{'fraction_non_alphanumeric': 0.0426606435894833, 'fraction_numerical': 0.01997259161506446, 'mean_word_length': 4.651893287435456, 'pattern_counts': {'":': 0, '<': 11, '<?xml version=': 0, '>': 1, 'https://': 1, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Amelioration of imiquimod-induced psoriasis-like dermatitis in mice by DSW therapy inspired hydrogel

Psoriasis is a long-lasting and recurrent autoimmune disease which is incurable so far. Dead Sea water (DSW) therapy is an effective approach to help control the symptoms of psoriasis due to the abundant mineral ions in DSW, which inspired the material formulation in this study. Rubidium–Sodium alginate/Polyacrylamide hydrogels (Rb-SA/PAAm gels) composed of sodium alginate and polyacrylamide interpenetrating network structure with different concentrations of rubidium and certain magnesium and zinc content were prepared for the treatment of psoriasis. The obtained results suggest the good mechanical properties of the Rb-SA/PAAm gels including toughness and swelling performance. In terms of in vitro tests, the Rb-SA/PAAm gels not only show nontoxicity to human keratinocyte cell line (Hacats) but also inhibits the activity against inflammatory NF-κβ signaling pathway. Meanwhile, they can release Rb+ which enable the Rb-SA/PAAm gels have better antibacterial ability to Streptococcus and Escherichia coli. The results obtained from in vivo tests indicate that these hydrogels could alleviate the symptoms of psoriasis caused by Imiquimod (IMQ) in mice by reducing the inflammatory factor in STAT3 pathway and therefore reduce the immune stimulation of the spleen. In conclusion, the 100Rb-SA/PAAm gel has demonstrated great potential to be a topical wettable dressing for psoriasis treatment.


