PJ‑001, a small‑molecule proteolysis‑targeting chimera, ameliorates atopic dermatitis‑like inflammation in mice by inhibiting the JAK2/STAT3 pathway and repairing the skin barrier
- Authors:
- Published online on: February 29, 2024 https://doi.org/10.3892/etm.2024.12464
- Article Number: 176
Abstract
Introduction
Atopic dermatitis (AD) is a common chronic, recurrent inflammatory skin disease with a complex pathogenesis. Its main symptoms include dry skin, chronic eczematous lesions and severe itching. Patients often experience allergic rhinitis, asthma and other specific diseases, which significantly impact their quality of life (1). One of the key mechanisms involved in AD is the disruption of skin barrier function. Keratin 17 (K17) is the main structural protein and characteristic component of keratinocytes (KCs) and other epithelial cells. As a marker of cell proliferation and inflammation, K17 is rarely expressed in healthy skin, but is highly expressed in skin with abnormal differentiation and inflammatory injury (2). Loss of function and mutation of the filaggrin (FLG) gene reduce the accumulation of keratin filaments in the skin, thus affecting the levels of natural moisturizing factor, causing dry skin and creating conditions associated with the occurrence of AD (3,4). Therefore, increasing the FLG content and controlling K17 expression may be a viable strategy for the treatment of AD.
The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway mediates the intracellular signaling of various cytokines, including AD-related cytokines, in particular, Th2 cytokines, including interleukin (IL)-4, IL-5, IL-13 and IL-31, and thymic stromal lymphopoietin, contribute to the symptoms of chronic inflammation and pruritus in AD. Furthermore, the JAK/STAT is involved in the regulation of the epidermal barrier and the modulation of peripheral nerves related to the transduction of pruritus. The pathogenesis of AD is related to activation of the downstream JAK/STAT pathway by its receptors (5). When IL-4 and IL-13 bind to their respective receptors, the JAK family is activated and phosphorylates STAT3 (6-8). STAT3, a versatile transcription factor, promotes excessive proliferation of KCs and endothelial cells upon phosphorylation, causing increased permeability of the skin barrier to pathogens (9). In addition, IL-31 induces STAT3 activation in KCs when it binds to its receptor, yielding the production of β-endorphin, which may contribute to peripheral pruritus in AD. IL-6, as a cytokine that activates STAT3, has been shown to regulate autophagy through inhibitory or stimulatory effects (10). Moreover, STAT3 can influence autophagy regardless of its phosphorylation status (11). Beclin 1 plays a crucial role in the regulation of the autophagic process and serves as a marker for the initiation of autophagy. Microtubule-associated protein 1 light chain 3 (LC3) is involved in the formation of autophagosomes and serves as specific protein marker of autophagosomes. The combination of Beclin 1 and LC3 enables accurate detection of changes in autophagic activity. Autophagy plays a critical role in the differentiation of keratinocytes and in their function as a skin barrier. Autophagy occurs continuously and actively in the epidermal layer. Various stresses applied to the epidermis can serve as triggers to activate autophagy, which contributes to keratinocyte differentiation. For example, mitochondrial reactive oxygen species produced during oxidative phosphorylation and released into the cytoplasm in suprabasal keratinocytes trigger autophagy, which is necessary for epidermal differentiation (12). Therefore, inhibiting cytokine, growth factor and hormone receptor signaling pathways may prove to be an effective treatment for AD.
Proteolysis-targeting chimera (PROTAC) technology is a novel approach in small-molecule drug development. Over the past two decades, targeted protein degradation using PROTAC molecules to harness the ubiquitin proteasome system has transitioned from research to industry (13,14). Several companies have now disclosed programs at the preclinical and early clinical development stages. With clinical proof-of-concept demonstrated against two well-known cancer targets (oestrogen receptor and androgen receptor), research is now exploring previously considered ‘undruggable’ targets (14). PJ-001 is a cereblon-based degrader of JAK2 synthesized by our laboratory using PROTAC technology. In our previous experiment, PJ-001 was used to induce degradation of JAK protein in Jeko-1 cells and it was observed that the half-maximal degradation concentration (DC50) was <100 nM (unpublished data). The present study evaluated the therapeutic effect of PJ-001 using the 2,4-dinitrofluorobenzene (DNFB)-induced mouse model of AD, providing experimental and theoretical evidence for the potential application of small-molecule protein digesters in the treatment of AD.
