RETRACTED: Lang-chuang-ding restores bone homeostasis in systemic lupus erythematosus associated osteoporosis by targeting NF-κB signaling: a network pharmacology and experimental study

TL;DR

This study shows that Lang-chuang-ding, a traditional Chinese medicine, helps reduce bone loss in lupus patients by targeting inflammation and bone-related pathways.

Contribution

The study is the first to investigate LCD's effects on SLE-associated osteoporosis and identifies its mechanism via NF-κB signaling.

Findings

LCD improved bone density and structure in lupus-prone mice.

LCD reduced inflammation by lowering IL-1β, TNF-α, and IL-6 levels.

LCD inhibited NF-κB pathway activation in bone tissue.

Abstract

Systemic lupus erythematosus (SLE) is frequently associated with secondary osteoporosis (OP), substantially compromising patients’ quality of life. Although Lang-chuang-ding (LCD), a traditional Chinese medicine formulation, has demonstrated efficacy in suppressing SLE progression, its therapeutic potential for SLE-associated OP remains uninvestigated. This study investigated the therapeutic effects and underlying pharmacological mechanisms of LCD on SLE-associated OP through in vivo experimental validation using MRL/lpr mouse model in conjunction with network pharmacology analysis. Our findings demonstrated that LCD significantly attenuated bone loss in the distal femur by improving bone morphometric parameters, including bone mineral density (BMD), trabecular number (Tb.N), and trabecular bone separation (Tb.Sp), while simultaneously suppressing osteoclast activity and promoting osteogenesis. Network pharmacological analysis identified 63 overlapping targets among LCD components, SLE-related genes, and OP-associated targets, with inflammatory mediators TNF-α, IL-6, and IL-1β emerging as pivotal hub targets. KEGG enrichment analysis revealed significant NF-κB pathway enrichment among the core therapeutic targets. Experimental validation demonstrated that LCD effectively suppressed inflammatory responses by markedly reducing pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 expression while simultaneously inhibiting NF-κB pathway activation through downregulation of p-IκB, P65, and p-P65 in the distal femur. Collectively, these findings demonstrate that LCD effectively ameliorates SLE-associated OP through modulation of inflammatory cytokine networks and the NF-κB signaling pathway, establishing its therapeutic potential as a mechanism-based intervention for SLE-associated OP.

Figures

LCD attenuates bone loss and improves trabecular architecture in MRL/lpr mice. (A) Representative μCT images of femurs, with yellow arrows indicating trabecular bone structure (n = 3 per group). (B) Quantitative analysis of bone morphometric parameters, including BMD, Tb.Sp, and Tb.N in (A). (C, D) Representative H&E staining (C) and MASSON staining (D) of distal femurs of 14-week-old mice. Black Yellow arrows in (C) indicate trabecular bone structures. Yellow arrows in (D) indicate mineralized bone matrix. Data are presented as mean ± SEM (n = 6). *indicates significant differences compared with BALB/c mice, P < 0.01. #indicates significant differences compared with MRL/lpr mice treated with Vehicle, #P < 0.05, ##P < 0.01.

LCD promotes osteoblast differentiation and suppresses osteoclast activity in the distal femur of MRL/lpr mice. (A) Representative images and quantitative analysis of ALP staining in the distal femur. Yellow arrows indicate ALP-positive osteoblasts. (B) Representative IF staining and corresponding quantification results of the expression of osteogenic markers, including RUNX2, OSTERIX, and OPG in the distal femur. (C) Representative IF staining and corresponding quantification results of the expression of osteoclastogenic markers, RANKL and CTSK in the distal femur. DAPI stained the cell nucleus blue. White arrows indicate positive cells. (D) Quantitative analysis of the OPG/RANKL ratio in the distal femur. Data are presented as mean ± SEM (n = 6 per group). *indicates significant differences compared with BALB/c mice, *P < 0.05, P < 0.01. #indicates significant differences compared with MRL/lpr mice treated with Vehicle, #P < 0.05, ##P < 0.01.

Network pharmacology analysis identifies key therapeutic targets of LCD in SLE-associated OP and experimental validation of inflammatory cytokines. LCD can inhibit the secretion of inflammatory factors promoted by SLE. (A) Venn diagram illustrating the intersection of LCD targets, SLE-related genes, and OP-related genes. A total of 63 overlapping targets were identified as potential therapeutic targets of LCD for SLE-associated OP. (B) Construct the PPI network with intersection targets. Node size and color intensity represent the degree of connectivity, with darker red nodes indicating hub targets with higher connectivity. Core targets including inflammatory mediators emerged as central nodes in the network. (C) Representative IF staining and corresponding quantification results of the expression of pro-inflammatory cytokines, IL-1β, TNF-α, and IL-6 in the distal femur. White arrows indicate positive IF signals. Data are presented as mean ± SEM (n = 6 per group). *indicates significant differences compared with BALB/c mice, P < 0.01. #indicates significant differences compared with MRL/lpr mice treated with Vehicle, ##P < 0.01.

