Saussurea involucrata oral liquid alleviates joint inflammation in CIA rats by modulating macrophage polarization and glucose metabolism

Abstract

Objective

To evaluate the therapeutic effects of Saussurea involucrata Oral Liquid (SIOL) in a collagen-induced arthritis (CIA) rat model.

Methods

This study was conducted from April 2023 to June 2024. Sixty male SPF SD rats were randomly divided into six groups: control, model, tripterygium glycosides tablet (TGT) group, and low, medium, and high doses of SIOL. CIA was induced by subcutaneous injection of bovine type II collagen (CII) and incomplete Freund’s adjuvant (IFA). Treatment groups received corresponding doses of SIOL or TGT. Therapeutic efficacy was assessed through body weight, arthritis index (AI), ankle diameter, and toe volume. Joint tissue pathology, serum and synovial inflammatory factors, macrophage polarization, and plasma metabolites were analyzed using H&E staining, ELISA, flow cytometry, Western blotting, and LC–MS.

Results

SIOL significantly reduced AI, ankle diameter, toe volume, and pathological scores compared to the model group. SIOL also decreased serum and synovial proinflammatory factors TNF-α, IL-6, and IL-1β while increasing anti-inflammatory IL-10 and IL-4. Medium and high doses of SIOL notably lowered M1 macrophage markers CD86 and iNOS, while elevating M2 markers CD163 and Arg-1. No significant differences were observed between the medium/high SIOL and TGT groups regarding therapeutic effects. Additionally, SIOL (high dose) markedly reduced glucose metabolism-related plasma metabolites, particularly key metabolites in the TCA cycle. A significant correlation was found between macrophage polarization, inflammatory markers, and metabolite levels.

Conclusion

SIOL alleviates joint inflammation in CIA rats, which is associated with modulation of macrophage polarization and alterations in glucose metabolism.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12865-026-00811-1.

Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune disease marked by chronic synovial inflammation, joint damage, and functional impairment, leading to severe disability [1]. Despite significant advances in therapeutic strategies, including non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), and biological agents, a considerable proportion of patients show inadequate responses or experience severe adverse effects, such as infections and liver toxicity [2]. Therefore, exploring novel therapeutic agents with high efficacy and safety profiles remains a critical need in RA management.

The pathogenesis of RA is multifactorial, including T cells, B cells, macrophages, and fibroblast-like synoviocytes (FLS) [3]. Among them, macrophages have garnered significant attention for their pivotal role in RA progression [4]. Macrophages are highly plastic, polarizing into pro-inflammatory M1 or anti-inflammatory M2 states depending on microenvironmental cues, influencing disease trajectory. M1 macrophages exacerbate inflammation by secreting tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), while M2 macrophages aid in inflammation resolution and tissue repair via interleukin-4 (IL-4) and interleukin-10 (IL-10) [5]. Consequently, rectifying the imbalance of macrophage polarization—specifically by suppressing M1 and promoting M2 polarization—has emerged as a promising therapeutic strategy for RA.

Recent advances in the field of immunometabolism have highlighted that macrophage polarization is intimately linked to metabolic reprogramming. Distinct metabolic pathways drive the functional phenotype of macrophages [6]. Similar to the "Warburg effect" observed in tumor cells, M1 macrophages rely primarily on enhanced aerobic glycolysis to rapidly generate ATP and biosynthetic precursors required for inflammatory responses, even under oxygen-sufficient conditions [7], whereas M2 macrophages depend on the tricarboxylic acid (TCA) cycle [8]. This metabolic flexibility suggests that targeting glucose metabolism could effectively modulate macrophage plasticity and control inflammation.

Saussurea involucrata (SI), a rare herb native to the western Tianshan Mountains in Xinjiang, China, has long been used to alleviate joint inflammation [9]. Modern pharmacological studies have indicated that SI possesses potent anti-inflammatory, antioxidant, and immunomodulatory properties. Our previous studies demonstrated that Saussurea involucrata oral liquid (SIOL) mitigates joint inflammation by modulating Th1/Th2-related cytokines and altering gut microbiota in collagen-induced arthritis (CIA) rats [10]. Furthermore, non-targeted metabolomics analysis in our preliminary work revealed that SIOL significantly impacts serum metabolites related to glucose metabolism [11], hinting at a potential metabolic regulatory mechanism. However, whether SIOL influences macrophage polarization through glucose metabolism to modulate inflammatory factors and alleviate RA remains unclear. Therefore, the specific objectives of this study were: 1) To evaluate the therapeutic efficacy of SIOL in a CIA rat model; 2) To determine the impact of SIOL on macrophage polarization and inflammatory cytokine levels; 3) To analyze plasma metabolic profiles, focusing on glucose metabolism; and 4) To explore the correlations between metabolic changes, macrophage polarization, and disease severity.

