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. 2025 Aug 18;15:121. doi: 10.1186/s13568-025-01931-4

Therapeutic potential of recombinant IL-22BP in psoriasis: suppression of IL-22/STAT3 signaling in mice

Xiuze Chen 1, Tao Zeng 1, Fang Fang 2, Qinyu Tian 1, Yingying Li 1, Wenlin Zhou 1, Binqian Gong 1, Na Tian 3, Jizhou Zhang 4, Yechen Xiao 1,✉
PMCID: PMC12361007  PMID: 40824430

Abstract

Psoriasis is a persistent immune-mediated inflammatory disorder that adversely affects the skin. Interleukin-22 (IL-22) is integral to the development and pathophysiology of psoriasis, and targeting IL-22 may serve as a promising therapeutic approach for treating the condition. IL-22 binding protein (IL-22BP) exhibits a binding affinity for IL-22 that far surpasses that of IL-22RA1 and functions as a natural antagonist of IL-22. Traditional IL-22BP production methods predominantly rely on eukaryotic animal cell expression systems, which generally require complex processes, resulting in low yield and high production costs. This study reports the expression of long-acting IL-22BP with a high yield and purity over 90% in Escherichia coli by fusion with the albumin-binding structural domain ABD. The biological functions of rhIL-22BP-ABD were assessed utilizing cell lines and a murine model. Our findings indicated that rhIL-22BP-ABD successfully suppressed IL-22-induced proliferation of HaCaT cells in vitro and alleviated imiquimod-induced psoriasis inflammation in mice. Furthermore, rhIL-22BP-ABD can effectively inhibit the signaling of its downstream signaling pathway STAT3 and the associated inflammatory factors by binding to IL-22, which is beneficial to the recovery of psoriasis. These findings provide a basis for forthcoming extensive studies on the rhIL-22BP-ABD protein for industrial manufacturing and pharmaceutical development.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13568-025-01931-4.

Keywords: IL-22, rhIL-22BP-ABD, Renaturation of inclusion body, Psoriasis

Introduction

Psoriasis is an immune-mediated inflammatory disorder marked by autoimmune pathology impacting the skin and joints. The worldwide frequency of psoriasis is believed to be between 2% and 3% (Gibbs 1996; Parisi et al. 2013). Psoriasis is defined by the rapid proliferation and atypical differentiation of keratinocytes, presenting clinically as erythematous squamous plaques of varying sizes. It is associated with numerous other health issues, including psoriatic arthritis, metabolic syndrome, cardiovascular disorders, diabetes, and various comorbidities (Owen et al. 2000; Perera et al. 2012; Schön and Boehncke 2005). Recent years have witnessed substantial advancements in the studies about the pathophysiology of psoriasis. Research indicates that psoriasis is regulated by intricate connections between external cytokine pathways and intracellular signaling molecules (Sieminska et al. 2024). A range of cytokines convey extracellular signals to the cell membrane, subsequently acknowledged by the appropriate receptors (Petit et al. 2021). This process activates intracellular signaling pathways and triggers a sequence of events, ultimately culminating in an inflammatory signal cascade. The TNF/IL-23/ IL-17 pathway plays a pivotal role in the development of psoriasis (Guo et al. 2023). A range of pharmacological inhibitors has been formulated to target this pathway, including adalimumab, etanercept, infliximab, IL-17 inhibitors, IL-23 inhibitors, and others. Notwithstanding the early effectiveness of these drugs, obstacles and restrictions in their utilization endure. This primarily results from certain patients’ lack of response to these treatments, resulting in treatment failure, the generation of neutralizing antibodies in patients after repeated use, and the exorbitant cost of therapeutic agents, which presents a significant challenge to both patients and the healthcare system (Lee and Kim 2023).

IL-22 is a member of the IL-10 family, an it can be produced by several immune cells, including Th17 cells, Th22 cells, γδT cells, and NK cells (Fujita et al. 2009; Tokura et al. 2010). Secreted IL-22 exerts its effects on target cells via binding to IL-22R1, which is primarily expressed on non-immune cells, such as keratinocytes (Wolk et al. 2004). IL-22 expression is heightened in psoriasis lesions and contributes to acanthosis nigricans by suppressing keratinocyte differentiation, representing a distinctive feature of psoriasis-like epidermal inflammation (Wolk et al. 2009). Consequently, IL-22 is considered a possible target for psoriasis treatment.

The IL-22 receptor is a heterodimeric complex of IL-22R1 and IL-10R2. It contains three major domains: extracellular, transmembrane, and intracellular signaling regions (Bleicher et al. 2008; Xie et al. 2000). IL-22 initially binds to the IL-22R1 subunit and the resulting complex undergoes a conformational change followed by IL-10R2 binding, thereby initiating a downstream signaling cascade (Li et al. 2004), after forming a complex with IL-10R2, activator of transcription 3 (STAT3 phosphorylation) is triggered by activated Janus kinase (JAK) and tyrosine kinase 2 (Tyk2). Not only that, IL-22 also activates the kinases p38, MEK/ERK1/2 and JNK (Lejeune et al. 2002; Nagalakshmi et al. 2004), which distinguishes it from other IL-10 related cytokines.

IL-22 binding protein (IL-22BP/IL-22RA2) is a secretory protein with a total length of 231 amino acids (Jones et al. 2008). Although structurally akin to the IL-22R1 chain, the affinity of IL-22BP for IL-22 is 20 to 1000 times higher than that of IL-22R1 (Khawar et al. 2016; Pelczar et al. 2016). Some studies showed that the dissociation constant between IL-22 and IL-22BP was 1 pM, and the dissociation constant between IL-22 and IL-22R1 was 20 nM. The corresponding dissociation rate constants of the two conjugates were 4.7 d and 7.4 min, respectively (Jones et al. 2008), therefore obstructing IL-22 interaction with IL-22R1 (Mühl and Bachmann 2019). Numerous studies have shown that a deficit in IL-22BP results in the upregulation of IL-22, consequently worsening psoriasis (Voglis et al. 2018). Research indicates that patients with psoriasis exhibit elevated levels of IL-22 and reduced levels of IL-22BP compared to healthy individuals (Martin et al. 2017). Moreover, the modulation of IL-22 signaling via autocrine IL-22BP synthesis in keratinocytes aids in the preservation of skin homeostasis (Fukaya et al. 2018). Research on animal models of skin inflammation has elucidated the protective function of IL-22BP, demonstrating a significant downregulation of IL-22BP mRNA in imiquimod-induced skin inflammation models, which therefore worsens the condition (Voglis et al. 2018). A comparable study demonstrated that administering a recombinant IL-22BP-Fc fusion protein to mice mitigated cutaneous inflammation and decreased the expression of inflammatory cytokine genes (Fukaya et al. 2018).

