LSG was prepared using a previously established method (18). SG was isolated from the red seaweed G. fisheri using a cold-water extraction method as described by Wongprasert et al (19). SG was stirred in 0.1 M HCl (RCI Labscan Ltd.; ratio 10:1) for 6 h at room temperature. The mixture was neutralized to pH 8 and precipitated with 95% ethanol (RCILabscan). The resulting pellet was collected, re-suspended in distilled water and dialyzed against distilled water in a dialysis bag for 24 h. LSG was obtained by freeze-drying overnight at -60˚C. The chemical structure and molecular weight of LSG were confirmed by fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC) analyses. LSG (2 mg) was analyzed using FTIR spectroscopy with an IRAffinity-1S (IR) and MIRacle 10 (ATR; Shimadzu). The compound was transferred to a film and analyzed. Spectral baselines were corrected to the 400-4,000 cm^-¹ range. For NMR analysis, LSG (10 mg) was dissolved in deuterium oxide (D₂O, 99.9%; 0.5 ml) in 5 mm NMR tubes and analyzed using a Varian Bruker 400 MHz spectrometer. For GPC, LSG was dissolved in deionized water (1:1 w/v), and 20 µl of the solution was analyzed using a Shimadzu LC-20AD system with an LC-20A oven column and a RID-10A detector, equipped with a TSKgel Guard PWH Size Exclusion Guard column. The column temperature was maintained at 60.0±0.1˚C.

Male BALB/c mice (n=48; age, 6-8 weeks; average weight, 23.96±1.34 grams) were purchased from Nomura International Siam (Bangkok, Thailand). All animals were housed under SPF conditions with controlled humidity (50-70%), temperature (25±2˚C) and a 12/12-h light/dark cycle. Mice had free access to a standard pellet diet and water ad libitum throughout the experiment. All experimental procedures were approved by the Ethical Committee of the University of Phayao, Phayao, Thailand (approval no. 1-024-65).

The dorsal hair of BALB/c mice was removed 1 day before starting the experiment. IMQ cream (5% IMQ; Aldara™, Ensign Laboratories Pty Ltd.) was applied daily at a topical dose of 62.5 mg for 7 consecutive days to establish an IMQ-induced psoriasis mouse model characterized by moderate-to-severe psoriasis-like skin lesions, including pronounced erythema, scale formation, epidermal thickening and inflammatory cell infiltration, corresponding to Psoriasis Area and Severity Index (PASI) scores of 2-3 for each parameter (20,21). MTX was used as a positive control because it is commonly used as a first-line treatment for moderate-to-severe plaque psoriasis (22). The concentration of 5.0 mg/kg LSG was selected based on previous studies (23,24). The mice were randomly divided into six groups (n=8/group) as follows: i) Normal control (NC), mice were intraperitoneally injected with 200 µl normal saline, followed by the topical application of petroleum cream (Chemipan Corporation Co., Ltd.); ii) methotrexate (MTX) control, mice were intraperitoneally injected with 200 µl 1.0 mg/kg MTX, followed by the topical application of petroleum cream; iii) LSG control, mice were intraperitoneally injected with 200 µl 5.0 mg/kg LSG, followed by the topical application of petroleum cream; iv) IMQ, mice were intraperitoneally injected with 200 µl normal saline, followed by the topical application of IMQ cream; v) MTX + IMQ, mice were intraperitoneally injected with 200 µl 1.0 mg/kg MTX, followed by the topical application of IMQ cream; and vi) LSG + IMQ, mice were intraperitoneally injected with 200 µl 5.0 mg/kg LSG, followed by the topical application of IMQ cream (Fig. S1). After 7 days of treatment, all mice were anesthetized with 5% isoflurane in oxygen and maintained at 1.5-3%, followed by cervical dislocation. The thoracic cavity was opened, and blood was collected via cardiac puncture. Skin lesions, lymph nodes, spleen, kidney and liver were harvested for further analysis.

The severity of skin lesions was assessed using a modified human scoring system based on PASI (21,25). Mice were evaluated daily for erythema, scaling and thickening. Each parameter was scored independently on a scale from 0 to 4 (0, none; 1, mild; 2, moderate; 3, marked; 4, obvious; Table I) (21). The cumulative score was used to evaluate the overall severity of skin inflammation.

Body weight was recorded on days 1, 3, 5 and 7 of administration. On day 8, the mice were euthanized, and right axillary lymph nodes and spleens were removed, cleaned and weighed. The weights of the right axillary lymph nodes and spleens were normalized to body weight to calculate the organ index.

