Introduction

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by chronic pain and joint deformities. In addition to the joints, inflammatory factors continuously activate immune cells and attack other tissues and organs, causing damage to the heart, kidneys, lungs, digestive system, eyes, skin, and the nervous system.^1,2 RA-related pain and morning stiffness lead to impairments in quality of life, such as sleep deprivation.³ The prevalence of rheumatoid arthritis can reach as high as 1% in some areas, making it a global economic and social burden.⁴

The specific pathogenesis of RA is not fully understood; however, risk factors, such as genetics, smoking, obesity, infections, periodontal disease, and the gut microbiota, are believed to contribute to its development.⁵ The diagnosis is made with the help of clinical features and laboratory investigations. RA is characterized by the presence of rheumatoid factor (RF) and other autoantibodies against posttranslationally modified proteins, such as anti-citrullinated protein antibodies (ACPAs), which form immune complexes and lead to the extended activation of immune cells.⁶

In recent years, overactivation of adaptive cells has been recognized as essential during RA progression, and the function of innate immune cells has received increasing attention. Circulating neutrophils in RA patients actively participate in the production of high levels of reactive oxygen species (ROS), cytokines, proteases with delayed apoptosis, and the formation of neutrophil extracellular traps (NETs).^7,8 During NET formation (NETosis), citrullinated histone H3 (CitH3), which is mediated by peptidyl arginine deiminase 4 (PAD4), is exposed to T cells and B cells in autoimmune diseases, leading to the emergence of autoantibodies.⁹ Secreted proteases such as myeloperoxidase (MPO) and neutrophil elastase (NE) exacerbate oxidative stress and matrix degradation in joints. NET formation is induced by autophagy and ROS production.^10,11 The simultaneous expression of CitH3 has been detected, accompanied by increased autophagy during NET formation.¹² Conversely, the lack of autophagy-related 5 (Atg5), a gene encoding a protein essential for autophagosome formation, impedes NET formation.¹³ During NET formation, the phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (AKT) pathway is activated.¹⁴ NADPH oxidase, PAD4, autophagy, and PI3K inhibitors significantly prevent NET formation.^13,15–17

Conventional therapeutic strategies, including disease-modifying antirheumatic drugs (DMARDs), such as methotrexate, and other immunosuppressive agents, such as glucocorticoids, are recommended as the primary treatment for patients with RA.¹⁸ Some recombinant biomolecules that target specific cell surface receptors or extracellular molecules have also provided another treatment option.¹⁹ These targeted synthetic DMARDs or biological DMARDs, such as TNF-α inhibitors and Janus kinase (JAK) inhibitors, showed improved efficacy in patients who failed to achieve ideal outcomes with treatment with methotrexate (MTX) or other conventional DMARDs, but the cost and side effects significantly increased.²⁰ The period in which elevated autoantibodies and RA-associated risk factors are identified is referred to as pre-RA.^21,22 According to recent clinical trials, intervention with T-cell costimulation modulation in the preclinical phase effectively reduces the progression to RA.^23,24 Traditional Chinese medicine (TCM) efficiently alleviates arthralgia in patients with RA and pre-RA. Some patented TCM medicines have been recommended as clinical treatment strategies.^25,26 Decoctions and medicines in TCM may ameliorate RA by inhibiting inflammation, regulating immune balance, and modulating the intestinal flora and metabolism.^27–29

Si Miao San (SMS), a traditional formula documented in the medical literature in ancient China, has been used as the core component for dispelling “damp heat syndrome” in RA. It is composed of Phellodendron amurense (Huang Bai), Atractylodes lancea (Cang Zhu), Achyranthes bidentata (Niu Xi), and Coix lacryma-jobi (Yi Yiren). Different combinations of these herbal drugs are referred to as a series of classic formulas with distinct characteristics for the treatment of RA. Huang Bai and Cang Zhu are called Er Miao San, and Er Miao San with the addition of Niu Xi is called San Miao San. Experiments on rats with complete Freund’s adjuvant induced arthritis (AA) showed that, compared with Er Miao San and San Miao San, Si Miao San had greater efficacy, especially in terms of bone recovery.³⁰ Multiomics combined with network pharmacology analysis has demonstrated its anti-inflammatory, analgesic, and immunomodulatory effects.³¹ In animal experiments, SMS effectively mitigated RA progression by lowering proinflammatory cytokine levels and reducing oxidative stress.³² Moreover, SMS has been shown to ameliorate RA-induced lung fibrosis both theoretically and experimentally.^33,34