KEGG pathway enrichment analysis reveals NF-κB signaling pathway as a key mechanism and experimental validation of NF-κB pathway modulation by LCD. (A) KEGG pathway enrichment analysis of the 63 common targets identified from network pharmacology. Bubble plot displays the top enriched pathways, with bubble size representing gene count and color intensity indicating statistical significance. The NF-κB signaling pathway emerged as one of the significantly enriched pathways. (B-D) Representative IF staining and corresponding quantification results of NF-κB pathway components, including p-i-κB (B), P65 (C), and p-P65 (D), in the distal femur. White arrows indicate positive IF signals. Data are presented as mean ± SEM (n = 6 per group). *indicates significant differences compared with BALB/c mice, P < 0.01. #indicates significant differences compared with MRL/lpr mice treated with Vehicle, ##P < 0.01.

Schematic illustration of the proposed mechanism by which LCD ameliorates SLE-associated OP through modulation of the NF-κB signaling pathway-mediated inflammatory responses and bone remodeling dysregulation.

Tables

Table 1. Detailed information on antibodies used in this study.

Antibody	Host Species	Dilution	Catalog#	Company
RUNX2	Rabbit	1:500	Bs-1134R	Bioss
OSTERIX	Rabbit	1:500	Bs-1110R	Bioss
OPG	Rabbit	1:500	RLT3466	Ruiying Biological
RANKL	Mouse	1:500	23408-1-AP	Proteintech
CTSK	Rabbit	1:500	11239-1-AP	Proteintech
IL-1β	Rabbit	1:500	Bs-6319R	Bioss
TNF-α	Rabbit	1:500	RLT4689	Ruiying Biological
IL-6	Rabbit	1:500	R1412–2	HuaBio
p-i-κB	Rabbit	1:500	RLT2419	Ruiying Biological
P65	Rabbit	1:500	8242S	Cell Signaling Technology
p-P65	Rabbit	1:500	3033S	Cell Signaling Technology

Full text

Introduction

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disorder characterized by pathogenic autoantibody accumulation and immune complex deposition that drive inflammatory responses across multiple organ systems, including cardiovascular, musculoskeletal, integumentary, hepatic, vascular, and renal systems (1). Accumulating clinical evidence has established a significant association between SLE progression and elevated risks of skeletal complications (2), representing a major clinical concern that substantially impacts patient morbidity and quality of life. Cohort analyses reveal alarming prevalence rates of bone pathology in SLE populations, with osteopenia affecting 24-74% and osteoporosis (OP) occurring in 1.4-68.7% of patients (3). Beyond the inherent disease pathophysiology, prolonged glucocorticoid (GC) therapy—a cornerstone of SLE management—has been identified as a critical contributor to accelerated bone mineral density reduction in these patients (4). This dual pathogenic mechanism, involving both inflammatory disease processes and iatrogenic factors, underscores the urgent need for therapeutic strategies that can concurrently address autoimmune dysregulation while preserving bone metabolism in SLE management.

Traditional Chinese medicine has demonstrated unique therapeutic advantages in chronic disease intervention through its characteristic multi-target regulatory mechanisms (5). Lang-chuang-ding (LCD), a herbal formulation developed based on the traditional theory of “detoxification, blood stasis removal, and Yin nourishment”, has demonstrated significant clinical therapeutic efficacy for SLE management (6). Importantly, both short-term and long-term LCD exhibits fewer adverse effects compared to conventional GC and immunosuppressive therapies (7). A prospective cohort study involving 147 female SLE patients demonstrated that LCD adjuvant therapy significantly attenuated GC-induced OP progression while enhancing bone mineral density (BMD) through synergistic pharmacological interactions (6). Despite these promising clinical observations, the molecular mechanisms underlying SLE-associated OP pathogenesis and LCD’s therapeutic effects remain insufficiently characterized.