Materials and methods
Reagents and instruments
DNFB was obtained from MilliporeSigma. Total RNA Extractor (Trizol), Tween-20, TBS buffer, Premixed Powder (1X) and TBE Buffer, Premixed Powder (1X) were purchased from Shanghai Shenggong Biology Engineering Technology Service, Ltd. Bradford protein concentration determination kit (detergent-compatible), bovine serum albumin (BSA) and RIPA buffer (cat. no. 89900) were purchased from Thermo Fisher Scientific Inc. BeyoRT™ III cDNA first-strand synthesis premix (5X), Easy-Load™ PCR Master Mix (Green), BeyoECL Moon, agarose, nucleic acid green, antibodies and primers were purchased from Beyotime Institute of Biotechnology. Crisaborole ointment was purchased from Pfizer, Inc. Compound dexamethasone ointment was purchased from China Resources Sanjiu Medical & Pharmaceutical Co., Ltd.
Drug formulation
A 0.5% DNFB solution was prepared by adding 51 µl DNFB solution (purity: 98%) to 9.949 ml acetone/olive oil solution (4:1 ratio by volume). The mixture was uniformly suspended by vortex shaking, resulting in a final volume of 10 ml (15). For 4% PJ-001 solution preparation, the aqueous phase was prepared by weighing 4 g PJ-001 sample and adding to the following reagents: 15 g 70% ethanol, 10 g propylene glycol, 10 g glycerol, 33.9 g water and 0.3 g nipagin. The oil phase consisted of 8 g Vaseline, 2.5 g monoglyceride, 2.5 g peregal, 5.5 g octadecyl alcohol and 8 g liquid paraffin. The aqueous and oil phases were heated and melted, mixed at 80˚C and stirred well. The mixture was then cooled to 30-40˚C to produce a cream (100 g). According to the preparation method of crisaborole ointment (2% concentration), 2% PJ-001 was also prepared (16).
Experimental animals
A total of 42 specific pathogen-free grade BABL/c male mice (age, 6 weeks; weight, 18-22 g) were purchased from Charles River Laboratories, Inc. The mice were housed in individually ventilated cages maintained at constant temperature (20-26˚C) and humidity (40-60%). The animals were housed in a 12 h light/12 h dark cycle, with free access to water and food. Both the feed and water provided to the mice were sterilized. The animal license number was SYXK (Su) 2016-0045. At the end of the experiment, the mice were euthanized with CO2 (17). The mice were placed in a container, and the concentration of CO2 was gradually increased using a flow rate of 5.5 l/min to achieve a CO2 displacement rate of 30% (18) until their breathing stopped (3.0-3.5 min). Subsequently, bilateral pneumothorax chest opening was performed on the mice to prevent their recovery from asphyxia. It is crucial to allow an adequate interval between euthanasia procedures, 1-2 min, to ensure the dissipation of CO2 from the container. This precautionary measure guarantees that subsequent groups of animals are not suddenly exposed to high levels of CO2 gas. At the end of euthanasia, mouse spleens are collected and weighed. The skin was collected from the lesion site for further experiments.
Establishment of a mouse model of AD
All animal experiments were approved by the Ethics Committee of Experimental Animal Management and Animal Welfare of Jiangnan University [approval no. JN. No20210930b0801218(353)]; Wuxi, China). After 1 week of adaptive feeding, the mice acclimated to the unfamiliar surroundings and were than randomly assigned to the following six groups based on their body mass: Control group, model group, dexamethasone group, crisaborole group, 4% PJ-001 group and 2% PJ-001 group (n=7 mice/group). The weight deviation of each mouse was consistently around 20% of the average body weight. The experimental process is shown in Fig. 1. On the first and second days of the experiment, 100 µl acetone/olive oil (4:1) solution without DNFB was applied to the back of the control group mice once a day. In all mice, with the exception of the control group, 100 µl acetone/olive oil (4:1) solution containing 0.5% DNFB was applied to the back once a day. On days 3-6 of the experiment, the intervention was suspended. On days 7-28 of the experiment, the control group was administered the vehicle [acetone/olive oil (4:1) solution without DNFB], and the remaining mice were coated with 70 µl acetone olive oil (4:1) solution containing 0.5% DNFB every 2 days (15,19). At the end of the modeling process, 2 non-compliant mice (with skin broken during shaving or being over/underweight) were excluded from each group, and the remaining mice were subjected to 6 days of topical administration. Both the control and model groups were administered 100 µl saline, twice a day. The other groups were treated with 60 mg compound dexamethasone ointment, crisaborole ointment (19), 4% PJ-001 or 2% PJ-001, twice a day.
Changes in mouse skin lesions and scratching frequency
On days 0, 2, 4 and 6 of administration, changes in the specific dermatitis lesions were observed and the severity of mouse skin lesions was scored using the scoring standard provided in Table I (20). At 1 h after the last application administration, scratching frequency in mice was recorded over a duration of 10 min; for all the mice, scratching the back skin once was counted as one effective scratching incident, and continuous scratching for >3 sec was counted as two incidents.