Introduction
Psoriasis is an inflamed disease mediated by the immune system and very widespread all over the world. It was estimated that the psoriasis population accounts for about 1-2% of the world's population [1]. Psoriasis usually develops in late adolescence or early adulthood and is too difficult to be cured. The skin of the patient presents with scales and plaque-like skin damage accompanied by itching and pain [2]. The stratum corneum cells of psoriasis patients are found to be over-proliferating and incompletely differentiating. The deep-stage pathogenesis is still unclear, so the treatment strategy is not uniform yet [3]. Nowadays smear hormones, immunosuppressive agents, phototherapy, and ozone therapy are the most used therapeutic methods. However, these treatments have significant side effects or other disadvantages. For example, glucocorticoids must be used uninterruptedly, otherwise, it may cause "retaliatory rebound", which can lead to severe skin damage if discontinued [4]. Long-term use of immunosuppressant (eg. Methotrexate) and phototherapy can cause extensive liver fibrosis, cirrhosis [5,6], even skin cancer [7]. Many pieces of research have shown side effects of ozone therapy, because of its instability, easiness to decompose and difficulty to work in deep skin. It mainly plays the role of sterilization [7]. Consequently, finding a new way to treat psoriasis with fewer or no side effects is critically important for both doctors and patients.
In recent years, hydrogel has attracted significant attention. Hydrogel is a gel with water as a dispersion medium. It exhibits high swell ability in an aqueous environment and hydrogel scaffold also provides spatial and mechanical stability for new tissue formation. When the cells are contacted with hydrogel, the largely swollen hydrogel promotes the absorption of nutrients and the release of waste of cells. Accordingly, hydrogel has broad applications in tissue engineering, especially on the skin [8]. The good biocompatibility and strong water retention function of hydrogel help to retain the hydration and intact of the stratum corneum of the skin. As a result, it is widely used as a drug delivery carrier [9], therapeutic agent encapsulation materials [10], wound dressings [11], and tissue engineering scaffolds [12]. In skin tissue engineering, most of the traditional medical hydrogels used for wound treatment always lack viscosity, insufficient hardness, strength, and toughness [13]. For psoriasis, hydrogel must be durable and stress resistant in order to withstand the normal stress encountered during their application and handling which contributes to long-time drug delivery [14]. Appropriate toughness and stiffness mean that this kind of biomaterial can fit body undulation perfectly [15]. The moisturizing of the affected skin areas is beneficial for psoriasis treatment. Moreover, diseased parts of psoriasis tend to have thicker layers and larger areas, which makes the ordinary hydrogels cannot meet the long-term operating requirements.
The mechanical properties of hydrogel have a direct impact on its performance. Interpenetrating network hydrogels overcome the disadvantages of traditional hydrogels, especially the poor mechanical properties [16]. For instance, calcium cross-linked sodium alginate/ polyacrylamide interpenetrating network structure has the characteristics of high viscosity(3.5 times higher than the control) and large elasticity on the order of 100 kPa [17]. Iron cross-linked sodium alginate/polyacrylic acid interpenetrating network structure possesses high toughness and self-healing ability [18]. Polyacrylamide/polyvinyl alcohol interpenetrating network structure has extremely high toughness and elongation [19]. In practical application, hydrogels have to be loaded with antibiotics to achieve antibacterial activity [20] which may lead to drug resistance. Hence, improving the antibacteria ability of hydrogel by introducing multiple inorganic ions has attracted more attentions.
Recently, Dead Sea water (DSW) therapy for psoriasis is increasingly popular, with a high cure rate. After this treatment, the psoriasis severity index decreased by 81.5%, and 70% of patients were fully cured [21]. Previous studies have shown that the onset of psoriasis may be associated with the imbalance of the elements in the body (such as the lack of Zn 2+ and Mg 2+ ) [22,23]. The therapeutic effect of DSW on psoriasis comes from the rich minerals contained in the Dead Sea [24]. The concentration of Rb + in the DSW is 250 times that in the normal seawater, while the concentration of Mg 2+ and Zn 2+ is more than 40 and 30 times, respectively [25]. Studies have shown that Mg 2+ can regulate the balance between cyclic adenosine phosphate (cAMP) and cyclic guanosine phosphate (cGMP). The excessive proliferation of cells caused by the increase of cGMP is one of the main causes of psoriasis, so magnesium can inhibit the abnormal proliferation of Hacats [26,27]. The reason of the choice of such specific human cells is that keratinocytes in the skin lesions of patients with psoriasis tend to differentiate and proliferate abnormally [28], and human skin immortalized keratinocytes (Hacats) are highly similar to normal human keratinocytes in differentiation and biological characteristics, so they are often used to replace keratinocytes [29]. Hacats have been widely used to study psoriasis and related lesions [30]. Besides, as a cofactor for many enzymes, Mg 2+ can inhibit protein synthesis during the development of psoriasis [31,32]. Lack of Zn 2+ will lead to abnormal keratinization, resulting in incomplete or excessive keratinization and rough thickened skin [33]. Rb + can regulate the propagation and differentiation of epithelial cells [34]. Previous studies have shown that Rb + can alleviate psoriasis to some extent [35] and the content of Rb + in patients' bodies who treated with DSW therapy was significantly increased [36].
Meanwhile,it has been reported that psoriasis and atherosclerosis have similar immune-mediated the inflammatory responses, and increasing the content of Rb + in human blood can inhibit atherosclerosis [37], which implies that Rb + may inhibit psoriasis by inhibiting inflammatory response. Mg 2+ and Zn 2+ are both divalent ions that can cross-link alginate to form gels with specific shapes. Zn 2+ and Mg 2+ were added into alginic gels, which can improve the effectiveness of the transmission of Zn 2+ and Mg 2+ through the ionic electroosmosis mechanism, and also can capture abundant water with abundant hydrogen bonds, promoting the local treatment of psoriasis [38]. However, Alginic gels only linked by ions have shortcomings of poor swelling and mechanical properties [17,18]. Polyacrylamide hydrogel can alleviate psoriasis to some extent due to its high flexibility, biocompatibility, and air permeability. If acrylamide was added into the alginate system, the alginate/polyacrylamide interpenetrating network structure formed by in-situ generation polymerization can have the advantages of alginate and polyacrylamide, such as the excellent biocompatibility and mechanical properties [17,39].
Thus, in this study, inspired by DSW therapy, Rubidium-Sodium alginate/Polyacrylamide hydrogels (Rb-SA/PAAm gels) interpenetrating network structure has been synthesized by light curing method, in which an in-situ formed and physical combined zinc-magnesium-alginate hydrogel decorated with polyacrylamide. The SA/ PAAm hydrogels containing Rb + demonstrate good physical properties and superior biological response will meet the clinical need and bring hope for the psoriasis treatment. The fabrication of porous Rb-SA/PAAm gels were adulterating different concentrations of Rb into SA adopting in situ synthesis as modified the method mentioned in previous publication [17] (Fig. 1). Samples were defined as 0/10/50/100 Rb-SA/PAAm gels, respectively, according to their own Rb/(Rb + Na) proportion (0, 10, 50 and 100 at %). In short, Rb 2 CO 3 and AA were dissolved in water with a mass ratio of 1:1 to synthesize rubidium alginate (RA). Then ZnCO 3 and MgCO 3 were added to (SA and RA) mixtures, mixed and vortexed for 20 min, the mass concentration ratio of n (ZnCO 3 + MgCO 3 )/n (SA + RA) was 2:1. Then GDL and AAm were dissolved in the above solution. AAm weighed 0.168 times as much as water. The ZnCO 3 and MgCO 3 to GDL molar ratio of was always maintained at 0.5 to achieve a neutral pH. The mixture were stirred for 40 min at 25°C until the solution was well mixed. Then the mixture was blended with MBAA, TEMED and APS orderly, which were weighted 0.001, 0.01 and 0.01 times of AAm, respectively. Then the mixture was placed in a glass mold and cured with UV (20 W, wavelength 365 nm) for 3 min.