However, the therapeutic mechanisms of SMS in RA remain unclear owing to the multicomponent, multitarget, and multipathway nature of Chinese herbal remedies. This study focused on the underlying effects of SMS on neutrophils and the regulatory mechanism through which NET formation is modulated.

Materials and Methods

Animals

The experimental animals used in this study were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. [licence number: SCXK (jing)-2021-0006], including SPF-grade C57BL/6N mice (6–7 weeks old, weighing approximately 20 g) and SD rats (weighing approximately 250 g). The animals were housed in the specific pathogen free (SPF) experimental animal centre of Beijing University of Traditional Chinese Medicine (temperature, 22 ± 2 °C; humidity, 50 ± 5%; natural circadian rhythm) without dietary restriction. Before the start of the experiments, the mice were randomly grouped for one week of adaptive feeding.

Drugs and Chemicals

Herbs in the SMS decoctions were purchased from Beijing Tong Ren Tang Co., Ltd. They were mixed at a ratio of 1:1:1:1 and boiled for 60 min. The decoctions were filtered through gauze and concentrated to a final concentration of 1.5 g/mL. Methotrexate (MTX) was obtained from Shanghai Pharmaceutical Xinyi Pharmaceutical Co. Ltd. Antibodies against AKT (4685), p-AKT (4060), PI3K (4257), p-PI3K (4228), and β-actin (4970S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against α-tubulin (66031-1-Ig), ATG5 (10,181-2-AP), and PAD4 (17,373-1-AP) were purchased from Proteintech Group Inc. (Wuhan, HB, CHN). Antibodies against MPO (ab9535), CitH3 (ab281584), NE (ab68672), Beclin1 (ab207612), Alexa Fluor^® 647 (ab150079), and HRP-conjugated goat anti-rabbit IgG (ab6721) were obtained from Abcam (Cambridge, MA, USA). Antibodies against microtubule-associated protein 1 light chain 3B (LC3B) (A19665) were obtained from ABclonal Biotechnology Co. Ltd. Phorbol 12-myristate 13-acetate (PMA) (P1585) were obtained from Sigma Chemicals (Louis, MO, USA). SC79 (305,834–79-1) and MK2206 (1,032,350–13-2) was purchased from Selleck Chemicals (Houston, USA). Stripping buffer (PS107) was purchased from Epizyme (Shanghai, China).

Mouse AA Model and Drug Treatment

The mice were then randomly divided into six groups: the normal control group (Control), the adjuvant-induced arthritis group (AA), the Si Miao San low-dose administration group (SMS-L), the Si Miao San medium-dose administration group (SMS-M), the Si Miao San high-dose administration group (SMS-H), and the MTX-treated group. Joint inflammation was induced as previously described.³⁵ After anaesthetization with isoflurane, 100 μL of complete Freund’s adjuvant (CFA) was injected into the ankle joint cavity of the left hindfoot of C57BL/6 mice.³⁵ From the 4th to the 31st day after CFA immunization, the mice in the SMS-L, SMS-M and SMS-H groups were administered SMS decoction at concentrations of 6.24, 12.48, and 24.96 g/(kg·d), respectively. The dosage was calculated according to previous publications.^32,33 The mice in the MTX group were orally administered MTX every two days at a concentration of 1.73 mg/kg. On the 4th day after CFA injection, the ankle joint diameters were recorded every three days, and the joints were scored according to a five-grade scoring system.³⁶

SMS Mass Spectrometry Detection

LC‒MS/MS analysis was performed using an Agilent ultrahigh-performance liquid chromatography (HPLC) 1290 UPLC system. A Q Exactive Focus mass spectrometer coupled with Xcalibur software (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to obtain MS and MS/MS data in the IDA acquisition mode.