The albumin-binding domain (ABD) is derived from streptococcal protein G and is a compact three-helix protein domain consisting of 46 amino acids, with a molecular weight of approximately 5.2 kilodaltons (Altai et al. 2018; Jonsson et al. 2008). It can interact with human serum albumin (HSA), the predominant plasma protein with a half-life of roughly 19 days. Numerous studies have proven the importance of ABD fusion in extending the half-life of polypeptide and protein therapeutics (Liu et al. 2022). This study involved the production of a long-acting rhIL-22BP-ABD fusion protein, which incorporates IL-22BP and ABD, utilizing the E. coli expression system. The resultant fusion protein demonstrates prolonged in vivo lifespan and exhibits significant biological activity both in vitro and in vivo. These findings lay the groundwork for extensive industrial manufacturing and pharmacological advancement of rhIL-22BP-ABD protein in the future.

Materials and methods

Materials, strains and plasmids

Tryptone and yeast extract were acquired from Oxoid (Basingstoke, UK). Restriction endonucleases Nde I and Xho I, ampicillin antibiotic, Ni-NTA agarose resin, and SDS-PAGE precast gel kit were acquired from Yeasen (Shanghai, China). Imidazole and Isopropyl-β-D-thiogalactopyranoside (IPTG) were acquired from Solarbio (Beijing, China). BCA assay kit and HRP-labeled goat anti-rabbit secondary antibody were procured from Beyotime (Shanghai, China). A three-color pre-stained protein marker was acquired from Thermo (Waltham, MA, USA). Human IL-22 (Cat# HY-P7039) was acquired from MCE (New Jersey, USA). Anti-histidine antibody (Cat# T0009), anti-IL-22 antibody (Cat# DF8343), anti-phosphorylated STAT3 antibody (Cat# AF3293), and anti-STAT3 antibody (Cat# AF6294) were acquired from Affinity (Cincinnati, Ohio, USA). TNF-α ELISA kit (Cat# EK282HS) and IL-6 ELISA kit (Cat# EK206) were purchased from MultiSciences Biotech Co., Ltd (Hangzhou, China). E. coli BL21 (DE3) and E. coli DH5α were preserved and manufactured in our laboratory.

Construction of recombinant plasmids

Based on the protein sequence of interleukin-22 receptor subunit alpha-2 (NP_851826) in the NCBI database, rhIL-22BP-ABD was synthesized utilizing the preferred codon of E. coli (Supplementary file 1: Data 1, Data 2) and subsequently cloned between the Nde I and Xho I cleavage sites of the pET-20b(+) vector by IGE Biotechnology Co. (Guangzhou, China). Utilizing rhIL-22BP-ABD as a template, rhIL-22BP was amplified via PCR and subsequently cloned into the plasmid pET20b to create the recombinant plasmid pET20b-rhIL-22BP (Supplementary file 1: Data 3). The recombinant plasmid was introduced into E. coli BL21 (DE3) cells, and the recombinant strain was cultivated on LB solid medium at 37 ℃ for 12–16 h. Individual colonies were isolated from the solid medium and cultivated in LB liquid media by shaking at 37 ℃ at 200 rpm overnight. The activated bacterial solution was inoculated into LB liquid medium at a 1:100 ratio and incubated at 37 ℃ with shaking at 200 rpm until the OD600 value reached 0.6–0.8. IPTG was introduced to achieve a final concentration of 0.5 mM and incubated at 37 ℃ for 5 h. The expression of recombinant proteins was evaluated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Soluble assay of rhIL-22BP-ABD and rhIL-22BP

The engineered bacteria harboring pET20b-rhIL-22BP-ABD and pET20b-rhIL-22BP were inoculated and cultured in LB liquid medium at 37 ℃ in a shaker until the OD600 value attained 0.6–0.8. IPTG was introduced at a final concentration of 0.5 mM, and the induction incubation was conducted at 37 ℃ for 5 h. The recombinant bacteria underwent centrifugation and sonication to disrupt and release the proteins into a 20 mM Tris-HCl buffer. The supernatant containing soluble proteins was centrifuged at 12,000 rpm for 15 min at 4 ℃. The resultant precipitate was subsequently resuspended in an equivalent volume of buffer. The samples were subsequently analyzed for soluble protein expression using SDS-PAGE.

Optimization of the expression conditions induced by rhIL-22BP-ABD

To determine the best concentration of IPTG while keeping other variables constant, varying final concentrations of IPTG (0, 0.3, 0.5, 0.7, 1 mM) were introduced to the LB medium and incubated for 5 h at 37 ℃. The total protein of the stimulated bacteria was assessed via 12% SDS-PAGE to ascertain the appropriate IPTG concentration. Correspondingly, various temperatures (16 ℃, 20 ℃, 30 ℃, 37 ℃) were established to elicit the expression of recombinant protein, and samples were subjected to electrophoresis post-expression to ascertain the optimal induction temperature. To ascertain the best induction duration, IPTG was introduced to a final concentration of 0.5 mM, and the recombinant bacteria were incubated at 37 ℃ for 3, 4, 5, 6, 7, and 8 h. The expression of recombinant proteins was evaluated via 12% SDS-PAGE to ascertain the appropriate induction duration.

Acquisition and denaturation of inclusion bodies

Under ideal circumstances for inclusion body protein expression (OD value of 0.6–0.8, induced by 0.5 mM IPTG at 37 ℃ for 6 h), the organisms were harvested via centrifugation at 12,000 rpm for 10 min post-induction, followed by ultrasonic disruption at 500 W for 30 min. The supernatant was removed, and the precipitate was resuspended in the designated volume of inclusion body washing solution. It was agitated for 20 min at 4 ℃, followed by centrifugation at 12,000 rpm for 15 min, after which the supernatant was discarded. This process was done three times. The precipitate was rewashed with a suitable volume of 20 mM Tris-HCl buffer (pH 8.0), centrifuged at 12,000 rpm for 30 min at 4 ℃, and the supernatant was discarded to isolate pure protein inclusion bodies. The inclusion bodies were denatured and solubilized in 6 M or 8 M urea solutions by repeated agitation, denatured for 4 h at 4 ℃, and subsequently centrifuged at 12,000 rpm for 30 min to determine the optimal urea concentration for denaturation. The supernatant was collected to obtain the soluble protein solution for further purification.