Characterization of Rb-SA/PAAm gels
The pore structure, element distribution, surface morphology of Rb-SA/PAAm gel were characterized by scanning electron microscopy (SEM, Nova NanoSEM, USA), as well as energy-dispersive spectrometry (EDS, EDAX, USA). Before SEM observation, the hydrogels were freezedried and coated with a thin platinum film to improve the image quality. The phase and crystallinity were detected by X-ray diffraction (XRD, Brooke Advance D8, Swiss). The surface roughness was measured via atomic force microscopy (AFM, Nanosurf Easyscan 2, Swiss) in an area of 10 × 10 μm 2 under the tapping model. The freeze-dried hydrogel samples mixed with KBr were then pressed onto a transparent sheet and functional groups were examined by a Thermo Scientific Nicolet nexus Fourier transform infrared (FTIR) spectrometer.

Mechanical and rheological properties
The uniaxial mechanical properties of Rb-SA/PAAm gels were tested by a universal testing machine (235 MTS Insight, USA) at room temperature. The crosshead speeds of the tensile tests and the compression test were 50 mm/min and 1 mm/min. In the tensile tests, the length of the sample is 20 mm and the diameter is 3.2 mm./While for the compression tests, samples were prepared in a height of 12 mm and a diameter of 8 mm in diameter (The interesting compression process is shown in video 1). Three independent measurements were conducted with a fresh sample per case.
Supplementary video related to this article can be found at https:// doi.org/10.1016/j.bioactmat.2020.08.007 To evaluate the hydrogels' rheology property, the storage modulus (G′) and the loss modulus (G″) were measured by a rheometer (AR2000EX, TA Instruments, UK). In this test, the sweeping angular frequency was from 0.1 to 650 rad/s and the angular frequency function was at strain = 2% at room temperature.

Swelling behavior and ion release of Rb-SA/PAAm gels
Test the swelling kinetics, cylindrical-shaped Rb-SA/PAAm gels were freeze-dried (diameter = 10 mm, height = 2 mm). The weight values before soaking were measured and recorded as the dry weight (W a ). Then the samples were immersed in phosphate buffer solutions (PBS, pH = 5.0). The mass of each sample after different soaking time was obtained and defined as W b . Meanwhile, ion release rates were measured using inductively coupled plasma atomic emission spectrometry (ICP, ThermoFisher, iCE-3500, USA). All measurements were repeated three times. The swelling ratio (%) was calculated according to the formula shown in Equation (1).

. Response of cells to Rb-SA/PAAm gels
Human skin immortalized keratinocytes (Hacats) have been widely used to study psoriasis and related lesions [40]. They were cultured on the Rb-SA/PAAm gels. Each group has four parallel samples. The steps were followed as described in previous studies [41,42]. In short, the Rb-SA/PAAm gel was sterilized with ethyl alcohol (75%), before those cells were inoculated. After being soaked in dulbecco's modified eagle medium (DMEM), the hydrogels were expanded to equilibrium state. The cells on the hydrogels were stained with Thermo Scientific HER-Acell (150i, U.S.A.) and then photographed by laser scanning confocal microscope using (TCSSP5, Leica, Germany). As described in previous work, CCK8 method was used to quantify the cell growth on hydrogels [43].

qRT-PCR
The qRT-PCR was used for studying the effect of Rb-SA/PAAm gels on the expression of inflammatory factors (IL-1, IL-22, IL-23, etc) that are related to psoriasis. The detailed sequence was shown in Table S1. After collecting cells, RNA of cells was extracted with Trizol reagent (Invitrogen). The OD 260/280 ratio was between 1.8 and 2.0 by Na-noDrop 2000 spectrophotometer (Thermo). Complementary DNA (cDNA) was generated by using reverse transcriptase kit (Invitrogen), and amplificated by SYBR Green PCR Master Mix (Thermo). Actin was used as an internal reference to compare the relative expression of these genes. The cycle threshold (Ct) values were determined using CFX384 Real-Time System (Bio-Rad, USA).
2 −ΔΔCt reflects the ratio of the target gene expression level of each sample to that of the control group.

