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

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.

Keywords: Lang-chuang-ding; SLE-associated osteoporosis; bone homeostasis; inflammatory cytokines; mechanism.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.