Purification and renaturation of rhIL-22BP-ABD inclusion body

The denatured supernatant of rhIL-22BP-ABD was purified using Ni-NTA affinity chromatography. The Ni-NTA resin was rinsed with lysis buffer (pH 8.0) containing 10 mM imidazole and 8 M urea until the OD280 value aligned with the baseline level. The protein supernatant was incubated with the Ni-NTA column for 0.5 to 1 h. Thereafter, nonspecifically bound heteroproteins were eliminated using a wash buffer composed of 40 mM imidazole and 8 M urea at pH 8.0. The target protein was ultimately eluted using an elution buffer of 250 mM imidazole and 8 M urea at pH 8.0. The purity of the recombinant proteins was assessed using 12% SDS-PAGE, and their quantity was quantified with a BCA protein assay kit.

To investigate the optimal complexation scheme, the supernatant of the purified denatured proteins was divided into two equal portions. The first portion was placed in a dialysis bag (with a cut-off molecular weight of 3500 D) and subsequently immersed in a 5-fold volume of gradient complexation buffer containing 6, 4, 2, 1, and 0 M urea (20 mM Tris-HCl, 50 mM NaCl, 0.5 mM GSSG, 1.0 mM GSH, pH 8.0), and dialyzed at 4 ℃ for a minimum of 12 h for each gradient. At the conclusion of each gradient, 40 µL of the sample was aliquoted for preparation and reserved, and following dialysis, all gradient samples underwent 12% SDS-PAGE electrophoresis to assess complexation efficiency. The second protein supernatant was transferred into a dialysis bag (with a cut-off molecular weight of 3500 Da) and subsequently placed into a urea gradient complexation buffer comprising 4 M (with 2 mM DTT) and 0 M urea gradient complexation buffer (with 0.5 mM DTT) at a volume five times greater. Dialysis was conducted at 4 ℃ for a minimum of 12 h for each gradient, and at the conclusion of each gradient, 40 µL of the sample was extracted for preparation and reserved. Post-dialysis, all gradient samples underwent 12% SDS-PAGE to assess complexation efficiency. The protein acquired post-complexation was subjected to centrifugation at 4 ℃ and 12,000 rpm for 15 min, after which the supernatant was filtered and sterilized, subsequently distributed and preserved in a -80 ℃ freezer.

Identification of rhIL-22BP-ABD

Western blotting was conducted to ascertain the purified rhIL-22BP-ABD. Protein samples mixed with 5×protein loading buffer were subjected to boiling in a water bath and subsequently separated using 12% SDS-PAGE. The isolated proteins were subsequently transferred to a PVDF membrane and incubated with 5% skim milk for 2 h to inhibit non-specific binding sites. Thereafter, the membrane was treated with His-tag polyclonal antibody (1:1000 dilution), followed by washing and incubation with horseradish peroxidase-conjugated secondary antibody (1:1000 dilution). Immunoreactive bands were seen utilizing an ECL chemiluminescence detection kit.

The amino acid content of rhIL-22BP-ABD was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) by APTBIO in Shanghai, China.

In vitro activity assay of rhIL-22BP-ABD

To evaluate the biological activity of rhIL-22BP-ABD, HaCaT cells were incubated at 37 ℃ in a 5% CO2 atmosphere using DMEM media supplemented with 10% fetal bovine serum. Cultured cells were inoculated into 96-well plates (3,000–5,000 cells per well) with varying doses of rIL-22, and cell proliferation was assessed using the CCK8 kit after 12 h of incubation. Following the identification of the optimal concentration for IL-22 stimulation, HaCaT cells were grown in 96-well plates (3000–5000 cells per well) with 10 ng/mL rIL-22 and varying concentrations of rhIL-22BP-ABD. The DMEM medium group devoid of rIL-22 and rhIL-22BP-ABD served as a blank control, whereas the DMEM medium group containing rIL-22 without rhIL-22BP-ABD functioned as a negative control. The cell proliferation was assessed using the CCK8 kit following 12 h of incubation.

Determination of kinetic binding parameters of rhIL-22BP-ABD and human serum albumin

Due to both rhIL-22BP-ABD and rhIL-22BP possessing a 6×His tag, His-Tag pull-down assays were employed to ascertain the interaction between rhIL-22BP-ABD and HSA. The His tag purification resin was first thoroughly washed five times and equilibrated with 20 mM Tris-HCl (pH = 8.0). The purified rhIL-22BP-ABD and rhIL-22BP were incubated with His tag purification resin in a rotating incubator at 4℃ for 30 min, after which the resin was collected and washed three times. Dissolve 0.2 mg/mL of HSA in the reaction buffer (20 mM Tris, 0.5 mM DTT, pH 8.0) and incubate with the column material in a rotary incubator at 4℃ for 30 min. Subsequent to the removal of the supernatant, the resin was subjected to four washes with the reaction buffer. The target protein was washed and separated using 10% SDS. The eluents were subsequently tested and detected using 12% SDS-PAGE.

The molecular interaction approach was utilized to evaluate the kinetic binding characteristics of rhIL-22BP-ABD and HSA by a BLI experiment employing the Octet system. All proteins were diluted in 20 mM Tris-HCl with 0.5 mM DTT. The his-tagged rhIL-22BP-ABD structural domain (100 µg/mL) was initially adsorbed on the biosensor (ForteBio) and equilibrated with 20 mM Tris-HCl. The biosensor was subsequently exposed to HSA and rinsed with 0 M urea buffer. Relevant parameters were set as follows: Baseline 1 (180 s), Loading (300 s), Baseline 2 (180 s), Association (600 s), Dissociation (600 s). The ultimate binding affinity (Kd) was determined using data analysis software (Octet® BLI Analysis 12). In brief, the Data were first processed in Data Correction using Y-axis alignment, inter-step correction, and Savitzky-Golay filtering. Dissociation and binding data were analyzed under Options. Secondly, the quality of fitting data was evaluated, and judged according to the quantitative difference between Ka (Association Constant), Kd (Dissociation Constant), and their corresponding error values, R2 (Measure the degree of conformity between the fitted curve and the actual measurement lines), X2 (Measure the difference between the fitted curve and the measured curve) and other parameters.