Antibacterial activity assay
According to the results of the above experiments, 0/50/100Rb-SA/ PAAm were further used in the following anti-bacterial tests. The platecounting method was used to test the antibacterial properties of Rb-SA/ PAAm gels by co-cultured with E.coli (ATCC27853) and hemolytic Streptococcus (CMCC32210) [20]. In short, 0.5 g samples were added into a 5 mL bacterial solution (5 × 10 4 CFU mL −1 ) in a tube at 37°C for half-day, shaking at 100 r/min. A 10 μL solution was diluted 10000 times and plated on a nutrient agar dish for a 12 h co-culture, and the number of viable bacteria in each dish was counted.
In order to understand the reason for antibacterial behavior, the pH value of sample extract was measured by a pH meter (Seven Excellence S500, mettler toledo, Switzerland).

Evaluation of psoriasis-like mouse model with Rb-SA/PAAm gels
The repair ability of Rb-SA/PAAm gels was measured by a previously modified method of building the psoriasis-like mouse model [44]. According to the results of cell culture experiments, 0/50/100 Rb-SA/PAAm samples were selected for in vivo study. The negative control is the group with IMQ treatment only, the positive control group is the untreated defects (only hair removed). All procedures were supported by the animal ethics association of the animal center of Central South University. Six-week-old female w/BC mice were treated with IMQ (from Xin Ming company of China) on the depilated back skin once a day for 6 days except for untreated defects, creating an experimental psoriasis-like mouse model. When the skin on the back of the mouse turns red, thickens and scales accumulate, it indicates that the modeling is successful. Except for the negative control group (IMQ treatment only), 0/50/100rbsa-PAAm gel(2*2.5*0.2 cm) was applied to the skin of animals in the other 4 groups for 1 time a day. The gel was cut into a uniform specification of 2*2.5*0.2 cm and fixed on the back of the mouse with gauze.

qRT-PCR and Western blot (WB)
After the mice were killed, the treatment part of their back skin was taken. The skin tissue was frozen quickly with liquid nitrogen, stored at −80°C, and then cut into small pieces and homogenized in a homogenizer. Total RNA was extracted with Trizol (Invitrogen). The extracted RNA was converted into cDNA by reverse transcriptase kit (Invitrogen). Then qRT-PCR procedure was conducted with SYBR Green PCR Master Mix (Thermo, USA) with β-actin as internal reference. The detailed sequence was shown in Table S2. The skin was cut up and homogenized using a homogenizer (Qiagen, Tissue Lyser II, German) with stainless steel ball-bearing. Tissue homogenate and primary keratinocyte lysate were obtained in Ripa lysate buffer with a mixture of 1× protease inhibitors. The insoluble substance was granulated at 12000 r/min and 4°C for 5 min, and then the supernatant liquid was taken, separated electrophoretically with sodium dodecyl sulfate-polyacrylamide gel and shifted to the polyvinylidene fluoride film. The expression of STAT3 was analyzed by primary rabbit anti STAT3 (Abcam) and secondary antibody Goat anti rabbit IgG (Abcam). The target protein was detected by the electrochemiluminescence (ECL) system and visualized by the Image Lab software. The same method was used to detect p-STAT3, P65, P50.

Histology, immunohistochemistry and immunofluorescence analysis
Fresh mouse skin samples were immersed in 4% paraformaldehyde solution for one day, then dehydrated and embedded in paraffin. After beingsliced, the samples were stained by hematoxylin and eosin. The expression of CD1α and CD68, CD4 and IL-17, CD20 and CD19 were detected in 5 mm paraffin embedded sections of mouse psoriatic skin with rat anti-CD4 and IL-17 primary antibodies (Abcam). Secondary Goat anti-rat IgG-TR antibodies and Goat anti-rabbit IgG FITC antibodies (Abcam) were used, respectively. The primary Abs to CD1α, CD68, CD20 and CD19 were connected with fluorescence directly. In the negative control group, the main antibody was omitted. Zeiss and Axio vr.a1 were used to examine and photograph the stained sections.

Evaluation of spleen expression in psoriasis-like mouse model
The weight of spleen was seen as the indicator to judge immune stimulation or depletion. The spleen was obtained after the mice were killed, then the spleen/body %(wt%) was counted. The spleens from different groups were stained with HE according to section 2.6.3.

Statistic analysis
Statistic analysis was conducted by Student's t-test and one-way analysis of variance with Tukey's post hoc test with GraphPad Prism 8.0. (GraphPad, San Diego, California, USA). All experiments were replicated 3 times and the data were shown as mean ± SD. p < 0.05 was taken for statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.

mechanical properties of Rb-SA/PAAm
It was observed from Fig. 2A, the Rb-SA/PAAm gel had a large tensile strain which displays its incredible toughness and stretch ability. Furthermore, the Rb-SA/PAAm gel exhibited excellent compression resistance. Once unloading pressure, Rb-SA/PAAm gel rapidly returned to its initial shape (Fig. 2B), showing excellent shape-recovery property. Typical compression stress-strain curve and typical tensile stress-strain curve of different kinds of hydrogels are embodied in Fig. 2C and D. It can be seen that as the Rb/Na + Rb proportion increases from 0 to 100 at. %, the tensile strength and toughness of SA/PAAm hydrogels decreased, which means that the incorporation Rb was closely related to PAAm chains and had a negative influence on the mechanical properties.