Bone homeostasis maintenance relies on precise coupling between osteoblast-mediated formation and osteoclast-driven resorption processes (8). Chronic inflammation fundamentally disrupts this delicate equilibrium, with pro-inflammatory cytokines—particularly TNF-α and IL-6—serving as key mediators of pathological bone remodeling (9). Our previous experimental investigations using SLE-prone MRL*/lpr* mice revealed elevated TNF-α and IL-6 expression across multiple tissues, with pronounced enrichment in vertebral bone marrow microenvironments (10–13). We speculate that this inflammatory cytokine dysregulation represents a key mechanism underlying the pronounced OP observed in MRL*/lpr* mouse vertebral bodies. Intriguingly, LCD administration demonstrated potent anti-inflammatory effects through NF-κB signaling inhibition in both systemic circulation and bone marrow-derived macrophages of MRL*/lpr* mice (14), suggesting potential regulatory effects on bone remodeling through inflammatory pathway modulation.

The NF-κB transcription factor complex, typically comprising p50/p65 subunits, serves as a central regulatory hub for inflammation, immunity, and cellular survival (15). In SLE pathogenesis, NF-κB hyperactivation promotes autoreactive B-cell persistence, enhances type I interferon responses, and amplifies pro-inflammatory cytokine cascades, thereby exacerbating autoantibody production and multi-organ damage (16). Furthermore, our previous investigations identified NF-κB pathway activation in MRL*/lpr* mouse articular cartilage correlating with accelerated arthritis progression (11). Transcriptomic profiling of LCD-treated splenic B cells revealed significant NF-κB signaling modulation, suggesting its pivotal role in mediating LCD’s therapeutic effects and highlighting this pathway as a potential therapeutic target (17).

The advancement of bioinformatics platforms and comprehensive databases has established a foundation for understanding complex drug-disease interactions through network theory and systems biology approaches (18). Network pharmacology, an emerging interdisciplinary approach, enables systematic exploration of the intricate relationships between multi-component herbal formulations and disease networks, providing mechanistic insights into traditional medicine’s therapeutic effects that would be difficult to achieve through conventional single-target approaches.

Therefore, this study utilized the well-established MRL*/lpr* mouse model, which closely recapitulates the key manifestations of human SLE, combined with comprehensive network pharmacology analysis to elucidate the potential mechanism of underlying LCD treatment for SLE-associated OP. Through integration of computational prediction with rigorous experimental validation, we aimed to demonstrate that LCD restores bone homeostasis in MRL*/lpr* mice by modulating NF-κB signaling pathways to inhibit inflammatory factor secretion in the distal femoral tissues, ultimately providing mechanistic insights for the clinical application of LCD in SLE-associated bone complications.

Materials and methods

Reagents and antibodies

The antibodies used in this study are listed in Table 1. Fluorescent secondary antibody was obtained from Sungene Biotech Co. (Tianjin, China). Vector^®^ TrueVIEW^®^ Autofluorescence Quenching Kit (SP-8500-15) containing DAPI was purchased from Vector Laboratories Inc (Newark, USA). The MASSON stain kit (#G1346) and Safranin O/Fast green (SO/FG) staining kit (#G1371) were purchased from Solarbio (Beijing, China). The ALP stain kit (#CW0051) was purchased from CWBIO (Beijing, China). Unless otherwise specified, all chemicals were sourced from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of LCD

The traditional Chinese medicine compound decoction was prepared based on the established formula (7, 14, 19, 20). Ten constituent herbs were provided from the pharmacy department of the first affiliated hospital of Zhejiang Chinese Medical University (Hangzhou, China). The herbal components include Herba Artemisia annua (Qing Hao), Trionycis carapax (Zhi Bie Jia), Rhizoma Cimicifugae (Sheng Ma), Herba Hedyotis Diffusae (She Cao), Herba Centellae asiaticae (Ji Xue Cao), Radix Rehmanniae (Shu Di Huang), Radix Paeoniae rubra (Chi Shao), Semen Coicis (Mi Ren), Citri sarcodactylis (Fo Shou) fructus, Radix Glycyrrhizae (Gan Cao), mixed in proportions of 2:2:1.5:2.5:2.5:2.5:2:2.5:1.5:1 (w/w). The decoction was prepared using traditional extraction methods: the herbal mixture was initially soaked in 12 volumes of distilled water (v/w) for 30 minutes to facilitate cellular penetration. Due to its hard texture requiring extended extraction time, Trionycis carapax was pre-boiled separately for 30 minutes before combining with the remaining herbs for the first decoction period of 1.5 hours. Following filtration, the herbal residue underwent a second extraction for 1.5 hours to maximize bioactive compound recovery. The combined filtrates were concentrated under reduced pressure to achieve a standardized concentration of 24 g/mL crude drug equivalent, representing the traditional clinical dosage, and stored at -20°C to maintain bioactivity until experimental use.