Materials and methods

Reagents

Immunization Grade Bovine Type II Collagen (CII) and Incomplete Freund's adjuvant (IFA) were purchased from Chondrex (Washington, USA).

ELISA kits for rat IL-1β, IL-6, IL-10, TNF-α, and IL-4 were obtained from Hangzhou Lianke Biotechnology Co., Ltd. (Hangzhou, China).

Primary antibodies were sourced as follows: F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); CD86 (Biolegend, San Diego, CA, USA); CD163 (Bioss, Beijing, China); β-Actin (Cell Signaling Technology, Danvers, MA, USA); iNOS and IL-4 (Proteintech, Wuhan, China); Arg-1 (Invitrogen, Carlsbad, CA, USA); and TNF-α, IL-1β, IL-6, and IL-10 (Abcam, Cambridge, UK).

General laboratory reagents included: 4% paraformaldehyde (Baisha, China); PBS buffer, electrophoresis buffer, and transfer buffer from Servicebio (Wuhan, China); TBS buffer from Solarbio (Beijing, China); Tween 20 (Merck, Darmstadt, Germany); PVDF membranes (Millipore, Billerica, MA, USA); Pre-stained Marker (10–180 KDa) (Thermo Fisher Scientific, Waltham, MA, USA); and non-fat milk (BD, Franklin Lakes, NJ, USA). Serum-free cell freezing medium was purchased from Procell (Wuhan, China).

Chemicals for metabolic analysis, including sodium hydroxide, methanol (−40 °C pre-cooled), acetonitrile, and acetic acid, were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Medicine

Saussurea involucrata Oral Liquid (Xinjiang Tianshan Lotus Pharmaceutical Co., Ltd., Lot# 20,220,301); Tripterygium glycosides tablets (TGT) (Zhejiang Deende Pharmaceutical Co., Ltd., Lot# 20,200,901).

Experimental animals

All animal experiments were conducted between April 2023 and June 2024 at the Animal Experiment Center of Xinjiang Medical University. Sixty specific pathogen-free (SPF) male Sprague–Dawley (SD) rats (6–7 weeks old, 200 ± 20 g) were purchased from the Animal Experiment Center of Xinjiang Medical University. The rats were housed under SPF conditions (temperature 22–24 °C, humidity 50–60%, 12-h light/dark cycle) with free access to food and water.

Animal ethics approval

All animal experiments were approved by the Laboratory Animal Use Committee of Xinjiang Medical University (Approval No. IACUC-JT-20230321–98) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, USA) and relevant institutional guidelines and regulations. This study was conducted and reported in accordance with the ARRIVE guidelines 2.0.

Preparation of RA model rats

After seven days of adaptive feeding, rats were immunized with an emulsion of bovine type II collagen (CII) and incomplete Freund's adjuvant (IFA) (CII/IFA, 1:1 ratio). 0.2 mL was injected subcutaneously at the base of the tail, 1.5 cm from the tip, for the first immunization. After seven days, a second subcutaneous injection of 0.1 mL CII/IFA emulsion was administered to induce the CIA model [12]. To ensure objectivity, the assessment was conducted in a blinded manner by two independent observers who were blinded to the experimental allocation. The severity of arthritis in each paw was quantified on a scale of 0–4 based on the extent of erythema and edema, as detailed in Table 1. The Arthritis Index (AI) scoring criteria were strictly aligned with established protocols described in previous literature [13]. Model success was indicated by swelling in at least one foot (including the ankle), with an AI score reaching 4. The model success rate was 87%.

Table 1.

Criteria for arthritis index (AI) scoring

Score	Clinical Manifestations
0	No evidence of arthritis
1	Erythema and swelling of a single digit
2	Erythema and swelling involving two joints
3	Erythema and swelling involving more than two joints
4	Severe arthritis involving the entire paw and digits

The total Arthritis Index (AI) for each rat was calculated as the sum of the scores for all four paws, resulting in a maximum possible score of 16

Grouping and drug administration

Rats were randomly allocated into six groups (n = 10 each): normal control (non-arthritic), CIA model (untreated arthritic), positive control [Tripterygium Glycosides Tablets (TGT) group, 36 mg/kg/day] [14], and three SIOL treatment groups: SIOL low-dose (SLD), SIOL medium-dose (SMD), and SIOL high-dose (SHD). The normal control and CIA model groups received daily normal saline. The SIOL groups received 0.31, 0.62, and 1.24 g/kg/day for 28 consecutive days. The SIOL dosage was calculated based on human-to-rat body surface area conversion,wherein the equivalent dose is the medium dose of SIOL.