Hematoxylin and eosin (H&E) staining
Following euthanasia, skin from the back of the mice was collected and immersed in 10% (v/v) neutral formalin at 4˚C for 24 h; then, the tissues were embedded in paraffin and sliced into 5-µm slices. The samples were then placed in xylene for 5 min at room temperature for dewaxing. The xylene was then replaced with fresh xylene, followed by another 5 min of dewaxing. The samples were subsequently cleaned sequentially with different gradient concentrations of ethanol and distilled water to rehydrate them. Next, the sections were stained with hematoxylin solution for 5 min at room temperature. The excess staining solution was washed off by soaking the samples in water for ~10 min, followed by another wash in distilled water. The samples were then stained with eosin staining solution for 2 min and washed twice with water. Dehydration and permeabilization were performed using absolute ethanol and xylene sequentially, and the slices were sealed with neutral gum. Finally, the slides were placed under an optical microscope for image collection and analysis (15).
Western blotting
Mouse skin tissue was cut into small pieces and mixed with precooled RIPA lysis buffer. The mixture was then centrifuged at 4˚C and 9,500 x g for 10 min. The resulting supernatant was collected as the protein lysate. Protein quantification was performed using the Bradford protein concentration assay kit, and the proteins were separated on 10% gels using SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were then blocked with 5% BSA at room temperature for 1 h and incubated with the following primary antibodies: Toll-like receptor 4 (TLR4; 1:1,000; cat. no. AF8187), nuclear factor-κB (NF-κB; 1:1,000; cat. no. AF1234), JAK2 (1:1,000; cat. no. AF1489), STAT3 (1:1,000; cat. no. AF1492), Beclin 1 (1:1,000; cat. no. AF5123), LC3 (1:1,000; cat. no. AL221) and GAPDH (1:10,000; cat. no. AF1186) at 4˚C overnight. Subsequently, the membrane was incubated with secondary antibodies (1:10,000; cat. no. A0208) at room temperature for 1 h. The aforementioned primary antibodies were all anti-rabbit polyclonal/monoclonal antibodies from Beyotime Institute of Biotechnology. A 5% BSA solution and antibodies were prepared using TBS buffer containing 1% Tween-20. Beyo-ECL Moon Kit was used to develop the membrane and a Gel Imaging System (Tanon Science and Technology Co., Ltd.) was used to capture images. The gray value was analyzed using ImageJ (1.46r) software (National Institutes of Health) (21). The following formula was used for semi-quantification: Protein expression (% of control)=(GA-target/GA-GAPDH)/(GC-target/GC-GAPDH) x100. G indicates grayscale value; A-target indicates target protein of the model and treatment group; A-GAPDH indicates GAPDH protein of the model and treatment group; C-target indicates target protein of the control group; C-GAPDH indicates GAPDH protein of the control group.
Reverse transcription (RT)-PCR
Mouse skin tissue was cut into small pieces and precooled total RNA extractor (TRIzol) was added, according to the manufacturer's protocol. The extracted RNA sample was then placed into a MK-20 dry thermostat and heated at 70˚C for 5 min. The concentration and purity of the RNA sample were measured using a micro nucleic acid protein analyzer. RNA was then subjected to RT to obtain cDNA, and the reaction procedure was as follows: 42˚C for 60 min and 80˚C for 10 min. Then, primers were added to amplify the target gene; PCR conditions were as follows: 94.0˚C for 3 min; followed by 35 cycles at 94.0˚C for 30 sec, 56.0˚C for 30 sec and 72.0˚C for 1 min; and a final step at 72.0˚C for 1 min, finally maintained at 4˚C. Primer sequences are shown in Table II. The amplified products were subjected to 2% agarose gel electrophoresis, and the results were analyzed by ImageJ software with GAPDH as an internal control. The agarose gel was prepared using TBE buffer, then microwaved and mixed with Nucleic Acid Green.
Statistical analysis
Data were analyzed using GraphPad Prism 8.0.1 statistical software (Dotmatics), and the experimental results are presented as the mean ± standard deviation and median. Statistical analyses and comparisons among groups were performed using one-way analysis of variance followed by Tukey's post hoc test for parametric data, and the Kruskal-Wallis test followed by the Dunn-Bonferroni post hoc test for non-parametric data. P<0.05 was considered to indicate a statistically significant difference.
Results
Comparison of scratch frequency and skin lesion severity scores in mice
As shown in Fig. 2A, the number of scratches in the model group was significantly higher than that in the control group. However, the number of scratches in the compound dexamethasone group, crisaborole group and 2% PJ-001 group was significantly reduced compared with that in the model group (P<0.01). This finding indicated that 2% PJ-001 had anti-pruritic properties. The skin lesion score of the model group mice was higher than that of the control group (Fig. 2B). As expected, the crisaborole group, 4% PJ-001 group and 2% PJ-001 group exhibited a decrease in skin lesion scores.