Histopathology Analysis

Joint paraffin sections were prepared as described previously.³⁵ Haematoxylin and eosin (H&E) staining, toluidine blue staining, safranin O-fast green staining, periodic acid-Schiff staining (PAS) and TUNEL staining were performed according to standard procedures. Pathological changes in the ankle joints of the mice were observed and scored according to specific criteria.³⁷

Immunohistochemistry (IHC) Analysis

After dewaxing and hydration, the tissue sections were incubated with 0.05% Triton X-100 at 37 °C for 30 min for permeabilization. After rinsing three times with PBS, 3% H₂O₂ was used to remove endogenous peroxidase. Antigen retrieval was performed as previously described.³⁸ After blocking, the sections were incubated with the primary antibody against the target protein at an appropriate concentration at 4 °C overnight and then with the secondary antibody at 37 °C for 30 min. The target proteins were finally developed with 3,3’-diaminobenzidine (DAB) and analysed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) by quantifying the IOD of the positive area.

Immunofluorescence Assays (IFAs) of Tissue Sections

An opal 3-plex anti-Rb manual detection kit (Cat#: NEL840001KT, Akoya Biosciences, Co., Ltd, Delaware, USA) was used to simultaneously detect the expression of target proteins. All the experiments were performed in accordance with the guidelines. Finally, the nuclei were counterstained with DAPI. The slides were mounted and imaged using a confocal microscope. The fluorescent signals were analysed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Microcomputed Tomography (Micro-CT) Analysis

Mouse ankles were collected and fixed in paraformaldehyde, and microcomputed tomography (micro-CT) specimens were scanned using a Bruker Micro-CT Skyscan 1276 system (Bruker, Germany). Analyses were performed using the manufacturer’s evaluation software. Reconstruction was performed using NRecon (Brucker, version 2.2.0.6, Bruker, Germany), and 3D images were obtained from 2D images using a method based on the grayscale original image distance transform (CTvox, version 3.3.0.0, Bruker, Germany). 2D and 3D analyses were performed using CT Analyzer software (version 1.20.8.0, Bruker, Germany). ROIs were analysed using CT Analyzer software (version 1.20.8.0, Bruker, Germany). The bone volume (BV), bone volume density (BV/TV), and bone surface area (BS) were calculated using CT Analyzer software (Bruker, Germany) to evaluate the morphology, microarchitecture, and bone quality of representative mice.

Cytometric Bead Arrays of Serum Cytokines

The experimental procedures were performed according to the instructions provided for the cytometric bead array (CBA) kit (Cat#: 560485, BD Biosciences, New Jersey, USA). Standards were prepared according to the manufacturer’s instructions. Th1/Th2/Th17 capture beads (40 μL) were added to each well of the 96-well plate. Standard diluents or samples (50 μL) were added, followed by 40 μL of detection reagent. The 96-well plate was sealed with a membrane and incubated at room temperature in the dark for 2 h. After washing, the beads were transferred to a new 96-well plate and were then subjected to fluorescence-activated cell sorting (FACS) and analysed using FCAP Array software (BD Biosciences, New Jersey, USA).

Preparation of Drug-Containing Serum

SD rats were randomly divided into normal control and SMS administration groups. SMS was orally administered at a dosage of 34.944 g/(kg·d). The control group was given water for 7 consecutive days. After the last gavage, blood was collected from the abdominal aorta, and the serum was collected after centrifugation at 4 °C and 3000 rpm for 20 min. After heating at 57 °C for 30 min, the SMS-containing and control sera were then filtered through a 0.22 μm diameter microporous membrane and stored at - 80 °C for future use.