Measurement of rat body weight, arthritis index, and ankle swelling

During the modeling and treatment periods, body weight was monitored, and AI scores were recorded on days 21, 28, 35, 42, and 49 post-immunization. Ankle diameter was measured using a digital vernier caliper (resolution 0.01 mm). Toe volume was measured by water displacement using a plethysmometer (PV-200; Chengdu Taimeng Software Co., Ltd., China), based on Archimedes’ principle. The displacement of water, which equals the volume of the immersed paw, was recorded. Measurements were taken at the widest point of the ankle joint, specifically the transverse diameter at the level of the medial and lateral malleoli (malleolus). To ensure consistency, the rats were restrained in a prone position, and the ankle joint was maintained at a 90° flexed position during the measurement. Each measurement was performed in triplicate, and the mean value was recorded.

Histopathological examination of the ankle joint

The left hind ankle was collected on week 4 of SIOL intervention for istopathological evaluation. Ankle joints were fixed in 4% paraformaldehyde for 24 h, followed by decalcification in 10% EDTA. After dehydration, embedding, sectioning, and HE staining, sections were examined under a 100 × microscope to assess synovial hyperplasia, inflammatory cell infiltration, cartilage destruction, and bone erosion [1]. All histological assessments were conducted by an independent pathologist who was blinded to the experimental groups.

Enzyme-linked immunosorbent assay (ELISA)

At the end of the experiment, a dosage of 50 mg/kg of sodium pentobarbital solution was administered intraperitoneally to induce anesthesia in rats [15]. After confirming that the animals were fully anesthetized and unconscious, 5 mL of blood was collected from the abdominal aorta. Sodium pentobarbital was selected for its rapid onset and reliable depth of anesthesia, which are essential for minimizing pain and distress during the procedure. Following blood collection, the rats were humanely euthanized via exsanguination while under deep anesthesia. The blood was left to stand for 2 h, followed by centrifugation at 4,000 r/min for 20 min, and the serum was stored at −80 °C. Levels of TNF-α, IL-6, IL-1β, IL-4, and IL-10 were measured using ELISA kits in accordance with the manufacturer’s instructions.

Flow cytometry

A sample of 0.5 mL of abdominal aortic blood was collected and incubated with F4/80, CD86, and CD163 antibodies at 25 °C for 20 min in the dark. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended, filtered, and analyzed by flow cytometry. M1 macrophages (F4/80 +/CD86 +) and M2 macrophages (F4/80 +/CD163 +) were quantified using CellQuest software. Spleen tissue samples were homogenized, filtered, and processed into single-cell suspensions. Subsequently, 50 μL of spleen single-cell suspension was stained with F4/80, CD86, and CD163 antibodies at 4 °C in the dark for 30 min and analyzed by flow cytometry to determine the levels of CD86 and CD163 in spleen macrophages.

Western blotting(WB)

Ankle synovial tissues were washed with PBS, lysed with buffer containing PMSF (100:1), and centrifuged (4 °C, 12,000 r/min, 5 min). Supernatants were stored at −80 °C. Protein concentrations were determined using a BCA kit, and samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk for 2 h, incubated with primary antibodies overnight at 4 °C (TNF-α, IL-6, IL-1β, IL-4, IL-10, iNOS, Arg1, and β-actin), followed by secondary antibody incubation at room temperature for 2 h. Proteins were visualized using enhanced chemiluminescence (Thermo Fisher Scientific) and quantified using an Amersham Imager 600 (GE, Fairfield, CT, USA). β-actin served as a loading control.

LC–MS analysis of metabolites in plasma

Plasma samples were collected, frozen in liquid nitrogen, and stored at −80 °C. During analysis, samples were thawed in an ice water bath, vortexed for 30 s, and 100 μL of each sample was processed with 300 μL of −40 °C pre-cooled methanol. The mixture was vortexed for 30 s, kept for 15 min in an ice water bath under sonication, incubated for an hour at −40 °C, and centrifuged for 15 min (12,000 r/min, 4 °C). Supernatants (320 μL) were spin-dried, resuspended with 160 μL of ultrapure water, and analyzed by HPIC-MS/MS analysis.