Skin lesions were collected, fixed in 4% paraformaldehyde for 48 h at 4˚C and embedded in paraffin. Paraffin blocks were sectioned at 4 µm thickness using a rotary microtome. Tissue sections were deparaffinized with xylene and rehydrated through a descending graded ethanol series. Sections were stained with Mayer's hematoxylin (cat. no. 05-06002/L) and Eosin Y alcoholic solution (cat. no. 05-11007/L; both Bio Optica Milano SpA). Sections were dehydrated, cleared, mounted and cover slipped for histopathological observation under a light microscope. Epidermal and dermal thickness were measured in four randomly selected areas of view/section at 10X magnification using ImageJ software version 1.32j (National Institutes of Health). To visualize blood vessels in the skin lesions, Periodic Acid-Schiff (PAS) staining was performed using a PAS staining kit (cat. no. 1.01646.0001, Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol (26). Additionally, Giemsa staining was performed using Giemsa solution (cat. no. RA-002-05, Biotechnical Co., Ltd.), following the method described by Pudgerd et al (27). Stained sections were observed and photographed under a light microscope. Mast cells were counted manually in four randomly selected fields of view/section at 10X magnification.

Skin lesions were fixed in 4% paraformaldehyde for 48 h at 4˚C and embedded in paraffin. Paraffin blocks were sectioned at 4 µm thickness. Tissue sections were deparaffinized with xylene and rehydrated through a descending graded ethanol series. Tissue sections were incubated in 3% H₂O₂ for 15 min to block endogenous peroxidase activity, followed by antigen retrieval using Tris-EDTA buffer (pH 9.0) at 100˚C for 20 min, and washed with 1X PBS-T (0.05% Tween 20). Tissue sections were incubated with protein blocking solution (0.5% BSA, 0.5% casein in PBS; cat. no. AB64226, Abcam) for 1 h at room temperature. Sections were incubated with primary antibodies against CD4⁺ (rabbit monoclonal anti-CD4⁺, cat. no. ab183685, Abcam; 1:50) and Ki67 (rabbit polyclonal anti-Ki67, cat. no. ab15580, Abcam; 1:100) for 3 h at room temperature. Following washing with 1X PBS-T, sections were incubated with peroxidase-conjugated secondary antibody (AffiniPure goat anti-rabbit IgG H&L, cat. no. 111-035-003, Jackson ImmunoResearch Laboratories, Inc.; 1:500) for 1 h at room temperature. Positive signals were developed using NOVA Red substrate (cat. no. SK-4800, Vector Laboratories, Inc.; Maravai LifeSciences) and counterstained with Mayer's hematoxylin for 20 sec at room temperature. For quantitative analysis, slides were imaged at 10X magnification under a light microscope. CD4⁺ and Ki67-positive cells were counted manually in four randomly selected areas of view/section.

Skin tissue and blood serum samples were collected to assess cytokine levels using ELISA kits. Dorsal skin was excised and any attached connective tissue was removed. A 0.1 cm² section of tissue was homogenized in 1 ml normal saline using a tissue homogenizer. The homogenate was centrifuged at 2,500 x g for 15 min at 4˚C. The supernatant was collected and stored at -20˚C until analysis. For blood serum, samples were allowed to clot at room temperature for 30 min, followed by centrifugation at 2,500 x g for 15 min at 4˚C. The serum was separated and stored at -20˚C until use (27). Cytokine concentrations including IL-1β (ELISA MAX™ Deluxe Set Mouse IL-1β kit, cat. no. 432604), TNF-α (cat. no. 430904), IL-17A (cat. no. 432504), and IL-23 (cat. no. 433704), were measured using commercial ELISA kits, according to the manufacturer's protocols (all BioLegend, Inc.). Absorbance was measured at 450 nm using a VersaMax™ microplate reader and data were analyzed with SoftMax Pro software version 6 (Molecular Devices, LLC).

Total RNA was extracted from skin tissue using TRI Reagent^® (Molecular Research Center, Inc., cat. no. TR118) according to the manufacturer's instructions. A total of 1 µg total RNA was reverse-transcribed into cDNA using the iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories, Inc.; cat. no. 1708840). All RT-qPCR assays were conducted using the QIAquant Real-Time PCR Thermal Cycler (Qiagen, Inc.) with the SensiFAST™ SYBR^® No-ROX Kit (Bioline, cat. no. BIO-98050), according to the manufacturer's protocols. Thermocycling conditions were as follows: Initial denaturation at 95˚C for 2 min, followed by 40 cycles of 95˚C for 5 sec, 60˚C for 10 sec of annealing, and 72˚C for 20 sec of extension. Relative gene expression of IL-1β, TNF-α, keratin 6, 16 and 17, involucrin, JAK1, 2 and 3, STAT1, 2 and 3, BCL2 and CCND1 was normalized to β-actin and calculated using the 2^-ΔΔCq method (28). Primer sequences for all target genes are listed in Table II.

