C-Phycocyanin derived from Spirulina maxima attenuates the symptoms of psoriasis in mouse models

Background: Psoriasis is a chronic autoimmune disease caused by the abnormal proliferation of keratinocytes in the epidermis. The ecacy of c-phycocyanin, a photosynthetic pigment isolated from Spirulina maxima, was assessed via histological examinations, and anti-inammatory properties were investigated in mouse models of imiquimod-induced psoriasis (BALB/C-nu and BALB/C). Result: C-Phycocyanin lowered epidermal thickening, as well as immune cell clustering in the dermis. Furthermore, C-phycocyanin modulated the levels of inammatory cytokines (tumor necrosis factor-α, interleukin (IL)-6, cyclooxygenase-2, IL-1b) in the BALB/C-nu mouse model and psoriasis-related cytokines (IL-17a, interferon-gamma, calcitonin gene-related peptide) in BALB/c mice. Conclusion: We show that C-Phycocyanin could be developed as a natural pharmaceutical against psoriasis.


Background
Psoriasis is a chronic autoimmune disease characterized by the abnormal proliferation of keratinocytes in the epidermis [1]. Although the exact cause of psoriasis remains unknown, immune factors, environmental factors, infection, and psychological stress have been implicated in its pathogenesis. [2][3][4][5][6]. Plaque psoriasis or psoriasis vulgaris is the most common type observed worldwide. Plaque psoriasis is typically characterized by the presence of plaques with silver scales. As a symptom of psoriasis, the epidermal granular layer disappears and the cells in the stratum corneum retain their nuclei. The stratum corneum becomes thicker and unaligned. Hence, owing to these changes, the epidermis loses its role as a moisture barrier [7].
Several drugs demonstrating anti-in ammatory, antihistaminic, and antioxidant properties have been used to treat psoriasis [8][9][10]. Despite the numerous drugs and treatments available, natural pharmaceutical products or treatment strategies are gaining momentum, as synthetic drugs could have adverse effects on the psoriatic tissue where immune cells are locally activated [11][12][13].
C-Phycocyanin (C-PC), a natural pigment from cyanobacteria, is known to possess potent antioxidant, anti-in ammatory, and antihistaminic properties [19]. Thus, C-PC has widely been investigated as a bioactive pharmaceutical compound for several diseases, including cancer, central nervous system (CNS) injury, or stroke [20][21][22]. Especially, in skin disorders, C-PC has demonstrated e cacy in atopic dermatitis, as well as allergic in ammation models [23]. Nevertheless, C-PC has rarely been utilized in a psoriasis model.
In psoriasis, immune dysfunction involves the interaction of immune and non-immune cells. Currently, there is no objective parameter to measure the severity of psoriasis, but several studies have reportedly measured the severity of psoriasis by monitoring the levels of various cytokines [14][15][16][17][18]. In this study, we evaluated the levels of pro-in ammatory and T cell-related cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-17, IL-6, IL-1b, interferon (IFN)-γ, cyclooxygenase (COX)-2, and calcitonin generelated peptide (CGRP) for monitoring the severity of psoriasis.
Additionally, the activity of C-PC was assessed using a mouse model (BALB/c-nu, BALB/c) of psoriasis, successfully established after treatment with imiquimod cream, and the e cacy of C-PC was assessed by histological and molecular analyses.
(South Korea), respectively. The following methodologies and relevant kits were used in this study: centrifugation (Supra 25K, Hanil, South Korea); anion exchange chromatography (HiTrap Q HP, GE Healthcare, USA); dialysis (MEMBRA-CEL MC18 X 100 CLR; Serva, Germany); AKTA start puri cation system with Frac30 fraction collector (GE Healthcare, USA); spectroscopy with a microplate reader, equipped with a 50 W xenon ash lamp (Varioskan LUX, Thermo Scienti c, USA); RNeasy Mini kit plus (Takara Bio, Japan); QuantiFasT Reverse Transcription kit (Qiagen, Germany); Rotor-Gene Q PCR (Qiagen, Germany); ultra-centrifugation (Vivaspin 500, MWCO 10000, Sartorius Germany) 2.2 Extraction and puri cation of the c-phycocyanin complex from Spirulina To obtain C-PC, we utilized Spirulina maxima from Jeju (Republic of Korea). The cell wall was lysed using a lysis buffer (1 M Tris (pH 8.0), 0.5 M EDTA, and 20% (w/v) sucrose) and 50 mM piperazine buffer (pH 6.0) for 1 h at 4℃. Cell debris was removed by centrifugation at 10000×g, for 15 min at 4℃. After removing the proteins precipitated at low concentrations of ammonium sulfate (0-30% (w/v)), the proteins in the supernatant were precipitated using high concentrations of ammonium sulfate (30-50% saturation) as previously described [24]. Then, precipitated proteins were dialyzed and further puri ed using anion exchange chromatography. To elute C-PC, 0.05 M piperazine (pH 6.0) containing 0.3 M sodium chloride was used. Chromatography was performed using the AKTA start puri cation system with the Frac30 fraction collector.