Recruitment, Isolation and Culture of Mouse Peritoneal Neutrophils

Peritoneal neutrophils were isolated as previously described.³⁹ Briefly, after peritoneal injection of 10% proteose peptone (prepared in saline) twice, the cells were recovered and resuspended in the cell culture medium. Neutrophils were isolated using Percoll (Cat#: P8370, Solarbio, Beijing, China) and cultured under different treatments.

CCK8 Assay

A Cell Counting Kit 8 (CCK8) was purchased from Beijing Aoqing Biotechnology Co., Ltd. (Cat#: AQ308). Primary neutrophils were inoculated into 96-well plates as previously described.³⁵ Different concentrations of SMS-containing serum were added and cultured for 4 h. CCK8 solution (10 μL/well) was added to each well and incubated for 1 h. The absorbance was measured at 450 nm using a microplate reader.

Immunofluorescence Assays in Cell Culture

Neutrophils (1×10⁶) were seeded in a laser confocal culture dish and cultured with or without PMA or SMS-containing sera. This procedure was performed as previously described.³⁵ In brief, after fixation and permeabilization, the cells were blocked and incubated with primary antibodies at 4 °C overnight. The next day, after rinsing with PBS, secondary antibodies were added, and the cells were incubated with DAPI. A laser confocal microscope was used to observe the staining, and photographs were taken. The fluorescence intensity of the target protein and the area of the nucleus were analysed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Detection of Reactive Oxygen Species From Periarticular Tissue and Cell Culture

The joint tissues were dissociated using a skeletal muscle dissociation kit (Cat#:130–098-305, Miltenyi Biotec, Cologne, Germany). The supernatant was discarded by centrifugation at 800 × g for 10 min. The pellets were subsequently resuspended in 1 mL of PBS and counted. The DCFH-DA probe from an ROS assay kit (Cat#: E004-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was then added and incubated with the cells at 37 °C for 40 min in the dark. FITC fluorescence was detected by fluorescence-activated cell sorting (FACS), as suggested by the guidelines.

For the production of ROS from neutrophils in vitro, 1×10⁶ neutrophils were seeded in a laser confocal dish and incubated with diluted DCFH-DA after different treatments. The cells were then washed three times with serum-free cell culture medium to remove DCFH-DA from the supernatant. The fluorescence intensity was observed directly using a laser confocal microscope.

Total Protein Extraction From Mouse Tissues and Neutrophils

Radioimmunoprecipitation assay lysis buffer (RIPA) was purchased from Solarbio (Beijing, China). Periarticular tissues were collected and lysed using a tissue homogenizer after the addition of RIPA complete buffer (with 1 mM PMSF). The tissue samples were subsequently incubated on ice for 15–30 min to facilitate lysis and the supernatants were collected after centrifugation at 12,000 rpm for 10 min at 4 °C. After the addition of loading buffer, the protein samples were heated at 95 °C for 5 min and stored for future use.

Isolated neutrophils were cultured in six-well plates at 5×10⁶ cells per well and labelled as the NC, NC + PMA, SMS, SMS+PMA, and SMS+PMA+SC79 groups. After four-hour treatment, the supernatant and the neutrophils at the bottom of the wells were collected. After centrifugation, RIPA buffer (with 1 mM PMSF) was added to the pellets. The lysates were centrifuged at 12,000 × g for 10 min, and the supernatant was collected. After the addition of loading buffer, the protein samples were heated at 95 °C for 5 min.

Western Blot Analysis

Protein samples were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 3% bovine serum albumin (BSA) in TBST buffer (Tris-buffered saline with 0.05% Tween-20) for 2 h, the membranes were incubated with primary antibodies overnight at 4 °C. The membranes were then incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. Proteins were detected with ECL reagent (Cat#: 36208ES, Yeasen Biotechnology Co., Ltd., Shanghai, China) and visualized. The grey values of the target bands were subsequently analysed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the results were recorded. After WB detection, the membrane was stripped and re-probed with another antibody.

