WA (cat. no. HY-N2065), DMSO (cat. no. HY-Y0320) and lipopolysaccharide (cat. no. HY-D1056) were purchased from MedChemExpress. The antibodies were as follows: Mouse α-smooth muscle active (SMA; cat. no. ab7817), TNF-α (cat. no. ab183218), IL-1β (cat. no. ab283818), kidney injury molecule-1 (KIM-1; cat. no. ab47635), paraoxonase 1 (Pon1; cat. no. ab92466), peroxisome proliferator-activated receptor (PPAR)-α (cat. no. ab126285), PPAR-γ (cat. no. ab45036), Bax (cat. no. ab32503), BCL-2 (cat. no. ab182858) and GAPDH (cat. no. ab8245; all Abcam). The mouse anti-nuclear antibody (ANA)/extractable nuclear antigen (ENA) Igs (total A + G + M; cat. no. 5210) and mouse anti-double-stranded DNA Igs (total A+G+M) ELISA kit (cat. no. 5110) were obtained from Alpha Diagnostic Intl Inc.

To identify potential therapeutics for LN, scRNA-seq data derived from human peripheral blood were obtained from the Gene Expression Omnibus database. (ncbi.nlm.nih.gov/gds). Differentially expressed genes (DEGs) in B lymphocytes from GSE135779, GSE162577 and GSE142016 were identified by comparing patients with SLE with healthy donors (adjusted P<0.05). To represent the disease-specific expression profile, the top 150 upregulated and top 150 downregulated genes were selected as the gene signature for the CMAP/L1000 database using the CLUE web platform (https://clue.io/), based on their rank in log₂ Fold Change magnitude. Connectivity scores were calculated and the highest-ranking compounds from the CMAP/L1000 analysis were selected for further investigation.

MRL/lpr mice (n=10; weight, 27.71±0.86 g) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. and maintained under specific pathogen-free conditions at the Animal Center of Gansu University of Chinese Medicine (Lanzhou, China). Mice were housed at 22±2°C and relative humidity of 50±10%, with a 12/12-h light/dark cycle. Female mice (age, 8 weeks) were randomly allocated into two groups (n=5 per group). The treatment group received intraperitoneal injection of 2 mg/kg WA dissolved in DMSO and the control group was treated with 2% DMSO once/day. All animals had free access to food and water throughout the study. After 8 weeks of treatment, mice were euthanized by CO₂ asphyxiation with a chamber fill rate of 60% of total volume/min, in accordance with the AVMA Guidelines for the Euthanasia of Animals (18), followed by sample collection for downstream analyses including spleen, kidney and lymph gland. All experimental procedures were approved by the Animal Experimental Ethical Inspection of Gansu University of Chinese Medicine (approval no. SY2024-257) and performed in compliance with institutional guidelines for animal welfare and use.

According to the manufacturer's instructions, serum creatinine (cat. no. EIASCR) and urea nitrogen (cat. no. BC1535) were measured using commercial kits from Thermo Fisher Scientific, Inc. and Beijing Solarbio Science & Technology Co., Ltd., respectively. Urinary protein levels were determined using the Pierce™ Bradford Plus Protein Assay kit (cat. no. 23236; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

The concentrations of ANA and dsDNA antibodies in mouse serum were quantified using the aforementioned ELISA kits, following the manufacturer's instructions. In brief, samples and controls were incubated at 37°C for 60 min. HRP-conjugated anti-mouse Ig horseradish peroxidase (1:100) and tetramethylbenzidine substrate were added for 30 and 15 min at 37°C, respectively. Absorbance was measured at 450 nm using a microplate reader.

Tissue samples were fixed in 4% paraformaldehyde prepared in 1X phosphate-buffered saline (pH 7.5) at room temperature overnight, dehydrated through a graded series of ethanol and embedded in paraffin. 3-μm-thick sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) using commercial staining kits (cat. no. G1120 and G1281 for PAS, respectively; both Solarbio) at room temperature (hematoxylin for 5 min and eosin for 2 min. For PAS staining, sections were oxidized in periodic acid for 15 min, incubated with Schiff's reagent for 15 min in the dark, and counterstained with hematoxylin for 1 min. to evaluate renal structural damage and pathological changes. The pathological changes in the tissue were observed under the light microscope (Zeiss Axio Imager M2) and analyzed by ImageJ (version 1.53, National Institutes of Health). Glomerulosclerosis was assessed using a semi-quantitative scale as follows: 0, normal; 1, mesangial expansion and slight glomerular damage involving <25% of the glomerulus; 2, mild sclerosis involving 25-49% of the glomerulus; 3, moderate sclerosis involving 50-74% of the glomerulus; and 4, severe sclerosis involving ≥75% of the glomerulus (19).

