteristics. 31 The aim of this study was to evaluate the influences of etanercept on LPS-induced human keratinocyte HaCaT cells and IMQ-induced psoriasis-like mouse model. Moreover, the possible molecular mechanism related to the HMGB1 signaling pathway was also analyzed. Our findings may be helpful for understanding the therapeutic effects of etanercept in psoriasis.

Cell culture
HaCaT cells were obtained from the Guangzhou Cellcook Biotech

Cell treatment and transfection
HaCaT cells were starved in a serum-free medium for 12 h before treatment and transfection. Next, HaCaT cells were exposed to dif-

Cell viability assay
In accordance with the manufacturer's instructions, Cell Counting

Cell cycle analysis
Cell cycle analysis was performed with cell cycle assay kit reagents

Western blot analysis
The total proteins were extracted from tissues and HaCaT cells using RIPA lysis buffer (Beyotime Biotechnology, China). Protein concentration was determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific

2.9
Enzyme-linked immunosorbent assay After the indicated treatment, cell culture supernatants from HaCaT cells were collected and stored at -20˚C for cytokine measurements. staining, and skin samples of four mice were used to perform RT-qPCR and western blot assay.

H&E staining
The skin of mice was collected and fixed in 10% neutral-buffered formalin (Sigma-Aldrich). Next, a graded series (70%, 80%, 95%, and 100%) of alcohol solutions were used to dehydrate, and then the samples were embedded in paraffin. Subsequently, the tissue samples were sliced into 5 μm sections, and the sections were dewaxed with xylene and subjected to gradient dehydration. Finally, the sections were stained with hematoxylin and eosin (Merck KGaA, Darmstadt, Germany), and pathological changes were evaluated under a light microscope (Zeiss, Germany). 44 Image-Pro Plus software (NIH) was made by use of analyzing the epidermal thickness.

Statistical analysis
GraphPad Prism7.0 software (USA) and SPSS software were used for statistical analysis, and all data were presented as the mean ± standard deviation from three independent experiments. Statistical significance was analyzed using a one-way analysis of variance followed by Tukey's post hoc test. p-Values less than 0.05 were considered statistically significant.

Etanercept inhibits proliferation and induces cell cycle arrest and apoptosis in HaCaT cells
To  Figure 1E). We then measured the expression of apoptosis-related proteins (cleaved caspases-9 and cleaved caspases-3) using western blot analysis. As shown in Figure 1F

Etanercept inhibits proliferation and promotes cell cycle arrest and apoptosis in LPS-induced HaCaT cells
To investigate the effects of etanercept in psoriasis, we stimulated HaCaT cells with LPS to generate cellular psoriatic changes. As shown in Figure 2A Figure 2D).

Etanercept decreases the production of inflammatory cytokines and inhibits the HMGB1 signaling pathway in LPS-induced HaCaT cells
The effect of etanercept on the inflammatory cytokines (IL-8, IL-6, and TNF-α) in LPS-induced HaCaT cells were measured using commercial ELISA kits and RT-qPCR assay. As shown in Figure 3A Figure 4I). In addition,

Etanercept ameliorates inflammation and inhibits HMGB1 signaling pathway in an IMQ-induced psoriasis-like mouse model
To further analyze the anti-psoriatic effect of etanercept in vivo, we established an IMQ-induced psoriasis-like mouse model. As shown in Figure 5A, IMQ increased the epidermal thickness, induced the formation of rete ridges, and was accompanied by inflammatory cell infiltration, while MTX (as a reference agent) and etanercept reduced epidermal thickening and inhibited the formation of rete ridges and inflammatory cell infiltration induced by IMQ. In addition, the mRNA expression of IL-8, IL-6, and TNF-α was measured using RT-PCR assay.
The results showed that the mRNA expression of IL-8, IL-6, and TNF-α was significantly higher in IMQ-induced mice than in those from con-

DISCUSSION
Psoriasis is a common immune-mediated skin disease, which causes complications (such as kidney dysfunction and hepatic inflammation) and brings great damage to the quality of patients' life. 41,[45][46][47] The pathogenesis of psoriasis is involved in the hyperproliferation of keratinocytes and infiltration of immune cells (such as T cells, neutrophils, and macrophages) in the skin. 48 In the present study, the aim was   76,77 In the immune response, NF-κB is a key downstream signal pathway of TLR regulation. 78 The activation of the NF-κB pathway causes the release of proinflammatory cytokines, such as TNF-α and IL-1β. 79  However, there is a limitation in the study. The animal study was only divided into control, Model, Model+MTX, and Model+Etanercept group. In order to make the experiment more complete, we should add the Normal group (healthy mice without any treatment) and the Model+vehicle group.

CONCLUSIONS
Overall

ACKNOWLEDGMENTS
Not applicable.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

FUNDING INFORMATION
No funding.

DATA AVAILABILITY STATEMENT
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The experimental protocol of our study was performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by Taizhou People's Hospital.