Transmission Electron Microscopy for Detection of Neutrophils

After the different treatments were performed in vitro, the cell culture medium was replaced with 2.5% glutaraldehyde, and the cells were fixed for 24 h. The glutaraldehyde solution was then removed, and the cells were washed with PBS for 6 h. Subsequently, the cells were postfixed with 1% osmium tetroxide. Dehydration was performed using a graded ethanol series, and the cells were stained with uranyl acetate dissolved in 70% ethanol during dehydration. The dehydrated samples were then infiltrated with a 1:1 mixture of propylene oxide and epoxy resin and embedded in pure epoxy resin. The embedded samples were polymerized in an oven at 45 °C for 12 h and then at 72 °C for 24 h. After curing, the resin blocks were trimmed, and ultrathin sections (approximately 70 nm thick) were prepared. Sections were collected on copper grids and stained with lead for imaging. The final images were captured using a transmission electron microscope (JEOL Ltd., Tokyo, JEM-1400).

Molecular Docking

The components of SMS were acquired from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.php). The active compounds were filtered based on oral bioavailability (OB) ≥ 30% and a drug-likeness (DL) ≥ 0.18. The corresponding targeted genes of the active ingredients were also retrieved and compared with the names in the UniProt database (https://www.uniprot.org/) to ensure unique gene nomenclature. Cytoscape 3.9.1 software (Cytoscape Consortium) was used to create the network diagram of herbs, active ingredients and targeted genes, from which the “Degree” values were calculated based on the number of edges directly connecting a node to other nodes. The 3D structures of the small molecule compounds (Degree≥30) were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The molecular structures of the agents and the human AKT protein (Protein Data Bank ID: 1GZO) were subsequently constructed and preprocessed using Autodock Tools 2.4.6 (The Scripps Research Institute, California, USA). Molecular docking was performed, and the docking results were visualized using PyMOL 3.1 (Schrödinger, LLC, New York, USA).

Statistical Analysis

Statistical analyses were performed using SPSS Statistics 20.0 (IBM, Armonk, New York) and GraphPad Prism 7 software (Insightful Science, San Diego, California). The data were presented as the means ± standard deviations (SDs). For normally distributed data, one-way analysis of variance (ANOVA) was used for comparisons among multiple groups, and least significant difference (LSD) tests were used for pairwise comparisons. Nonnormally distributed data were analysed using nonparametric tests. Statistical significance was defined as P < 0.05.

Results

Improvement of Arthritis in RA Mice by Si Miao San

The chemical fingerprinting results of the SMS decoctions (Supplementary Figure 1) identified specific compounds listed in Supplementary Tables 1 and 2. The active ingredients include quercetin, wogonin, berberine, palmatine and kaempferol, which were also identified as the principal active components of SMS in other studies.^40–42

The AA model was established on Day 0. Treatment with high, medium, and low doses of SMS and MTX was initiated on Day 4. Joint diameters and scores were recorded every 3 days (Figure 1A). Statistical analysis revealed that the joint scores and diameters in the high-dose SMS and MTX groups were significantly lower than those in the AA group. No significant differences were observed between the low- and medium-dose SMS groups and the AA group (Figure 1B and C). Compared with the mice in the control group, the mice in the model group presented severe symptoms, such as joint swelling, which was significantly alleviated by high doses of SMS and MTX (Figure 1D). However, no significant difference in body weight was observed between the groups (Figure 1E). The splenic index was calculated using the following formula: Splenic index = spleen weight (g)/body weight (g). SMS-H and MTX significantly reduced the spleen indices of the AA mice (Figure 1F). H&E staining revealed typical pathological states in the ankle joints of the AA group, including synovial cell hyperplasia and inflammatory cell exudation within the joint cavities. The degree of inflammatory cell infiltration was visualized using H&E staining, and the joints of arthritic mice were subsequently scored on an inflammation scale ranging from 0–4. In contrast, these pathological states were mitigated in the SMS-H and MTX groups, as reflected by decreased histological scores. However, no significant changes in the local ankle joint pathological state were observed between the SMS-L and SMS-M groups and the AA group (P > 0.05) (Figure 1G and H).