Correlation analysis of ankle diameter, toe solvent, joint histopathological score, inflammatory cytokines, metabolite levels, and macrophage expression

Correlation between ankle diameter, toe volume, joint histopathology scores, cytokine levels, metabolite levels, and macrophage expression was assessed using Pearson analysis via the Origin. Heatmaps visualized correlations (*P < 0.05; **P < 0.01), with red indicating positive and blue indicating negative correlations.

Statistical analysis

Data were analyzed using SPSS 23.0. Results were expressed as mean ± SD. One-way ANOVA was used for multi-group comparisons, with LSD t-tests for pairwise comparisons. A P-value < 0.05 indicated statistical significance.

Results

SIOL treatment reduced ankle swelling, arthritis scores, ankle diameter, toe volume, and inhibited weight loss in CIA rats

Compared to the control group, rats in the model group exhibited significant redness, swelling, and deformation of the metacarpophalangeal joints. The model group also showed slower weight gain, increased ankle diameter and toe volume, and significantly elevated arthritis scores (P < 0.01). In contrast, rats in the low- (SLD), medium- (SMD), and high-dose (SHD) SIOL groups, as well as the TGT group, experienced an improvement in redness and swelling (Fig. 1A). Additionally, their ankle diameters, toe volumes, and arthritis scores were significantly reduced, and weight loss was inhibited (P < 0.01) (Fig. 1B-E).

Fig. 1.

SIOL treatment reduced ankle swelling, arthritis scores, ankle diameter, toe volume, and inhibited weight loss in CIA rats. A SIOL improved joint morphology. B SIOL reduced ankle diameter. C SIOL decreased AI. D SIOL decreased toe volume. E SIOL prevented weight loss. n=10 per group, data were expressed as mean±standard deviation (±s). ## P < 0.01, #P < 0.05 vs. control; **P < 0.01, *P < 0.05 vs. model

SIOL significantly improved synovial tissue damage in CIA rats

H&E staining revealed distinct histopathological differences among the groups. In the Control group, the articular cartilage surface was smooth, with chondrocytes arranged in an ordered manner and exhibiting normal cellularity (black arrow in Fig. 2A). The synovial structure was clear with distinct layers, and no obvious inflammatory cell infiltration was observed (red arrow in Fig. 2A).

Fig. 2.

Histopathological evaluation of ankle joints in rats (H&E staining). A Control group: The articular cartilage surface is smooth with ordered chondrocytes (black arrow), and the synovial structure is clear without inflammatory infiltration (red arrow). B Model group: Severe cartilage loss and pannus formation caused by fibrous and inflammatory cell proliferation are observed (red arrow), along with necrotic tissue debris in the joint cavity (black arrow). C TGT group: Joint structure is significantly improved. D SIOL low-dose group: Disordered cartilage structure with reduced chondrocytes (black arrow) and synovial hyperplasia forming pannus (red arrow). E SIOL medium-dose group: Relatively intact cartilage with mild pannus. F SIOL high-dose group: Intact synovial structure with an uneven cartilage surface in some areas (black arrow)

In contrast, the Model group exhibited loss of the cartilage layer and necrosis or degeneration of chondrocytes. Extensive proliferation of fibrous tissue and inflammatory cells covered the cartilage surface, indicating pannus formation (red arrow in Fig. 2B). Additionally, a large amount of necrotic tissue debris was visible within the joint cavity (black arrow in Fig. 2B).

Compared with the Model group, the joint structure in the TGT group was significantly improved. The SIOL low-dose group showed disordered cartilage structure in some areas with a reduced number of chondrocytes (black arrow in Fig. 2D), as well as synovial hyperplasia covering the cartilage surface to form pannus (red arrow in Fig. 2D). The SIOL medium-dose group presented a relatively intact articular cartilage structure with only a small amount of pannus visible. In the SIOL high-dose group, the synovial structure was relatively intact without hyperplasia, although the cartilage surface remained uneven in some areas (black arrow in Fig. 2F). (P < 0.01). However, scores decreased significantly in the TGT and SIOL groups (P < 0.01, P < 0.05, Fig. 3C).

Fig. 3.