In vivo half-life determination of rhIL-22BP-ABD

All animal experiments conducted in this article were approved by the Laboratory Animal Ethics Committee of Guangdong Medical University. To detect pharmacokinetic analysis of rhIL-22BP-ABD, five female BALB/c mice (18–20 g) were taken for a single subcutaneous injection of 5 mg/kg rhIL-22BP-ABD, and post-injection at 0.2, 1, 2, 4, 8, 12, 24, 36, 48 and 60 h blood collection. After standing at room temperature for 1 h, the serum was centrifuged at 4 ℃ and 3500 rpm, and the serum level of rhIL-22BP-ABD was detected by the human IL-22BP enzyme-linked immunosorbent assay kit (Byabscience, Nanjing, China). rhIL-22BP was used as a negative control. The half-life was calculated by using the Microsoft Excel plug-in “PKSolver”.

Therapeutic effects of rhIL-22BP-ABD on psoriasis in mice

The psoriasis mouse model was developed in accordance with the pertinent literature (Li et al. 2022). Psoriasis was generated with the application of 5% imiquimod cream on the depilated dorsal skin for 9 consecutive days, alongside intraperitoneal injections of varying concentrations of rhIL-22BP-ABD or 0 M urea in a compounded solution for the same duration. In brief, twenty female BALB/c mice (18–20 g) were randomly allocated into four groups (n = 5): control group (coated with vaseline, i.p. 0 M urea in replica solution), model group (coated with 5% imiquimod cream, i.p. 0 M urea in replica solution), cream + 10 mg/kg rhIL-22BP-ABD group (coated with 5% imiquimod cream, i.p. 10 mg/kg rhIL-22BP-ABD), and cream + 5 mg/kg rhIL-22BP-ABD group (coated with imiquimod cream, i.p. 5 mg/kg rhIL-22BP-ABD). The alterations in body weight and dorsal dander attributes of the mice were meticulously observed and documented on a regular basis during the trial. At the conclusion of the experimental phase, the mice were killed to obtain dorsal skin samples for subsequent study. The spleens were weighed, photographed, and the spleen index was computed. PASI scores for the modeled skin on the backs of all mice in each group were assessed and recorded daily. To be precise, erythema, scaling and thickeness were independently scored on a scale of 0 to 4 (0, none; 1, mild; 2, moderate; 3, marked; 4, very marked), and the cumulative score is the total score (scale 0–12). The severity of the disease was assessed by pathology technical experts in a blinded manner. At the conclusion of the administration, the in vivo efficacy of rhIL-22BP-ABD was evaluated by measuring spleen size and associated organ indices in the mice, as well as assessing the dorsal modeling skin using western blot and HE staining.

Histopathological analysis

To evaluate the degree of skin tissue damage, dorsal skin samples were obtained from each group of mice post-euthanasia. Skin tissues measuring approximately 0.5 cm² were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin wax, sectioned to a thickness of 5 μm, and stained with hematoxylin-eosin (H&E). A pathologist conducted histologic investigation of the stained sections in a single-blind way. The extent of skin injury was evaluated with a double-blind methodology.

Analysis of inflammatory factors by ELISA

Before the mice were sacrificed, blood was collected from the eye socket into clean centrifuge tubes. The tubes were left at 4℃ for 1 to 2 h, then centrifuged at 3000 rpm for 15 min to obtain the serum. The expression levels of inflammatory factors IL-6 and TNF-α in the mouse serum were detected according to the instructions of the ELISA assay kit (MultiSciences Biotech Co.,Ltd., China).

Western blot analysis

Small slices of skin tissue, weighing around 50–100 mg, were excised, frozen in liquid nitrogen, and subsequently lysed in RIPA protein lysis buffer. The supernatant was centrifuged at 12,000 rpm for 20 min at 4 ℃, and the protein content was measured using a BCA kit. The protein was extracted using 12% SDS-PAGE and subsequently transferred from the gel to a PVDF membrane (Millipore, USA). The non-specific binding was inhibited by incubating the membrane with 5% skimmed milk for 2 h. Following blocking, the membranes were treated with particular antibodies overnight at 4 ℃, subsequently washed, and then incubated with secondary antibodies. The pertinent bands were generated with an ECL luminescence kit.

Statistical analysis of data

All experimental data were statistically analyzed using GraphPad Prism 9.0 software. The statistical method was analysis of one-way analysis of variance(ANOVA) and the results were expressed as mean ± standard deviation (SD). A P value less than 0.05 was considered statistically significant.

Results

The inducible expression of rhIL-22BP-ABD in E. coli

The recombinant plasmid comprising pET20b-rhIL-22BP-ABD and pET20b-rhIL-22BP was constructed and manufactured as depicted in Fig. 1A, respectively. The two accurately identified recombinant plasmids were transformed into E. coli BL21(DE3) and stimulated with IPTG at 37 ℃ at a final dose of 0.5 mM for 5 h. The empty vector pET20b served as a negative control concurrently. The protein expression was evaluated using 12% SDS-PAGE, revealing that rhIL-22BP-ABD (35 kDa) and rhIL-22BP (25 kDa) were specifically expressed in contrast to the empty vector; however, these proteins predominantly manifested as inclusion bodies in the precipitate following bacterial lysis (Fig. 1B-D).

Fig. 1.

Fig. 1

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The Design and expression of rhIL-22BP-ABD and rhIL-22BP in E. coli. A Schematic diagram of the construction of rhIL-22BP-ABD and rhIL-22BP recombinant plasmids. B The induced expression of rhIL-22BP-ABD. M represents marker, lane 1 represents the total protein of blank bacteria containing pET20b before induction, lane 2 represents the total protein of blank bacteria containing pET20b after induction, and lane 3 represents the total protein of recombinant bacteria containing pET20b-rhIL-22BP-ABD before induction, lane 4 represents the total protein of recombinant bacteria containing pET20b-rhIL-22BP-ABD after induction. lane 5 represents the supernatant of recombinant bacteria containing pET20b-rhIL-22BP-ABD after induction and ultrasonic disruption and lane 6 represents the precipitate of recombinant bacteria containing pET20b-rhIL-22BP-ABD after induction and ultrasonic disruption. C The induced expression of rhIL-22BP. M represents marker, lane 1 represents the total protein of the blank bacteria containing pET20b before induction, lane 2 represents the total protein of blank bacteria containing pET20b after induction, lane 3 represents the total protein of recombinant bacteria containing pET20b-rhIL-22BP before induction, and lane 4 represents the total protein of recombinant bacteria containing pET20b-rhIL-22BP after induction. lane 5 represents the supernatant of recombinant bacteria containing pET20b-rhIL-22BP after induction and ultrasonic disruption, and lane 6 represents the precipitate of recombinant bacteria containing pET20b-rhIL-22BP after induction and ultrasonic disruption. D Comparison diagram of rhIL-22BP-ABD and rhIL-22BP inclusion bodies by SDS-PAGE. M represents marker, lane 1 represents rhIL-22BP-ABD inclusion body, lane 2 represents rhIL-22BP inclusion body