The psoriatic mouse model
The in vivo e cacy of C-PC was evaluated using female BALB/c-nu and female BALB/c mice (Orient Bio, Korea). The study was approved by the Institutional Animal Care and Use Committee of Inha University (Incheon in South Korea, Approval Number, INHA 150924-381). Animals were housed in a climatecontrolled environment (25 ± 2°C, 45% relative humidity) with a 12 h light/dark cycle and provided ad libitum access to food and water. The animals of each type were divided into seven groups (Table. 1). Each group has 3 animals and a total of 21 mouse of each species were used. BALB/c mice were depilated using a hair removal cream. For 12 days, 5% imiquimod cream was applied every morning to the dorsal skin of the mice. From the 7 th day, C-PC was administered to the remaining four groups(Group , Group , Group , Group ) similar to the positive control, once a day in the evening. The negative control group was treated with the buffer used to purify the C-PC and maintained as the induced disease control group. BALB/c-nu and BALB/c mice were euthanized on the 12 th day using CO2, and the skin, spleen, and ears were collected from each group.

Measurement of in ammation severity scores
The severity of in ammation and redness was classi ed based on the presence of scaling, thickening, lesions, and erythema on the dorsal and ear skin. The score was individually determined for all groups from 0 to 9 as follows: 0; none, 1-3; slight, 4-6; moderate, 7-9; severe.

Skin morphology (H&E staining) and measurement of epithelial thickness
Samples of the dorsal skin were xed using 4% neutral buffered formalin. The samples were xed onto a 15 mL Falcon tube, and for the dehydration of the xed sample, each sample was dipped in a 30% sucrose solution in a 24-well plate overnight. On immersing the samples, the procedure was changed to a 50% sucrose solution. After generating the block using optimal cutting temperature compound (O.C.T. compound), the samples were cut to a thickness of 40 µm using a microtome. To remove O.C.T, the samples were washed with distilled water, followed by immersion in a hematoxylin solution for 8 min.
Using distilled water in a squeeze bottle, the samples were washed until the dye was released. After eosin staining for 1 min, the samples were washed until the dye was released. The cover glass was placed on the sample and observed using a microscope (Micros, Austria). Epithelial thickness was measured using ImageJ (ImageJ 1.49V).

Quantitative real-time PCR (qRT-PCR)
The mRNAs from the epidermis, ear, and spleen samples of mice in which psoriasis was induced were quanti ed using qRT-PCR according to the manufacturer's instructions. Each sample was homogenized, and total RNA was puri ed using a RNeasy MiNi kit plus. cDNA was synthesized using a Transcript All-in-One First strand cDNA synthesis SuperMix (TransGen, Germany). qRT-PCR was performed using the QuantiFast SYBR Green PCR Kit (QIAGEN, Germany); Rotor-Gene Q PCR (QIAGEN, Germany) was used to quantify the expression of markers.

Statistical analysis
All experiments were repeated thrice to validate the reliability of the results obtained. The relationship between the standard deviation and mean value of the experimental data was con rmed by statistical analysis using the t-test in the SigmaPlot program.

Results
Both spleen and ear obtained from BALB / c-nu were used for qRT-PCR analysis, and skin tissue obtained from both species was used for Histological analysis and qRT-PCR analysis.