For the detection of glomerular immune complex deposition, kidney tissues were embedded in OCT compound and frozen at −20°C. Frozen sections with a thickness of 5 μm were prepared. Following being blocking with 5% bovine serum albumin (HY-D0842, MedChemExpress) for 1 h at room temperature, the sections were incubated with primary antibodies, including rabbit anti-complement C3 (1:200; cat. no. ab97462; Abcam), overnight at 4°C. Sections were incubated with secondary antibodies, such as Cy5-conjugated goat anti-mouse IgG (H+L; 1:500; cat. no. 771421ES; Yeasen) or Alexa Fluor-labeled antibodies (both 1:500; cat. no. A-11012; Thermo Fisher Scientific), for 1 h at room temperature. Images were captured using a microscope (LSM 800, Zeiss) and analyzed using ImageJ (version 1.53, National IH). Semiquantitative analysis of glomerular complement C3 deposition was performed as follows: 0, no staining; 1, barely detectable at high magnification; 2, moderately visible; and 3, strongly and clearly visible (20).

Renal α-SMA expression was evaluated via IHC. Briefly, kidney tissues were fixed in 4% paraformaldehyde for 24 h at 4°C and embedded in paraffin. Sections were cut at a thickness of 3-5 μm. Following deparaffinization in xylene and rehydration through a descending alcohol series, antigen retrieval was performed by heating in 10 mM citrate buffer, pH 6.0 at 95°C for 15 min. To block endogenous peroxidase activity, sections were treated with 3% H₂O₂ for 10 min at room temperature. Following blocking with 5% normal goat serum (Beijing Solarbio) for 1 h at room temperature, sections were incubated with primary anti-α-SMA antibodies (1:2,000; cat. no. ab124964; Abcam) overnight at 4°C. Following four washes in PBS, sections were incubated with HRP-conjugated secondary antibodies (1:500; cat. no. GB23303; Servicebio) for 1 h at room temperature. The signal was developed using a DAB substrate kit and counterstained with hematoxylin for 2 min at room temperature. Images were acquired using the light microscope (Zeiss Axio Imager M2). Positive staining area was measured using ImageJ software (version 1.53).

To prepare RNA-seq samples, kidneys were harvested from the SLE model mice following treatment with either DMSO or WA. All tissues were immediately snap-frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted using the GeneJET RNA Purification kit (cat. no. K0731l Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, followed by DNase I treatment to eliminate genomic DNA contamination. RNA quality and quantity were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) to ensure an RNA Integrity Number >7.0 prior to cDNA library construction. Sequencing was performed on an DNBSEQ-T7 platform (MGI, Inc.) by Sangon Biotech Co., Ltd. The sequencing is performed in 150 bp paired end using DNBSEQ DNB Reagent Kit (cat. no. 1000028453; MGI Inc.). The loading concentration of the final library is 18.6 pM. Transcript abundance was quantified using the transcripts per million methods, which normalizes gene expression based on exon models, enabling cross-gene and cross-experiment comparability. Differential expression analysis was performed using the DESeq2 R package (v1.12.4) (https://www.r-project.org), applying a significance threshold of |fold-change|>2 and an adjusted P-value of <0.05. Based on the differentially expressed genes (DEGs), hierarchical clustering and heatmap visualization were performed using R software gplots package (2.17.0). Functional annotation of DEGs was performed using Gene Ontology (GO) (21) enrichment analysis using R software topGO package (2.24.0) (22). Pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (21) via R software clusterProfiler package (3.0.5) (23). Terms with a P-value <0.05 were considered significantly enriched.

For primary B cell isolation, spleens were obtained from MRL/lpr mice. Each spleen was placed in a 70-μm cell strainer and dissociated using a syringe plunger while rinsing with RPMI-1640 medium (cat. no. 11875093; Gibco; Thermo Fisher Scientific, Inc.) to collect cells into a tube. Following centrifugation at 300 × g for 5 min at 4°C, erythrocytes were lysed by resuspending the pellet in erythrocyte lysis buffer (cat. no. R1010; Solarbio) for 15 min at room temperature. The cells were collected at 450 × g for 10 min at 4°C and then washed, resuspended in PBS and adjusted to 1×10⁷ cells/ml. Cells were incubated with CD19 MicroBeads (cat. no. 130-121-301; Miltenyi Bio.; 10 μl/1×10⁷ cells) for 15 min at 4°C and CD19⁺ B cells were isolated using a magnetic separation column (cat. no. 130-042-401; Miltenyi Bio.) according to the introductions.