Phase analysis
In order to understand the influence of rubidium addition to SA/ PAAm gels on the phase and functional group of the system, the XRD and FTIR techniques were used. It can be found in XRD patterns that all the samples have no obvious crystal peaks, and each diffraction peak is wide and low. There was a slight decrease on intensity of the 27.5°p eaks doped with Rb + . In addition, the span of the strongest peak widened, which is similar to our previous research [45]. It was suggested the Rb + cross-linking effect contributes to amorphous structure of SA/PAAm hydrogel (Fig. 3A). It is found that the higher the Rb concentration is, the difference of Δν (-COO-) of Rb-SA/ PAAm is close to but slightly greater than Δν (-COO-) Na and Δν (-COO -) Rb. The results of Nakamoto et al. [46] show that when as (-COO-) shifts red, s (-COO-) shifts blue, and Δν (-COO-) metal ions ≈ Δν (-COO-) Na, the metal ions form a coordination bond with -COO-on the SA segment. It is inferred that the form of cross-linking of two kinds of metal ions (Mg 2+ and Zn 2+ ) with SA and RA is coordination crosslinking. With the increase of Rb content, Δν decreases gradually, which indicates that the increase of Rb content reduces the overall crosslinking density of Rb-SA/PAAm gel. In addition, amidation reaction can occur between acrylamide and alginate, which will enhance the corresponding C-O (1040), C-N (1020). The addition of rubidium has little effect on these peaks, indicating that rubidium mainly affects the ion crosslinking process of alginate rather than controlling the amidation crosslinking. Meanwhile, hydrogen bonds can be formed between -NH and -OH of acrylamide and alginate, which is manifested in the broadening and coupling among the peaks of as (N-H), s (N-H), and ν (O-H).

Rheological properties of Rb-SA/PAAm gels
The storage and loss modulus are important factors affecting the gelation of hydrogels. Fig. 3C and D displayed the results of rheological behaviors. The value of G" is lower than that of G' at any angular frequency, implying that these samples have better elasticity than viscosity. The slope of G′ with frequency is small, which indicates that the polymer has a typical cross-linked network structure, and no gel-sol transition occurred at the experimental frequency. By comparing the rheological results of hydrogels with different Rb contents, it can be observed that the elastic modulus and loss modulus of Rb-SA/PAAm were inversely proportional to Rb content. It indicates that Rb could decrease the degree of cross-linking and make the polymer network structure more loose, which is consistent with the results of SEM and FTIR detection.

Swelling/ion release Capacity of Rb-SA/PAAm gels
Swelling property plays a crucial part in the application of hydrogels in skin tissue engineering. For understanding the swelling behavior, we recorded the uptake in phosphate buffer saline (PBS, pH = 5.0) at 37°C. Four kinds of Rb-SA/PAAms were cultured in PBS, (pH = 5.0) media for 24 h. Fig. 4A demonstrated the equilibrium swelling values. The swelling ratios of all the Rb-SA/PAAm gels increased rapidly with time in the first 3 h and achieved stability after 8 h (All gels swelled more than 800%) which is similar to the recent study [39]. The incorporation of Rb + has a few impacts on their swelling ability. As shown in Fig. 4A, swelling rate achieves a balance after 24 h.The traditional view is that the equilibrium degree of swelling is inversely proportional to cross-linking density of the hydrogels [39,47]. The release of Rb + was controllable, and the sample groups with higher content of Rb released more Rb + . In Fig. 4B, after more than 7 days concentration measurement, the sample groups with higher content of Rb released more Rb + which represents high correlation within the A) The Rb-SA/PAAm gel could restore to initial length after stretching to four times its original length. B) Digital images showing that the Rb-SA/PAAm gel could recover its initial shape after compressing to a strain of 95%.Stress-strain curve of C) typical compression and D) typical tensile. amount of Rb added and released, so that the amount of Rb + released were able to be controlled by adjusting the contents of Rb in the hydrogel. The concentrations of Mg2 + and Zn2 + ions of the 4 groups make no difference in Fig. 4C and D due to the fixed amount added in every sample.