Animals and experimental grouping

Female MRL*/lpr* mice, a well-established lupus-prone murine model that typically manifests lupus symptoms at 12 weeks of age (21). Twelve female MRL*/lpr* mice (6 weeks old) were randomly assigned to two groups: the LCD treatment group received LCD at 20 g/kg daily (dosage used for mice was determined based on human equivalent dose conversion following standard interspecies scaling guidelines), and the vehicle control group received distilled water (0.2 mL/day). Six female BALB/c mice served as healthy controls, as this strain did not develop SLE, which received a single daily dose of distilled water (0.2 mL/day). All mice were sacrificed after 8 weeks of intervention, and femurs were harvested for subsequent analysis.

All mice were obtained from Zhejiang Chinese Medical University Laboratory Animal Research Center, and housed under specific pathogen-free conditions at 23 ± 2°C with 40–60% humidity and a 12 h light/dark cycle, with unrestricted access to food and water. All experimental procedures were approved by the Ethics Committee for the Use of Experimental Animals at Zhejiang Chinese Medical University (No. IACUC-20211101-04). All animal experiments were obedient to the ARRIVE guidelines and conducted in accordance with the UK Animals (Scientific Procedures) Act, 1986, and related guidelines, EU Directive 2010/63/EU for animal experiments.

Micro-CT analysis

Following paraformaldehyde fixation, femurs were scanned using μCT equipment (Skyscan 1176, Bruker μCT N.V., Kontich, Belgium) at 50 kV voltage, 500 μA current, and 9 μm pixel resolution. Image reconstruction and quantitative morphological analyses were performed using NRecon v1.6 and CTAn v1.15 software respectively. Three-dimensional images were generated using CTVol v2.2 software. Coronal images of the femoral subchondral bone were selected for bone morphometric parameter analysis, including bone mineral density (BMD), trabecular number (Tb.N), and trabecular bone separation (Tb.Sp), as described before (22).

Histological staining

Femurs were fixed in 4% paraformaldehyde for 48 hours, decalcified with 14% EDTA solution for 21 days, then embedded in paraffin, and sectioned into 5 μm thickness. Sections were deparaffinized and rehydrated through graded alcohols, followed by hematoxylin-eosin (H&E) staining, MASSON staining, and ALP staining according to the manufacturer’s instructions.

Immunofluorescent analysis

For IF analysis, tissue sections were incubated overnight at 4°C with primary antibodies against RUNX2, OSTERIX, OPG, RANKL, CTSK, IL-1β, TNF-α, IL-6, p-i-κB, P65, and p-P65. Fluorescent secondary antibody was applied in the dark for 60 minutes, followed by DAPI counterstaining. Vector^®^ TrueVIEW^®^ Autofluorescence Quenching Kit was applied for 20 minutes according to the manufacturer’s instructions to reduce background fluorescence. Finally, sections were mounted with a neutral mounting medium and examined using a Carl Zeiss fluorescence microscope (Gottingen, Germany). Quantitative analysis was performed in a blinded manner using Image-Pro Plus software (version 6.0, Media Cybernetics Inc, Rockville, Maryland, USA).

Screening for active compounds of LCD and target prediction

Active compounds from LCD were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database using oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 as screening criteria4. The identified compounds were imported into the PubChem database to obtain SMILES identifiers, followed by target prediction using the Swiss Target Prediction and Super-PRED databases. All predicted targets were integrated to establish the comprehensive LCD target profile. Target names were standardized using the UniProt database.

Screening of potential targets for SLE and OP

Potential targets related to SLE and OP were systematically screened from DrugBank, Genecards, and Online Mendelian Inheritance in Man (OMIM) databases. After eliminating duplicate targets, unique target sets for both conditions were obtained for subsequent analysis.

Protein-protein interaction network and KEGG enrichment analyses

To clarify functional interactions among the identified potential proteins, all targets were imported to the String database to construct a PPI network. Then the resulting network was analyzed using Cytoscape 3.8 software, with key hub proteins identified by the CytoNCA software. KEGG pathway enrichment analyses were performed through R software to identify core biological processes and signaling pathways. The top 20 KEGG pathways were visualized as bubble plots.

Statistical analysis

Data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism software 9.0 (San Diego, California, USA). Data were assessed for normal distribution and homogeneity of variance prior to analysis. Statistical comparisons were conducted using one-way analysis of variance (ANOVA) followed by appropriate post-hoc tests. A P-value < 0.05 was considered statistically significant.