Human proximal tubular human kidney-2 (HK-2) cells (cat. no. CTCC-002-0018; MeisenCTCC) were cultured in DMEM supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin streptomycin, under a humidified atmosphere of 5% CO₂ at 37°C. To investigate the therapeutic potential of WA, HK-2 cells were pretreated with 2.5 μM WA for 1 h at 37°C prior to the induction of inflammation using 40 μg/ml lipopolysaccharide (LPS) for 48 h at 37°C.

To inhibit PON1 expression, HK-2 cells were transfected with either PON1-specific small interfering RNA (si-PON1; Table SI) or a PON1 overexpression plasmid (Ribobio). Cells were transfected with 1 pmol siRNA using Lipofectamine™ RNAiMAX (cat. no. 13778100; Thermo Fisher Scientific, Inc.) at 37°C for 24 h before the medium was replaced with fresh complete medium and treated with 1 μg/ml LPS at 37°C for 24 h. For overexpression, the human PON1 full-length cDNA was cloned into the pcDNA3.1 plasmid vector (Ruibiotech). Cells were transfected with 0.2 μg of either the PON1 plasmid or the empty vector using Lipofectamine™ 3000 (Thermo Fisher Scientific, Inc.) at 37°C for 48 h. For PON1 activation, cells were exposed to 5 μM zinc phytate (ZnPA; cat. no. 529505; Sigma-Aldrich) for 48 h at 37°C followed by 1 μg/ml LPS exposure for 24 h at 37°C. Cells were subsequently harvested for downstream protein or RNA analysis.

HK-2 cells were seeded into 96-well plates at a density of 1×10⁴ cells/well. Following the respective treatments, the culture medium was aspirated and replaced with 100 μl of fresh medium containing Cell Counting Kit-8 (CCK-8) solution (cat. no. CA1210, Solarbio) in serum-free RPMI-1640. The plates were then incubated at 37°C for 1-4 h in the dark, according to the manufacturer's instructions. The absorbance at 450 nm was measured using a microplate reader.

Total RNA was isolated from kidney tissues or cultured cells using the GeneJET RNA Purification kit (cat. no. K0732; Thermo Fisher Scientific, Inc.). cDNA was synthesized from 500 ng of total RNA using the RevertAid First Strand cDNA Synthesis kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR was performed in 20 μl reaction mixtures containing 2 μl of cDNA, 0.3 μM of each gene-specific primer (Table SII) and 10 μl of SYBR Green qPCR Master Mix (cat. no. K0251; Thermo Fisher Scientific, Inc.). Amplification was carried out on an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the following thermocycling conditions: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. All reactions were performed in triplicate. The relative levels of target genes were calculated using the 2^−ΔΔCq method (24), normalized to GAPDH.

ROS levels were measured using a commercial kit (cat. no. EEA019; Thermo Fisher Scientific, Inc.). Briefly, following ZnPA treatments or PON1 overexpression, cells were loaded with 10 μM DCFH-DA in serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37°C for 30 min in the dark. Cells were thoroughly washed with PBS to remove excess probe and resuspended with serum-free medium for detection. The fluorescence intensity was measured using a microplate reader (excitation/emission, 488/525 nm). Results are expressed as a percentage relative to the untreated control group.

Lactate dehydrogenase (LDH) activity was assessed using a commercial kit (cat. no. BC0625; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol. Following si-PON1 transfection and incubation with WA for 24 h at 37°C, the culture supernatant was mixed with the substrate solution. The mixture was incubated in the dark at room temperature for 30 min. After adding the stop solution, the absorbance was measured at 450 nm using a microplate reader. LDH activity was calculated based on the standard curve. Intracellular levels of MDA and superoxide dismutase (SOD) were assessed using commercial assay kits (cat. nos. BC6415 and BC5165, respectively; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. The absorbance for each assay was measured at 532 and 600 nm for MDA or 450 nm for SOD using a microplate reader.