Assessment of C-PC in the BALB/c-nu mice psoriasis model
To induce plaque psoriasis in BALB/c-nu mice, imiquimod cream was applied. These mice exhibited characteristic scaling, redness, and thickness with silver scales. In this study, mometasone furoate (Novasone TM cream), used as a positive control, eliminated silver scales. Similarly, C-PC eliminated the presence of silver scales from the psoriatic skin mice in which psoriasis was induced (Fig. 1a). With respect to the severity scores, the experimental group with imiquimod-induced psoriasis demonstrated higher scores in both ear and dorsal skin when compared to the experimental group treated with 1 µg and 5 µg C-PC. (Fig. 2a). These results showed a more positive than effect of mometasone furoate. However, the image and severity scores were negatively affected when the animals were treated with more than 10 µg C-PC.
3.1.1. Downregulation of IL-6, IL-1b, COX-2, and TNF-α mRNA expression in BALB/c-nu mice IL-1b is expressed in activated macrophage cells and is involved in cell proliferation, differentiation, and apoptosis. Following the induction of psoriasis, IL-1b levels increase, which then induce the expression of COX-2 and COX-2 in psoriasis [15,17]. Furthermore, the levels of the pro-in ammatory factor, IL-6, and in ammatory factor, TNF-α, are reportedly increased following the induction of psoriasis [14]. We treated imiquimod-induced psoriatic BALB/c-nu with C-PC and mometasone, and collected the dorsal and ear skin, as well as the spleen. After isolating the tissue RNA, qRT-PCR was performed to measure the expression of four cytokines (IL-6, IL-1b, COX-2, and TNF-α). In the ear skin, the levels of TNF-α continuously decreased as the C-PC concentration increased; COX-2 expression was also decreased (Fig.   5). However, treatment with 10 µg C-PC marginally increased the expression of TNF-α and COX-2 when compared to the experimental group treated with 5 µg C-PC. Similarly, for tissues derived from the spleen and dorsal skin, qRT-PCR demonstrated that the expression of IL-1b, COX-2, IL-6, and TNF-α decreased following C-PC treatment (Fig. 5).

Histological analysis of C-PC in the BALB/c-nu psoriasis model
Following the induction of psoriasis, the stratum corneum thickened, with cell clusters observed in the epidermal layer [7,25]. In normal skin tissue (sham), the epidermis consists of 2-3 epithelial layers, with a stack of stratum granulosum, stratum spinosum, and stratum basale; the dermis is located below the epidermis, and is composed of hair follicles, sebaceous glands, and the extracellular matrix. However, tissues from the imiquimod-treated animals exhibited 4-16 thickened epidermal layers (Fig. 3). In the sham, psoriasis, and mometasone-treated groups, the epidermal thickness was 33.534 μm, 88.727 μm, and 31.338 μm, respectively (Table. 2). The C-PC-treated group demonstrated an epidermal thickness that was comparable to that observed in the mometasone-treated group; however, histological differences were not signi cant among C-PC treated samples.

Assessment of C-PC in the BALB/c mouse psoriasis model
Psoriasis was induced in BALB/c mice using imiquimod cream. Similar to that in the BALB/c-nu mice, the degree of redness and scaling was lowered in all groups treated with mometasone and C-PC when compared to the imiquimod-treated group (Fig. 1b). However, in the 10 μg C-PC treated group, psoriasis symptoms were more severe than those observed in the imiquimod-treated group. Similar scores were observed in terms of the severity of in ammation (Fig. 2b). 3.3.1. Downregulation of IL-1b, COX-2, TNF-α, IL-17a, IFN-γ, and CGRP mRNA expression in BALB/c mouse Reportedly, the expression of COX-2, IL-1b, TNF-α, IFN-γ, IL-17a, and CGRP mRNA is known to increase in psoriasis [14][15][16][17]. In the BALB/C psoriasis model, the negative group treated with imiquimod demonstrated an increased expression of all six factors when compared with the sham and positive groups. The groups treated with 1 μg, 2 μg, and 5 μg C-PC showed a similar or more effective decrease in COX-2 and IL-1b expression when compared with the positive group (Fig. 6). Following treatment with 1 μg C-PC, the expression of TNF-α and CGRP was lowered compared to that observed in the sham group; however, the other C-PC concentrations failed to effectively decrease the expression of these molecules.
IFN-γ expression was decreased in the mometasone-treated group, but no signi cant change was observed in the C-PC treated groups. In the 1 μg, 2 μg, and 5 μg C-PC treated groups, the expression of IL-17a was altered to levels observed in the sham group. Following treatment with 10 μg C-PC, the expression of all factors was higher than that observed in the negative group.