Figure 1 SMS attenuated the severity of arthritis in AA mice. (A) Schematic representation of the animal experiment. (B) Joint swelling was assessed by measuring the ankle joint diameter (n = 8). (C) Arthritis severity was graded on a scale of 0–4. (D) Representative images of arthritic paws from each group at the end of the experiments. (E) Changes in the body weights of the mice (n = 8). (F) Mouse spleen index (n = 8). (G) H&E images. Scale bars, 100 and 200 μm. (H) Histological scores (n = 5). The values are presented as the means ± SDs. *P<0.05, **P<0.01, nsP > 0.05.

PAS staining (Supplementary Figure 2) and TUNEL staining (Supplementary Figure 3) of liver and kidney tissues revealed no damage to the liver or kidneys in any of the groups, indicating the safety of SMS when administered at different dosages.

Subsequently, relevant pathological tests were performed on the ankle joints in each group. Safranin O-fast green and toluidine blue staining demonstrated that cartilage damage in the ankle joints was effectively protected in the high-dose SMS and MTX groups compared with the AA group (Figure 2A-C). Furthermore, the bone mineral densities of the mice in the control, AA, SMS-H, and MTX groups were analysed using micro-CT, which confirmed that the bone surface erosion induced by joint inflammation was alleviated by SMS-H and MTX treatment (Figure 2D and E). The changes in the serum levels of inflammatory cytokines indicated that the TNF-α and IL-6 levels were significantly elevated in the AA group compared with those in the control group, and were markedly reduced by SMS-H and MTX treatment (Figure 2F and G).

Figure 2 Bone destruction in mice with RA was ameliorated by SMS. Representative joints with (A) safranin O-fast green staining and (B) toluidine blue staining. Scale bars, 100 and 200 μm. (C) Histological scores (n = 5). (D) Representative micro-CT images of the mice. The red arrows indicate bone damage. (E) BV/TV of each group (n = 5). (F) IL-6 concentration in the serum of each group (n = 5). (G) TNF-α concentration in the serum of each group (n = 5). The values are presented as means ± SDs. *P<0.05, **P<0.01, nsP > 0.05.

Local NET Formation in Joints Was Inhibited by SMS

Increasing evidence indicates that neutrophils play a significant role in regulating both innate and adaptive immunity⁴³ and that inhibiting NET production can ameliorate RA symptoms.^44–46 SMS has been reported to decrease MPO activity in the joint tissues of mice with gouty arthritis.⁴⁷ To explore whether SMS alleviated RA by regulating NET formation, the activity of MPO, an essential enzyme of neutrophils, was first detected (Figure 3A and B). PAD4 was also detected using IHC (Figure 3C and D). Compared with the control group, the AA group presented significantly elevated levels of MPO and PAD4, which were reduced by high-dose SMS. Additionally, immunofluorescence analysis of NE and CitH3 was performed to confirm NET formation (Figure 3E–G). High doses of SMS and MTX significantly reduced CitH3 and NE protein expression, suggesting that SMS inhibits NET production in mice with RA.

Figure 3 SMS inhibited local NET formation in joints. (A) IHC was used to assess MPO protein expression in mouse ankle joints. Scale bars, 100 and 200 μm. (B) Quantitative analysis of (A) (n = 3). (C) IHC was used to assess PAD4 protein expression in mouse ankle joints. Scale bars, 100 and 200 μm. (D) Quantitative analysis of (C) (n = 3). (E) Immunofluorescence analysis was used to detect the expression levels of CitH3 and NE proteins in mouse joints. (F) Quantitative analysis of NE (n = 3). (G) Quantitative analysis of CitH3 (n = 3). The values are presented as the means ± SDs. **P<0.01.