Histopathological changes in mouse skin
As shown in Fig. 3B, compared with the control (Fig. 3A), the model group exhibited excessive and incomplete keratinization, thickening of the spinous layer, mild sponge edema, decreased hair follicles, dilated capillaries and a substantial presence of inflammatory cell infiltration. In comparison to the model group, the crisaborole group demonstrated a marked thinning of the epidermal layer and an increase in hair follicles, although incomplete keratinization and a small amount of inflammatory cell infiltration persisted (Fig. 3D). The 2% PJ-001 group exhibited a notable increase in the structure of hair follicles and a slight decrease in the thickness of the spinous layer; however, severe edema was observed (Fig. 3F). In the 4% PJ-001 group, the corneum and spinous layer became thinner, cell edema decreased and inflammatory cell infiltration decreased slightly (Fig. 3E). The compound dexamethasone group showed no marked difference compared with the control group, with only a slightly thicker epidermal layer and minimal infiltration of inflammatory cells. Cellular edema could still be observed (Fig. 3C).
Effects of PJ-001 on the JAK2/STAT3 pathway in mouse skin tissue
As shown in Fig. 4A-C, the protein expression levels of JAK2 and STAT3 in the AD model group were significantly increased compared with those in the control group (P<0.001). However, in the compound dexamethasone group, crisaborole group, 4% PJ-001 group and 2% PJ-001 group, the expression levels of JAK2 were significantly decreased compared with those in the model group (P<0.001). The expression levels of JAK2 in the 4 and 2% PJ-001 groups decreased by 65.2 and 61.3%, respectively. Moreover, PJ-001 also inhibited STAT3 expression. The expression levels of STAT3 in the 4 and 2% PJ-001 groups were significantly decreased by 53.4 and 29.8%, respectively (P<0.001).
Effects of PJ-001 on TLR4/NF-κB in mouse skin tissue
As shown in Fig. 4A, D and E, the protein expression levels of TLR4 and NF-κB were significantly higher in the AD model group than those in the control group (P<0.001). Compared with those in the model group, the expression levels of TLR4 were significantly downregulated in the compound dexamethasone group, crisaborole group and PJ-001 groups (P<0.01). Specifically, the protein expression levels of TLR4 in the 4 and 2% PJ-001 groups were decreased by 41.1 and 34.7%, respectively. In addition, the protein expression levels of NF-κB were significantly decreased in the compound dexamethasone group, crisaborole group, 4% PJ-001 group and 2% PJ-001 group (P<0.001). Specifically, the protein expression levels of NF-κB were downregulated by 46.6% in the 4% PJ-001 group and 39.9% in the 2% PJ-001 group. These findings indicated that the inflammatory pathway involving TLR4/NF-κB was inhibited by PJ-001.
Effects of PJ-001 on Beclin 1 and LC3 in mouse skin tissue
As shown in Fig. 4A, F and G, compared with those in the control group, the expression levels of Beclin 1 and LC3 (LC3II/LC3I) were significantly increased in the model group (P<0.001). The protein expression levels of Beclin 1 in the compound dexamethasone group, crisaborole group, 4% PJ-001 group and 2% PJ-001 group were significantly lower than those in the model group (P<0.01). Specifically, the expression levels of Beclin 1 were decreased by 36.2% in the 4% PJ-001 group compared with that in the model group, whereas they were decreased by 22.0% in the 2% PJ-001 group. Furthermore, the expression levels of LC3 were significantly decreased in the crisaborole group and 2% PJ-001 group compared with those in the model group (P<0.01).
Effects of PJ-001 on the mRNA expression levels of K17 and FLG in mouse skin tissue
As shown in Fig. 5A and B, the mRNA expression levels of K17 in the AD model group exhibited a significant increase (P<0.001) compared with those in the control group. The positive controls, dexamethasone and crisaborole, demonstrated a notable suppressive effect on K17 mRNA (P<0.001). However, the PJ-001 treatment was ineffective in suppressing the expression and transcription of K17 (Figs. 4H and 5B). The mRNA expression levels of FLG in the AD model group were significantly reduced by PJ-001 group (P<0.001; Fig. 5A and C). By contrast, the groups treated with compound dexamethasone, 4% PJ-001 and 2% PJ-001 showed a significant increase in the expression levels of FLG (P<0.001). Specifically, the 4% PJ-001 group exhibited an 847% increase, whereas the 2% PJ-001 group showed a 911% increase (Fig. 5C).