Histological analysis in C-PC-treated BALB/c psoriasis model
In the sham, psoriasis, and mometasone-treated groups, the epidermal thickness was 23.86 μm, 57.06 μm, and 38.98 μm, respectively (Table.3). In the 1 μg treated C-PC group, the results were similar to those observed in the mometasone-treated group (Fig. 4). However, treatment with more than 2 μg of C-PC demonstrated minimal effects on the thickness of the epidermal layer.

Discussion
Psoriasis is characterized by abnormally proliferating keratinocytes induced by T lymphocytes; this condition is associated with atopic dermatitis. Previously, C-PC has been demonstrated to have antioxidant and anti-in ammatory properties [26,27]. Hence, the present study was performed to assess whether C-PC is effective in an animal model of psoriasis. We performed histological and in ammatory investigations to elucidate the positive effects of C-PC. In histological ndings, the psoriasis mouse model showed typical epidermal thickening, as well as plaques of silver scales. In the C-PC treated model, the removal of silver scales was explicitly observed. A dramatic decrease in epidermal thickness is a positive indication of C-PC e cacy against psoriasis. The abnormal proliferation of keratinocytes in the epidermal layer causes psoriatic, epidermal thickening. Thus, it can be postulated that C-PC modulates keratinocyte proliferation. In the dermis, psoriasis is characterized by a cluster of immune cells. In this study, the cell clusters in the dermis of the psoriasis model comprised immune cells. Notably, C-PC works well in the dermal layer of psoriasis, as demonstrated by the reduced number of immune cells when compared with those observed in the psoriasis model (Fig. 3, 4). With respect to the in ammatory ndings, the psoriasis model demonstrated elevated expression of all 6 factors (IL-1b, COX-2, TNF-α, IL-6, CGRP, IL-17a) when compared with the sham group. In BALB/c-nu mice, the levels of these in ammation-related factors tended to decrease as the C-PC concentration increased.
In BALB/c-nu mice with FOXN1 gene knockout, the T cell-mediated response remains incomplete. Therefore, in the BALB/c-nu psoriasis model, the anti-in ammatory effect of C-PC is expected to lower the expression of in ammatory factors, thereby alleviating psoriatic symptoms. Conversely, in the BALB/c mouse model, in which the T cell-mediated response operates normally, C-PC demonstrates more than just an anti-in ammatory effect. Here, C-PC promoted the production of IFN-γ from T cells, with the generated IFN-γ increasing the activity of immune cells, including macrophages [29]. However, considering the decreased expression of several other in ammatory factors, in ammatory factors associated with psoriasis could be lowered to normal levels following C-PC treatment at an appropriate concentration.

Conclusion
In the present study, the potential e cacy of C-PC to treat skin psoriasis was investigated using a mouse model and analyzing tissue histology and mRNA expression levels of in ammatory factors. We successfully induced psoriasis in both models, presenting characteristic silver scales, a thickened epidermis, and clustered immune cells in the dermis. C-PC treatment resulted in silver scale removal, reduced epidermal thickness, and a reduced number of immune cells in the dermis. Furthermore, C-PC modulated the mRNA expression of in ammatory factors. These results demonstrated the potential application of C-PC as a natural pharmaceutical agent against psoriasis.
6. Abbreviations   Table 3. Measurement of epidermis thickness in BALB/c mouse model. Figure 1 In vivo test images of psoriasis mouse models for sham, mometasone, imiquimod, and C-PC, respectively; (a) BALB/c-nu (b)BALB/c