Mechanistic study of compound Muniziqi granule on lupus nephritis in MRL/lpr mice: insights from transcriptomics and experimental validation

Abstract

Background

Lupus nephritis (LN) is a severe and prevalent complication of SLE. Compound Muniziqi granule (CMG), a traditional remedy, is commonly used to treat immune system-related disorders. However, its efficacy in treating LN remains unassessed. This study aims to evaluate the therapeutic potential of CMG for LN and to explore its molecular mechanisms.

Methods

CMG was evaluated for therapeutic effects on LN in Murphy Roths Large (MRL)/lymphoproliferation (lpr) mice. Renal tissues from mice in the Control, Model and CMG treatment groups were collected for transcriptomic analysis to identify the potential pathways involved in CMG’s action in LN. We then examined the effects of CMG on inflammatory cell infiltration, complement system activation and B-cell accumulation in the renal tissues of MRL/lpr mice. Additionally, we assessed the impact of CMG on the cluster of differentiation 40 (CD40)/Nuclear Factor-kappa B p65 (NF-κB p65) signalling pathway in these tissues.

Results

CMG treatment reduced the spleen index in MRL/lpr mice, decreased serum levels of antibodies and inflammatory cytokines and improved renal function markers. It also alleviated renal pathological damage and inflammatory infiltration. Transcriptomic analysis indicated that CMG’s therapeutic effects may be linked to ‘SLE’ and the ‘NF-κB signalling pathway’. Further investigation revealed that CMG downregulated a wide array of complement-related and inflammation-associated genes. Notably, CMG reduced the gene expression of CD40 and CD40L. In addition, CMG decreased inflammatory cytokine levels. Preliminary immunofluorescence analysis indicated that CMG reduced immune complex deposition and diminished B-cell infiltration in the renal tissues of MRL/lpr mice. Preliminary western blot analysis indicated that CMG downregulated B Lymphocyte-Induced Maturation Protein 1 (BLIMP1), CD40 and tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) expression, as well as the phosphorylation of NF-κB p65.

Conclusion

CMG effectively mitigates inflammatory infiltration and renal damage in MRL/lpr mice. Preliminary evidence indicates that this protective effect may be associated with reduced activity of the CD40/NF-κB p65 signalling pathway, along with decreased B-cell activation and complement system activation.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Compound Muniziqi granule (CMG), a traditional remedy to treat immune system-related disorders. However, its efficacy in treating lupus nephritis remains unassessed.

WHAT THIS STUDY ADDS

• CMG effectively alleviates renal injury and inflammatory infiltration in MRL/lymphoproliferation mice. These therapeutic benefits appear to be associated with the mitigation of B-cell activation and complement system dysfunction and the downregulation of the cluster of differentiation 40 (CD40)/Nuclear Factor-kappa B p65 (NF-κB p65) pathway.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Our study offers a crucial foundation for the further development and clinical application of CMG.

Introduction

SLE is a chronic, multiorgan autoimmune disorder marked by autoantibody production, deposition of immune complexes and activation of the complement system.¹ SLE global prevalence has increased to 43.7 per 100 000 individuals and continues to rise.² Among its many complications, lupus nephritis (LN) is the most severe manifestation. Approximately 60% of adults with SLE will develop renal dysfunction over the course of the disease.³ High morbidity and mortality are associated with LN, largely due to its potential progression to end-stage renal disease (ESRD) and the adverse effects of treatment.⁴ Within 10 years, one-third of patients with severe LN progress to ESRD, and their mortality rate is eight times higher than that of the general population.⁵ At present, there is no definitive cure for LN. First-line treatments include mycophenolate mofetil, hydroxychloroquine, glucocorticoids, cyclophosphamide and biologics such as belimumab, which targets B-cell-activating factors. The primary treatment goals are to relieve symptoms, prevent flare-ups and slow the progression of renal damage. Despite a growing array of therapeutic options for both SLE and LN, many patients remain refractory to treatment, experiencing poor responses and significant side effects.⁶ As such, there is an urgent need for the development of new, safe and effective therapies to address this unmet medical need in LN management.

