Allogeneic mesenchymal stromal cells ameliorate lupus nephritis by regulating lipid metabolism via ASGR1

Abstract

Background

Systemic lupus erythematosus (SLE) is an autoimmune disease in which disruptions in lipid metabolism contribute to immune dysregulation, inflammation, and organ damage. This study aims to elucidate the role of hepatic asialoglycoprotein receptor 1 (ASGR1) in lipid metabolism dysregulation in lupus nephritis, and to investigate the therapeutic potential of mesenchymal stromal cells (MSCs) in restoring lipid homeostasis via ASGR1 modulation.

Method

A high-fat diet (HFD)-fed MRL/lpr lupus mouse model was used to assess the impact of lipid dysregulation on lupus pathology. ASGR1 expression and cholesterol metabolism-related genes were evaluated using molecular and histological analyses. Liver-specific Asgr1 knockdown was achieved using adeno-associated virus. The therapeutic effects of MSCs were evaluated both in vivo and in vitro, with a focus on ASGR1 regulation and downstream metabolic signaling.

Results

In MRL/lpr mice, HFD exacerbated lipid accumulation, renal injury, and immune imbalance. ASGR1 expression was significantly upregulated in the liver, leading to suppressed cholesterol efflux and enhanced lipid synthesis. Asgr1 knockdown ameliorated these metabolic disturbances. MSCs treatment reduced ASGR1 expression, restored lipid homeostasis, and improved lupus phenotypes by modulating mTOR, LXRα, and AMPK pathways. Overexpression of Asgr1 reversed the protective effects of MSCs in vitro.

Conclusions

Lipid metabolism dysregulation contributes to SLE pathogenesis through ASGR1-mediated pathways. Targeting ASGR1, particularly via MSCs therapy, offers a promising strategy to restore metabolic balance and attenuate disease severity in lupus nephritis.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13287-026-05167-2.

Introduction

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disorder characterized by the deposition of aberrant autoantigens and immune complexes, leading to multiple organs involvements. SLE is most common in women and in those of non-white ethnicity. The clinical presentation of SLE is heterogeneous, can involve one or more organs, including the skin, kidneys, joints, and nervous system, and often follows a chronic or relapsing - remitting disease course [1, 2]. Among the many comorbidities associated with SLE, lipid metabolism disorders—particularly dyslipidemia—are increasingly recognized as key contributors to disease progression and adverse outcomes [3]. Dyslipidemia in SLE patients, often marked by elevated cholesterol and triglyceride levels, is strongly associated with heightened disease activity and increased cardiovascular risk [4, 5]. Persistent lipid imbalance not only contributes to atherosclerosis and cardiovascular disease—leading causes of mortality in SLE—but may also exacerbate inflammation and immune dysfunction. While statins remain the cornerstone of dyslipidemia treatment, emerging therapies targeting specific metabolic regulators are under investigation.

Cholesterol imbalance is central to lipid metabolic disorders and contributes to the pathogenesis of non-alcoholic fatty liver disease (NAFLD), diabetes, and cardiovascular disease [6]. Recently, asialoglycoprotein receptor 1 (ASGR1) has gained attention as a pivotal regulator of cholesterol homeostasis. ASGR1 is a prominent subunit of the asialoglycoprotein receptor complex (also known as Ashwell-Morell receptor), which is highly conserved across mammalian species [7, 8]. It is predominantly expressed in hepatocytes, where it mediates the clearance of desialylated glycoproteins from circulation. Specifically, ASGR1 recognizes terminal galactose (Gal) or N-acetylgalactosamine (GalNAc) residues that become exposed after the removal of sialic acid, thereby maintaining serum protein homeostasis and exerting various biological functions [9]. A large-scale genetic study from Iceland found that a loss of function variant in ASGR1 is associated with lower cholesterol and a reduced risk of cardiovascular disease [10]. Further studies support these findings, showing that inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion [11].

In parallel, mesenchymal stromal cells (MSCs) have emerged as promising therapeutic agents for metabolic and inflammatory diseases [12, 13]. MSCs possess potent immunomodulatory and regenerative properties and have been shown to influence both immune and metabolic processes. Previous studies have demonstrated that MSCs transplantation can regulate the secretion of cytokines and chemokines, enhance regulatory T cell function, reduce dyslipidemia, stabilize atherosclerotic plaques and improve vascular pathology [14]. Given their multifaced biological activities, MSCs represent a compelling candidate for treating complex conditions such as dyslipidemia and its associated complications.

In this study, we investigated whether MSCs could modulate cholesterol metabolism in the context of SLE by targeting the ASGR1 signaling axis. Focusing on ASGR1 as a mechanistic link between lipid dysregulation and lupus progression, we conducted in vivo and in vitro experiments to evaluate how MSCs influence ASGR1 expression and its downstream metabolic pathways. The aim of this study was to provide experimental evidence supporting MSCs therapy as a novel strategy for correcting lipid metabolism abnormalities and mitigating disease severity in SLE.

Materials and methods

Clinical data

Patients diagnosed with SLE and treated at the Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, between January and December 2024, were enrolled in this study. SLE diagnosis was based on the 2019 European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) classification criteria [15]. Clinical parameters, including the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores [16], and laboratory results were recorded using standardized forms and entered into an electronic database. Hyperlipidemia was defined by elevated levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C), along with decreased levels of high-density lipoprotein cholesterol (HDL-C), in accordance with established criteria [17]. Peripheral blood samples and clinical data were collected from healthy controls, SLE patients, and hyperlipidemic SLE patients who received MSCs therapy, both before and after treatment. Samples were obtained at baseline (prior to MSCs administration) and at 3 and 6 months after a single intravenous infusion of allogeneic human umbilical cord-derived MSCs (2 million cells per kilogram of body) from a healthy donor.