Microstructures and surface properties
As showed in SEM images of Fig. 5A-D, all the lyophilized Rb-SA/ PAAms had analogous porous structures (hole diameter = 30-70ìm), which is representative characteristic of lyophilized SA/PAAms, which has an association with ice crystals [48]. Interestingly, the pores of SA/  PAAm without Rb are denser and more uniform than the Rb-SA/PAAm, which also supports the hypothesis that crosslink density played a crucial role in the stable and regular structure [49]. In 0Rb-SA/PAAm gel, pore diameter is 35.5 ± 11.1 mm (Fig. 5E). Due to Rb added, crosslink density increases with smaller pore sizes (Fig. 5F). Then increasing contents of Rb result in decrease of crosslink density so pore sizes become large( Fig. 5G and H). Fig. 5I-L demonstrated that Rb + was scaffold-incorporated. AFM was applied to study the surface structures of Rb-SA/PAAm gels, which confirmed the rubidium content can affect the surface roughness of the hydrogel. The average area roughness (Ra) of four different Rb-SA/PAAm membranes were measured by AFM ( Fig. 5M − P) it turned out that the Ra of 0/10/50/100Rb-SA/PAAm membranes were 23.7 ± 2.1, 33.4 ± 1.2, 48.5 ± 2.6 and 81.9 ± 4.8 nm, respectively. From the above results, doping Rb ions into the gel increased the surface roughness of the Rb-SA/PAAm. Within a certain range, the greater the surface roughness of biomaterials, the better the wound healing. Rough surfaces enhance the ability to produce initial proinflammatory cytokines, thus producing higher concentrations of anti-inflammatory and immunomodulatory factors [50].

Antibacterial performance of the composite hydrogel
The causes of psoriasis are complex, including infections of bacteria such as E. coli and Streptococcus pyogenes. E. coli has been reported as one of the related microorganisms that may cause psoriasis [51].
Streptococcus is a well-recognized reason for acute guttate psoriasis [52], which is highly correlated with first onset/relapse in individuals who are prone to psoriasis [53]. So some scholars believe that longterm prevention and treatment of Streptococcus pyogenes infection are very important in the management of chronic plaque psoriasis [53]. An expected function of composite hydrogels is the antibacterial activity. Number of colonies counted by a plate count and presented as a histogram ( Fig. 6I and J). The results confirm the hypothesis and show that the antibacterial rate of SA/PAAm composite hydrogel in extraction and direct contact method was above 80% (Fig. 6I and J). In comparison, the SA/PAAm hydrogel without rubidium showed a little inhibition on the growth of Streptococcus and E. coli. As shown in Table 1, all the SA/PAAm composite hydrogels were weakly acidic without obvious difference resulted by rubidium, Mg 2+ or Zn 2+ . The 0Rb-SA/PAAm gels have a certain degree of antibacterial activity (Fig. 6B,F), owing to the effect of both Mg 2+ and Zn 2+ [54]. The hydrogels with more Rb have better antibacterial capability (Fig. 6C, D, G, H) with the prerequisite of same content of Mg 2+ and Zn 2+ in every sample.The results is in accordance with previous study [55], which further validated the potent antibacterial properties of Rb.

In vitro bioactivity of Rb-SA/PAAm gels on Hacats
The CCK8 was used to determine whether these hydrogels have unspecific cytotoxicity on Hacats. The result (Fig. 7A) indicates that Rb-SA/PAAm did not have significant inhibition effect on the proliferation of Hacats. Cell stretch is a significant clue to assess the cellular affinity of biomaterials. The aim of the current work is to analyze the Hacats cultivated on the four hydrogels for 12 h by CLSM (Fig. 7B). The rhodamine-phalloidin reagent (red), and diamidine phenyl indole (DAPI, blue) were used for the staining of F-actin and nucleus, respectively. The immunofluorescent micrographs shows the cells on the Rb-SA/ PAAm gels grew vigorously and had typical cell morphological characteristics. Meanwhile, the spreading areas on the different Rb-SA/ PAAm hydrogels are similar, indicating good cell adhesion and spreading. By electrostatic interactions, the proteins are immobilized on the hydrogel, which in turn forces the cells to adhere firmly to it. This hydrogel contains a large number of cations, and many vital proteins of the extracellular matrix, such as vitronectin, collagen, and fibronectin, are overall negatively charged (pH < 7) at physiological conditions [56]. So, they can attract each other, which is conducive to the attachment of cells to the hydrogel. In addition, compared with other ordinary hydrogels, higher stiffness value of this hydrogel is helpful [57]. Good adhering state of Hacats is revealed from Fig. 7B on account of its adherent growth. Above all, all the results are further evidence of unspecific cytotoxicity of Rb-SA/PAAm hydrogels. From Fig. 7C, it can be observed that the Rb-SA/PAAm can down-regulate TNF-α, IFN-γ, STAT3, JAK, IL-1β, IL-4, β-TrCP, IKK gene expression. NF-κB which is composed of p65 and P50 proteins can regulate the expression of many genes which are involved in the response of cells to external stimuli. It is a signal pathway of many kinds of immunity and inflammation and a key regulator in cell proliferation, differentiation and apoptosis, but also an important mediator of psoriasis [58,59].The NF-κb signaling pathway arising from extracellular stimulation leads to a series of downstream reactions. The receptor protein first activates the IκB kinase (IKK), which decomposes the NF-κB·IκB complex and releases free NF-κB. NF-κB rapidly enters the nucleus and starts the transcription process. NF-κB promotes the production of IL-1 β, IL-4, IFN -γ and other important inflammatory factors, leading to the developing of psoriasis. In addition, many cytokines like IFN-γ, IL-12, IL-22 and IL-23,13 also through JAK-STAT signaling pathway to play a role in promoting psoriasis [60]. In the IMQ treated mice, the Wnt/β-catenin signal pathway was destroyed obviously, which elevated the expression of IL-2, abated the IL-10 expression and aggravated the inflammation progress [61]. In the IMQ treated mice, we found that the content of β-TrCP increased significantly, and β-TrCP could degrade the β-catenin, which may be the reason why Wnt/β-catenin signal pathway was inhibited in the IMQ treated mice. Through the treatment of Rb-SA/ PAAm gels, the expression level of β-TrCP decreased significantly.