The optimization of inducible expression conditions of rhIL-22BP-ABD by SDS-PAGE analysis

To attain elevated expression levels of rhIL-22BP-ABD, the induction conditions were optimized across four dimensions: selection of high-expression strains, induction temperature, IPTG concentration, and induction duration. Figure 2A illustrates that three individual colonies were chosen from the successfully transformed plates for induction expression under identical conditions. The results indicated that strain No. 2 exhibited a superior expression level and was utilized as the primary strain for subsequent condition optimization. Figure 2B indicates that optimal protein expression occurred at 37 ℃ under 0.5 mM IPTG induction for a duration of 6 h. In a similar manner, maintaining constant induction temperature and time while establishing a series of inducer concentration gradients revealed that the optimal expression of the target protein occurred at an IPTG concentration of 0.5 mM (Fig. 2C). The peak expression of the target protein was seen at 37 ℃ by varying the induction temperature under identical conditions (Fig. 2D). To ascertain if low temperature enhances the soluble expression of recombinant proteins, we further reduced the induction temperature to 16 ℃, which revealed that the target proteins remained in the inclusion bodies (Fig. 2E). These tests revealed that the optimal expression of the target protein rhIL-22BP-ABD occurred at 37 ℃ with a 0.5 mM IPTG concentration for 6 h.

Fig. 2.

Fig. 2

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The optimization of induced expression conditions of rhIL-22BP-ABD by SDS-PAGE analysis. A Screening of optimal expression strains. Lane 1, 3 and 5 represent the total protein of bacterial strains No. 1, 2 and 3 before induction, respectively; lane 2, 4, and 6 represent the total protein of bacterial strains No.1, 2 and 3 after induction, respectively. B The effect of induction time on the expression of recombinant protein. M represents protein markers, lane 1 represents total protein before IPTG induction, and lanes 2 to 7 represent total protein after 0.5 mM IPTG induction at 37 ℃ for 3, 4, 5, 6, 7 and 8 h, respectively. C The effect of IPTG concentration on the expression of recombinant protein. M represents protein markers; lane 1 represents total protein before IPTG induction; lanes 2 to 6 represents total protein after 0, 0.3, 0.5, 0.7 and 1 mM IPTG induction, respectively. D The effect of induction temperature on the expression of recombinant protein. M represents protein markers, lane 1 represents total protein before IPTG induction, and lanes 2 to 4 represent total protein after 0.5 mM IPTG induction at 20℃, 30℃ and 37℃, respectively. E The expression of recombinant proteins at a low temperature of 16℃. M is protein markers, lane 1 represents the total protein of the bacteria before induction, lane 2 represents the total protein of the bacteria after induction, lane 3 represents the supernatant of recombinant bacteria containing pET20b-rhIL-22BP-ABD after induction and ultrasonic disruption, and lane 4 represents the precipitate of recombinant bacteria containing pET20b-rhIL-22BP-ABD after induction and ultrasonic disruption

The treatment and purification of rhIL-22BP-ABD inclusion bodies

Given that rhIL-22BP-ABD manifests as inclusion bodies at an expression temperature of 37 ℃, these inclusion bodies must be denatured into soluble proteins prior to further investigations. Figure 3A illustrates that the concentration of denatured supernatant protein derived from an 8 M urea denaturation treatment exceeded that obtained from an initial treatment of the inclusion body with 6 M urea. The concentration of the protein solution after renaturation with 8 M urea is greater than that of the protein solution after renaturation with 6 M urea. We employed Ni-NTA affinity chromatography to purify denatured rhIL-22BP-ABD, owing to the presence of a 6×His tag at its C-terminus. Figure 3B depicts the elution of rhIL-22BP-ABD from the Ni-NTA column utilizing a Tris-HCl buffer with 250 mM imidazole (pH = 8.0), achieving a protein purity of around 90%.

Fig. 3.

Fig. 3

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The inclusion body treatment and purification of rhIL-22BP-ABD. A Denaturation effect of different concentrations of urea on inclusion bodies. M represents protein markers, lane 1 represents the untreated inclusion bodies, lane 2 represents 6 M denatured and renatured to 0 M urea protein precipitation, lane 3 represents the 6 M-denatured protein supernatant refolded to 0 M urea, lane 4 represents the protein precipitation of inclusion bodies denatured to 6 M urea, lane 5 represents the protein supernatant of inclusion bodies denatured to 6 M urea, lane 6 represents the protein precipitation of 8 M denatured to 0 M urea, lane 7 represents the protein supernatant of 8 M denatured to 0 M urea. Lane 8 represents the protein precipitation of inclusion bodies denatured with 8 M urea, and lane 9 represents the protein supernatant of inclusion bodies denatured with 8 M urea. B SDS-PAGE analysis of the purification of recombinant rhIL-22BP-ABD by Ni-NTA. M represents protein markers; lane 1 represents protein before purification; lanes 2 to 4 represent protein after purification by 40 mM iminazole elution; lanes 5 to 8 represent protein after purification by 250 mM iminazole elution. C The reduction of rhIL-22BP-ABD using GSSG/GSH redox. M represents protein marker, lane 1 represents the denatured supernatant of rhIL-22BP-ABD, lanes 2–5 represent purified rhIL-22BP-ABD and 4, 2, 1 and 0 M urea renatured rhIL-22BP-ABD, respectively. D The reduction of rhIL-22BP using GSSG/GSH redox. M represents protein marker, lane 1 represents the denatured supernatant of rhIL-22BP, lanes 2–5 represent rhIL-22BP and 4, 2, 1 and 0 M urea renatured rhIL-22BP, respectively. E Graphs of the results of DTT renaturation of rhIL-22BP-ABD and rhIL-22BP. M represents protein marker, lanes 1–3 represent rhIL-22BP-ABD after purification, rhIL-22BP-ABD after renaturation with 4 M urea, rhIL-22BP-ABD after renaturation with 0 M urea, respectively. Lanes 4–6 represent rhIL-22BP after purification, rhIL-22BP after renaturation with 4 M urea, and rhIL-22BP after renaturation with 0 M urea, respectively