Mouse splenic weight and IL-10 mRNA expression levels
As shown in Fig. 6C, splenic weight was significantly increased in the model group compared with that in the control group (P<0.001). However, the compound dexamethasone group exhibited a significant decrease in splenic weight (P<0.001). By contrast, there was no notable difference observed in the crisaborole group, 4% PJ-001 group and 2% PJ-001 group relative to the model group. Furthermore, the mRNA expression levels of IL-10 in the splenic tissues of mice in the model group were significantly lower than those in the control group (P<0.001; Fig. 6A and B). In the 4% PJ-001 group, the mRNA expression levels of IL-10 were significantly increased by 282.2% (P<0.001) compared with those in the model group. The compound dexamethasone group, crisaborole group and the 2% PJ-001 did not succeed in inducing IL-10 expression.
Discussion
The pathogenesis of AD is a complex process that involves various factors, including the external environment, genetic susceptibility, skin barrier dysfunction and immune response (4). Currently, there are several drugs available for treating AD, primarily systemic drugs such as topical corticosteroids and oral antihistamines; however, these drugs have limited efficacy and notable side effects. Compound dexamethasone ointment, a long-term glucocorticoid, has the ability to suppress the synthesis of inflammatory factors, reduce exudation, relieve itching, and exhibit anti-inflammatory and anti-allergic effects (22). However, as a hormone-based drug, prolonged usage can lead to skin atrophy, capillary dilation, pigmentation and secondary infections (23). Crisaborole ointment is a new small-molecule drug used to treat AD. Its main component, crisaborole, functions as a phosphodiesterase 4 inhibitor. Although this ointment has demonstrated high efficacy and safety, without causing skin atrophy, it exhibits a slow onset and does not provide immediate relief from itching, which may reduce medication compliance among patients with AD (24). To enhance treatment options for patients with AD, it is crucial to continue the development of drugs that provide faster onset and effective relief from itching, with fewer side effects.
In recent years, PROTAC drugs have advanced to the clinical research stage, representing a significant development from a conceptual standpoint. Unlike traditional small-molecule inhibitor drugs, PROTAC drugs utilize the ubiquitin-proteasome system to degrade target proteins, showing immense potential in targeting ‘non-drug targets’ (25). KT-474 is a potential first-in-class IRAK4 degrader that is being developed for the treatment of TLR/IL-1R-driven immune inflammatory diseases, such as AD (26). The utilization of PROTAC technology holds promise as a novel approach in drug development for the treatment of inflammatory diseases. PJ-001 is a degrader of JAK synthesized by our laboratory using PROTAC technology. The original purpose of the present study was to develop a PROTAC molecule with the potential to alleviate AD. To evaluate the effectiveness of the molecule, a comparison with clinically recommended drugs, namely dexamethasone and crisaborole, was performed. The severity of skin lesions in AD mice was assessed by comparing the number of scratches and severity scores among different groups. The results demonstrated that PJ-001 can effectively reduce the severity of skin lesions, particularly when used at a concentration of 2%. However, the results did not conform to the expected dose-effect relationship, as lower doses showed a more significant effect than higher doses. The potential explanation for this is that each drug has an upper limit, which is known as the ‘hook effect’ that PROTAC drugs need to overcome. When the concentration is higher than the DC50 value, PROTAC molecules can saturate binding sites on either the target protein or the E3 ligase, inhibiting the formation of the required ternary complex and resulting in the generation of an unproductive binary complex. This results in loss of efficacy at higher concentrations (27). Additionally, analysis of mouse H&E pathological sections revealed that PJ-001 could reduce the thickness of the spinous layer in AD mice and alleviate excessive or incomplete epidermal keratinization. Notably, the application of 2% PJ-001 demonstrated a marked restoration of hair follicle structure, whereas 4% PJ-001 exhibited a substantial reduction in the infiltration of inflammatory cells. These findings provide direct evidence that PJ-001 effectively mitigates inflammatory infiltration, thereby improving skin itching and epidermal keratinization.
FLG, an essential component of the outer membrane of KCs, is produced during the process of KC differentiation. Its main function is to maintain epidermal homeostasis by facilitating the regular gathering of keratin filaments. Additionally, mutations or abnormal expression of FLG-related genes can result in a decrease in ceramides and antimicrobial peptides, while also causing an increase in water loss through the epidermis (3,28). Consequently, this leads to increased infiltration of environmental stimuli, allergens and microorganisms, ultimately contributing to the development of AD (29). The expression of FLG has been observed to decrease in the epidermis of patients diagnosed with AD (30). In the present study, a significant decrease in FLG mRNA expression was observed in the skin lesions of AD mice. However, following treatment with PJ-001, FLG mRNA expression was upregulated ~9 times compared with that in the model group. These findings suggested that PJ-001 has the ability to promote the expression of FLG and to enhance the resistance of the stratum corneum. As a result, it may effectively prevent the penetration and invasion of harmful substances and allergens.