Histology and psoriasis area and severity index (PASI) evaluation
The back skin of five groups of mice is shown in Fig. 8. Compared with the control group, the typical symptoms of psoriasis can be seen on the back of mice treated with IMQ only, with severe inflammatory reaction and white, scaly and thickened skin. In HE staining results, it can be observed that it is consistent with the skin macroscopic view. In addition to basal and suprabasal hyperplasia, abnormal differentiation, hyperkeratosis and incomplete keratosis of keratinocytes are also found. After treatment with different hydrogels containing Rb + , especially with 50/100Rb-SA/PAAm gels, the symptoms of inflammatory  grade improvement. For 50Rb-SA/PAAm and 100 Rb-SA/PAAm gel, the scores of the two indexes were much lower (p < 0.05). The total PASI scores for the five groups are shown in Fig. 9D. After 6 days of treatment, the total PASI score of IMQ+100Rb-SA/PAAm treatment group (4.6 ± 0.30) was the lowest followed by IMQ + 50Rb-SA/PAAm gel and pure SA/PAAm gel. However, the highest total PASI score (9.80 ± 0.41) was seen from the IMQ alone group. It could be indicated that moisturizing effect of 0SA/PAAm gel presents a positive effect on psoriasis. The Rb + from 50/100Rb-SA/PAAm is beneficial for treating psoriatic skin lesions.

Evaluation mechanism of Rb-SA/PAAm gels in vivo
IL-23/IL-17 A cytokine axis has a close relationship with the pathogenesis of psoriasis which evidenced by clinical experiment and IMQ causes psoriasis-like dermatitis skin lesions [62]. The mRNA levels of related cytokines of mouse skin were researched by qRT-PCR analysis.The result is shown in Fig. 10. In comparison with normal group, mRNA levels of IL-1β; IL-22; IL-23; stat3; IFN-γ; IL-17F; IL-6 and TNF-α noticeably elevated in the skin of IMQ only group (p < 0.05). However, the mRNA expression levels of these eight cytokines considerably decreased in the 50/100Rb-SA/PAAm gel compared to the pure IMQ group (p < 0.05). However, pure SA/PAAm gel treatment did not show apparent effect. The results (Fig. 10I) demonstrated that Rb-SA/ PAAm gels downregulated protein secretion of STAT3, p-STAT3, p65 and p50 which further certificated that Rb-SA/PAAm gel could inhibit the IL-23/IL-17 A by inhibiting the two signal pathways of STAT and NF-κB.

Immunofluorescence staining
It is believed that psoriasis is caused by the lack of Langerhans(LC) cell. In this experiment, we use immunofluorescence double staining (CD1α and CD68) to label LC cells (Fig. 11A). In normal mouse skin, many cells can express CD1α and CD68 at the same time. However, in IMQ skin, even there are some green fluorescence and red fluorescence, there are few overlaps. In other words, LC cells in IMQ only group almost disappear. With the addition of rubidium, the number of other immune cells in corresponding tissues reduced, but the number of LC cells increased. In lesions from psoriatic patients, IL-17-producing cells are considered to be key pathology-driving factors which influence the psoriasis [63]. Finally, the changes in CD4 + IL-17 + T lymphocytes (Th17) were observed by immunofluorescence in mice back skin lesions. Compared with the IMQ only group, Rb-SA/PAAm prevents the invasion of Th17 in the back skin (Fig. 11B).The outcomes illustrated that Rb-SA/PAAm could reduce the local penetration of Th17 lymphocytes in mice. Th17 cells can produce IL-17 A, IL-17 A can induce keratinocytes to express CCL20, and recruit more Th17 cells and dendritic cells, which can increase the level of Th17 in psoriatic lesions [64]. Through immunofluorescence double staining of Th17 cell markers CD4 and IL-17, it can be observed that rubidium-containing hydrogel can effectively inhibit the infiltration of Th17 into the lesion site, thereby alleviating the related symptoms. The mechanism of B cell inhibition of mouse psoriasis induced by IMQ may be through the regulation of Th1/Th2 balance or the interaction with regulatory T cells [65]. By immunofluorescence labeling different groups of B cells (Fig. 11C), it can be observed that the IMQ group significantly reduced the B cells in the lesion area, and the rubidium-containing hydrogel dramatically increased the number of B cells in the lesion site.