SIOL modulated cytokine expression in CIA rats. A SIOL decreased pro-inflammatory cytokines in serum. B SIOL increased anti-inflammatory cytokines in serum (n = 10). C SIOL reduced ankle joint pathology scores (n = 6). Data are presented as ±s. ##P < 0.01 and #P < 0.05 vs. control; **P < 0.01 and *P < 0.05 vs. model

SIOL modulated cytokine expression in CIA rats

ELISA results indicated that in the model group, serum concentrations of TNF-α, IL-6, and IL-1β were significantly elevated compared to the control group, while IL-10 and IL-4 levels were significantly reduced (P < 0.01). In the TGT and SIOL-treated groups (low, medium, high doses), TNF-α, IL-6, and IL-1β levels were significantly decreased (Fig. 3A), while IL-10 and IL-4 levels were increased (Fig. 3B). These inflammatory cytokines showed similar changes in the synovium of the ankle joint, as detected by Western blot analysis (Fig. 5A, D-H).

Fig. 5.

SIOL modulated macrophage and cytokine expression in ankle synovium of CIA rats. CIA, collagen-induced arthritis; SIOL, Saussurea involucrata Oral Liquid. The treatment groups are designated as follows: SLD, SIOL low-dose; SMD, SIOL medium-dose; SHD, SIOL high-dose. A-H SIOL decreased iNOS, TNF-α, IL-1β, and IL-6 expression, while increasing Arg-1, IL-10, and IL-4 in the ankle synovium. Quantitative data are presented as ±s (n = 3). ##P < 0.01 and #P < 0.05 vs. control; **P < 0.01 and *P < 0.05 vs. model.Full-length blots/gels are presented in Supplementary materials

SIOL regulated macrophage expression in peripheral blood, spleen, and ankle synovium of CIA rats

Flow cytometry revealed that CD86 expression was upregulated in the peripheral blood of rats in the model group (P < 0.01), while CD163 expression was downregulated (P < 0.05). In the TGT group and the high-dose SIOL group, CD86 expression decreased, while CD163 expression increased (P < 0.05) (Fig. 4A, C, D). Similar changes were observed in spleen macrophages (Fig. 4B, E, F). Western blot results further showed that in the model group, iNOS expression in the synovium was upregulated, while Arg-1 expression was downregulated (P < 0.01). The TGT and SIOL (medium and high dose) groups exhibited decreased iNOS expression and increased Arg-1 expression compared to the model group (Fig. 5A-C).

Fig. 4.

SIOL modulated macrophage expression in peripheral blood and spleen of CIA rats. A, C, D SIOL reduced CD86 and increased CD163 expression in peripheral blood. B, E, F SIOL reduced CD86 and increased CD163 expression in the spleen (n = 4). Data are presented as ±s. ##P < 0.01 and #P < 0.05 vs. control; **P < 0.01 and *P < 0.05 vs. model

SIOL affected glucose metabolism-related plasma metabolites in CIA rats

Metabolomic analysis quantified 56 sugar metabolism-related metabolites in rat plasma, identifying a total of 46 (Fig. 6A). In the model group, the expression of cis-aconitic acid, isocitrate, indoleacetic acid, picolinic acid,and quinolinate (P < 0.05) was downregulated. Compared to the model group, SIOL treatment upregulated the expression of cis-aconitic acid, isocitrate, indoleacetic acid (P < 0.01), fumaric acid, malic acid, α-ketoisovaleric acid, picolinate,homogentisic acid,quinolinate, and methylmalonic acid (P < 0.05) (Fig. 6D). KEGG pathway analysis revealed that these metabolites were significantly enriched in the TCA cycle (Fig. 6B, C).

Fig. 6.

SIOL modulated glucose metabolism-related metabolites in CIA rat plasma (n = 6). A Heat map of identified metabolites. B, C Enriched pathways of differentially expressed metabolites between the model and the high-dose SIOL groups. D Glucose metabolism-related metabolite changes across groups. ##P < 0.01 and #P < 0.05 vs. control; **P < 0.01 and *P < 0.05 vs. model

Correlation between macrophage expression and ankle diameter, toe volume, joint pathology scores, inflammatory cytokines, and metabolite levels

Pearson correlation analysis was performed to investigate the relationships among ankle diameter, toe volume, joint histopathological scores, inflammatory cytokines, metabolite levels, and macrophage expression across the control, model, and high-dose SIOL groups. The expression of CD86 in peripheral blood and spleen, as well as iNOS expression in the synovium, exhibited a positive correlation with the majority of the pathological indices. In contrast, CD163 expression in peripheral blood and spleen, along with Arg-1 expression in the synovium, demonstrated a negative correlation with most pathological indices (Fig. 7A).Moreover, a significant correlation was observed between the levels of glucose metabolism-related metabolites and the polarization state of macrophages. Specifically, the expression of CD86 and iNOS was negatively correlated with metabolite levels, whereas the expression of CD163 and Arg-1 was positively correlated with metabolite levels (Fig. 7B).