To investigate the ideal conditions for protein renaturation, we employed two renaturation methodologies. The first involved a gradient renaturation of urea utilizing the redox pairs of GSH and GSSG, resulting in a renaturation rate of 60–70% for rhIL-22BP-ABD, whereas rhIL-22BP achieved only a 10–20% renaturation rate (Fig. 3C, D). The secondary denaturation method involves gradient denaturation utilizing urea in conjunction with the reducing agent dithiothreitol (DTT). This denaturation protocol can elevate the denaturation rates of the two proteins to about 100% (Fig. 3E). The final denaturation conditions consisted of 8 M urea, maintained at 4 ℃ for 4 h, whereas the reduction conditions involved a gradient from 4 M urea to 0 M urea utilizing DTT as the reducing agent.

The identification of rhIL-22BP-ABD

Western blot analysis verified the authenticity of rhIL-22BP-ABD by demonstrating its specific binding to the anti-His-tag antibody, hence confirming effective expression of the histidine tag in the fusion protein (Fig. 4A). LC-MS/MS was employed to examine the amino acid composition of rhIL-22BP-ABD, revealing a coverage of 91.2% for the amino acid sequence of rhIL-22BP-ABD (Fig. 4B), thereby demonstrating that the synthesized rhIL-22BP-ABD corresponded with the pre-designed protein sequence. To assess the biological activity of rhIL-22BP-ABD, we initially confirmed that IL-22 may stimulate the proliferation of HaCaT cells in vitro by a dose-response analysis (Fig. 4C). rhIL-22BP-ABD had no inhibitory effect on the proliferation of HaCaT cells; however, it strongly inhibited cell proliferation triggered by 10 ng/ml IL-22 (Fig. 4D-E), and this inhibitory effect demonstrated a dose-dependent relationship (Fig. 4E).

Fig. 4.

Fig. 4

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The identification of rhIL-22BP-ABD. A rhIL-22BP-ABD was performed on immunoblotting with anti-His-tag antibody by Western blot. B Determination of rhIL-22BP-ABD peptide map by mass spectrometry. C The effect of 5, 10 and 50 ng/mL IL-22 on the proliferation of HaCaT cells was detected by CCK8 assay. D The 50 μg/mL rhIL-22BP-ABD inhibits in vitro HaCaT cell proliferation stimulated by 10 ng/mL IL-22. E The inhibitory effect of 10, 50 and 100 μg/mL rhIL-22BP-ABD on HaCaT cell proliferation stimulated by 10 ng/ml IL-22. Quantitative data are presented as mean ± standard deviation (n = 4). Statistical significance is indicated as follows:*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns means no significant difference

The half-life analysis of rhIL-22BP-ABD

To characterize the half-life of rhIL-22BP-ABD, the His-tag pull-down method was employed to assess the binding affinity between rhIL-22BP-ABD and HSA. The results indicated that rhIL-22BP-ABD exhibits a superior binding affinity for Ni-NTA at equivalent concentrations, while rhIL-22BP-ABD demonstrates a measurable binding affinity with HSA, in contrast to rhIL-22BP (Fig. 5A-C). The molecular interaction method was employed to evaluate the kinetic binding parameters of rhIL-22BP-ABD and HSA via BLI assay utilizing the Octet system. The results indicated that rhIL-22BP-ABD exhibits a superior binding affinity for HSA, with a dissociation constant of Kd = 1.146 µM, whereas rhIL-22BP demonstrates negligible binding to HSA (Fig. 5D).

Fig. 5.

Fig. 5

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Specific binding of rhIL-22BP-ABD to HSA and determination of its half-life. A Washed clutter diagram of rhIL-22BP His-tag pull-down experiments. M represents the protein marker, lanes 1–4 represent the washed sample after rhIL-22BP binding to Ni-NTA, and lanes 5–9 represent the washed sample after HSA binding to rhIL-22BP. B Wash diagram of rhIL-22BP-ABD his tag pull-down experiments. M represents the protein marker, lanes 1–4 represent the washed sample after rhIL-22BP-ABD was bound to Ni-NTA, and lanes 5–9 represent the washed sample after HSA was bound to rhIL-22BP-ABD. C rhIL-22BP and rhIL-22BP-ABD his-tag pull-down results. M represents protein marker; lane 1 represents unbound rhIL-22BP-ABD, lane 2 represents unbound rhIL-22BP, lane 3 represents unbound HSA, lane 4 represents bound rhIL-22BP-ABD and HSA with Ni-NTA, and lane 5 represents bound rhIL-22BP and HSA with Ni-NTA. D The interaction of different concentrations HSA with the same concentration of rhIL-22BP-ABD. E The half-life assay of IL-222BP-ABD and rhIL-22BP

To ascertain the in vivo pharmacokinetics of rhIL-22BP-ABD, both rhIL-22BP-ABD and rhIL-22BP were administered intraperitoneally at a dosage of 5 mg/kg in female BALB/c mice. The concentration of recombinant protein in serum at various time intervals post-injection was assessed using ELISA. The findings indicated that the serum level of rhIL-22BP-ABD peaked at 1 h, with a maximum concentration of 342.1 ng/mL, followed by a rapid decline from 1 to 8 h and a gradual reduction from 8 to 48 h (Fig. 5E). Conversely, the in vivo half-life of rhIL-22BP devoid of the ABD domain is rather brief, rendering it undetectable in less than two hours. The pharmacokinetic parameters were assessed utilizing the Microsoft Excel plug-in “PKSolver,” revealing that the in vivo half-life of rhIL-22BP-ABD was 17 h.

rhIL-22BP-ABD weakens the inflammatory phenotype of the mouse psoriasis model induced by imiquimod

To evaluate the in vivo therapeutic efficacy of rhIL-22BP-ABD on psoriasis, we developed an imiquimod-induced psoriasis mouse model by administering 5% imiquimod cream to the depilated dorsal skin of BALB/c mice for nine consecutive days, as depicted in the image (Fig. 6A). The mice received intraperitoneal injections of recombinant protein at several concentrations starting from the initial day of modeling. In comparison to the model group, rhIL-22BP-ABD at dosages of 5 mg/kg and 10 mg/kg diminished dorsal skin lesions and spleen size in psoriatic mice (Fig. 6B, C) and reinstated the spleen index (Fig. 6D). The PASI scores of both groups of mice administered rhIL-22BP-ABD were markedly diminished on the 8th and 9th days in comparison to the model group (Fig. 6E).