Evaluation of spleen response to Rb-SA/PAAm gels
The spleen is one of the biggest organs in human/animal body. If the ratio of spleen to body wt% augmented, there are more immune cells in the spleen, that is to say, the immune response is more serious [66]. After the IMQ treatment for 6 days, the percentage of spleen/body weight (1.05 ± 0.37) in the IMQ only group was more than 2 times higher than that in the normal mice group (0.5 ± 0.19) after 6 days of administration, indicating that the number of spleen cells increased ( Fig. 12A and B). What is more, this discovery marks the immune activation of psoriasis-like mice. The spleen/body wt% values of the three hydrogel groups decreased to varying degrees in comparison to the IMQ group. In the three groups, 100Rb-SA/PAAm group had the lowest spleen/body wt%. The spleen/body wt% of Rb-SA/PAAm gel treatment group was observably smaller than that of IMQ group and 0Rb-SA/ PAAm group (p < 0.05). However, there was no significant variation between 50/100 Rb-SA/PAAm gel treatment groups (p > 0.05). The white pulp of the spleen is a site of immune response and may expand when exposed to proinflammatory signaling molecules [67]. In this study, we investigated the result of the ratio of white pulp expansion of the spleen is similar to spleen/body wt% ( Fig. 12C and D) which indicates that a better therapeutic efficacy by the Rb-SA/PAAm gel.

Conclusions
As the above research shows, Rb-SA/PAAm gels were in-situ synthesized through dual-ion cross-linking with Zn 2+ and Mg 2+ . The chemical, physical and biological behaviors were investigated in detail. The results show that the amount of rubidium will affect certain physical properties of the hydrogel. Freeze-dried hydrogels have a porous structure of 30 μm-70 μm, and the decrease of Rb content improves the crosslink density of Rb-SA/PAAm.The addition of Rb could slightly decrease the rheological properties and mechanical properties of SA/ PAAm gels, while increase the swelling and roughness of the material. In addition, although the incorporation of Rb + into SA/PAAm gels did not have an inhibitory effect on the proliferation and spreading of Hacat, it increases the ability to inhibit inflammation in NF κB/STAT signaling pathway. Rb-SA/PAAm gels also had an excellent antibacterial activity to Streptococcus and E.coli, which is influenced by Rb + release instead of the pH change.Moreover, the strong moisturizing effect of SA/PAAm gels is beneficial to alleviate skin lesions. Rb-SA/ PAAm gels induce the inhibition of IL-23/IL-17 via STAT3 and NF-κB pathway of IMQ-induced psoriasis skin. Meanwhile, the incorporation of Rb + into SA/PAAm gels also reduces the immune response of skin and spleen of mice. Therefore, the 100Rb-SA/PAAm gels may represent a high-efficiency strategy for psoriasis lesions. This study opened up an innovation for the development of multi-ions cross-linking materials which derived to make daily chemical supplies such as shower gel, shampoo and multifunctional spray for the psoriasis treatment.

Declaration of competing interest
The authors declare no competing financial interest. Fig. 11. A) Immunofluorescence staining of infiltrated CD1α+CD68 (LC) Langerhans cell in mouse back lesions. Red shows anti-CD1α Ab; green shows anti-CD68Ab; yellow shows CD1α and CD68 merged; blue shows DAPI.B) Immunofluorescent staining of CD4+IL-17 + (Th17) lymphocyte infiltrated from the diseased region of mouse back. Red shows anti-CD4 Ab; green shows anti-IL-17 blue shows DAPI. C) Immunofluorescent staining of infiltrated CD20 + CD19 (B)B cells in mouse back lesions. Red shows anti-CD20 Ab; green shows anti-CD19Ab; yellow shows CD20 and CD19 merged; blue shows DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) The corresponding statistical bar chart of the ratio of spleen weight to body weight. C) H&E staining of spleen was used to observe the distinctions of the treatment with different groups. D) the corresponding statistical bar chart of the ratio of white pulp to the whole of spleen. Mean ± SD; n = 5. (*p < 0.05; **p < 0.01).