Mice and interventions

Female MRL/MPJ-FAS^lpr (MRL/lpr) mice aged 4–6 weeks were obtained from SPF (Beijing) Biotechnology Co., Ltd. The experimental animals were housed under specific pathogen-free (SPF) conditions in individually ventilated cages at the Experimental Animal Center of Nanjing Drum Tower Hospital. MRL/lpr mice were fed either a normal chow diet (NCD) or high-fat diet (HFD) throughout the study. For gene knockdown, an AAV2/8 vector carrying shRNA targeting Asgr1 (AAV2/8-shRNA-Asgr1) or a control shRNA (AAV2/8-shRNA-Ctrl) was purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China). Mice received slow tail vein injection of vectors (100 µL, 1 × 10¹² vg/mL) using a 29-gauge insulin syringe (BD Biosciences). For MSCs transplantation, human umbilical cord-derived MSCs were provided by the Stem Cell Center of Jiangsu Province. The isolation and culture procedures have been described in detail in our previous work [18]. After 12 weeks of HFD feeding, MRL/lpr mice received a single intravenous injection of MSCs (5 × 10⁵ cells in 200 µL PBS) via the tail vein. Control mice received an equivalent volume of PBS. Samples were harvested 4 weeks post-treatment. Mice were euthanized by gradual-fill CO₂ inhalation, with cervical dislocation to confirm death before tissue collection. All procedures complied with institutional ethical regulations and were reported in accordance with the ARRIVE 2.0 guidelines. Six groups were included (n = 6 for each group, as commonly used in comparable mechanistic studies [19]): NCD vs. HFD, AAV-shCtrl vs. AAV-shAsgr1, PBS vs. MSCs.

Primary hepatocytes isolation and cell culture

Primary hepatocytes (PHC) were isolated from HFD-fed female MRL/lpr mice using a two-step collagenase perfusion technique [20, 21]. Isolated PHC and Huh7 cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. For coculture experiments, hepatocytes were cocultured with MSCs using a Transwell system.

Lentiviral vector construction and transduction

The coding sequence of Asgr1 was amplified and cloned into the lentiviral transfer plasmid pLV4ltr-PGK-ZsGreen1(2 A) Puro-CMV-c3×FLAG. For lentivirus production, HEK293T cells were co-transfected with the transfer plasmid along with the packaging plasmids psPAX2 and pMD2.G using transfection reagent (Corues Biotechnology Co. #1001CR01). Viral supernatants were harvested at 48- and 72-hours post-transfection, filtered through a 0.45 μm membrane, and concentrated by ultracentrifugation. Overexpression efficiency was validated by qRT-PCR and western blot analysis.

Serum and tissue biochemistry

Serum and liver concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were quantified using enzymatic colorimetric kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China; Cat. Nos. A111-1-1, A110-1-1, A113-1-1, and A112-1-1, respectively). Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine, and blood urea nitrogen (BUN) were measured using commercial assay kits from the same manufacturer (Cat. Nos. C009-2-1, C010-2-1, C011-2-1, and C013-2-1, respectively). All assays were performed according to the manufacturer’s protocols.

ELISA for sh2a and anti‑dsDNA antibodies

Human serum sh2a levels were measured using the KAIABIO ELISA kit according to the manufacturer’s instructions. Pre-coated microplate strips were equilibrated at room temperature for 20 min. Standards (10-0.3125 ng/mL) and samples (50 µL) were added, followed by 100 µL of HRP-conjugated antibody. After incubation at 37 °C for 60 min, plates were washed five times with 350 µL wash buffer per well, and incubated with substrates A and B in the dark for 15–30 min. Absorbance was measured at 450 nm, and concentrations were determined from the standard curve. Anti-dsDNA antibodies were quantified using the WUHAN HUAMEI BIOTECH ELISA kit, following a similar protocol.

Histological analysis

At the study endpoint, liver and kidney tissues were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome for histological evaluation. Liver tissues from mice across treatment groups were processed similarly and stained with H&E, Masson’s trichrome, and Oil Red O to assess tissue architecture, fibrosis, and lipid accumulation. All histopathological evaluations were conducted in a blinded manner by two independent pathologists to ensure unbiased assessment.

Immunofluorescence

Snap-frozen kidneys were sectioned and subjected to immunofluorescence to detect IgG and C3 deposition in the glomeruli. Sections were fixed in acetone and incubated with anti-mouse IgG and Alexa Fluor 488-conjugated anti-mouse C3 antibodies (Santa Cruz, sc-58926 AF488). Kidney sections were incubated with antibodies against F4/80 (Cell Signaling Technology, 70076) together with either CD86 (Cell Signaling Technology, 19589) or CD206 (Abcam, ab300621), followed by incubation with fluorophore-conjugated secondary antibodies. Liver sections were stained with anti-rabbit ASGR1 followed by Alexa Fluor 488-conjugated secondary antibodies. Fluorescence imaging was performed using FV3000 microscope (Olympus, Tokyo, Japan).

RNA Isolation and quantitative real-time PCR (RT–qPCR)

Total RNA was extracted from liver, kidney tissues or cultured cells (grown in 6 cm dishes) using Trizol Reagent, followed by DNase I treatment to remove genomic DNA. Reverse transcription was performed on 2 µg of total RNA using M-MLV Reverse Transcriptase. Quantitative PCR was carried out using the ChamQ Universal SYBR qPCR Master Mix (High ROX) on a Bio-Rad CFX384 or CFX96 system. Gene expression levels were calculated using the 2^−ΔΔCt method. Primer sequences are provided in Table S1.