Total protein was extracted from kidney tissue or cultured cells using RIPA lysis buffer (cat. no. R0010; Beijing Solarbio) supplemented with protease and phosphatase inhibitors (cat. no. 78440; Thermo Fisher Scientific), and the protein concentration was quantified using the Bradford assay (cat. no. PC0010; Solarbio). A total of 20 μg of protein lysate were separated via 4-20% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h] at room temperature and incubated overnight at 4°C with anti-PON1 (1:1,000; cat. no. ab92466; Abcam), anti-TNF-α (1:1,000; cat. no. ab183218; Abcam), anti-KIM-1 (1:1,000; cat. no. ab323414; Abcam), anti-IL-1β (1:1,000; cat. no. ab283818; Abcam), anti-Bcl-2 (1:1,000; cat. no. ab182858; Abcam), anti-Bax (1:1,000; cat. no. ab32503; Abcam), anti-PPAR-α (1:1,000; cat. no. ab61182; Abcam), anti-PPARγ (1:1,000; cat. no. ab45036; Abcam) and anti-GAPDH (1:2,000; cat. no. ab8245; Abcam) as the internal reference control. After washing three times with TBST with 0.1% Tween-20, the membranes were incubated with HRP-conjugated secondary antibodies (1:5,000; cat. no. HA1001; HuaBio) for 1 h at room temperature. Protein bands were visualized using an ECL detection kit (cat. no. 32209; Thermo Fisher Scientific, Inc.). Quantification of the protein bands was performed using ImageJ software (version 1.54g).

Data are expressed as the mean ± standard error of the mean of ≥3 independent experimental repeats. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using GraphPad Prism 8 (Dotmatics).

To identify potential therapeutic agents for SLE-associated LN, the present study analyzed transcriptomic profiles of peripheral immune cells from the public datasets GSE135779, GSE162577 and GSE142016. Given the key role of B cells in SLE pathogenesis (25), DEGs were identified in B lymphocytes from patients with SLE compared with those from healthy donors (adjusted P<0.05). Moreover, the top 150 up-and downregulated genes, ranked by log₂ fold-change, were selected as gene signatures (Table SIII) and submitted to the CMAP platform to screen candidate compounds (Fig. 1A). All output compounds were ranked by CMAP score and the top 10 candidates were identified (Fig. 1B). Among these, WA ranked first (Fig. 1C), suggesting its potential as a therapeutic agent for SLE-associated LN.

The present study assessed the effects of WA on SLE-associated LN in MRL/lpr mice. Following 8 week treatment, mice administered WA showed no significant difference in body weight compared with that of the control group (Fig. 2A). However, WA significantly improved renal function in lupus-prone mice, as demonstrated by significant decreases in urinary protein excretion and serum creatinine levels (Fig. 2B and C). Additionally, a significant decrease in blood urea nitrogen was observed in the WA-treated group (Fig. 2D). Notably, spleen weight was significantly lower in WA-treated mice, whereas liver and kidney weight remained comparable between the two groups (Fig. 2E-G).

SLE involves splenic immune cell dysfunction that contributes to multi-organ pathology (26). SLE model mice displayed marked splenomegaly and enlarged axillary lymph nodes, both of which were attenuated by WA treatment (Fig. 3A). WA administration downregulated the splenic expression of pro-inflammatory cytokines, including monocyte chemoattractant protein (MCP)-1, TNF-α and IL-1β (Fig. 3B-D), while upregulating the anti-inflammatory transcription factor forkhead box P3 (Fig. 3E). WA treatment significantly decreased serum levels of ANA and dsDNA autoantibodies (Fig. 3F and G). Collectively, these results demonstrated that WA ameliorates splenic immune dysfunction in murine lupus.

Renal pathological changes were assessed using H&E and PAS staining. Kidney sections from SLE model mice exhibited glomerular abnormality, including diffuse endothelial cell proliferation, segmental sclerosis and tubular atrophy. These pathological features were markedly attenuated by WA, consistent with the observed improvements in renal function (Fig. 4A-D). To evaluate glomerular injury, immunofluorescence staining was performed, which revealed that WA significantly decreased glomerular deposition of IgG and C3 in SLE mice (Fig. 4E-G).

Immune cell-mediated sustained inflammation contributes to the pathogenesis of renal injury in lupus (27). In the present study, WA ameliorated lupus-induced renal pathology, as demonstrated by a decrease in α-SMA positive areas (Fig. 5A and B). RT-qPCR and western blotting demonstrated that WA abrogated the upregulation of key proinflammatory mediators, including IL-1β, TNF-α and MCP-1, in the kidney of SLE mice (Fig. 5C-F). In addition, the tubular injury was also attenuated, demonstrated by a decrease in the expression of tubular injury marker KIM-1 (Fig. 5F). Collectively, these findings indicated that WA attenuated tubular injury by suppressing the intrarenal inflammatory response.