Fig. 7.

Correlation analysis between macrophage expression, ankle diameter, toe volume, joint pathology, inflammatory cytokines, and metabolites. A Pearson analysis (heat map) of ankle diameter, toe volume, joint pathology, inflammatory cytokines, and macrophage expression. B Pearson analysis (heat map) of glucose metabolism-related metabolites and macrophage expression. Red indicates a positive correlation, blue indicates a negative correlation. The darker the red/blue, the higher the correlation value. *P < 0.05, **P < 0.01

Discussion

The pathogenesis of RA involves a complex interaction of various immune cells, including T and B lymphocytes, macrophages, and FLS [16, 17]. Among these, macrophages play a central role in the development and progression of RA [18, 19]. These cells are primarily derived from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells first differentiate into monocytes, which then enter the bloodstream and migrate to tissues, where they further differentiate into tissue-specific macrophages [20]. Depending on their environment, macrophages acquire unique phenotypes and functions, enabling them to respond to the specific needs of different tissues. In RA, macrophages in peripheral blood are attracted by inflammatory signals, migrating to the spleen and joint synovium, where they polarize first in the spleen and further into M1 or M2 macrophages in the synovium, contributing to RA pathogenesis. M1 macrophages promote synovial inflammation and joint destruction by secreting TNF-α, IL-1β, and IL-6 [21], while M2 macrophages help suppress inflammation and protect joint tissue integrity by secreting IL-4 and IL-10 [5]. In immunological research, F4/80 is commonly used to confirm macrophage presence, phenvanotype, and activation status [22], with CD86 and iNOS serving as M1 markers, and CD163 and Arg-1 as M2 markers [23].

In this study, model group rats exhibited increased ankle diameter, toe volume, AI, and a significant rise in synovial hyperplasia and inflammatory cell infiltration as seen in HE staining, consistent with CIA model pathology [24, 25], confirming successful model establishment. SIOL treatment at all doses significantly reduced ankle diameter, toe volume, toe volume, and AI. TGT was chosen as a positive control due to its recognized immunomodulatory and anti-inflammatory properties [26], Our results showed that medium and high-dose SIOL achieved therapeutic efficacy comparable to TGT in mitigating joint swelling and histological damage. While the low-dose SIOL group also showed improvement, the efficacy was statistically lower than that of TGT, suggesting a dose-dependent therapeutic effect.

At the cytokine level, the model group showed a marked increase in pro-inflammatory factors (TNF-α, IL-6, IL-1β) and a decrease in anti-inflammatory factors (IL-10, IL-4) in serum and ankle synovium. SIOL treatment significantly reduced pro-inflammatory factor levels and increased anti-inflammatory factor levels. No significant differences in cytokine expression were observed among the SIOL dose groups compared to TGT. In terms of macrophage expression, CD86 expression in peripheral blood and spleen was significantly upregulated, while CD163 expression was downregulated in the model group. Similarly, iNOS expression was upregulated in the ankle synovium, and Arg-1 expression was downregulated, consistent with Cutolo et al.'s findings on macrophage polarization imbalance in the CIA model [27]. These results suggest that macrophages originate in the bone marrow, enter the bloodstream, migrate to the spleen for differentiation, and eventually reach the synovium to participate in RA inflammation. SIOL treatment, particularly at medium and high doses, significantly regulated these markers, reducing CD86 and iNOS expression while increasing CD163 and Arg-1 expression. No significant differences were observed in M1/M2 macrophage expression between the TGT group and the medium/high-dose SIOL groups, aligning with Wan et al.'s findings on TGT's role in macrophage regulation [28].

While SIOL and TGT demonstrated comparable therapeutic efficacy in alleviating joint inflammation and regulating cytokine expression in CIA rats, it is crucial to recognize that this "equivalence" in outcome does not necessarily indicate identical underlying mechanisms. TGT, as a broad-spectrum immunosuppressant, primarily exerts its anti-inflammatory effects through multiple pathways including NF-κB inhibition, T cell suppression, and reduction of multiple pro-inflammatory mediators [29, 30]. Although recent studies suggest that TGT can influence macrophage function [28], this is likely a secondary consequence of its broader immunosuppressive activity rather than a primary targeted mechanism.