Western blot

Western blotting was conducted to assess protein expression. Liver or cell lysates were separated by SDS-PAGE and transferred to a methanol-activated PVDF membrane under cold, constant current conditions. Membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, followed by 1-hour incubation with HRP-conjugated secondary antibodies at room temperature. Signals were detected using ECL and visualized with a chemiluminescence imaging system. Primary antibodies used in this study included: anti-ASGR1 (Abcam, ab254262; 1:1000), anti-Actin (Abclonal, AC006; 1:5000), anti-ABCA1 (Proteintech, 26564-1-AP; 1:1000), anti-SREBP2 antibody (Proteintech, 28212-1-AP; 1:1000), anti-ABCG8 (Proteintech, 24453-1-AP; 1:1000), anti-CYP7A1 (Proteintech, 18054-1-AP; 1:1000), anti-mTOR (Proteintech, 28273-1-AP; 1:1000), anti-Phospho-mTOR antibody (Proteintech, 67778-1-Ig; 1:1000), anti-AMPK antibody (PTM Bio, PTM-6739; 1:1000), anti-Phospho-AMPK antibody (PTM Bio, PTM-7428; 1:1000), and anti-LXRα (Abclonal, A3974; 1:5000).

Flow cytometry

Briefly, single-cell suspensions of splenic cells were prepared by mincing mouse spleens in PBS containing 0.5% FBS and 1% EDTA. PBMCs were isolated from EDTA-treated peripheral blood using Ficoll density gradient centrifugation. Erythrocytes were removed with ACK lysis buffer, and the resulting cell suspension was washed and filtered through 70-µm meshes. The cells were then counted and prepared for FACS analysis following standard flow cytometry protocols. Cells were first stained for surface markers (live-APC-Cy7, CD45-AF700, TCR-β-BV421, CD4-FITC, CD8-BV510, CD25-APC, CD138-BB515, F4/80-PE, CD86-BV421), fixed, and permeabilized using a fixation/permeabilization kit (eBioscience, San Diego, CA). Subsequently, intracellular cytokine markers (IFNγ-BV785, Foxp3-PE, IL4-PE-Cy7) were applied. All samples were analyzed using a FACS Calibur flow cytometer (BD Biosciences) and processed with FlowJo software (Tree Star, Ashland, OR).

Statistical analysis

Data were analyzed using GraphPad Prism 9.0, ImageJ, and SPSS 25.0. For continuous variables, normality was assessed. Normally distributed data with equal variance were expressed as mean ± SEM and compared using t-tests for group comparisons. Non-normally distributed data were reported as median and interquartile range (Q1, Q3) and analyzed with the Mann-Whitney U test. Categorical variables were presented as percentages and compared using the chi-square test. Pearson correlation analysis was used for continuous variable correlations. All experiments were repeated independently at least three times. A p-value < 0.05 was considered statistically significant.

Results

ASGR1 upregulation is associated with lipid metabolism disorders in SLE

ASGR1 has been well-characterized and has emerged as a critical modulator of cholesterol homeostasis [11], though its role in autoimmune-associated metabolic disorders remains unexplored. In this study, we provide the first evidence linking ASGR1 to SLE-associated lipid dysregulation. Analysis of the NCBI GEO dataset (GSE135251) showed significant upregulation of hepatic Asgr1 in non-alcoholic fatty liver disease (NAFLD) patients compared to healthy donors (p < 0.001; Fig. 1A), with further increase in active disease (p < 0.01; Fig. 1B), highlighting its involvement in metabolic stress. Extending to SLE, we found that the serum levels of the soluble ASGR1 (sh2a) were markedly higher in SLE patients compared to matched controls (Fig. 1C). Notably, sh2a levels positively correlated with atherogenic lipids (TG, TC, LDL-C) and negatively with HDL-C (Fig. 1D), mirroring the dyslipidemia commonly seen in SLE.

Fig. 1.

Increased ASGR1 expression involves lipid metabolism disorders in SLE. A Analysis of Asgr1 expression in liver tissues of healthy controls and NAFLD patients from the GSE135251 dataset. B Hepatic Asgr1 expression levels in NAFLD patients stratified by NAS scores. C Serum sh2a levels in healthy controls and SLE patients. D Correlation analysis between serum sh2a levels and TG, TC, LDL-C, and HDL-C in SLE patients. E Hepatic Asgr1 mRNA levels in MRL/lpr mice fed NCD or HFD (n = 6 per group). F ASGR1 protein levels in the liver of NCD- and HFD-fed mice (n = 6 per group). G Representative immunofluorescence images of hepatic ASGR1 expression of NCD- and HFD-fed mice (scale bar = 50 μm). (H) The quantification of hepatic ASGR1 fluorescence intensity (n = 6 per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. HC, healthy controls

To model this metabolic disturbance in vivo, MRL/lpr mice, a well-established lupus model, were fed either a NCD or a HFD for 12 weeks (n = 6 per group; Fig. S1A). Compared with NCD-fed controls, HFD-exposed MRL/lpr mice displayed significant weight gain (Fig. S1B). Gross liver examination revealed hepatomegaly, with features such as thickened edges, pallor, parenchymal softening, and a greasy surface, indicating steatosis-like changes (Fig. S1C). Correspondingly, serum levels of ALT and AST were significantly elevated in HFD-fed mice (Fig. S1D). Comprehensive lipidomic profiling revealed profound dyslipidemia, with marked increases in TG, TC, and LDL-C, alongside a reduction in HDL-C in both serum and hepatic tissue (Fig. S1E-F). Histopathological assessment further corroborated these findings. H&E staining demonstrated extensive micro- and macro vesicular steatosis, with pronounced lipid vacuolization indicative of hepatocyte injury (Fig. S1G). Oil Red O staining confirmed pervasive neutral lipid deposition within hepatocytes, while Masson’s trichrome staining showed minimal collagen deposition, indicating early-stage steatohepatitis without significant fibrosis. Collectively, these data confirm that chronic HFD exposure induces hepatic lipid accumulation and metabolic disturbances in MRL/lpr mice, successfully recapitulating the dyslipidemic phenotype observed in SLE patients.