Results

LCD ameliorates the OP progression in MRL/lpr mice

To explore the effect of LCD on SLE-associated OP, we administered LCD to MRL*/lpr* mice, and comprehensive bone morphometric analyses were conducted using μCT and histological examinations. μCT analysis revealed that MRL*/lpr* mice exhibited profound trabecular bone loss in the distal femur compared to BALB/c controls, as evidenced by substantially reduced BMD and Tb.N, corroborating the bone deterioration associated with SLE pathogenesis. Notably, LCD intervention significantly preserved bone mass, with BMD and Tb.N showing marked improvements and Tb.Sp showing significant reduction compared to Vehicle-treated MRL/lpr mice (Figures 1A, B).

*LCD attenuates bone loss and improves trabecular architecture in MRL/lpr mice. (A) Representative μCT images of femurs, with yellow arrows indicating trabecular bone structure (n = 3 per group). (B) Quantitative analysis of bone morphometric parameters, including BMD, Tb.Sp, and Tb.N in (A). (C, D) Representative H&E staining (C) and MASSON staining (D) of distal femurs of 14-week-old mice. Black Yellow arrows in (C) indicate trabecular bone structures. Yellow arrows in (D) indicate mineralized bone matrix. Data are presented as mean ± SEM (n = 6). *indicates significant differences compared with BALB/c mice, *P < 0.01. #indicates significant differences compared with MRL/lpr mice treated with Vehicle, #P < 0.05, ##P < 0.01.

Histological analysis using H&E staining demonstrated deteriorated trabecular architecture in MRL*/lpr* mice, characterized by significantly increased trabecular separation, while LCD treatment effectively restored trabecular microarchitecture (Figure 1C). In parallel, MASSON staining revealed compromised collagen organization, which was reversed by LCD intervention (Figure 1D). These findings suggest that LCD can attenuate bone mass loss and improve trabecular architecture, thus ameliorating the OP development caused by SLE exacerbation.

LCD promotes osteoblast differentiation while suppressing osteoclast activity

Previous studies have demonstrated impaired osteogenic differentiation and enhanced osteoclastic activity in the vertebral bodies in MRL*/lpr* mice (23). To determine whether similar alterations occur in the distal femur and evaluate LCD’s effects on bone cell activities, we assessed osteoblast and osteoclast function through ALP staining and IF analysis. Consistent with histological findings, ALP staining revealed markedly diminished osteoblasts in the distal femur of MRL*/lpr* mice, with ALP expression reduced to 24.9% of the BALB/c mice, and LCD administration significantly restored osteoblast function, elevating ALP activity to 66.7% of control levels (Figure 2A), indicating enhanced osteoblastic bone formation.

*LCD promotes osteoblast differentiation and suppresses osteoclast activity in the distal femur of MRL/lpr mice. (A) Representative images and quantitative analysis of ALP staining in the distal femur. Yellow arrows indicate ALP-positive osteoblasts. (B) Representative IF staining and corresponding quantification results of the expression of osteogenic markers, including RUNX2, OSTERIX, and OPG in the distal femur. (C) Representative IF staining and corresponding quantification results of the expression of osteoclastogenic markers, RANKL and CTSK in the distal femur. DAPI stained the cell nucleus blue. White arrows indicate positive cells. (D) Quantitative analysis of the OPG/RANKL ratio in the distal femur. Data are presented as mean ± SEM (n = 6 per group). *indicates significant differences compared with BALB/c mice, *P < 0.05, *P < 0.01. #indicates significant differences compared with MRL/lpr mice treated with Vehicle, #P < 0.05, ##P < 0.01.

To validate these observations, we evaluated key osteogenic markers (RUNX2, OSTERIX, and OPG) in the distal femur using IF analysis. MRL*/lpr* mice exhibited substantially reduced expression of critical osteoblast differentiation factors, including RUNX2, OSTERIX and OPG, in the distal femur, while LCD treatment significantly upregulated all examined osteogenic markers (Figure 2B), demonstrating its capacity to promote osteoblast differentiation and bone formation.

Conversely, analysis of osteoclast-related proteins revealed elevated expression of RANKL and CTSK in the distal femur of MRL/lpr mice, indicating enhanced osteoclastic bone resorption (Figure 2C). LCD intervention effectively suppressed both RANKL and CTSK expression, suggesting reduced osteoclast activity and bone resorption. Furthermore, quantitative analysis of the OPG/RANKL ratio, a critical indicator of the balance between bone formation and resorption, revealed that MRL/lpr mice exhibited a dramatically reduced ratio of only 3.3% compared to BALB/c controls, while LCD treatment substantially restored this balance to 56.5% of control levels (Figure 2D), indicating significant improvement in the osteoblast-osteoclast regulatory axis.

These findings collectively demonstrate that LCD effectively rebalances bone homeostasis by promoting osteoblast differentiation while simultaneously inhibiting osteoclast activity, thereby mitigating bone loss in SLE-associated OP.