The pathogenesis of LN primarily involves an aberrant immune response against self-antigens, with B-cell activation playing a critical role in initiating the process. On activation, B cells produce excessive antibodies, such as anti-double-stranded DNA (anti-dsDNA) antibodies, targeting nuclear components of self-cells. These antibodies bind to their corresponding antigens, forming circulating immune complexes that deposit in tissues, particularly the glomerular basement membrane and the mesangial region, thereby activating the complement system. The resulting complement activation recruits a substantial number of inflammatory cells, driving further inflammation and tissue damage.⁷ Studies have shown that LN patients exhibit elevated levels of anti-complement antibodies, which correlate strongly with disease activity.⁸ Moreover, therapeutic strategies aimed at depleting or neutralising B cells have demonstrated significant efficacy in improving LN outcomes.⁹

Traditional Chinese medicine (TCM) offers a unique therapeutic approach in managing LN, particularly through the modulation of B-cell function. A meta-analysis revealed that Jinshuibao Capsule and Huangkui Capsule are effective in treating LN, while Xinganbao Capsule enhances complement system function.¹⁰ Furthermore, Chailingtang has been shown to suppress B-cell activity in Murphy Roths Large (MRL)/lymphoproliferation (lpr) mice, leading to improvements in LN.¹¹ Other formulations, such as Langchuangping Granule and JieduQuyuZishen Prescription, have emerged as promising candidates for LN treatment due to their ability to inhibit B-cell-activating factors.^{12 13} Investigating the therapeutic potential and mechanisms of TCM in LN provides valuable insights that could help inform and refine current treatment strategies for this challenging condition.

Compound Muniziqi granule (CMG) is a distinctive TCM known for its ability to regulate immune system balance, consisting of seeds of Peganum harmala, Nigella glandulifera, Dracocephalum moldavica, Ocimum basilicum, Cichorium intybus and Althaea rosea; fruits of Pimpinella anisum; roots of Glycyrrhiza uralensis, C. intybus and Apium graveolens; root bark of Foeniculum vulgare and herbs (aerial part) of Cymbopogon caesius and Matricaria chamomilla. Previous studies have demonstrated that CMG exhibits anti-inflammatory, antioxidant and anti-apoptotic properties.14,16 It has been used effectively to treat various immune-related disorders, including vitiligo, eczema and urticaria.¹⁷ Building on this foundation, we aim to evaluate, for the first time, the therapeutic potential of CMG in the treatment of LN and to explore its underlying molecular mechanisms. This investigation could provide valuable insights for the development of new therapeutic strategies for LN. In this study, we initially assessed the therapeutic effects of CMG on female MRL/lpr mice. We then performed transcriptomic analysis on renal tissues from these mice and, based on these results, evaluated CMG’s impact on the complement system and its effects on cluster of differentiation 40 (CD40)/Nuclear Factor-kappa B p65 (NF-κB p65) signalling pathway and B-cell activation. Our findings aim to establish a strong foundation for the future application of CMG in the treatment of LN.

Methods

Reagents

Detailed information regarding the experimental reagents used is provided in online supplemental materials. Online supplemental table S1 provides a comprehensive overview of all biochemical and molecular assays performed in this study.

Qualitative analysis of CMG constituents

CMG (batch No.: 230976) was purchased from Xinjiang Uyghur Pharmaceutical (National drug approval No. Z65020166). We performed qualitative analysis of the major constituents of CMG using Ultra-Performance Liquid Chromatography-Quadrupole-Time of Flight-Mass Spectrometry (UPLC-Q-TOF-MS); detailed methods and results are provided in the online supplemental materials. The main components identified in CMG include harmaline, harmine, liquiritin, isoliquiritin, sieboldianoside A, glycyrrhizic acid, chlorogenic acid, caffeic acid, ferulic acid and apigenin 7-O-glucoside (online supplemental figure S1 and table S2).

Animal grouping and drug administration

Female MRL/lpr mice aged 8 weeks old and age-matched female MRL/Mpj mice (weighing 20±2 g) were purchased from Changzhou Cavens Laboratory Animal (Licence No.: SCXK (Su) 2021–0013). All mice were housed in a Specific Pathogen Free (SPF) facility, maintained at a controlled temperature and humidity with a 12-hour light-dark cycle. They were provided with ad libitum access to food and water. All animal procedures were approved by the Ethics Committee of Xinjiang Medical University (Ethics Number: 20241126–01).