In this well-established model, we observed significant upregulation of Asgr1 at both the transcriptional and protein levels (Fig. 1E-H). ASGR1 induction was accompanied by downregulation of cholesterol efflux genes (Abca1, Abcg1, Abcg8), and bile acid synthesis enzyme Cyp7a1, as well as decreased Ldlr expression, alongside increased expression of genes promoting cholesterol biosynthesis (Hmgcr, Srebp2) and lipogenesis (Srebp1, Scd1) (Fig. S1H). These molecular changes were corroborated by protein-level analyses, which confirmed reduced expression of ABCA1, ABCG8, and CYP7A1, along with increased SREBP2 (Fig. S1I-J). Collectively, these results identify ASGR1 as a previously unrecognized factor associated with SLE-related lipid dysregulation. Based on its known metabolic function, we hypothesize that ASGR1 may contribute to this process through a bidirectional mechanism, simultaneously suppressing cholesterol elimination and enhancing its synthesis, thereby aggravating metabolic perturbations in SLE.

Lipid metabolism disorders exacerbate lupus pathogenesis

To further clarify the link between lipid metabolism disturbances and lupus-related organ damage, we analyzed SLE patient clinical data and found serum TG levels were significantly positively correlated with serum creatinine but inversely with estimated glomerular filtration rate (eGFR) (Fig. 2A, B). Serum TC showed analogous correlations (Fig. 2C, D), highlighting the clinical relevance of dyslipidemia in lupus nephritis.

Fig. 2.

Lipid metabolism disorders exacerbate lupus nephritis in SLE. A Correlation analysis between Scr and TG in SLE patients. B Correlation analysis between eGFR and TG in SLE patients. C Correlation analysis between Scr and TC in SLE patients. D Correlation analysis between eGFR and TC in SLE patients. E Representative images illustrating skin lesions and hair loss in MRL/lpr mice fed with NCD or HFD. F Representative images of the spleen and spleen-to-body weight ratio (n = 6 per group). G Representative images of cervical lymph nodes and lymph node-to-body weight ratio (n = 6 per group). H Serum levels of anti-dsDNA antibody (n = 6 per group). I Urinary protein levels (n = 6 per group). J Blood urea nitrogen levels (n = 6 per group). K Serum levels of creatinine (n = 6 per group). L Representative H&E and Masson’s trichrome staining of kidney from NCD- and HFD-fed mice (scale bar = 100 μm). (M) Representative immunofluorescence staining of C3 and IgG deposition in kidney (scale bar = 20 μm). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Consistent with this, HFD-fed MRL/lpr mice displayed exacerbated lupus phenotypes. Cutaneous lesions were more severe, particularly in postauricular and facial regions, with concomitant alopecia (Fig. 2E). These mice also developed pronounced splenomegaly and lymphadenopathy (Fig. 2F-G), as well as elevated anti-dsDNA antibody titers (Fig. 2H). In addition, flow cytometry analysis showed elevated pro-inflammatory Th1 and plasma B cells, reduced Treg populations, and an elevated macrophage CD86 MFI indicative of an altered M1 phenotype (Fig. S2), indicating enhanced autoimmune activation. Renal assessment revealed significantly elevated proteinuria, serum BUN, and creatinine, confirming impaired renal function (Fig. 2I-K). Histopathological analysis revealed aggravated glomerulonephritis featuring hypercellularity, basement membrane thickening, and crescent formation, accompanied by extensive interstitial inflammation and fibrosis (Fig. 2L). Immunofluorescence staining confirmed enhanced glomerular deposition of IgG and C3 (Fig. 2M), suggesting exacerbated immune complex-mediated renal injury.

Taken together, these findings demonstrate that HFD-induced dysregulation accelerates lupus progression through systemic immune activation and worsening of renal pathology. This underscores metabolic disturbance as a critical disease modifier in SLE and suggests that ASGR1 may represent a mechanistic link between altered lipid metabolism and autoimmune exacerbation.

Liver-specific Asgr1 knockdown alleviates lipid dysregulation and renal injury in HFD-fed MRL/lpr mice

Building on our findings that ASGR1 is upregulated in lupus-associated metabolic dysregulation and may contribute to cholesterol imbalance, we next investigated whether selective hepatic suppression of Asgr1 could reverse these abnormalities and mitigate disease severity. To this end, MRL/lpr mice maintained on HFD for 12 weeks were administered adeno-associated virus vectors (AAV) carrying either control sequences (AAV-Ctrl) or Asgr1-targeting shRNA (AAV-Asgr1) via tail vein injection (Fig. 3A). Effective gene silencing was confirmed by significant reductions in hepatic Asgr1 mRNA and protein levels in the AAV-Asgr1 group (Fig. 3B, C).

Fig. 3.

ASGR1 knockdown reverses metabolic dysregulation to restore renal function. A Schematic representation of the Asgr1 knockdown protocol in HFD-fed MRL/lpr mice. B Hepatic Asgr1 mRNA levels in AAV-shCtrl and AAV-shAsgr1 groups. C Hepatic ASGR1 protein levels. D Hepatic TG, TC, LDL-C, and HDL-C levels. E Representative images of liver morphology. F Blood urea nitrogen levels. Serum levels of creatinine. G Representative images of renal histopathology in AAV-shCtrl and AAV-shAsgr1 mice, assessed by H&E and Masson’s trichrome staining (scale bar = 100 μm). H Representative immunofluorescence staining of renal C3 and IgG deposition I in AAV-shCtrl and AAV-shAsgr1 mice (scale bar = 20 μm). Data are presented as mean ± SEM (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001

Liver-targeted Asgr1 knockdown led to marked improvements in hepatic phenotype. Treated mice exhibited normalized liver morphology, reduced body weight, and significantly decreased serum ALT and AST levels, indicating resolution of hepatic injury (Fig. 3E; Fig. S3D-E). Lipidomic profiling demonstrated substantial reversal of HFD-induced dyslipidemia, with reduced TC, TG, LDL-C and restored HDL-C in both serum and liver tissue (Fig. 3D; Fig. S3H). Histopathological evaluation showed amelioration of steatosis in knockdown mice, evidenced by diminished lipid droplet accumulation in H&E and Oil Red O staining (Fig. 3G), while Masson’s trichrome staining revealed unchanged collagen deposition. These findings illustrate that targeted Asgr1 suppression effectively restores lipid homeostasis and alleviates steatohepatitis-like pathology in lupus-prone mice under metabolic stress.