In contrast, our data suggest that SIOL may operate through a more specific metabolic-immunological axis. The targeted metabolomics analysis revealed that SIOL significantly upregulated key TCA cycle intermediates (cis-aconitic acid, isocitric acid, fumaric acid, and malic acid), which showed significant correlations with macrophage polarization markers. This metabolic reprogramming appears to be a central mechanism by which SIOL modulates the M1/M2 balance. Furthermore, the traditional use of Saussurea involucrata in regulating "cold-heat" imbalance in Traditional Chinese Medicine may align with its capacity to restore metabolic homeostasis, a concept gaining recognition in modern immunometabolic research [11, 31, 32].

Therefore, we propose that SIOL and TGT represent convergent but mechanistically distinct therapeutic approaches: TGT achieves anti-inflammatory effects through broad immunosuppression that secondarily affects macrophage behavior, whereas SIOL appears to directly target metabolic pathways that govern macrophage polarization, thereby restoring immune balance in a more physiologically oriented manner. This distinction is clinically significant, as metabolic modulation may offer advantages in terms of preserving host defense mechanisms and reducing long-term immunosuppressive complications. The unique value of SIOL lies not merely in matching TGT's efficacy, but in potentially achieving comparable therapeutic outcomes through a fundamentally different and potentially safer mechanistic route. Future comparative mechanistic studies examining the differential effects of SIOL and TGT on glucose metabolism, mitochondrial function, and immune cell subset distribution would further clarify these mechanistic distinctions.

To better understand the biological basis of these findings, it is worth noting that glucose metabolism disturbance is a key factor in RA pathogenesis [33, 34]. Under normal conditions, glucose breaks down into pyruvate, which enters mitochondria, proceeds through the TCA cycle via acetyl-CoA, and produces ATP [35], supporting anti-inflammatory M2 macrophage polarization [36]. In RA's inflammatory environment, macrophages often experience hypoxia, leading them to rely on glycolysis for rapid energy production [37]. This heightened glycolytic state not only fuels the inflammatory response but also promotes M1 polarization, exacerbating synovial inflammation and destruction [3, 38]. TCA cycle intermediates like cis-aconitic acid, isocitrate, α-ketoglutarate, fumarate, and malate are crucial for cellular energy metabolism [39]. Cis-aconitic acid accumulation can inhibit citrate synthase, reduce acetyl-CoA utilization, and impair energy metabolism [40]. The conversion of isocitrate to α-ketoglutarate by isocitrate dehydrogenase is a key regulatory step in the TCA cycle, influencing the cycle's rate and energy output [41, 42]. Fumarate is converted to malate by fumarate hydratase [43], and malate is converted to oxaloacetate by malate dehydrogenase in the final TCA cycle step. Oxaloacetate combines with acetyl-CoA to boost ATP production, supporting cell repair and anti-inflammatory responses [44, 45]. Decreased levels of these metabolites weaken the overall efficiency, hindering M2 macrophage polarization [46], and diminishing anti-inflammatory and tissue repair signaling. Recent studies suggest that targeting these metabolic pathways can alleviate RA inflammation [47].

In this study, SIOL significantly alleviated joint inflammation in CIA rats, decreased inflammatory factor expression, and regulated macrophage activation phenotypes, with the high-dose group showing the most significant therapeutic effect. Consequently, the SIOL high-dose group was selected for HPIC-MS/MS target metabolomics analysis. Results revealed significant downregulation of cis-aconitic acid and isocitric acid in the model group compared to controls. However, high-dose SIOL treatment significantly upregulated cis-aconitic acid, isocitric acid, indoleacetic acid, α-ketoisovaleric acid, fumaric acid, and malic acid. KEGG pathway analysis indicated that these metabolites were primarily enriched in the "TCA cycle" suggesting that SIOL may exert therapeutic effects by modulating glucose metabolism, particularly enhancing the TCA cycle. Pearson correlation analysis demonstrate that the expression of the M1 macrophage marker CD86 in peripheral blood and spleen, is positively correlated with inflammatory indicators in the CIA rats, while the expression of the M2 macrophage marker CD163 is negatively correlated with these indicators. These findings are consistent with existing literature [48], which suggests that M1 macrophages promote inflammatory responses, whereas M2 macrophages aid in the resolution of inflammation and tissue repair. Moreover, the expression patterns of iNOS and Arg-1 in the ankle synovium further support the close association between macrophage polarization states and joint inflammation. The correlations we observed between glucose metabolism-related metabolite levels and macrophage polarization states indicate that these metabolites may play a role in modulating macrophage functions and inflammatory responses. These results are in line with the discoveries in the emerging field of immunometabolism [49], which emphasize the critical role of metabolic reprogramming in the activation and polarization of immune cells.