Fig. 6.

Fig. 6

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rhIL-22BP-ABD ameliorates imiquimod-induced psoriasis-like dermatitis. A Schematic diagram of psoriasis model and administration cycle. B Representative images of skin lesions on the back of mice in each group on day 4 and 8. C Representative images of spleen size of mice in each group. D Comparison of spleen index in each group. E PASI scores of the dorsal skin of each group of mice throughout the modeling process. Quantitative data are presented as mean ± standard deviation (n = 5). Statistical significance is indicated as follows: ****P < 0.0001

H&E staining of mouse skin was used to evaluate the anti-inflammatory impact of rhIL-22BP-ABD in psoriatic mice. Figure 7A illustrates that the model group exhibited neutrophil aggregation abscesses in the stratum corneum, a markedly thickened epidermis, elevated eosinophil levels, and heightened infiltration of dermal inflammatory cells relative to the non-model group, confirming the efficacy of the imiquimod-induced mouse psoriasis model. In the 5 mg/kg rhIL-22BP-ABD group, epidermal thickening occurred exclusively, dermal inflammatory cell infiltration diminished, and the incidence of epidermal abscesses improved. In the 10 mg/kg rhIL-22BP-ABD group, the skin exhibited keratinization just in the stratum corneum, the epidermis became hypertrophied, the infiltration of dermal inflammatory cells diminished, and the incidence of epidermal abscess formation was significantly reduced (Fig. 7A). In summary, rhIL-22BP-ABD attenuates the inflammatory phenotype in the murine psoriasis model in a dose-dependent manner.

Fig. 7.

Fig. 7

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rhIL-22BP-ABD decreases the expression of pro-inflammatory cytokines and STAT3 signaling pathway in psoriatic mice. A The representative images of the H&E-stained skin sections of different treatment groups. B Expression of IL-6 in different treatment groups by ELISA (n = 5). C Expression of TNF-α in different treatment groups by ELISA (n = 5). D The expressions of IL-22, IFN-γ, IL-1β and p-STAT3 proteins in different treatment groups were detected by Western blot. Quantitative data are presented as mean ± standard deviation (n = 5). Statistical significance is indicated as follows: ****P < 0.0001, and ns means no significant difference

rhIL-22BP-ABD decreases the expression of pro-inflammatory cytokines and STAT3 signaling pathway in psoriatic mice

To explore the inhibition of rhIL-22BP-ABD on the expression of pro-inflammatory cytokines in psoriasis mice, the levels of IL-6 and TNF-α in the serum of psoriasis mice were detected by ELISA kits, and the results showed that rhIL-22BP-ABD can significantly inhibit the increase of IL-6 and TNF-α in the serum of the inflammatory model, and the inhibitory effect of an injection dose of 10 mg/kg on IL-6 and TNF-α is superior to that of an injection dose of 5 mg/kg, indicating that this inhibitory effect shows a certain dose effect (Fig. 7B, C).

To define the molecular mechanism of rhIL-22BP-ABD’s anti-inflammation effect, Western blot was performed to detect expression of IL-22 and its downstream STAT3 signal molecular in dorsal skin, and the results showed that 10 mg/kg dose of rhIL-22BP-ABD reduced the expression of IL-22, IFN-γ, IL-1β and p-STAT3 in dorsal skin proteins compared with the model group (Fig. 7D). It is worth noting that the 5 mg/kg dose of rhIL-22BP-ABD has no reducing effect on the expression of related inflammatory factor proteins in dorsal skin proteins and the expression of inflammatory factors in serum, although it at a dose of 5 mg/kg can significantly reduce the spleen index and PASI score.

Discussion

Psoriasis is an autoimmune dermatological condition, distinguished by clearly demarcated erythematous plaques adorned with white scales (Boehncke and Schön 2015; Nestle et al. 2009). Significant advancements have been achieved in comprehending the fundamental mechanics of psoriasis, resulting in the creation of diverse therapeutic alternatives. Contemporary therapy approaches for psoriasis focus on alleviating symptoms, enhancing quality of life, and inhibiting disease progression (Armstrong and Read 2020; Kaushik and Lebwohl 2019). This encompasses topical therapies, phototherapy, systemic medicines, and biologics aimed at specific immune pathways. Notwithstanding progress in psoriasis treatment, numerous difficulties and unmet requirements persist (Lee and Kim 2023). Since 2000, biologics have been pivotal in the treatment of psoriasis (Reid and Griffiths 2020), with the primary biologics available still functioning by blocking the pathways linked to the principal pathogenic signaling axis, TNF/IL-17/IL-23. While presently available TNF-α inhibitors, IL-17 inhibitors, and IL-23 inhibitors can effectively manage psoriasis to some degree, their adverse effects need consideration. Adalimumab (anti-TNF-α antibody) is presently the preferred first-line biologic for managing psoriasis with psoriatic arthritis; however, it remains linked to adverse effects in rare conditions, including multiple sclerosis, congestive heart failure, opportunistic infections (e.g., tuberculosis), and lupus (Reid and Griffiths 2020). Ustekinumab and other agents are presently available as IL-23 inhibitors for psoriasis treatment. The primary adverse effects are nasopharyngitis, upper respiratory tract infection, headache, and weariness (Farhi 2010; Nogueira and Torres 2019; Tokuyama and Mabuchi 2020; Witjes et al. 2020). Consequently, the creation of an innovative medicine that effectively treats psoriasis without adverse effects is a pressing task in the biopharmaceutical sector.