Network pharmacology identifies key therapeutic targets and pathways

To systematically elucidate the potential therapeutic mechanisms underlying LCD’s effects on SLE-associated OP, we employed comprehensive network pharmacology analysis to identify common targets among LCD components, SLE-related genes, and OP-associated targets. Venn diagram analysis identified 63 overlapping targets (Figure 3A), representing potential therapeutic intervention points for LCD in SLE-associated OP. PPI network analysis revealed multiple hub targets, with inflammatory mediators including IL-1β, TNF-α, and IL-6 emerging as central nodes with high connectivity (Figure 3B), suggesting their pivotal roles in the therapeutic mechanism. Based on these computational predictions, we performed experimental validation using IF analysis to examine the expression of key inflammatory markers (IL-1β, TNF-α, and IL-6) in the distal femur. The results confirmed that MRL/lpr mice exhibited significantly elevated expression of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in the distal femur compared to BALB/c controls, while LCD treatment effectively suppressed inflammatory responses, reducing IL-1β, TNF-α, and IL-6 expression by 4.18-fold, 6.41-fold, and 4.24-fold, respectively (Figure 3C), demonstrating LCD effectively ameliorates the inflammatory microenvironment within the trabecular bone region of the distal femur, providing mechanistic insight into its therapeutic efficacy against SLE-associated OP.

*Network pharmacology analysis identifies key therapeutic targets of LCD in SLE-associated OP and experimental validation of inflammatory cytokines. LCD can inhibit the secretion of inflammatory factors promoted by SLE. (A) Venn diagram illustrating the intersection of LCD targets, SLE-related genes, and OP-related genes. A total of 63 overlapping targets were identified as potential therapeutic targets of LCD for SLE-associated OP. (B) Construct the PPI network with intersection targets. Node size and color intensity represent the degree of connectivity, with darker red nodes indicating hub targets with higher connectivity. Core targets including inflammatory mediators emerged as central nodes in the network. (C) Representative IF staining and corresponding quantification results of the expression of pro-inflammatory cytokines, IL-1β, TNF-α, and IL-6 in the distal femur. White arrows indicate positive IF signals. Data are presented as mean ± SEM (n = 6 per group). *indicates significant differences compared with BALB/c mice, *P < 0.01. #indicates significant differences compared with MRL/lpr mice treated with Vehicle, ##P < 0.01.

LCD modulates SLE-associated OP through NF-κB signaling pathway inhibition

To identify the key signaling pathways mediating LCD’s therapeutic effects, we performed KEGG enrichment analysis of the core targets identified from network pharmacology. The results indicated significant enrichment of NF-κB signaling pathway among the top-ranked pathways (Figure 4A), suggesting its central role in LCD’s mechanism of action. To experimentally validate this computational prediction, we examined key components of the NF-κB signaling cascade in the distal femur tissues. IF analysis demonstrated significant activation of the NF-κB pathway in MRL/lpr mice, as evidenced by elevated expression of p-IκB, p65, and p-P65 compared to controls. LCD treatment markedly suppressed NF-κB pathway activation, significantly reducing the expression of p-IκB, P65, and p-P65 (Figures 4B, D), indicating effective inhibition of NF-κB nuclear translocation and transcriptional activity. These findings collectively demonstrate that LCD ameliorates SLE-associated OP through targeted inhibition of the hyperactivated NF-κB signaling pathway, thereby interrupting the inflammatory cascade that drives bone destruction in SLE (Figure 5).

*KEGG pathway enrichment analysis reveals NF-κB signaling pathway as a key mechanism and experimental validation of NF-κB pathway modulation by LCD. (A) KEGG pathway enrichment analysis of the 63 common targets identified from network pharmacology. Bubble plot displays the top enriched pathways, with bubble size representing gene count and color intensity indicating statistical significance. The NF-κB signaling pathway emerged as one of the significantly enriched pathways. (B-D) Representative IF staining and corresponding quantification results of NF-κB pathway components, including p-i-κB (B), P65 (C), and p-P65 (D), in the distal femur. White arrows indicate positive IF signals. Data are presented as mean ± SEM (n = 6 per group). *indicates significant differences compared with BALB/c mice, *P < 0.01. #indicates significant differences compared with MRL/lpr mice treated with Vehicle, ##P < 0.01.

Schematic illustration of the proposed mechanism by which LCD ameliorates SLE-associated OP through modulation of the NF-κB signaling pathway-mediated inflammatory responses and bone remodeling dysregulation.