The JAK/STAT pathway is a major signaling pathway regulated by cytokines; it serves a crucial role in priming innate immunity, coordinating adaptive immune mechanisms, and ultimately suppressing inflammation and immune responses (31). NF-κB is present in almost all animal cell types and participates in cell responses to various stimuli, such as stress, cytokines, free radicals, heavy metals, ultraviolet radiation, and bacterial or viral antigens. NF-κB is essential in regulating the immune response to infection (32). Both JAK/STAT and NF-κB pathways are connected through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. Receptors phosphorylated by JAKs recruit PI3K, which activates the PI3K/Akt pathway. This leads to the phosphorylation of inhibitor κB, enabling the activation of NF-κB. Once activated, NF-κB enters the nucleus, interacts with DNA, and initiates or inhibits the transcription of related genes (33). PJ-001 is a PROTAC molecule designed to target the JAK protein. The present study indicated that PJ-001 can effectively decrease JAK/STAT3 and TLR4/NF-κB protein expression to alleviate skin damage in AD mice. Moreover, PJ-001 induced a significant downregulation of the autophagy factors Beclin 1 and LC3, thereby inhibiting excessive autophagy in the inflammatory tissues.
IL-10, an important anti-inflammatory cytokine, has a controversial role in the pathogenesis of AD. A previous study detected high expression of IL-10 in the serum of patients with AD (34). However, Hussein et al (35) found no significant abnormality in the serum of IL-10 in patients with atopy. By contrast, Zhang et al (36) demonstrated that IL-10 levels in the supernatants of peripheral blood culture were lower in patients with severe AD compared with those in normal controls. In the present study, it was observed that 4% PJ-001 significantly increased the mRNA expression levels of IL-10 in the spleens of AD mice. We hypothesize that PJ-001 can inhibit JAK/STAT signaling, thereby modulating macrophage activation to suppress inflammatory diseases. This modulation may promote the repolarization of inflammatory (M1) macrophages into anti-inflammatory (M2) macrophages, increasing the transcription of IL-10(37). However, evidence has suggested that IL-10 may have a dual role, as it can stimulate the immune response and potentially trigger diseases instead of suppressing them (38). In future studies, we aim to evaluate the molecular mechanism of PJ-001 in alleviating AD by inhibiting the JAK/STAT pathway using inhibitor interference. Additionally, we will utilize mature PROTACs for comparison to ensure a more comprehensive evaluation of our test compound.
In conclusion, PJ-001 can reduce the inflammatory response in a mouse model of AD by inhibiting the activation of inflammatory pathways. In addition, it may alleviate excessive autophagy in skin lesions and increase the expression of skin barrier factors. These findings suggested that targeted inhibition of the JAK2/STAT3 and TLR4/NF-κB signaling pathways represents a potential therapeutic approach for AD.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by the National Natural Science Foundation of China (grant no. 82004027).
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
PL and ZC drafted the final manuscript. PL, ZC and JL made substantial contributions to the conception and design of the study. PL and JL critically revised the manuscript and provided constructive feedback. YL and HS participated in animal experiments and assisted in interpreting data. PL and JL confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
The present study was performed according to the guidelines laid out by the Ethics Committee for Animal Experiments of Jiangnan University (Wuxi, China) and was approved by the Ethics Committee of Experimental Animal Management and Animal Welfare of Jiangnan University [approval no. JN. No20210930b0801218(353)].