Following a 4-week acclimation period, we used MRL/lpr mice to establish the spontaneous SLE model, with reference to prior methodologies.¹⁸ 40 female MRL/lpr mice were randomly divided into four groups (n=10 per group): Model, prednisone (PRE), CMG low-dose (CMG-L) and CMG high-dose (CMG-H). 10 female MRL/Mpj mice were assigned to the control group. Mice in the PRE group received a daily dose of 9 mg/kg PRE via oral gavage.¹⁹ The CMG-L and CMG-H groups were treated with daily gavages of CMG at 2.5 g/kg and 5 g/kg, respectively.¹⁴ Mice in the control and model groups were gavaged with an equal volume of normal saline. The treatment period lasted 8 weeks. Afterwards, the body weights of the mice were recorded. All animals were euthanised, and blood samples were collected for serum separation and subsequent biochemical analyses. Spleens were weighed to calculate the spleen index, while kidneys were either frozen for storage or fixed for further analysis.

Biochemical index detection

We collected serum samples from all mice groups for analysis of several biomarkers. Levels of ANA, anti-dsDNA-immunoglobulin G (IgG), total IgG, interleukin-1 beta (IL-1β), IL-6, tumour necrosis factor-alpha (TNF-α), blood urea nitrogen (BUN), creatinine (Cr), complement component 3a (C3a), complement component 5a (C5a) and total haemolytic complement (CH50) activity were measured according to the instructions provided with the corresponding reagent kits. To assess renal function further, we collected 24-hour urine samples from each group using metabolic cages. 24-hour urine total protein (UTP) was quantified using a urinary protein detection kit. Renal tissue samples were harvested from mice in all experimental groups. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay method. The levels of IL-1β, TNF-α, interferon-gamma (IFN)-γ, complement component 1q (C1Q), intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL2 were then quantified according to the reagent kit protocols.

Pathological staining

We fixed renal tissues in 4% paraformaldehyde, dehydrated them through a graded ethanol series and cleared them with xylene. The tissues were then embedded in paraffin and sectioned. The resulting sections were stained with H&E for general histological evaluation. Renal pathology was assessed using the revised National Institutes of Health (NIH) LN activity index.²⁰ In brief, the activity index comprises six activity scoring parameters: endocapillary hypercellularity (0–3), neutrophils/karyorrhexis (0–3), fibrinoid necrosis (0–3, weighted 2), hyaline deposits (0–3), cellular/fibrocellular crescents (0–3, weighted 2) and interstitial inflammation (0–3). The scoring criteria for each parameter: 1,<25%; 2, 25%–50%; 3,>50% of glomeruli/tubules/cortical interstitium involved. The total activity index score is calculated by summing all parameter scores, with a maximum possible score of 24.

To evaluate fibrosis, sections were stained with Masson’s trichrome and examined under a light microscope. Additionally, periodic acid-Schiff (PAS) staining was performed to assess the extent of renal damage. The entire evaluation process was conducted under a blinding design.

Transcriptomics

Six renal tissue samples were collected from each group of mice, respectively, and total RNA was extracted using the Total RNA Isolation phenol (TRIzol) reagent according to the manufacturer’s protocol. High-throughput sequencing of the RNA samples was performed on the Illumina HiSeq platform by Novogene (Beijing, China). The raw sequencing data were processed and aligned to the Mus musculus reference genome (mus_musculus_GRCm39_release106) using HISAT2 V.2.0.5 software. Differential gene expression was analysed using DESeq2 V.1.20.0, with a threshold set for |log₂FC|>1 and p_adj <0.05 to identify genes that were significantly differentially expressed. To further investigate the biological functions of these genes, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (online supplemental tables S3 and S4). Based on the KEGG screening results, we focused on the SLE and NF-κB signalling pathway for an indepth analysis of their differentially expressed genes (DEGs).

Immunofluorescence

Paraffin-embedded tissue sections were dewaxed and rehydrated through a series of ethanol washes. Antigen retrieval was performed, followed by blocking with BSA for 30 min at room temperature. The sections were then incubated overnight at 4 °C with primary antibodies specific to the target proteins. After washing, fluorescently labelled secondary antibodies were applied, and the sections were incubated for 1 hour at room temperature. For nuclear staining, 4',6-diamidino-2-phenylindole (DAPI) was added. The sections were then incubated at room temperature in the dark for 10 min. Autofluorescence was quenched using an appropriate solution, followed by a 5 min incubation. Finally, we mounted sections with an anti-fade mounting medium and examined them under a fluorescence microscope. The entire evaluation process was conducted under a blinding design. Images were captured and fluorescence intensity was quantified using ImageJ software.