Discussion

SLE, a chronic autoimmune disorder characterized by dysregulated immune responses and sustained systemic inflammation, exhibits marked female predominance and increasing recognition of musculoskeletal complications in clinical practice (24, 25). Meta-analysis data indicate that OP prevalence in SLE patients reaches 16%, with a 2.03-fold increased risk compared to the general population, primarily attributed to systemic chronic inflammation and GC utilization (26, 27). The challenge of maintaining bone homeostasis while effectively managing SLE represents a critical unmet clinical need that demands innovative therapeutic approaches targeting the intersection of autoimmune inflammation and bone metabolism. This study provides compelling evidence that LCD effectively ameliorates SLE-associated OP progression through coordinated modulation of the NF-κB signaling pathway, inflammatory cytokine networks, and bone remodeling processes in the well-established MRL/lpr mouse model.

The MRL*/lpr* murine model, recognized as the gold standard for SLE research, effectively recapitulates critical human disease features including immune complex glomerulonephritis, elevated anti-dsDNA autoantibodies, and progressive proteinuria (28), providing a robust platform for evaluating therapeutic interventions. Multiple previous studies have confirmed LCD’s ability to improve SLE symptoms in MRL*/lpr* mice through various mechanisms, including reducing IRAK1 activation and downstream inflammatory signaling (7, 14, 29, 30), while also ameliorating disease progression by inhibiting effector B cell activation and regulating T cell metabolic reprogramming (31, 32), establishing a strong foundation for the current investigation. Our findings that LCD maintains weakened bone homeostasis by regulating osteoblast and osteoclast activities in MRL/lpr mice suggest significant potential for treating SLE-associated OP through addressing root pathological mechanisms rather than merely treating downstream consequences. Importantly, the formula design of LCD reflects a strategic integration of herbs with complementary therapeutic properties targeting both inflammation and bone remodeling. Several components possess potent anti-inflammatory effects: Hedyotis diffusa (33), Artemisia annua (34), Glycyrrhiza uralensis (35), Paeonia lactiflora (36), Semen Coicis (37) and Citri sarcodactylis (38) collectively suppress inflammatory cascades and cytokine production. Concurrently, other herbs demonstrate bone-protective properties: Rehmannia glutinosa (39), Centella asiatica (40), Trionycis carapax (41) and Cimicifugae rhizome (42) promote osteogenesis and enhance bone remodeling capacity. This dual-action approach enables LCD to simultaneously target autoimmune inflammation and bone metabolism dysfunction, representing a paradigm shift from current therapeutic strategies that typically address these complications separately, potentially offering superior clinical outcomes with reduced adverse effects compared to conventional immunosuppressive approaches.

The restoration of bone homeostasis observed in LCD-treated mice reflects fundamental changes in bone cell biology mediated through inflammatory cytokine modulation. Our network pharmacology analysis identified TNF-α, IL-6, and IL-1β as core targets, which was experimentally confirmed through dramatic 4.18-6.41-fold reductions in these inflammatory mediators following LCD treatment. The inflammatory microenvironment represents a key regulatory factor of bone homeostasis, with TNF-α and IL-6 serving as critical mediators of pathological bone remodeling (43). Mechanistically, TNF-α inhibits the expression of essential osteoblast transcription factors RUNX2 and OSTERIX through NF-κB-dependent mechanisms (44), while IL-6 disrupts the OPG/RANKL axis by promoting RANKL expression while inhibiting the production of OPG (45). Our previous research demonstrated significant TNF-α and IL-6 recruitment in MRL*/lpr* mouse vertebrae, where these cytokines exacerbated osteoblast ferroptosis (23). The simultaneous enhancement of osteoblast differentiation markers and suppression of osteoclastogenic factors observed in our study indicates that LCD treatment effectively reverses these pathological processes through coordinated cellular reprogramming. Notably, the restoration of the OPG/RANKL ratio from 3.3% to 56.5% of control levels demonstrates that LCD’s primary mechanism of osteoclast inhibition is achieved indirectly through enhanced osteoblast-derived regulatory signals rather than direct osteoclast suppression. This intercellular communication pathway represents a more physiologically relevant therapeutic approach, as it restores the natural bone remodeling coupling mechanism. Collectively, these findings indicate that LCD treatment creates a permissive microenvironment for bone regeneration rather than merely preventing further destruction, establishing a foundation for sustained bone health improvement.