Multiple studies have shown that IL-22 plays a role in the development and pathophysiology of psoriasis, suggesting that targeting IL-22 could serve as a promising therapeutic approach for treating the condition (Hao 2014; Martin et al. 2017). IL-22BP is a soluble receptor homolog that exhibits a higher affinity for IL-22 than the membrane-bound receptor, suggesting its potential therapeutic significance in diminishing IL-22 levels in psoriasis (Zenewicz 2021). In this study, we successfully engineered a recombinant bacterium that expresses a long-acting rhIL-22BP-ABD protein, comprising human IL-22BP and an ABD domain. This work demonstrated that recombinant protein was expressed in inclusion bodies, which can significantly facilitate future protein purification stages. Nonetheless, recombinant proteins present as inclusion bodies lack biological activity. Consequently, we utilized high-concentration urea-denatured protein, purified it, and delineated the parameters for protein reduction. We employed two strategies for protein gradient reduction: GSH/GSSG redox couples and DTT supplementation. In contrast to the GSH/GSSG redox pairs, DTT offers the benefit of requiring only two gradients of 4 M and 0 M urea for its reduction gradient, resulting in a total replication time that is less than that needed for the GSH/GSSG redox pair. Coversely, the renaturation rates of rhIL-22BP-ABD and IL-22BP proteins approached 100% with DTT, whereas the renaturation rates using GSH/GSSG redox were 60–70% and 10–20%, respectively, yielding a protein concentration insufficient to meet the requirements for subsequent molecular interactions. In conclusion, we ultimately selected DTT for protein renaturation. However, there are differences in biological activity among different batches of this protein in its inclusion body form, which may affect the further verification of its biological activity. During this process, without the assistance of genetic regulation, the protein’s folding will have a certain degree of deviation from its natural conformation. Currently, we have independently prepared three batches of protein samples, all of which show certain biological activity. However, at the same concentration, they did not have a consistent inhibitory effect on the proliferation of the same batch of HacaT cells. Furthermore, when using the Escherichia coli expression system to produce the target protein, the immunogenicity of the protein and the control of endotoxin contamination are the key factors that affect its biological application value. Even a small amount of residual endotoxin can trigger a strong inflammatory response through the activation of the Toll-like receptor 4 (TLR4) signaling pathway (Ciesielska et al. 2021; Fu et al. 2021), thereby non-specifically enhancing the immunogenicity of the protein. Currently, we thoroughly wash and perform affinity chromatography purification of the inclusion bodies when obtaining them, removing some endotoxin. In the future, we will further control the endotoxin content according to relevant standard requirements to reduce its impact on the immunogenicity of the target protein.

Research has established that IL-22 can promote the in vitro proliferation of HaCaT cells (Ma et al. 2023; Wang et al. 2018), which have been utilized as in vitro models for psoriasis in numerous research (Gao et al. 2020; Ma et al. 2023; Wang et al. 2018). Consequently, we hypothesize that the biological activity of rhIL-22BP-ABD can be assessed by measuring the proliferation rate of IL-22-stimulated HaCaT cells. Our results indicated that rhIL-22BP-ABD did not exhibit an inhibitory effect on the proliferation of HaCaT cells; nevertheless, it dramatically inhibited IL-22-stimulated cell proliferation by antagonizing the binding of IL-22 to its receptor (Fig. 4D-E).

The half-life of human IL-22BP has yet to be documented in the literature. Our findings indicated that the half-life of rhIL-22BP is notably brief, under 2 h (Fig. 5E). Consequently, the development of a long-acting IL-22BP protein is essential for IL-22BP to fulfill its biological function. The albumin-binding domain (ABD) has demonstrated the ability to extend the half-life of molecular pharmaceuticals (Mester et al. 2021; Nikravesh et al. 2022). We synthesized the fusion protein rhIL-22BP-ABD, comprising IL-22BP and ABD, using a prokaryotic expression system. We verified that the half-life of rhIL-22BP-ABD was markedly prolonged compared to rhIL-22BP through molecular interaction and half-life assessments, indicating a substantial enhancement in the drug’s stability and efficacy. Despite an 80.8% homology between human IL-22 and mouse IL-22 (Wolk and Sabat 2006), the homology between human IL-22BP and mouse IL-22BP was merely 67.1% (Wei et al. 2003), suggesting that the binding affinity of rhIL-22BP-ABD to mouse IL-22 is limited. Consequently, the half-life of rhIL-22BP-ABD in the human body may exceed our experimental findings.

Any protein can serve as a double-edged sword, and IL-22BP is no exception. Certain findings indicated that elevated levels of IL-22BP result in numerous adverse consequences (Fachi et al. 2024). IL-22 enhances intestinal barrier integrity by encouraging epithelial cells to activate defensive mechanisms against enteric infections. In IL-22-deficient animals, IL22BP enhances vulnerability to acute dextran sulfate sodium (DSS) and T-cell-mediated colitis (Zenewicz 2021). Conversely, a lack of IL22BP augments the gut microbiota’s capacity to defend against enteric infections (Fachi et al. 2024). IL-22 enhances bacterial host defense in the lung by maintaining epithelial integrity and inducing antimicrobial peptides, while its activity is decreased by IL-22BP, indicating that IL-22BP has a pro-inflammatory role and compromises epithelial barrier function, presumably through its binding to IL-22 (Abood et al. 2019). Consequently, we performed an initial safety evaluation of rhIL-22BP-ABD. Administration of 10 mg/kg of rhIL-22BP-ABD into healthy BALB/C mice, followed by the assessment of serum AST and ALT levels, along with histological examination of the kidneys, revealed no significant variation in enzyme levels compared to the normal group, as indicated by the renal pathological analysis (Supplementary file 2: Data 1), suggesting a high safety profile for rhIL-22BP-ABD. We will enhance the safety assessment of this medicine in future study. In conclusion, this study effectively produced long-acting rhIL-22BP-ABD using a prokaryotic expression method. This protein can significantly reduce psoriasis inflammation and suppress the IL-22/STAT3 signaling pathway and associated inflammatory factors in mice induced by imiquimod, indicating that rhIL-22BP-ABD holds substantial promise as a potential therapeutic agent for psoriasis treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (333.5KB, doc)
Supplementary Material 2 (109.6KB, docx)

Acknowledgements

None.

Author contributions

XZC performed the experiments, analyzed the data and wrote the manuscript. TZ, QYT, YYL, WLZ and BQG performed the experiments. FF, NT and JZZ revised the manuscript. YCX design the project, revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by Basic and Applied Basic Research Foundation of Guangdong Province (2022A1515010028, 2024A1515012937).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal experiments conducted in this article were approved by the Laboratory Animal Ethics Committee of Guangdong Medical University.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (333.5KB, doc)
Supplementary Material 2 (109.6KB, docx)

Data Availability Statement

No datasets were generated or analysed during the current study.

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