Western blot

We extracted proteins from renal tissues of each mice group using radioimmunoprecipitation assay (RIPA) lysis buffer. Then, loading buffer was added to the samples, which were denatured at 95 °C for 5 min. The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred using the wet transfer method to membranes. These membranes were then blocked with non-fat dry milk and incubated with primary and secondary antibodies. Chemiluminescent substrate was applied to the membranes, which were then exposed and imaged using a gel imaging system. The intensity of protein bands was quantified using ImageJ software. β-actin was used as an internal reference to normalise the target protein. In order to validate the stability of β-actin, the grey value of β-actin among groups was quantified and presented in online supplemental table S5. All renal tissue samples were subjected to precise protein quantification and normalisation using the BCA assay prior to Western blot loading, ensuring consistent sample loading across experimental groups. Regarding P65 detection, we analysed phospho-P65 and total P65 separately.

Statistical analysis

Statistical analysis was conducted using SPSS V.20.0 software. Data normality was assessed using the Shapiro-Wilk test. All data followed a normal distribution and are presented as the mean±SD (x±s). One-way analysis of variance was used to compare differences among multiple groups, followed by Bonferroni test. Statistical analyses were conducted only where the number of independent experiments, n>3. No statistical analysis was performed for n=3 per group and these results were regarded as preliminary. We considered a p<0.05 to be statistically significant.

Results

Therapeutic effects of CMG on MRL/lpr mice

Compared with MRL/Mpj mice, MRL/lpr mice exhibited a significantly elevated spleen index, which was notably reduced following CMG treatment (figure 1A). Serum analysis revealed that MRL/lpr mice had significantly higher levels of ANA, anti-dsDNA-IgG, total IgG, IL-1β, IL-6 and TNF-α when compared with MRL/Mpj mice (figure 1B–G). CMG treatment effectively lowered these levels, demonstrating its potential to modulate immune responses. Furthermore, we observed that CMG-H treatment demonstrated disease-ameliorating effects similar to those of PRE.

Figure 1. Therapeutic effects of CMG on MRL/lpr mice. (A) CMG reduces spleen indexes in MRL/lpr mice; (B, D) CMG decreases serum levels of ANA, anti-dsDNA-IgG and IgG in MRL/lpr mice; (E–G) CMG reduces serum levels of IL-1β, IL-6 and TNF-α in MRL/lpr mice. (H–J) CMG reduces 24-hour UTP, serum BUN and serum Cr levels in MRL/lpr mice; (K) H&E staining and LN activity index scoring; (L) MASSON staining and fibrosis assessment; (M) PAS staining and renal injury assessment. n=10 per group; *p<0.05, **p<0.01. anti-dsDNA, anti-double-stranded DNA; BUN, blood urea nitrogen; CMG, compound Muniziqi granule; CMG-H, CMG high-dose; CMG-L, CMG low-dose; Cr, creatinine; IgG, immunoglobulin G; IL-1β, interleukin-1 beta; LN, lupus nephritis; lpr, lymphoproliferation; MRL, Murphy Roths Large; PAS, periodic acid-Schiff; PRE, prednisone; TNF-α, tumour necrosis factor-alpha; UTP, urine total protein.

We also assessed CMG’s effects on renal damage in MRL/lpr mice. These mice exhibited significantly elevated levels of 24-hour UTP, BUN and Cr, indicating impaired renal function. CMG treatment notably reduced these markers, suggesting an improvement in renal function (figure 1H–J). Histopathological analysis suggested that MRL/lpr mice had extensive renal damage, including glomerular sclerosis, crescent formation, inflammatory cell infiltration and tubular atrophy. Additionally, the LN activity index score, fibrotic area and PAS-positive area were significantly higher in MRL/lpr mice than in MRL/Mpj mice (figure 1K–M). However, CMG treatment reduced these pathological alterations to varying extents. Notably, the therapeutic effects observed in the high-dose CMG group—such as BUN, Cr, the lupus LN index score and fibrotic area—showed no significant differences compared with those of PRE.