Intriguingly, our investigation revealed unexpected tissue-specific inflammatory patterns that provide new insights into the heterogeneous nature of SLE-associated bone pathology. While our previous studies showed no significant changes in IL-1β expression in MRL/lpr vertebral tissues (23), these cytokines were markedly elevated in distal femoral tissues examined in the current study. This unexpected discovery likely reflects the functional and metabolic heterogeneity of different skeletal sites, with the high expression of IL-1β in the distal femur suggesting that pyroptosis may play an important role in site-specific bone pathology. This finding has significant implications for understanding the complexity of SLE-associated bone disease and suggests that therapeutic interventions may need to account for anatomical variations in inflammatory responses. The dramatic reduction in inflammatory burden within the distal femoral bone microenvironment following LCD treatment indicates profound modulation of this pathological landscape, creating conditions favorable for normal bone remodeling while interrupting the inflammatory cascades driving bone destruction.

Our network pharmacology analysis and experimental validation converged on the NF-κB signaling pathway as the central mechanistic node mediating LCD’s therapeutic effects, consistent with extensive evidence demonstrating NF-κB hyperactivation in both lupus nephritis and OP pathogenesis (46, 47). The transcription factor NF-κB serves as a master regulator of immune and inflammatory responses (48), with multiple studies confirming its significant activation during lupus nephritis progression and osteoporotic bone loss. Previous investigations have observed upregulated IκKα expression and enhanced NF-κB activation in lupus nephritis patients (49), while NF-κB kinase activity inhibition increased survival rates and reduced renal pathological changes in NZB/W F1 mice, another spontaneous SLE model (50). Similarly, NF-κB activation mediates systemic chronic inflammation and reactive oxygen species accumulation to promote osteoclast formation (51). Our demonstration of marked suppression of p-IκB, P65, and p-P65 expression following LCD treatment indicates that this formulation effectively disrupts the pathological NF-κB cascade that links autoimmune inflammation to osteodestructive processes. Importantly, the multi-component nature of LCD enables synergistic targeting of NF-κB signaling through complementary mechanisms. Individual herbal components, including Artemisia annua (52), Hedyotis diffusa (53), Centella asiatica (54), Rehmannia glutinosa (55), Paeonia lactiflora (56), Buddha’s Hand (57) and Glycyrrhiza uralensis (58), have all demonstrated NF-ĸB inhibitory functions across different disease models. This convergent targeting suggests that LCD’s therapeutic effects represent synergistic actions of multiple bioactive compounds rather than single-target mechanisms, with each herb potentially targeting different nodes within the NF-κB signaling network to achieve more potent and comprehensive pathway suppression than any individual component could accomplish alone.

The clinical implications of our findings extend beyond the immediate therapeutic potential of LCD to encompass broader questions about integrating evidence-based traditional medicine approaches into modern treatment paradigms. Previous clinical studies have confirmed LCD’s ability to prevent and treat SLE-associated OP, though the underlying mechanisms remained unclear until now (6). Our mechanistic elucidation provides a scientific foundation for these clinical observations and offers opportunities for optimizing therapeutic applications. The multi-target nature of LCD’s effects, combined with its demonstrated safety profile and reduced side effects compared to glucocorticoids and immunosuppressants, positions this formulation as a potentially transformative intervention that could reduce fracture risk and improve long-term skeletal outcomes in SLE patients while minimizing treatment-related complications.

While our findings provide compelling evidence for LCD’s therapeutic potential, several important limitations merit acknowledgment and future investigation. Although the MRL/lpr mouse model effectively reflects human SLE manifestations, validation in other lupus-susceptible models such as NZB/W F1 mice would help determine the universality of our research results and strengthen translational relevance. Additionally, our study relied exclusively on in vivo experimental verification in MRL/lpr mice, and future investigations incorporating in vitro cell experiments and clinical samples from SLE patients would facilitate a deeper understanding of pharmacological mechanisms and confirm LCD’s therapeutic potential in human disease contexts. The long-term effects of LCD treatment on bone remodeling dynamics, optimal dosing strategies, potential drug interactions, and individual patient response variability require systematic clinical evaluation. Furthermore, investigation of specific contributions from individual herbal components could enable formulation optimization and quality standardization for clinical applications.

Conclusion

In summary, this comprehensive investigation combining network pharmacology analysis with rigorous in vivo experimental verification has elucidated the potential pharmacological mechanisms underlying LCD’s treatment of SLE-associated osteoporosis. Our results demonstrate that LCD reduces inflammatory responses by inhibiting NF-κB signaling pathway activation, thereby restoring bone loss caused by SLE through coordinated effects on inflammatory cytokine networks and bone cell function. These findings have enriched our understanding of the mechanistic connections between SLE and OP while providing a promising new therapeutic candidate for treating SLE-associated bone complications. The integration of traditional medicine wisdom with modern molecular pharmacology approaches demonstrated in this study offers a valuable framework for developing more effective, mechanism-based therapeutic strategies for complex autoimmune diseases and their complications.