The PI3K/AKT Signalling Pathway, ROS Production, and Autophagy Generation Were Inhibited by SMS in vivo

Neutrophil activation leads to increased ROS production, which in turn induces NET formation through mechanisms such as DNA damage and the NADPH oxidase complex.^48,49 ROS not only act as antimicrobial effectors but also serve as signalling molecules that regulate nuclear factor kappa-B (NF-kB) transcriptional activity and promote NET production. Furthermore, activation of the PI3K/AKT signalling pathway enhances ROS production, contributing to increased NET generation and thus playing a crucial role in neutrophil function.^50,51

As shown in Figure 4A and B, SMS-H and MTX significantly reduced the ROS levels in the cells of the joints. The elevated ROS levels in the peri-ankle tissues of the mice in the AA group were consistent with the increased NET formation in the AA group. The levels of phosphorylated AKT and PI3K in the ankle joints were examined by WB analysis (Figure 4C-F). SMS treatment significantly reduced the phosphorylation levels of AKT and PI3K, indicating the inhibition of the PI3K/AKT signalling pathway by SMS. In addition, ROS are known to influence autophagy processes.⁵² Enhanced neutrophil autophagy has been observed in patients,^53,54 and the inhibition of autophagy has been reported to reduce NET production.^55,56 To assess the level of autophagy in RA, the expression of LC3B in mouse ankles was analysed using IHC. As shown in Figure 4G and H, high-dose SMS treatment greatly reduced LC3B expression in the ankle joints of AA mice. In summary, SMS appears to alleviate RA by suppressing the PI3K/AKT signalling pathway, reducing ROS production, attenuating autophagy in peri-ankle tissues, and reducing NET formation.

Figure 4 SMS inhibited local NET formation in joints through PI3K/AKT/ROS/autophagy axis. (A) FACS analysis of ROS production. (B) Quantitative analysis of (A). (C) WB analysis of the expression of p-AKT and AKT in periarticular muscle tissues. (D) WB analysis of the expression of p-PI3K and PI3K in periarticular muscle tissues. (E) Quantitative analysis of p-AKT in (C) (n = 3). (F) Quantitative analysis of p-PI3K in (D) (n = 3). (G) IHC analysis of LC3B protein expression. Scale bars, 100 and 200 μm. (H) Quantitative analysis of LC3B in G (n = 3). The values are presented as means ± SD. *P<0.05, **P<0.01.

Effects of SMS-Containing Serum on the PI3K/AKT Signalling Pathway and NET Formation in Neutrophils in vitro

Our previous reports revealed that the activation of PI3K/AKT leads to the autophagy of neutrophils.^35,57 MK2206, an AKT inhibitor, was reported to inhibit ROS production and NET formation.⁵⁸ MK2206 suppressed ROS production (Supplementary Figure 4A and 4B), as well as NET formation and autophagy (Supplementary Figure 4C-F), indicating that inhibition of AKT has led to the regulation of ROS/autophagy/NETs axis.

To further investigate the effect of SMS on NET production, primary neutrophils from the peritoneal cavities of mice were used for in vitro experiments. CCK8 assays were performed to determine the concentration of drug-containing serum in the cell culture medium. The addition of 1% serum of control rats and SMS-containing serum had no significant effect on cell viability (Supplementary Figure 5). NET formation was induced in vitro using PMA and the effects of SMS-containing serum were examined. SC79, an AKT activator that promotes AKT phosphorylation, was used to assess the involvement of the PI3K/AKT pathway in this process. As expected, PMA significantly increased p-AKT levels in neutrophils cultured with serum from control rats, whereas SMS-containing serum reduced AKT phosphorylation. SC79 treatment further increased AKT phosphorylation (Figure 5A and B). Similar trends were observed for p-PI3K levels (Figure 5C and D). Cellular immunofluorescence (IF) analysis revealed that PMA stimulation elevated the expression of CitH3 (Figure 5E and I), MPO (Figure 5F and J), NE (Figure 5G and K), and PAD4 (Figure 5H and L) in neutrophils in the control serum, whereas significant reductions were observed following treatment with SMS-containing serum. SMS-containing serum also significantly reduced the neutrophil nuclear area after PMA stimulation (Figure 5M–P). However, SC79 eliminated the suppressive effect of SMS on NET formation.