The effects of CMG on genes in renal tissues of MRL/lpr mice

To investigate the underlying mechanism through which CMG alleviates renal injury in LN, we conducted transcriptomic analysis of renal tissues from mice in the control, model and CMG-H groups. DEGs were identified in the model versus control and CMG-H versus model groups, using the thresholds of |Log₂FC| >1 and p_adj <0.05. The results were visualised in volcano plots (figure 2A,B). Following this, we performed KEGG pathway enrichment analysis on the DEGs from both comparisons. The data related to KEGG pathways are included in online supplemental tables S3 and S4. The analysis revealed significant enrichment in pathways related to ‘SLE’ and the ‘NF-κB signalling pathway’ (figure 2C,D). Based on these findings, we hypothesise that the therapeutic effects of CMG on renal injury in LN may be primarily mediated through modulation of these two key pathways.

Figure 2. The effects of CMG on the renal tissue transcriptome of MRL/lpr mice. (A, B) Differentially expressed genes in model versus control and CMG-H versus model; (C, D) KEGG enrichment analysis of differentially expressed genes in model versus control and CMG-H versus model; n=6 per group. CMG, compound Muniziqi granule; CMG-H, CMG high-dose; Fc, fragment crystallisable; IL, interleukin; JAK, Janus kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; lpr, lymphoproliferation; MRL, Murphy Roths Large; NF, nuclear factor; STAT, signal transducer and activator of transcription; Th, T helper.

The effects of CMG on complement system activation and B-cell activation in renal tissues of MRL/lpr mice

We first examined genes associated with ‘SLE’ and identified a significant downregulation of several complement-related genes, including C1, C3, C4, C5, C6 and C7, following CMG intervention. These findings suggest that CMG regulates the complement system (figure 3A). Furthermore, CMG treatment reduced the expression of key B-cell marker genes, including CD40 and its ligand CD40 ligand (CD40L) (figure 3B). Given the interconnection between B-cell activation and complement system activation, we hypothesise that CMG’s mitigating renal injury may be related to modulating both processes.

Figure 3. The effects of CMG on complement system activation and B-cell activation in renal tissues of MRL/lpr mice. (A) Heatmap of differentially expressed genes related to ‘SLE’; (B) Schematic diagram showing changes to related factors in ‘SLE’ after CMG intervention; (C–F) CMG decreases levels of IL-1β, TNF-α, IFN-γ and C1Q in renal tissues of MRL/lpr mice; (G–I) CMG reduces IgG, C3 and CD19 positive expression in renal tissues of MRL/lpr mice; (J) CMG decreased BLIMP1 expression in renal tissues of MRL/lpr mice. (K‒M) CMG reduced C3a and C5a content, while increasing the CH50 content in serum of MRL/lpr mice. n=6 for A, n=10 for C‒F, K‒M, n=3 for G‒J. *p<0.05, **p<0.01. Note: data in panels G–J represent preliminary findings based on n=3 per group without statistical analysis. BLIMP1, B lymphocyte-induced maturation protein 1; C3, complement component 3; CD19, cluster of differentiation 19; CH50, total haemolytic complement; CMG, compound Muniziqi gGranule; CMG-H, CMG high-dose; IFN-γ, interferon-gamma; IgG, immunoglobulin G; IL-1β, interleukin-1 beta; KEGG, Kyoto Encyclopedia of Genes and Genomes; lpr, lymphoproliferation; MRL, Murphy Roths Large; TNF-α, tumour necrosis factor-alpha.

To validate this hypothesis, we performed additional experiments. Compared with MRL/Mpj mice, MRL/lpr mice exhibited significantly elevated levels of inflammatory factors such as IL-1β, TNF-α, IFN-γ and C1Q in renal tissues. CMG intervention effectively reversed these elevated levels (figure 3C–F). Preliminary immunofluorescence analysis revealed a marked increase in the deposition of IgG and C3, and the positive expression of CD19 in the renal tissues of MRL/lpr mice. CMG treatment reduced the deposition of both IgG and C3 and decreased the positive expression of CD19 (figure 3G, H). Additionally, preliminary western blot analysis indicated that CMG intervention downregulated B Lymphocyte-Induced Maturation Protein 1 (BLIMP1) expression in the renal tissues of MRL/lpr mice (figure 3I, J). Moreover, CMG reduced C3a and C5a content, while it increased the CH50 content in serum of MRL/lpr mice (figure 3K–M). These further support the potential of CMG in modulating B-cell activity and complement system involvement in LN.