Total protein from skin tissue was extracted using lysis buffer containing 20 mM Tris-HCL, 100 mM NaCl, 5 mM phenylmethylsulfonyl fluoride and 100X protease inhibitor solution. Protein concentration was determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). A total of 50 µg protein/lane was separated on a 12.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with 2% BSA (cat. no. 112018, Merck KGaA) in TBS-T (100 mM Tris-base, 150 mM NaCl, 0.1% Tween 20) for 2 h at room temperature and incubated overnight at 4˚C with primary antibodies (1:1,000) against mouse anti-Ki67 (cat. no. 14569982, Invitrogen, Thermo Fisher Scientific, Inc.), rabbit anti-TNF-α (cat. no. 3707S), mouse anti-IL-6 (cat. no. 12019S), rabbit anti-IL-10 (cat. no. 12163S; all Cell Signaling Technology Inc.) and rabbit anti-β-actin (cat. no. 4970S, Cell Signaling Technology, Inc.). After three washes with TBS-T for 10 min each, the membranes were incubated with HRP-conjugated secondary antibody (1:2,000; cat. nos. 31460 and 626520; Invitrogen, Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Membranes were washed with TBS-T and immunoreactivity was visualized using the Clarity™ Western ECL substrate kit (Bio-Rad Laboratories, Inc.). Protein bands were detected using the Amersham™ ImageQuant™ 800 biomolecular imager (Cytiva). Relative protein expression was quantified using ImageJ software version 1.32j (National Institutes of Health), with band intensities normalized to β-actin.

All data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc multiple comparison test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using GraphPad Prism software, version 10.3.1.509 (Dotmatics).

LSG had a molecular weight of 7.87 kDa and consisted of complex structures with alternating 3-linked β-D-galactopyranose and 4-linked 3,6-anhydro-α-L-galactopyranose or α-L-galactopyranose-6- sulfate units (Fig. S2) (18).

To investigate whether LSG ameliorated skin lesions in psoriasis, IMQ was applied to induce a psoriasis-like mouse model, with and without LSG treatment, and compared with MTX treatment. Mice treated with IMQ exhibited rapid proliferation of immune cells and keratinocytes, leading to increased cytokine production. This mimics the features of human plaque psoriasis, which is characterized by erythema, skin thickening, scaling, and epidermal changes associated with increased inflammation and vascular alterations (29). Symptoms of the psoriatic condition were observed over 7 days of continuous IMQ application in the IMQ group. However, LSG + IMQ group showed a decrease in IMQ-induced psoriatic traits, similar to MTX + IMQ group (Figs. 1A and S3).

Subcutaneous vasculature in the lesional area was visualized post-mortem to evaluate inflammatory angiogenesis. Mice in the IMQ group displayed dense and complex vascular networks, characterized by prominent branching and bifurcation, indicative of increased neovascularization and vascular remodeling commonly associated with psoriatic inflammation (8,29). By contrast, both the MTX + IMQ and LSG + IMQ groups exhibited decreased vascular complexity, suggesting that these treatments effectively suppressed IMQ-induced angiogenic responses. NC, MTX and LSG groups exhibited sparse and minimally branched vasculature, consistent with normal skin lacking inflammatory stimulation (Fig. 1B and C).

PASI scores were calculated to provide a qualitative assessment of disease progression and therapeutic efficacy (Figs. 1D and S4). Mice in the LSG + IMQ group showed significantly lower PASI scores in all parameters compared with the IMQ group, indicating a marked decrease in psoriatic severity. There were significant differences between the LSG + IMQ and MTX + IMQ groups. Collectively, the PASI data supported the protective effect of LSG against IMQ-induced psoriatic skin inflammation.

Histopathological analysis of dorsal skin was performed to evaluate the effect of LSG in IMQ-induced psoriasis. Mice in the IMQ group developed typical psoriasis-like changes, including hyperkeratosis, parakeratosis and thickening of the epidermal stratum spinosum, whereas the LSG + IMQ group showed decreased psoriasis-like alterations (Fig. 2A). Quantitative analysis confirmed that epidermal thickness was significantly decreased in the LSG + IMQ group compared with the IMQ group (Fig. 2B). No significant difference in epidermal thickness was observed between the LSG + IMQ and MTX + IMQ groups. Although dermal thickness did not differ significantly between the IMQ-treated groups, both the MTX + IMQ and LSG + IMQ groups showed decreased dermal thickness compared with the IMQ group (Fig. 2C).