In line with this, a recent findings on Saussurea involucrata injection indicate that this herb modulates macrophage polarization via glyoxylate and dicarboxylate metabolism, a pathway closely linked to the TCA cycle [32], thereby providing independent support for our findings.

Limitations

This study has several limitations. First, the absence of in vitro experiments limits our understanding of SIOL's direct cellular effects on macrophage polarization. Second, the sample size constrains comprehensive analysis of macrophage subtype interactions and their specific inflammatory roles. Third, we did not assess long-term efficacy or safety—critical for potential clinical translation in chronic diseases like rheumatoid arthritis. Fourth, while H&E staining (Fig. 2) clearly demonstrated that SIOL alleviated cartilage necrosis and pannus formation, we lacked specific Safranin O-Fast Green staining to quantify proteoglycan loss. This was due to the prioritization of limited rat ankle joint tissues for multi-omics analysis (Western blot and Metabolomics) to elucidate the immunometabolic mechanisms. Future studies will include in vitro experiments, larger cohorts, long-term follow-up, and comprehensive histological assessments including specific cartilage staining to validate SIOL's structural protective effects.

Conclusion

In conclusion, our findings suggest that SIOL may reduce inflammatory responses and tissue damage in CIA rats, which is accompanied by the modulation of glucose metabolism-related metabolites, the inhibition of M1 macrophage pro-inflammatory activity, and the promotion of M2 macrophage anti-inflammatory function. Future research should aim to further elucidate the molecular mechanisms by which SIOL regulates glucose metabolism and macrophage polarization through in vitro experiments and broader metabolic analyses. These studies will help to provide a stronger scientific foundation for the potential clinical application of SIOL and may offer new insights into therapeutic strategies for the treatment of rheumatoid arthritis.

Abbreviations

AI

Arthritis index

Arg-1

Arginase-1

ATP

Adenosine triphosphate

BCA

Bicinchoninic acid

CD86

Cluster of differentiation 86

CD163

Cluster of differentiation 163

CIA

Collagen-induced arthritis

CII

Bovine type II collagen

EDTA

Ethylenediaminetetraacetic acid

ELISA

Enzyme-linked immunosorbent assay

F4/80

EGF-like module-containing mucin-like hormone receptor-like 1

FLS

Fibroblast-like synoviocytes

H&E

Hematoxylin and eosin

HPIC-MS/MS

High-performance ion chromatography-tandem mass spectrometry

IFA

Incomplete Freund's adjuvant

IL-1β

Interleukin-1 beta

IL-4

Interleukin-4

IL-6

Interleukin-6

IL-10

Interleukin-10

iNOS

Inducible nitric oxide synthase

KEGG

Kyoto Encyclopedia of Genes and Genomes

LC-MS

Liquid chromatography-mass spectrometry

PBS

Phosphate-buffered saline

PMSF

Phenylmethylsulfonyl fluoride

PVDF

Polyvinylidene fluoride

RA

Rheumatoid arthritis

SD

Sprague-Dawley

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SI

Saussurea involucrata

SPF

Specific pathogen-free

SIOL

Saussurea involucrata oral liquid

SLD

SIOL low dose

SMD

SIOL medium dose

SHD

SIOL high dose

TCA

Tricarboxylic acid

TGT

Tripterygium glycosides tablets

TNF-α

Tumor necrosis factor-alpha

WB

Western blotting

Authors’ contributions

S.M, W.M and Z.Z contributed to the manuscript drafting and statistical analysis, S.C and X.Z contributed to the curation of data and supervision, P.J and L.L contributed to the study design and critical revision of the manuscript.

Funding

This work was supported by National Natural Science Foundation of China (82160841),the Key Program of Natural Science Foundation from Xinjiang Uyghur Autonomous Region, China (2022D01D65), and the Youth Scientific Research Launch Special Fund Project of the First Affiliated Hospital of Xinjiang Medical University(2022YFY-QKMS-02).

Data availability

All data generated or analyzed during this study are included in this article (and in the supplementary information files). The original datasets analyzed are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University, with the ethical approval number: IACUC-JT20230321-98.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.