Beyond metabolic improvement, Asgr1 knockdown also conferred systemic protection. AAV-Asgr1-treated mice displayed preserved skin integrity, lacking the alopecia and erythematous lesions observed in controls (Fig. S3A). Indicators of systemic autoimmune activity were also significantly reduced, including splenomegaly, lymphadenopathy, and circulating anti-dsDNA autoantibody titers (Fig. S3B-C, G). In AAV-Asgr1 group, flow cytometry revealed a reduction in pro-inflammatory Th1 and plasma B cells, an increased Treg frequency, and a decreased CD86 MFI compared with AAV-Ctrl (Fig. S4), indicating attenuated lupus activity and immune activation. Renal protection was particularly notable. Compared to AAV-Ctrl animals, AAV-Asgr1 mice exhibited significantly improved kidney function, with reduced proteinuria, serum creatinine, and BUN levels (Fig. 3F; Fig. S3F). Histopathological analysis of renal tissue demonstrated reversal of HFD-aggravated glomerular injury, including reduced glomerular hypertrophy, mesangial hypercellularity, and crescent formation, accompanied by diminished interstitial fibrosis (Fig. S3I). Immunofluorescence staining revealed decreased glomerular C3 and IgG deposition (Fig. 3H), indicating diminished immune complex-mediated injury.

Together, these findings demonstrate that liver-specific suppression of Asgr1 not only restores lipid balance but also mitigates autoimmune manifestations and renal damage in lupus-prone mice. These results highlight ASGR1 as a potential therapeutic target linking metabolic dysfunction to immune pathogenesis in SLE.

MSCs restore lipid metabolic homeostasis by inhibiting ASGR1

Given the pathogenic role of ASGR1 in SLE-associated dyslipidemia, we next evaluated whether MSCs, known for their immunomodulatory and regenerative properties, could therapeutically target this pathway. To assess mechanistic effects, we first utilized a customized co-culture system of MSCs and Huh7 cells. Co-culture with MSCs led to significant downregulation of Asgr1 mRNA and protein in Huh7 cells (Fig. 4A-B), as confirmed by immunofluorescence, which revealed diminished ASGR1 signal intensity and membrane localization (Fig. 4C). Concomitantly, RT-qPCR analysis showed that MSCs co-culture time-dependently modulated hepatic cholesterol metabolism in Huh7 cells, upregulating Abcg1 and Cyp7a1 while downregulating Srebp2 (Fig. 4A). These changes suggest enhanced cholesterol efflux and bile acid synthesis, alongside suppressed lipogenesis. Protein analysis confirmed increased ABCA1 and ABCG8, and decreased SREBP2 levels (Fig. 4B, Fig. S5F). These effects were accompanied by activation of key metabolic regulators—elevated LXRα and p-AMPK, and suppressed mTOR signaling (Fig. 4D)—suggesting MSCs restore lipid homeostasis via AMPK–LXRα–mTOR axis.

Fig. 4.

MSCs improve lipid metabolism in liver cells by inhibiting ASGR1. A The mRNA expression of cholesterol-related markers in Huh-7 cells co-cultured with MSCs (MSCs: Huh-7 co-culture ratio: 0, 1:10, 1:5, 1:1) compared to the control group. B Protein expression of cholesterol-related markers (MSCs: Huh-7 co-culture at a ratio of 1:5 for 48 h). C Representative immunofluorescence images of ASGR1 in Huh-7 cells from the control and MSCs co-culture groups (MSCs: Huh-7 co-culture at a ratio of 1:5 for 48 h) (scale bar = 40 μm). D Protein expression of downstream pathway markers of ASGR1 in Huh-7 cells following MSCs co-culture (MSCs: Huh-7 co-culture at a ratio of 1:5 for 48 h). E Asgr1 mRNA levels in Huh-7 cells from CTRL and OE groups. F ASGR1 protein levels in Huh-7 cells from CTRL and OE groups. G mRNA expression of cholesterol-related markers in Huh-7 cells from CTRL and OE groups. Data are presented as mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

To establish the dependence of these effects on ASGR1, we generated an Asgr1 overexpression (OE) model in Huh7 cells. Successful Asgr1 overexpression was confirmed by RT-qPCR and Western blot (Fig. 4E-F). MSCs-mediated improvements in lipid metabolism were reversed by Asgr1-OE, with reduced expression of cholesterol efflux genes (Abca1, Abcg1, Abcg5) and upregulation of Hmgcr and lipid synthesis regulators (Srebp1, Scd1) (Fig. 4G), confirming that MSCs improve hepatocyte lipid metabolism via ASGR1 inhibition.

These findings were further validated in primary hepatocytes isolated from HFD-fed MRL/lpr mice, which showed severe lipid accumulation and altered morphology. MSCs co-culture significantly reduced Oil Red O-positive lipid droplets and normalized hepatocyte appearance (Fig. S5A-B). Molecular analyses revealed that MSCs suppressed Asgr1 expression while upregulating genes involved in reverse cholesterol transport (Abca1, Abcg5, Abcg8, Ldlr) and Cyp7a1, and downregulating lipogenesis-related genes (Srebp1, Scd1, Fasn, Srebp2), suggesting coordinated activation of lipid clearance and suppression of synthesis (Fig. S5C-E).