Epidermal thickening in psoriasis results from increased keratinocyte proliferation, with Ki67 serving as a proliferation marker (30). Immunohistochemistry showed fewer Ki67-positive cells in the epidermis of the MTX + IMQ and LSG + IMQ groups compared with the IMQ group (Fig. 2A and D). Ki67 protein expression was significantly decreased in the LSG + IMQ compared with the IMQ group (Fig. 2E). To assess keratinocyte differentiation, the expression of keratin 6, 16 and 17 and involucrin (established biomarkers of keratinocyte differentiation) (7) was evaluated by RT-qPCR. Expression levels of keratin 6, 16 and 17 were significantly decreased in the LSG + IMQ compared with the IMQ group. No significant differences were observed between the LSG + IMQ and MTX + IMQ groups (Fig. 2F-H). Expression of involucrin, a structural protein involved in psoriatic plaque formation, was also decreased in the LSG + IMQ group compared with the IMQ group (Fig. 2I). Taken together, these findings indicated that LSG treatment reduces keratinocyte proliferation and differentiation.

Psoriasis is a T cell-mediated autoimmune skin disease characterized by overexpression of proinflammatory cytokines (31). TNF-α and IL-1β serve key roles in regulating inflammation and promoting Th17 cell development, which is central to the pathogenesis of psoriasis (32). In addition, IL-23 activates CD4⁺ T cells and promotes the release of IL-17A, which drives keratinocyte proliferation in psoriasis (4). ELISA and western blot analyses were performed to determine whether LSG modulates inflammatory cytokine expression during psoriasis development. ELISA showed that levels of TNF-α, IL-1β, IL-17A, and IL-23 were significantly decreased in the skin lesions of the LSG + IMQ group compared with the IMQ group. These effects were consistent with those observed in the MTX + IMQ group (Fig. 3A-H). Western blotting further demonstrated that protein expression of TNF-α and IL-6 proinflammatory cytokines was significantly decreased in the LSG + IMQ compared with the IMQ group. By contrast, IL-10, an anti-inflammatory cytokine, showed increased expression in the LSG + IMQ group (Fig. 3I-L). These findings highlight the efficacy of LSG in attenuating cutaneous inflammation in psoriatic mice.

By reducing levels of TNF-α, IL-1β, and IL-17A, LSG decreased the recruitment of inflammatory Th17 cells. To assess the effect of LSG on inflammatory cell infiltration in psoriatic skin lesions, immunohistochemical staining for CD4⁺ cells was performed. IMQ group demonstrated accumulation of CD4⁺ cells in psoriatic skin lesions. By contrast, CD4⁺ cell infiltration was significantly decreased in both the MTX + IMQ and LSG + IMQ groups compared with the IMQ group (Fig. 4A and B). Giemsa staining of skin sections revealed that mast cell infiltration was significantly lower in the MTX + IMQ and LSG + IMQ groups compared with the IMQ group (Fig. 5A and B). These findings suggested that LSG reduces inflammatory cell infiltration in psoriatic skin.

Proinflammatory cytokines are mediated by IL-17A and IL-23 via activation of the JAK/STAT signaling pathway (5). The decreased expression of IL-17A and IL-23 in the LSG + IMQ group may affect JAK/STAT pathway activation. To investigate this, RT-qPCR was used to analyze mRNA expression of JAK/STAT pathway components, demonstrating downregulation of JAK2, STAT2 and STAT3 (Fig. 6A-F). Consistent with these findings, expression of the JAK/STAT target genes BCL2 and CCND1 was also decreased in the LSG + IMQ group to similar levels observed in the MTX + IMQ group, compared with the IMQ group (Fig. 6G and H).

Psoriasis, as an inflammatory disease, leads to immune system activation and is associated with increased lymph node and spleen weight (33). To assess systemic inflammation, lymph nodes and spleens were collected and weighed. IMQ treatment resulted in increased size and weight of lymph nodes and spleens in the IMQ group compared with controls (NC, MTX, and LSG; Fig. 7). By contrast, the LSG + IMQ group showed a significant decrease in both lymph node (Fig. 7A and B) and spleen (Fig. 7C and D) size and weight compared with the IMQ group. No significant differences were observed between the MTX + IMQ and LSG + IMQ groups. These findings suggested LSG decreased systemic inflammation by suppressing IMQ-induced organomegaly.

To evaluate the potential toxicity of LSG on major internal organs, histopathological examination of the kidney and liver was performed using H&E staining (Fig. 8). Representative micrographs of renal (Fig. 8A) and hepatic tissue (Fig. 8B) showed preserved normal architecture without evidence of degeneration, necrosis, inflammatory infiltration or vascular congestion. Renal histology revealed intact glomeruli and renal tubules with no morphological abnormalities across all groups, including those receiving MTX or LSG with or without IMQ. Similarly, hepatic tissue exhibited normal lobular architecture with well-preserved hepatocyte morphology and no signs of hepatocellular damage. These findings suggested that LSG at the administered dose did not induce detectable hepatic or renal toxicity in IMQ-induced psoriatic mice, supporting its safety for systemic use.