The effects of CMG on CD40/NF-κB p65 pathway

We subsequently analysed genes associated with the NF-κB signalling pathway and observed a significant downregulation of several pro-inflammatory genes following CMG intervention. Notably, the gene expression of the B-cell marker CD40 and its ligand CD40L was markedly reduced (figure 4A). Previous studies have shown that CD40L activates the B-cell NF-κB p65 pathway via CD40, thereby promoting autoimmune inflammation.²¹ Based on these findings, we hypothesised that modulating B-cell activation by CMG may be related to regulating the CD40/NF-κB p65 pathway. Further investigation revealed that CMG treatment reduced the levels of inflammatory mediators, including ICAM1, VCAM1, CXCL1 and CXCL2, in the renal tissues of MRL/lpr mice (figure 4B–E). Preliminary western blot analysis suggested that CMG downregulated CD40 and TNF receptor-associated factor 6 (TRAF6) expression, as well as the phosphorylation of NF-κB p65 in renal tissues (figure 4F). These results provide preliminary evidence that CMG reduced the activation of the CD40/NF-κB p65 pathway, suggesting a potential mechanism underlying its therapeutic effects in LN.

Figure 4. The effects of CMG on the CD40/NF-κB p65 pathway. (A) Heatmap of differentially expressed genes related to the ‘NF-κB signalling pathway’; (B–E) CMG reduces levels of ICAM1, VCAM1, CXCL1 and CXCL2 in renal tissues of MRL/lpr mice; (F) CMG decreases expression of CD40, TRAF6 and p-p65/p65 in renal tissues of MRL/lpr mice. n=6 for A, n=10 for B‒E, n=3 for F. *p<0.05, **p<0.01. Note: data in (F) represent preliminary findings based on n=3 per group without statistical analysis. C1Q, complement component 1q; CD40, cluster of differentiation 40; CMG, compound Muniziqi gGranule; CMG-H, CMG high-dose; CXCL, chemokine (C-X-C motif) ligand; ICAM1, intercellular adhesion molecule 1; lpr, lymphoproliferation; MRL, Murphy Roths Large; NF-κB p65, nuclear factor-kappa B p65; TNF, tumour necrosis factor; TRAF6, TNF receptor-associated factor 6; VCAM1, vascular cell adhesion molecule 1.

Discussion

LN is one of the most severe and prevalent complications of SLE and remains the leading cause of mortality in SLE patients. The effectiveness of LN treatment directly influences the long-term prognosis and survival rates of these patients. Despite the widespread use of immunosuppressants in clinical practice, the outcomes remain suboptimal, often accompanied by significant adverse effects.⁶ This underscores the urgent need for novel therapeutic strategies and drugs for the prevention and treatment of LN. MRL/lpr mice carry the lpr gene²² and are widely recognised as an experimental model for spontaneous SLE, displaying many features similar to human SLE. These mice exhibit rapid disease onset, high incidence and severe disease progression, making them an ideal preclinical model for testing new LN therapies.²³ In MRL/lpr mice, the pathological manifestations include marked lymphadenopathy, an elevated spleen index, elevated levels of autoantibodies, deposition of immune complexes in the kidneys, inflammatory mediator synthesis, cellular infiltration, renal dysfunction and proteinuria.²⁴ Conversely, MRL/MpJ mice are highly genetically similar to MRL/lpr mice but do not carry the lpr gene. Therefore, we selected them as the control group.