MSCs ameliorate lipid dysregulation and lupus nephritis via ASGR1 inhibition in lupus mice and SLE patients

To determine whether MSCs confer therapeutic benefits in vivo, we transplanted MSCs into HFD-fed MRL/lpr mice, model of lupus complicated by metabolic stress (Fig. 5A). Hepatic Asgr1 mRNA and protein expression were significantly reduced following MSCs treatment, compared to PBS controls (Fig. 5B, C). Parallel improvements were observed across key lipid metabolic pathways: cholesterol efflux genes (Abca1, Abcg5, Abcg8) and cholesterol synthesis genes (Hmgcr, Srebp2). Moreover, Ldlr mRNA was elevated, lipogenic genes (Srebp1, Fasn, Scd1) were suppressed, and Cyp7a1 expression was enhanced (Fig. S6K). These molecular changes translated into robust systemic effects. MSCs-treated mice displayed significant reduction in body weight (Fig. 5D), normalized hepatic morphology (Fig. S6D) and reduced serum ALT and AST concentrations (Fig. S6F), indicating hepatoprotection. Lipid profiling revealed dramatic decreases in both serum and hepatic TG, TC, and LDL-C content, accompanied by elevated HDL-C levels (Fig. 5E; Fig. S6E). Histopathological analysis revealed alleviation of hepatic steatosis, as confirmed by H&E and Oil Red O staining, while fibrosis remained minimal across groups (Fig. S6G).

Fig. 5.

MSCs inhibit ASGR1 expression to alleviate lipid metabolic disorders and kidney injury in SLE. A Schematic representation of PBS or MSCs treatment in MRL/lpr mice fed with HFD. B Hepatic Asgr1 mRNA levels in PBS- and MSCs-treated mice (n = 6 per group). C Hepatic ASGR1 protein levels (n = 6 per group). D Body weight of mice in the PBS and MSCs groups (n = 6 per group). E Serum TG, TC, LDL-C, and HDL-C levels (n = 6 per group). F Serum levels of anti-dsDNA antibodies (n = 6 per group). G Representative kidney histology with H&E and Masson’s trichrome staining (scale bar = 100 μm). H Blood urea nitrogen levels (n = 6 per group). I Serum creatinine levels (n = 6 per group). J Representative immunofluorescence staining of renal C3 and IgG deposition (scale bar = 20 μm). K Line graphs depicting measurements of triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol at baseline, 3 months, and 6 months post-treatment. L Line graphs depicting measurements of serum creatinine and eGFR at baseline, 3 months, and 6 months post-treatment. M Serum Sh2a levels in SLE patients before and 24 h after MSCs treatment. Data are presented as mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Beyond metabolic correction, MSCs therapy significantly improved lupus pathology. Treated mice exhibited a complete resolution of cutaneous lesions (Fig. S6A), reduced splenomegaly and cervical lymphadenopathy (Fig. S6B-C), and decreased serum anti-dsDNA autoantibody production (Fig. 5F). MSCs treated mice exhibited a decline in Th1 and plasma B cell proportions, a heightened Treg frequency, and notably reduced CD86 MFI (Fig. S7), indicating suppression of autoimmune activity. Renal function was substantially preserved, with decreased proteinuria, serum creatinine, and BUN (Fig. 5H-I; Fig. S6H). Histological and immunofluorescence analysis confirmed reduced glomerular hypercellularity, interstitial inflammation, collagen deposition, and IgG/C3 immune complex deposition (Fig. 5G, J). Renal qPCR analyses demonstrated that MSCs treatment significantly reduced the expression of the pro-inflammatory M1-associated markers inos and Cd86, while increasing the expression of the anti-inflammatory/tissue-reparative M2-associated markers Arginase-1 and Cd206 (Fig. S6L). Immunofluorescence staining further showed that F4/80⁺ renal macrophages exhibited reduced CD86 expression and increased CD206 expression following MSCs treatment, consistent with a shift from a pro-inflammatory toward a protective phenotype (Fig. S6M-N). Together, these findings provide convergent evidence that MSCs treatment is associated with macrophage polarization toward a tissue-reparative state within the kidney.

To evaluate translational relevance, we conducted a prospective pilot study in 20 SLE patients with hyperlipidemia receiving MSCs transplantation. Serial monitoring over 6 months revealed significant improvements in lipid profiles: TG demonstrated progressive decline, TC showed rapid reduction by 3 months (p = 0.0012) that plateaued thereafter (p = 0.094), and LDL-C exhibited the same trend as TC. HDL-C levels remained stable throughout the observation period (p = 0.930) (Fig. 5K; Fig. S6I). Serum creatinine and eGFR were also improved following treatment (Fig. 5L), concomitant with a reduction in SLEDAI scores relative to baseline (Fig. S6J). Importantly, serum levels of sh2a were reduced following MSCs therapy (Fig. 5M), mirroring the effects observed in the murine model. Collectively, these data identify MSCs as a potent regulator of lipid homeostasis and immune-mediated injury in SLE via inhibition of ASGR1. MSCs-mediated downregulation of hepatic ASGR1 reprograms lipid metabolism, mitigates autoimmune pathology, and protects against lupus nephritis, both in lupus-prone mice and in SLE patients. These findings position ASGR1 as a therapeutically actionable target and MSCs as a promising intervention for SLE-associated metabolic complications.