The present study performed genome-wide transcriptomic sequencing on renal tissue from SLE mice with or without WA treatment to elucidate the molecular basis of its immunosuppressive effects. Notably, WA markedly reshaped the renal transcriptome (Fig. 6A), enhancing the expression of lipid metabolism genes [perilipin (PLIN)4, fatty acid-binding protein 4 (FABP4) and PLIN5] and suppressing genes associated with immunity [lymphocyte antigen 6 complex locus D, immunoglobulin heavy variable 5-9, C-X-C motif chemokine ligand (CXCL)10 and CXCL13], transcription (STAT4 and transcription factor 7) and apoptosis (growth arrest and DNA damage-inducible γ and serine/threonine kinase 17B; data not shown). GO and KEGG analyses demonstrated that WA activated lipid-centric and metabolic pathways while inhibiting pro-inflammatory signaling (Fig. 6B and C). Moreover, from the 161 DEGs identified by volcano plot (18 up- and 143 downregulated), Pon1 emerged as a lead candidate as the second most significantly upregulated transcript in the WA-treated group (Fig. 6D and E). Among the top upregulated genes identified by RNA-seq, Pon1 was selected for further functional validation due to its roles in alleviating oxidative stress and systemic inflammation (28-31), both of which are hallmark features of SLE-induced renal damage. Therefore, it was hypothesized that induction of Pon1 is a critical mechanism through which WA alleviates SLE pathology.

To assess the role of Pon1, the present study established an in vitro model of LN by stimulating HK-2 cells with LPS. WA significantly restored the impaired cell viability induced by LPS (Fig. 7A) and suppressed the upregulation of proinflammatory cytokines (IL-1β, TNF-α and MCP-1) at both the mRNA and protein levels (Fig. 7B-E). Notably, WA also upregulated Pon1 expression (Fig. 7F).

To determine whether Pon1 is essential for the effects of WA, the present study performed loss-of-function experiments using siRNA-mediated knockdown of Pon1. si-Pon1 effectively inhibited the expression of Pon1 (Fig. S1) and abrogated the protective effects of WA, as demonstrated by a reversal of the improved cell viability (Fig. 7A) and increased proinflammatory cytokine expression (Fig. 7B-E). Given previous findings that Pon1 mitigates cellular stress by activating PPARγ signaling (32) and the transcriptomic data in the present study indicating WA activates the PPAR pathway in vivo, this pathway was investigated. WA enhanced the expression of PPARα and PPARγ in LPS-treated HK-2 cells (Fig. 7G). This activation was associated with attenuated oxidative stress, demonstrated by decreased levels of LDH and MDA and an increase in SOD activity (Fig. 7H-J). WA modulated the expression of apoptosis-related proteins, as indicated by the downregulated expression of Bax and Bcl-2 (Fig. 7K). Notably, these beneficial regulatory effects of WA were nullified by Pon1 knockdown (Fig. 7G-K).

WA led to a decrease in supernatant IgG (Fig. 8A) and diminished expression of the B cell surface markers CD19, CD20, CD21 and CD22 (Fig. 8B-E), suggesting suppressed B cell differentiation and proliferation. Conversely, an increase in the apoptosis-associated marker CD24 was observed (Fig. 8F), indicating promotion of B cell apoptosis.

As Pon1 demonstrated a role in WA-induced effects, the present study assessed whether Pon1 activator showed similar effects induced by WA. ZnPA significantly enhanced the expression of Pon1 (Fig. 9A) in HK-2 cells. ZnPA increased the viability of HK-2 cells treated with LPS (Fig. 9B), which was accompanied by decreased expression of the proinflammatory cytokines IL-1β, TNF-α and MCP-1 (Fig. 9C). Notably, PPAR signaling, oxidative stress and apoptosis induced by LPS were markedly attenuated in HK-2 cells following ZnPA administration (Fig. 9D-F). Pon1 was overexpressed in HK-2 cells to evaluate whether higher levels of Pon1 led to similar kidney protection (Fig. 10A). Pon1 overexpression resulted in increased cell viability (Fig. 10B) and decreased inflammation (Fig. 10C-E) and ROS levels (Fig. 10F). Relative mRNA expression of Pon1 was compared between healthy donors and patients with LN in the public dataset GSE121239. Pon1 expression was significantly lower in the patients with LN than in healthy donors (Fig. S2), supporting the hypothesis that augmenting Pon1 expression represents a promising therapeutic strategy for LN.