In our study, MRL/lpr mice presented with a significantly increased spleen index, elevated serum antibody levels (including anti-dsDNA-IgG), increased inflammatory cytokines such as IL-1β and pronounced renal damage. This damage was characterised by increased levels of 24-hour UTP, BUN and Cr, as well as histopathological changes including glomerular sclerosis, crescent formation, inflammatory cell infiltration and tubular atrophy, along with elevated pathological scores. Notably, CMG treatment demonstrated disease-ameliorating effects similar to those of PRE—a positive control¹⁹—suggesting its therapeutic potential in LN. Although our preliminary studies have suggested CMG’s anti-inflammatory and antioxidant properties, potentially mitigating treatment-related tissue injury, the high dose of 5 g/kg/day employed in this study was based solely on prior research.¹⁴

Moreover, we observed that CMG significantly reduced the levels of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α. These cytokines originate not only from adaptive immune cells but also from innate immune sources—such as activated macrophages—suggesting a potential modulatory effect of CMG on innate immunity. Indeed, innate immune cells like macrophages are central to SLE/LN pathogenesis; their excessive activation can trigger macrophage activation syndrome, a rare but severe SLE complication marked by a dramatic TNF-α surge that promotes B-cell activation, differentiation, autoantibody production²⁵ and inflammatory amplification, thereby accelerating disease progression. ²⁶Emerging evidence suggests that modulating macrophage polarisation between the pro-inflammatory M1 and anti-inflammatory M2 phenotypes represents a promising therapeutic strategy for SLE/LN.²⁷

To investigate the molecular mechanisms through which CMG alleviates LN, we conducted transcriptomic analysis and identified significant enrichment in the ‘SLE’ and the ‘NF-κB signalling pathway’, suggesting that CMG’s therapeutic effects on renal injury may involve these pathways. Further examination of genes related to ‘SLE’ revealed a downregulation of several complement-related genes after CMG treatment. Notably, CMG intervention reduced the gene expression of CD40, a well-known marker for B cells, as well as its ligand, CD40L. These findings indicate that CMG’s treatment of LN may be associated with complement system activation and B-cell activation.

Previous studies have shown that complement system activation is crucial in the pathogenesis of both SLE and LN, leading to the abnormal expression of complement proteins.²⁸ In LN patients, renal biopsies often reveal immune complex deposition in the glomeruli, accompanied by decreased CH50 levels.²⁹ Once deposited, these immune complexes activate the complement cascade via the classical pathway, resulting in the release of complement proteins such as C3. Complement proteins such as C3 play a critical role in recruiting inflammatory cells and promoting the release of pro-inflammatory mediators, which contribute to renal injury.⁶ Inhibitory therapies targeting excessive complement activation, including pegcetacoplan³⁰ and narsoplimab,³¹ have shown promise in phase II clinical trials. Additionally, abnormal B-cell activation is closely linked to the pathogenesis of SLE and LN, leading to increased production of pathogenic autoantibodies and inflammatory cytokines.⁹ Immune complex deposition, driven by B-cell activation, further activates the complement system, amplifying the inflammatory response. Immunosuppressive treatments targeting B cells have demonstrated effectiveness in both animal models and clinical cases of LN. For instance, anti-CD20 monoclonal antibodies have alleviated renal impairment in MRL/lpr mice³² and show efficacy in treating refractory or frequently relapsing SLE and severe LN in patients.³³ In our study, CMG treatment significantly reduced inflammatory factors in renal tissues, decreased immune complex deposition and lowered B-cell infiltration in MRL/lpr mice. Simultaneously, CMG downregulated the expression of the B-cell activation-related protein BLIMP1³⁴ and reduced the contents of complement activation markers C3a and C5a, indicating that CMG regulates both B-cell activation and complement system activation. Notably, in SLE, reduced CH50 levels result from excessive complement consumption due to immune complex deposition, rather than insufficient complement synthesis. Our data show that CMG treatment restores CH50 level, which, together with decreased renal C3a and C5a deposition and reduced immune complex accumulation, indicates that CMG reduces the production of pathogenic autoantibodies and the subsequent immune complex formation, thereby alleviating complement consumption. This effect is linked to modulating the CD40/NF-κB p65 signalling pathway and attenuating aberrant B-cell activation. Furthermore, unlike agents that block specific complement components, CMG acts upstream by rebalancing dysregulated immune responses—thereby indirectly mitigating complement overactivation. This upstream, systems-level approach exemplifies a core principle of TCM: holistic regulation rather than single-target suppression, offering a distinctive therapeutic paradigm for complex autoimmune diseases like LN.