Discussion

Dysregulated cholesterol metabolism represents a fundamental pathophysiological mechanism underlying the lipid disorders frequently observed in SLE, which are closely linked to disease activity and progression [6]. Recent insights suggest that lipid disturbances in SLE are not merely comorbidities but integral to disease pathophysiology. Here, we identified ASGR1—a hepatocyte-specific endocytic receptor traditionally implicated in glycoprotein clearance—as a critical mediator linking metabolic dysfunction and immune activation in SLE. Our clinical and mechanistic investigations reveal that ASGR1 upregulation drives hepatic lipid accumulation and systemic inflammation, while its suppression via MSCs restores lipid homeostasis and ameliorates multi-organ damage.

SLE is a prototypic systemic autoimmune disorder characterized by aberrant lymphocyte activation, immune complex deposition, and multi-organ involvement [22]. Among its diverse clinical manifestations, dyslipidemia has garnered increasing attention for its high prevalence and strong association with cardiovascular comorbidities [17, 23]. In our clinical cohort, almost half of SLE patients exhibited hyperlipidemia, with serum lipid parameters showing a significant positive correlation with SLEDAI, reinforcing the clinical relevance of lipid imbalance in SLE pathogenesis (Data not shown). To investigate the mechanistic underpinning of this metabolic disturbance, we utilized MRL/lpr lupus-prone mice subjected to a HFD, effectively modeling lupus pathology in the context of metabolic stress. HFD-fed lupus mice displayed exacerbated skin lesions, lymphadenopathy, increased autoantibody production, and aggravated hepatic and renal injury. At the molecular level, HFD induced hepatic steatosis via upregulation of lipogenic enzymes and suppression of fatty acid oxidation, leading to lipid accumulation, mitochondrial dysfunction, oxidative stress, and systemic inflammation [24, 25]. Furthermore, previous studies have established that lipid-induced renal injury propagates systemic effects, contributing to damage in multiple organs via common pathological pathways, including ectopic lipid accumulation, mitochondrial cristae disruption, and impaired fatty acid oxidation [26–28]. These findings highlight dysregulated lipid metabolism as a potential contributor to multi-organ injury in SLE and underscore the immunometabolic axis as a critical driver of disease progression.

ASGR1, a C-type lectin receptor highly enriched in hepatocytes, facilitates clathrin-mediated endocytosis and lysosomal degradation of desialylated glycoproteins [29, 30]. Emerging evidence has extended its functional repertoire to include regulation of hepatic cholesterol uptake, intracellular trafficking, and excretion [11, 31]. In this study, ASGR1 expression was significantly upregulated in the livers of HFD-fed lupus mice. Clinically, circulating soluble ASGR1 (sh2a) was elevated in SLE patients and positively correlated with TG, TC and LDL-C levels, and negatively with HDL-C. These data position ASGR1 as a key metabolic node integrating lipid homeostasis and systemic immune activation. Importantly, hepatic knockdown of Asgr1 in HFD-fed lupus mice markedly alleviated lipid accumulation, reduced hepatic inflammation, and mitigated renal injury. Mechanistically, ASGR1 suppression promoted cholesterol efflux through upregulation of Abca1 and Abcg1, inhibited de novo lipogenesis via reduced nuclear localization of SREBP1/2 and downregulation of Fasn and Hmgcr, and enhanced bile acid synthesis through upregulation of Cyp7a1. These changes restored hepatic cholesterol homeostasis and mitigated downstream inflammation. Recent studies have implicated ASGR1 in immune regulation via modulation of lipid raft architecture and downstream signaling pathways, further supporting its role as an immune–metabolic integrator [32]. Genetic evidence from human populations reinforces this concept; loss-of-function variants in Asgr1 are associated with reduced plasma cholesterol and lower cardiovascular risk, underscoring its therapeutic potential [10]. Genetic ablation of Asgr1 activates liver X receptor α/β (LXRα/β) signaling, thereby promoting cholesterol efflux and ameliorating lipid metabolic disturbances in models of atherosclerosis [33, 34].

Given the lack of clinically approved ASGR1-targeting agents, we explored MSCs as a viable alternative for modulating this pathway. While MSCs are known for their immunomodulatory properties and have shown clinical promise in refractory SLE [35, 36], our study reveals that they also exert potent metabolic effects [37]. In lupus mice, MSCs reduced serum autoantibodies, restored renal function, ameliorated skin lesions, and—critically—corrected dyslipidemia. Consistent with these findings, serial lipid profiling in SLE patients treated with MSCs showed significant reductions in TG, TC, and LDL-C, accompanied by decreased circulating sh2a levels, suggesting a systemic suppression of ASGR1 activity. Mechanistically, we demonstrated that MSCs suppressed ASGR1 expression in both murine hepatocytes and human Huh7 cells. Overexpression of Asgr1 abrogated the metabolic benefits conferred by MSCs, confirming ASGR1 as a key downstream target. Further investigations revealed that MSCs inhibit the ASGR1–mTOR axis, activate AMPK, and induce LXRα signaling, thereby orchestrating transcriptional and translational control of lipid metabolism. This regulatory cascade resulted in downregulation of SREBP1 activity, suppression of lipogenic gene expression, and restoration of cholesterol efflux via the LXRα–ABCG5/8 axis [38–40]. Notably, ASGR1-mediated endocytosis of glycoproteins likely contributes to lysosomal amino acid release, a known activator of mTORC1 [11, 41–43]. Inhibition of ASGR1 by MSCs attenuated mTOR signaling and permitted AMPK-mediated restoration of lipid homeostasis, offering a selective means to target mTOR without invoking the adverse effects associated with systemic AMPK activation. Together, these results position MSCs as dual-action therapeutics capable of reprogramming both immune and metabolic dysfunction in SLE. Through targeted suppression of ASGR1 and modulation of the mTOR–AMPK–LXRα axis, MSCs restore lipid balance and mitigate organ injury, expanding their therapeutic utility beyond immunosuppression into the realm of metabolic intervention.

