Female BALB/c mice (6–8 weeks old, weighing 20 ± 1 g) were procured from the Animal Facility of Jeeva Life Sciences, Hyderabad, India. The animals were housed in individually ventilated cages (IVCs) under specific pathogen-free (SPF) conditions with a controlled environment (22 ± 2 °C, 12 h light/dark cycle, and 55% ± 10% humidity). Mice were provided with autoclaved bedding and had ad libitum access to autoclaved water and a standardized rodent diet (Teklad Global 18% Protein Rodent Diet, Envigo). All experimental procedures were conducted in accordance with the guidelines of the Committee for the Control and Supervision of Experiments on Animals (CCSEA), Government of India. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Jeeva Life Sciences, Hyderabad (Approval No: IAEC/JLS/2,023/09).

Female BALB/c mice (8 weeks old) were randomly divided into six groups (n = 6 per group) to evaluate the immunomodulatory potential of four peptides SK1203, SK1217, SK1260, and SK128 in a pristane-induced lupus model. Group 1 served as the normal control with no treatment, while Group 2 received pristane and was treated with vehicle (PBS). Groups 3 to 6 were injected with pristane to induce lupus-like disease and subsequently treated with SK1203, SK1217, SK1260, and SK1281 peptides, respectively. Pristane (0.5 mL) was administered intraperitoneally to Groups 2–6. After an 8-week induction period, peptide treatments were initiated at a dose of 2 mg/kg, three times per week, until the mice reached 30 weeks of age.

All four peptides SK1203, SK1217, SK1260, and SK1281 were synthesized and prepared following the protocol described by (30). Each peptide was dissolved in 1X Phosphate-Buffered Saline (PBS) to a final concentration of 2 mg/mL. Mice in the respective treatment groups received intraperitoneal injections of the assigned peptide at a dose of 2 mg/kg body weight, administered three times per week. The vehicle control group received 500 µL of sterile 1X PBS without peptide. Among the tested peptides, SK1217 demonstrated the most pronounced therapeutic effects in the pristane-induced lupus model and was selected for further dose-response assessment at 1 mg/kg and 2 mg/kg in subsequent experiments.

Serum samples were collected from the tail vein at 8, 16, and 30 weeks of age to measure the levels of autoantibodies. At 30 weeks (22 weeks post-pristane administration), the mice were euthanized using carbon dioxide. Nephritic tissues were excised for subsequent immunological detection and examined by light microscopy.

Serum IgG autoantibodies against single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), nuclear antigens, anti-nRNP/Sm, and anti-chromatin were quantified by indirect enzyme-linked immunosorbent assay (ELISA) as described by Wellmann et al. (2001), with minor modifications. Peripheral blood was collected from mice at weeks 8, 16, and 30, allowed to clot at room temperature, and centrifuged at 3,000 × g for 10 min at 4 °C to isolate serum, which was stored at −80 °C. High-binding 96-well ELISA plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 100 µL/well of antigen diluted in carbonate-bicarbonate buffer (0.05 M, pH 9.6), including ssDNA (Sigma-Aldrich), dsDNA (Sigma-Aldrich) at 10 µg/mL each, nuclear extract (Active Motif) at 5 µg/mL, nRNP/Sm, and chromatin antigens (EUROIMMUN) at 5–10 µg/mL. Plates were washed with PBS containing 0.05% Tween-20 (PBST) and blocked with 1% BSA in PBST (200 µL/well) for 2 h at room temperature. Serum samples were diluted 1:100 in blocking buffer, added in duplicate, and incubated for 2 h at room temperature, followed by washing and incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) at a 1:3,000 dilution for 1 h. TMB substrate (Thermo Fisher) was added for color development, and the reaction was stopped with 2N H₂SO₄. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad iMark, Cat# 1681130), and results were expressed as background-subtracted optical density (OD) values.

Serum and peritoneal lavage samples were collected from control, pristane-induced lupus, and SK1217-treated mice at the indicated time points and stored at −80 °C until analysis. Serum autoantibodies were detected by ELISA using alkaline phosphatase-conjugated anti-mouse IgG antibodies, followed by colour development with phosphatase substrate (Sigma-Aldrich). Absorbance was measured at 405 nm. Serum BAFF levels were quantified using a mouse BAFF ELISA kit (EEL082; Thermo Fisher Scientific), complement C3 concentrations were measured using a mouse C3 ELISA kit (ab157711; Abcam), serum creatinine (ab65340), and blood urea nitrogen (MBS2611085) according to the manufacturer's instructions. Serum levels of IL-6, IL-17, IL-1β, and TNF-α were determined using mouse ELISA kits from R&D Systems. Peritoneal lavage samples were centrifuged at 1,000 × g for 10 min to obtain cell-free supernatants, which were stored at −20 °C. IFN-γ levels in the peritoneal lavage were measured by ELISA using anti-mouse IFN-γ antibodies (R&D Systems).

Proteinuria was assessed using urine samples collected from mice. Urine samples were obtained by placing mice in metabolic cages for over 24 h. The protein concentration in the urine was measured using a commercially available proteinuria ELISA kit (AAA-Biotech, AAA16177) according to the manufacturer's instructions. The protein concentration was determined by comparing the absorbance values of the urine samples to a standard curve generated from known protein concentrations. The results were expressed as ng of protein per mL of urine.