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
Hadi HA, Tarmizi AI, Khalid KA, Gajdács M, Aslam A and Jamshed S: The epidemiology and global burden of atopic dermatitis: A narrative review. Life (Basel). 11(936)2021.PubMed/NCBI View Article : Google Scholar | |
Cornaghi L, Gagliano N, Preis FWB, Prignano F and Donetti E: Inside-out and outside-in organotypic normal human skin culture: JAK-STAT pathway is activated after pro-inflammatory psoriatic cytokine exposure. Tissue Cell. 74(101675)2022.PubMed/NCBI View Article : Google Scholar | |
Moosbrugger-Martinz V, Leprince C, Méchin MC, Simon M, Blunder S, Gruber R and Dubrac S: Revisiting the roles of filaggrin in atopic dermatitis. Int J Mol Sci. 23(5318)2022.PubMed/NCBI View Article : Google Scholar | |
De Vuyst E, Salmon M, Evrard C, Lambert de Rouvroit C and Poumay Y: Atopic dermatitis studies through in vitro models. Front Med (Lausanne). 4(119)2017.PubMed/NCBI View Article : Google Scholar | |
Welsch K, Holstein J, Laurence A and Ghoreschi K: Targeting JAK/STAT signalling in inflammatory skin diseases with small molecule inhibitors. Eur J Immunol. 47:1096–1107. 2017.PubMed/NCBI View Article : Google Scholar | |
Bao L, Zhang H and Chan LS: The involvement of the JAK-STAT signaling pathway in chronic inflammatory skin disease atopic dermatitis. JAKSTAT. 2(e24137)2013.PubMed/NCBI View Article : Google Scholar | |
Wang L, Xian YF, Loo SKF, Ip SP, Yang W, Chan WY, Lin ZX and Wu JCY: Baicalin ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice through modulating skin barrier function, gut microbiota and JAK/STAT pathway. Bioorg Chem. 119(105538)2022.PubMed/NCBI View Article : Google Scholar | |
Goenka S and Kaplan MH: Transcriptional regulation by STAT6. Immunol Res. 50:87–96. 2011.PubMed/NCBI View Article : Google Scholar | |
Tang J, Liu C, Liu S, Zhou X, Lu J, Li M and Zhu L: Inhibition of JAK1/STAT3 pathway by 2-methoxyestradiol ameliorates psoriatic features in vitro and in an imiquimod-induced psoriasis-like mouse model. Eur J Pharmacol. 933(175276)2022.PubMed/NCBI View Article : Google Scholar | |
Kang R, Tang D, Lotze MT and Zeh HJ: AGER/RAGE-mediated autophagy promotes pancreatic tumorigenesis and bioenergetics through the IL6-pSTAT3 pathway. Autophagy. 8:989–991. 2012.PubMed/NCBI View Article : Google Scholar | |
Li H, Chen L, Li JJ, Zhou Q, Huang A, Liu WW, Wang K, Gao L, Qi ST and Lu YT: miR-519a enhances chemosensitivity and promotes autophagy in glioblastoma by targeting STAT3/Bcl2 signaling pathway. J Hematol Oncol. 11(70)2018.PubMed/NCBI View Article : Google Scholar | |
Kim HJ, Park J, Kim SK, Park H, Kim JE and Lee S: Autophagy: Guardian of skin barrier. Biomedicines. 10(1817)2022.PubMed/NCBI View Article : Google Scholar | |
Li X and Song Y: Proteolysis-targeting chimera (PROTAC) for targeted protein degradation and cancer therapy. J Hematol Oncol. 13(50)2020.PubMed/NCBI View Article : Google Scholar | |
Békés M, Langley DR and Crews CM: PROTAC targeted protein degraders: The past is prologue. Nat Rev Drug Discov. 21:181–200. 2022.PubMed/NCBI View Article : Google Scholar | |
Xiong X, Huang C, Wang F, Dong J, Zhang D, Jiang J, Feng Y, Wu B, Xie T and Cheng L: Qingxue jiedu formulation ameliorated DNFB-induced atopic dermatitis by inhibiting STAT3/MAPK/NF-κB signaling pathways. J Ethnopharmacol. 270(113773)2021.PubMed/NCBI View Article : Google Scholar | |
Woo TE and Kuzel P: Crisaborole 2% ointment (Eucrisa) for atopic dermatitis. Skin Therapy Lett. 24:4–6. 2019.PubMed/NCBI | |
Tadvalkar G, Pal-Ghosh S, Pajoohesh-Ganji A and Stepp MA: The impact of euthanasia and enucleation on mouse corneal epithelial axon density and nerve terminal morphology. Ocul Surf. 18:821–828. 2020.PubMed/NCBI View Article : Google Scholar | |
Shomer NH, Allen-Worthington KH, Hickman DL, Jonnalagadda M, Newsome JT, Slate AR, Valentine H, Williams AM and Wilkinson M: Review of rodent euthanasia methods. J Am Assoc Lab Anim Sci. 59:242–253. 2020.PubMed/NCBI View Article : Google Scholar | |
Zheng BW, Wang BY, Xiao WL, Sun YJ, Yang C and Zhao BT: Different molecular weight hyaluronic acid alleviates inflammation response in DNFB-induced mice atopic dermatitis and LPS-induced RAW 264.7 cells. Life Sci. 301(120591)2022.PubMed/NCBI View Article : Google Scholar | |