Importantly, the mechanisms linking hepatic lipid remodeling to renal protection remain incompletely understood. Emerging evidence from the field of immunometabolism suggests that lipid metabolism serves as a central regulator of macrophage activation and polarization [44, 45]. Modulation of lipid-associated metabolic pathways, including PPAR signaling and lipid transport networks, has been shown to suppress pro-inflammatory macrophage responses and ameliorate lupus nephritis in murine SLE models [44, 46, 47]. In addition, specialized pro-resolving lipid mediators, such as resolvins and related bioactive lipid species, can directly induce metabolic reprogramming and promote anti-inflammatory, tissue-reparative M2-like macrophage phenotypes [48, 49]. Based on our findings, MSCs-mediated suppression of hepatic ASGR1 may remodel systemic lipid metabolism and influence renal macrophage polarization. Consistent with this possibility, MSC-treated lupus mice showed reduced expression of CD86 and increased expression of CD206 in the kidney. Although the underlying mechanism requires further investigation, these findings suggest a potential link between hepatic lipid regulation and renal protection.

The mechanisms through which MSCs regulate hepatic ASGR1 expression also warrant further consideration. Accumulating evidence suggests that the therapeutic effects of MSCs are mediated predominantly through paracrine signaling rather than long-term engraftment or direct cell replacement [50]. MSC-derived secretomes, including soluble trophic and immunomodulatory factors as well as extracellular vesicles (EVs), have been shown to regulate hepatocyte function, hepatic inflammation, and tissue repair [51–55]. Moreover, MSC-derived EVs can transfer proteins, mRNAs, and microRNAs to recipient hepatocytes, thereby modulating intracellular signaling pathways and transcriptional programs [56, 57]. Consistent with these observations, our in vitro experiments demonstrated that MSCs significantly suppressed ASGR1 expression in hepatocytes under Transwell coculture conditions, in which direct cell-cell contact was prevented while diffusible mediators were allowed to freely exchange between the two cell populations. These findings indicate that MSC-mediated downregulation of ASGR1 can occur through indirect intercellular communication and support a paracrine mode of action.

Building on our findings that MSCs exert dual immunoregulatory and metabolic effects through suppression of hepatic ASGR1, several limitations warrant consideration. First, the upstream regulators and post-transcriptional modifiers of ASGR1 under autoimmune and metabolic stress remain incompletely characterized, and broader systemic effects of MSCs on other metabolically active organs also require further investigation. Second, although our findings support that MSCs regulate hepatic ASGR1 through indirect paracrine communication, the specific mediators remain undefined, as conditioned medium and extracellular vesicle-based mechanisms were not experimentally dissected. Third, the clinical component of this study was a prospective observational pilot study with a limited sample size; therefore, the findings should be interpreted as exploratory and hypothesis-generating, and validation in larger randomized controlled studies is required to address causal inference and inter-individual variability.

Conclusion

We identify ASGR1 as a previously underappreciated hepatic driver of cholesterol metabolism and immune activation in SLE. Our study demonstrates that MSCs mitigate SLE-associated dyslipidemia and organ injury through suppression of ASGR1 and reprogramming of the mTOR–AMPK–LXRα signaling axis. These findings not only highlight ASGR1 as a compelling target for precision metabolic intervention but also broaden the therapeutic scope of MSCs to include metabolic modulation. Collectively, our work offers a mechanistic framework for the development of targeted therapies aimed at restoring immune and metabolic homeostasis in autoimmune diseases complicated by lipid dysregulation.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AAV

Adeno-associated virus

ALT

Alanine aminotransferase

ASGR1

Asialoglycoprotein receptor 1

AST

Aspartate aminotransferase

BUN

Blood urea nitrogen

EVs

Extracellular vesicles

HFD

High-fat diet

HDL-C

High-density lipoprotein cholesterol

LDL-C

Low-density lipoprotein cholesterol

MSCs

Mesenchymal stromal cells

NAFLD

Non-alcoholic fatty liver disease

NCD

Normal chow diet

SLE

Systemic lupus erythematosus

TC

Total cholesterol

TG

Triglycerides

Author contributions

YJW, LZ and SMG proposed conception and designed the study. YJW, LZ, SMG, YRS and YZ performed experiments, analyzed and interpreted data. YJW, LZ, XJH and XJT recruited and collected samples and clinical data. GHY, WWC and LYS supervised the project and provided critical reagents and analytical tools. YJW drafted the manuscript, LZ and SMG wrote part of the methods and results. WWC and LYS critically revised the manuscript, and approved the final version. The order of the co‑first authors was determined based on the extent of contribution and time devoted to the study. YJW, LZ and SMG contributed equally to this work. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the Key Program of the National Natural Science Foundation of China (82330055), the Joint Fund of the National Natural Science Foundation of China (U24A20380) and Jiangsu Innovative and Entrepreneurial Talent Program (JSSCBS20211493).

Data availability

All data supporting the findings of this study are included in this published article and Supplementary Materials. The raw data are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study protocol of patients was reviewed and approved by the Ethics Committee of Nanjing Drum Tower Hospital (Approval No. 2024-1034-01), and written informed consent was obtained from all participants. All experimental protocols of mice were reviewed and approved by the Experimental Animal Ethics Committee of Nanjing Drum Tower Hospital (Approval No.2024AE01101). Title of the approved project for human and animal studies: research on the mechanism of MSCs in the treatment of lupus nephritis, human umbilical cord MSCs and exosomes for the treatment of lupus brain injury and abnormal glucose and lipid metabolism. All procedures pertaining to animal experimentation in this study were conducted in strict compliance with the updated United Kingdom Animals (Scientific Procedures) Act 1986 and European Union Directive 2010/63/EU.

Consent for publication

All authors consent for publication.

Competing interests

The authors declare no competing interests.