At 30 weeks of age, following euthanasia, the kidneys were excised and fixed in 10% formalin for 24 h. Fixed tissues were then processed and embedded in paraffin. Tissue sections of 4–5 µm thickness were prepared and mounted on glass slides. For histological examination, the sections were stained with hematoxylin and eosin (H&E) to assess general tissue morphology. The stained sections were examined under a Nikon DS-Fi1 microscope (Shinagawa, Tokyo, Japan). Histopathological findings, including glomerular changes, tubular injury, and interstitial inflammation, were documented and scored according to standard histological grading systems.

Western blotting was performed to detect NF-κB p65, pJNK, JNK, p-p38, and p38 in kidney tissue samples. Proteins were extracted using RIPA buffer (Santa Cruz Biotechnologies, Inc.) supplemented with protease and phosphatase inhibitors, and their concentrations were determined using a BCA assay. Equal amounts of protein (30–50 µg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 3% BSA in TBST for 30 min and then incubated overnight at 4 °C with primary antibodies diluted in 3% BSA in TBST: NF-κB p65, pJNK, JNK, p-p38, and p38 (Cell Signaling Technologies) at 1:2,000 dilution, and GAPDH (Santa Cruz Biotechnologies, Inc.) at 1:1,000 dilution. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an ECL detection system.

Total peritoneal exudate cells (PEC), Ly6Chi monocytes, and granulocytes were quantified from peritoneal lavage samples. After euthanasia, peritoneal lavages were collected with ice-cold PBS. Cell suspensions were centrifuged at 300 × g for 10 min and resuspended in staining buffer. Cells were stained with the following fluorescently labeled antibodies: CD11b-APC to identify total PEC, Ly6C-FITC to detect Ly6Chi monocytes, and Ly6G-PE to identify granulocytes. Flow cytometric analysis was performed to determine the frequencies and absolute numbers of these cell populations (31). Splenic lymphocytes were isolated by gentle mechanical disruption of freshly excised spleens. The cell suspensions were washed with phosphate-buffered saline (PBS), and erythrocytes were removed using red blood cell lysis buffer. Following centrifugation at 450 × g for 10 min at 4 °C, the cells were collected and resuspended in complete culture medium. For surface staining, cells (5–10 × 10⁶ cells/mL) were incubated with TruStain FcX™ (BioLegend) at a 1:50 dilution on ice for 5–10 min to block Fc receptor–mediated nonspecific binding. Subsequently, cells were stained with PE-conjugated anti-mouse CD138 antibody (1:160, BioLegend, San Diego, CA) and FITC-conjugated anti-mouse CD40 antibody (1:50, BioLegend, San Diego, CA) for 30 min at 4 °C in the dark. After washing with staining buffer, the cells were analyzed using a flow cytometer, and data were processed with FlowJo software.

Statistical analysis was performed using GraphPad Prism software, version 8.0.2. Data are presented as mean ± standard error of the mean (SEM). Comparisons between groups were conducted using one-way analysis of variance (ANOVA). A p-value of less than 0.05 was considered statistically significant. Statistical significance is indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****)

Systemic lupus erythematosus (SLE) is characterized by the production of a broad spectrum of pathogenic autoantibodies that contribute to immune-complex formation and renal damage. To evaluate the effect of SK1217 on autoantibody generation, serum IgG autoantibodies against nuclear antigens were assessed at weeks 8, 16, and 30 following pristane induction. Progressive increases in anti-nuclear, anti-dsDNA, and anti-nRNP/Sm IgG autoantibodies were observed in lupus mice, confirming disease development (Supplementary Figures 2A–C). By week 30, vehicle-treated lupus mice exhibited a significant elevation in serum anti-nuclear, anti-dsDNA, and anti-nRNP/Sm antibody levels. In contrast, mice treated with SK1217 showed a marked reduction in these autoantibodies, indicating effective suppression of lupus-associated autoimmunity. Furthermore, SK1217 administration significantly decreased serum anti-chromatin and anti-ssDNA IgG levels compared with the vehicle-treated control group at week 30 (Figures 1A–E). Collectively, these findings demonstrate that SK1217 effectively mitigates autoantibody production in pristane-induced lupus, highlighting its potential to limit immune-complex–mediated tissue damage.

To investigate the anti-inflammatory effects of SK1217 in pristane-induced lupus, serum levels of key pro-inflammatory cytokines, including IL-1β, IL-6, IL-17, and TNF-α, were quantified. Vehicle-treated lupus mice exhibited a significant elevation in all measured cytokines compared with control animals, confirming systemic inflammatory activation in the SLE model (P < 0.01; Figures 2A–D). In contrast, treatment with SK1217 markedly reduced the serum concentrations of IL-1β, IL-6, IL-17, and TNF-α, restoring cytokine levels toward those observed in non-lupus controls (P < 0.01 vs. vehicle-treated SLE mice; Figures 2A–D). These results demonstrate that SK1217 effectively attenuates systemic inflammation in pristane-induced lupus, highlighting its potent anti-inflammatory and immunomodulatory activity.