Yang N, Shao H, Deng J, Yang Y, Tang Z, Wu G and Liu Y: Dictamnine ameliorates chronic itch in DNFB-induced atopic dermatitis mice via inhibiting MrgprA3. Biochem Pharmacol. 208(115368)2023.PubMed/NCBI View Article : Google Scholar | |
Gallo-Oller G, Ordoñez R and Dotor J: A new background subtraction method for Western blot densitometry band quantification through image analysis software. J Immunol Methods. 457:1–5. 2018.PubMed/NCBI View Article : Google Scholar | |
Schuler CFt, Billi AC, Maverakis E, Tsoi LC and Gudjonsson JE: Novel insights into atopic dermatitis. J Allergy Clin Immunol. 151:1145–1154. 2023.PubMed/NCBI View Article : Google Scholar | |
Yu T and Zhi-rong Y: Research and therapy progress on the mechanisms of pruritus in atopic dermatitis. Med J Peking Union Med. 13:473–479. 2022. | |
McDowell L and Olin B: Crisaborole: A novel nonsteroidal topical treatment for atopic dermatitis. J Pharm Technol. 35:172–178. 2019.PubMed/NCBI View Article : Google Scholar | |
Wen-xing L, Ming H and Yu R: Opportunities and challenges for PROTACs. Chinese J Med Chem. 30:745–764. 2020. | |
Therapeutics K: Kymera announces positive results from phase 1 clinical trial evaluating KT-474 in patients with HS and AD and Sanofi's decision to advance KT-474 into phase 2 clinical trials. Kymera Therapeutics, Inc., Watertown MS, 2022. | |
Madan J, Ahuja VK, Dua K, Samajdar S, Ramchandra M and Giri S: PROTACs: Current trends in protein degradation by proteolysis-targeting chimeras. BioDrugs. 36:609–623. 2022.PubMed/NCBI View Article : Google Scholar | |
Luger T, Amagai M, Dreno B, Dagnelie MA, Liao W, Kabashima K, Schikowski T, Proksch E, Elias PM, Simon M, et al: Atopic dermatitis: Role of the skin barrier, environment, microbiome, and therapeutic agents. J Dermatol Sci. 102:142–157. 2021.PubMed/NCBI View Article : Google Scholar | |
Stander S: Atopic dermatitis. N Engl J Med. 384:1136–1143. 2021.PubMed/NCBI View Article : Google Scholar | |
Zhao Y, Terron-Kwiatkowski A, Liao H, Lee SP, Allen MH, Hull PR, Campbell LE, Trembath RC, Capon F, Griffiths CE, et al: Filaggrin null alleles are not associated with psoriasis. J Invest Dermatol. 127:1878–1882. 2007.PubMed/NCBI View Article : Google Scholar | |
Cha B, Lim JW and Kim H: Jak1/Stat3 is an upstream signaling of NF-κB activation in Helicobacter pylori-induced IL-8 production in gastric epithelial AGS cells. Yonsei Med J. 56:862–866. 2015.PubMed/NCBI View Article : Google Scholar | |
Ahmad SF, Ansari MA, Zoheir KM, Bakheet SA, Korashy HM, Nadeem A, Ashour AE and Attia SM: Regulation of TNF-α and NF-κB activation through the JAK/STAT signaling pathway downstream of histamine 4 receptor in a rat model of LPS-induced joint inflammation. Immunobiology. 220:889–898. 2015.PubMed/NCBI View Article : Google Scholar | |
Ramadass V, Vaiyapuri T and Tergaonkar V: Small molecule NF-κB pathway inhibitors in clinic. Int J Mol Sci. 21(5164)2020.PubMed/NCBI View Article : Google Scholar | |
Lesiak A, Zakrzewski M, Przybyłowska K, Rogowski-Tylman M, Wozniacka A and Narbutt J: Atopic dermatitis patients carrying G allele in -1082 G/A IL-10 polymorphism are predisposed to higher serum concentration of IL-10. Arch Med Sci. 10:1239–1243. 2014.PubMed/NCBI View Article : Google Scholar | |
Hussein PY, Zahran F, Ashour Wahba A, Ahmad AS, Ibrahiem MM, Shalaby SM, El Tarhouny SA, El Sherbiny HM and Bakr N: Interleukin 10 receptor alpha subunit (IL-10RA) gene polymorphism and IL-10 serum levels in Egyptian atopic patients. J Investig Allergol Clin Immunol. 20:20–26. 2010.PubMed/NCBI | |
Zhang YY, Wang AX, Xu L, Shen N, Zhu J and Tu CX: Characteristics of peripheral blood CD4+CD25+ regulatory T cells and related cytokines in severe atopic dermatitis. Eur J Dermatol. 26:240–246. 2016.PubMed/NCBI View Article : Google Scholar | |
Quero L, Tiaden AN, Hanser E, Roux J, Laski A, Hall J and Kyburz D: miR-221-3p drives the shift of M2-macrophages to a pro-inflammatory function by suppressing JAK3/STAT3 activation. Front Immunol. 10(3087)2019.PubMed/NCBI View Article : Google Scholar | |
Amend A, Wickli N, Schäfer AL, Sprenger DTL, Manz RA, Voll RE and Chevalier N: Dual role of interleukin-10 in murine NZB/W F1 lupus. Int J Mol Sci. 22(1347)2021.PubMed/NCBI View Article : Google Scholar |