Subsequent analysis of genes associated with the ‘NF-κB signalling pathway’ revealed a significant downregulation of pro-inflammatory genes downstream of NF-κB p65 following CMG intervention. Notably, we observed that the B-cell marker gene Cd40 and its ligand Cd40lg were also enriched in the NF-κB pathway, with their expression markedly reduced after CMG treatment. This finding led us to hypothesise that interference with the CD40/NF-κB p65 pathway by CMG may reduce B-cell activation. CD40, a member of the tumour necrosis factor receptor superfamily, is predominantly expressed on the surface of B cells. It plays a critical role in activating NF-κB p65 and upregulating pro-inflammatory genes that depend on NF-κB p65.³⁵ CD40L, also part of the tumour necrosis factor superfamily, binds to CD40, forming the CD40-CD40L complex. This interaction accelerates B-cell activation, facilitates antibody class switching, promotes the release of antibodies and inflammatory mediators and amplifies the inflammatory cascade.³⁶ Elevated CD40 expression has been observed in renal biopsies from patients with LN, and preclinical studies have underscored the importance of the CD40-CD40L system in the pathogenesis of both SLE and LN.³⁷ Prior research has demonstrated that when CD40 binds to its ligand CD40L, it activates the NF-κB p65 signalling pathway in B cells via TRAF6, thereby driving autoimmune inflammation.^{21 35} Selective inhibition of the CD40-CD40L system has been shown to ameliorate LN in animal models,³⁷ and early-stage clinical studies also suggest its potential therapeutic benefit.³⁸ Furthermore, blocking NF-κB signalling has proven effective in mitigating renal injury in LN.³⁹ In our study, CMG treatment significantly reduced the levels of chemokines and downregulated the expression of CD40, TRAF6 and phosphorylated NF-κB p65. These findings suggest that CMG’s reduced the activation of the CD40/NF-κB p65 pathway.

Collectively, the therapeutic benefits of CMG in LN—manifesting as the alleviation of B-cell activation and complement system dysfunction—appear to be associated with the reduction of the CD40/NF-κB p65 pathway (figure 5).

Figure 5. Schematic diagram of the underlying mechanism of compound Muniziqi gGranule (CMG) on lupus nephritis. The protective effect of CMG on MRL/lymphoproliferation (lpr) mice was associated with the reduction of B-cell and complement system activation and the downregulation of the CD40/NF-κB p65 signalling pathway (the dashed lines are used to represent correlations). CD40, cluster of differentiation 40; lpr, lymphoproliferation; MRL, Murphy Roths Large; NF-κB p65, nuclear factor-kappa B p65; TNF, tumour necrosis factor; TRAF6, TNF receptor-associated factor 6.

However, several limitations need to be addressed. First, the immunofluorescence and Western blot analyses were based on n=3 per group; these results are considered preliminary and require validation in larger cohorts with full statistical analysis. Next, current evidence indicates that multiple bioactive constituents identified in CMG—including harmaline, harmine, liquiritin, isoliquiritin, sieboldianoside A, glycyrrhizic acid, chlorogenic acid, caffeic acid, ferulic acid and apigenin 7-O-glucoside—modulate key factors within the CD40/NF-κB p65 signalling pathway and B-cell activation (online supplemental figure S2). However, the lack of precise concentrations of these compounds in CMG hinders the reproducibility of CMG’s therapeutic effects and its underlying molecular mechanisms. Therefore, it is essential to conduct quantitative analysis of the concentrations of key compounds in CMG and their blood-entry components using techniques such as UPLC-Q-TOF-MS, and further carry out pharmacokinetic studies to facilitate the promotion and application of CMG. Third, systematic preclinical toxicity studies and comprehensive safety monitoring were beyond the scope of this initial efficacy study but are essential for future clinical translation. Moreover, further validations using comprehensive in vitro and in vivo studies are required to validate whether the regulatory effects of CMG on B-cell activation were through inhibiting the activation of CD40/NF-κB p65 pathway. Additionally, rescue experiments can serve as a powerful complement to validate the molecular mechanisms of CMG. Subsequent studies should integrate rescue experiment data, blood-entry component profiles and pharmacokinetic data to systematically identify the true targets of CMG in the targeted treatment of LN, providing mechanistic coordinates for its precise application.

Data availability statement

Data are available in a public, open access repository. Data are available upon reasonable request.