To determine whether SK1217 modulates B cell survival and differentiation pathways, serum BAFF levels were quantified, and splenic B cell activation markers were assessed by flow cytometry. Pristane (TMPD)-induced lupus mice exhibited significantly elevated serum BAFF levels compared to normal controls, indicating enhanced B cell survival signaling. Treatment with SK1217 at both 1 mg/kg and 2 mg/kg significantly reduced BAFF concentrations in lupus mice relative to vehicle-treated controls (Figure 3A). Flow cytometric analysis of splenocytes revealed a marked increase in CD40⁺ B cells in vehicle-treated lupus mice, reflecting heightened B cell activation. SK1217 administration significantly decreased the proportion of CD40⁺ cells in both treatment groups (Figure 3B). Furthermore, the frequency of CD138⁺ plasma cells was substantially elevated in the lupus model but was significantly reduced following SK1217 treatment at both doses (Figure 3C). Collectively, these findings demonstrate that SK1217 suppresses BAFF-mediated B cell activation and plasma cell differentiation, providing mechanistic support for the observed reduction in circulating autoantibodies.

To further evaluate renal protection and systemic immune dysregulation, serum creatinine, blood urea nitrogen (BUN), total IgG, and complement C3 levels were measured. Vehicle-treated lupus mice showed significantly elevated serum creatinine and BUN levels compared to normal controls, consistent with impaired renal function. SK1217 treatment at both 1 mg/kg and 2 mg/kg significantly reduced creatinine and BUN concentrations, indicating improved renal function (Figures 4A,B). Total serum IgG levels were markedly increased in lupus mice, reflecting hyperactive humoral immune responses. Administration of SK1217 significantly decreased IgG levels in both treatment groups (Figure 4C). In contrast, complement C3 levels were significantly reduced in vehicle-treated lupus mice, consistent with complement consumption secondary to immune complex deposition (Figure 4D). SK1217 treatment restored C3 levels toward those observed in normal controls, suggesting attenuation of complement activation. These results demonstrate that SK1217 not only modulates immune activation but also improves renal functional biomarkers and restores complement homeostasis in lupus.

Urinary protein excretion was quantified by collecting urine samples from individual mice housed in metabolic cages over 24 h. At week 30 (22 weeks after pristane injection), SK1217-treated mice exhibited significantly lower urinary protein excretion compared to the vehicle-treated model mice (Figure 5A). This reduction in proteinuria suggests that SK1217 offers protection against renal damage associated with SLE. To further assess renal damage, histopathological examination of kidney tissues was performed. Hematoxylin and eosin-stained sections of the kidneys from SK1217-treated mice showed reduced glomerular damage compared to the vehicle-treated lupus model mice (Figures 5B–E). These histopathological findings support the conclusion that SK1217 treatment alleviates renal injury and underscores its potential therapeutic benefit in mitigating SLE-related renal damage.

To elucidate the molecular mechanisms underlying the therapeutic effects of SK1217, key inflammatory signaling pathways were examined in renal tissues from pristane-induced lupus mice. Analysis revealed a marked downregulation of NF-κB p65 expression in the kidneys of SK1217-treated mice compared with vehicle-treated lupus controls, indicating suppression of NF-κB pathway activation (Figure 6). In parallel, SK1217 treatment significantly reduced the phosphorylation levels of JNK and p38 MAPK, two critical stress-activated kinases implicated in inflammatory signaling and lupus nephritis progression (Figure 6 and Supplementary Figure S3). The concurrent inhibition of NF-κB and JNK/p38 MAPK pathways suggests that SK1217 effectively disrupts key intracellular signaling cascades that drive renal inflammation in SLE. Collectively, these findings demonstrate that SK1217 exerts renoprotective and anti-inflammatory effects by attenuating NF-κB–and MAPK-mediated inflammatory signaling in the kidneys of pristane-induced lupus mice.

Treatment with SK1217 in the pristane-induced lupus mouse model resulted in distinct immunomodulatory effects on innate immune cell populations within the peritoneal cavity. Analysis of peritoneal lavage fluid demonstrated that the total number of peritoneal exudate cells (PECs) was not significantly altered between SK1217-treated mice and vehicle-treated lupus controls, indicating that SK1217 did not cause global depletion of immune cells (Figure 7A). In contrast, the frequency of Ly6C^hi inflammatory macrophages were significantly reduced in SK1217-treated mice, suggesting a selective suppression of pro-inflammatory macrophage subsets associated with lupus pathogenesis (Figure 7B). Consistent with this observation, SK1217 treatment significantly decreased IFN-γ levels. This key cytokine drives macrophage activation and promotes autoimmune inflammation (Figure 7D). Interestingly, granulocyte numbers were increased in the SK1217-treated group compared with vehicle-treated mice (Figure 7C), indicating a shift in innate immune composition rather than broad immunosuppression. Collectively, these findings demonstrate that SK1217 selectively reshapes innate immune cell populations and inflammatory cytokine production, contributing to reduced inflammatory responses and attenuation of autoimmune pathology in pristane-induced lupus.