Figure 5 SMS inhibited the PI3K/AKT signalling pathway and NET production in vitro. Neutrophils extracted from the peritoneal cavity of mice were subjected to different interventions in the following groups: NC, SMS, NC+PMA, SMS+PMA, and SMS+PMA+SC79. (A) WB analysis of the expression of p-AKT and AKT in the neutrophils of each group. (B) Quantitative analysis of the relative expression of p-AKT in (A). (C) WB analysis of the expression of p-PI3K and PI3K in the neutrophils of each group. (D) Quantitative analysis of the relative expression of p-PI3K in (C). (E) IF analysis of MPO expression. (F) IF analysis of NE expression. (G) IF analysis of PAD4 expression. (H) IF analysis of CitH3 expression. (I) Quantitative analysis of MPO in (E) (n = 3). (J) Quantitative analysis of NE in (F) (n = 3). (K) Quantitative analysis of PAD4 in (G) (n = 3). (L) Quantitative analysis of CitH3 in (H) (n = 3). (M)Neutrophil nuclear area of (E) (n = 20). (N) Neutrophil nuclear area of (F) (n = 20). (O) Neutrophil nuclear area of (G) (n = 20). (P) Neutrophil nuclear area of (H) (n = 20). The values are presented as the means ± SDs. *P<0.05, **P<0.01, nsP > 0.05.

Neutrophil ROS Production and Autophagy Were Inhibited by SMS-Containing Serum in vitro

PMA activates protein kinase C, which, in turn, activates the NADPH oxidase complex, leading to increased ROS production.⁵⁹ Figure 6A and B shows that ROS significantly increased after PMA stimulation in vitro, whereas SMS-containing serum effectively reduced ROS generation. SMS also decreased the expression levels of autophagy-associated protein 5 (ATG5), Beclin-1, and LC3B II (Figure 6C-F). Reduced LC3B levels were also observed by IF (Figure 6G and H). Additionally, autophagosomes in neutrophils were examined using TEM. PMA stimulation increased autophagosome formation in neutrophils, which was subsequently reduced by SMS treatment (Figure 6I). The inhibition of autophagy by SMS-containing serum corresponded with reduced NET formation, which is consistent with the finding that the inhibition of autophagy prevents intracellular chromatin disaggregation, thereby inhibiting the PMA-stimulated formation of NETs in neutrophils.⁶⁰ The addition of the AKT activator SC79, which activates the AKT signalling pathway, further increases ROS production and autophagy in neutrophils and abolishes the inhibitory effect of SMS serum on ROS and autophagy, indicating that SMS regulates ROS and autophagy by inhibiting the activation of AKT.

Figure 6 SMS inhibited ROS production and autophagy through AKT signalling in vitro. Neutrophils extracted from the peritoneal cavity of mice were subjected to different interventions in the following groups: NC, SMS, NC+PMA, SMS+PMA, and SMS+PMA+SC79. (A) IF images of neutrophils from each group after being stained with DCFH-DA for 4 h. (B) Quantitative analysis of ROS in (A) (n = 3). (C) WB analysis of the expression of ATG5, Beclin-1, and LC3B in the neutrophils of each group. (D) Quantitative analysis of ATG5 in (C) (n = 3). (E) Quantitative analysis of Beclin-1 in (C) (n = 3). (F) Quantitative analysis of LC3B in (C) (n = 3). (G) IF analysis of the expression of LC3B in the neutrophils of each group. (H) Quantitative analysis of LC3B in (G) (n = 3). (I) Transmission electron microscopy results for each group. The Orange arrows indicate autophagosomes. The values are presented as the means ± SDs. *P<0.05, **P<0.01.

Network pharmacology analysis and molecular docking were utilized to explore the potential direct effect of SMS on AKT. As shown in Supplementary Figure 6, the four herbs of SMS contained 41 active ingredients, corresponding to 228 potential targeted proteins, which included AKT. The top 10 active ingredients according to “Degree” value are listed in the Supplementary Table 3. Wogonin, berberine, kaempferol, palmatine and quercetin were also identified by LC‒MS/MS analysis. Their binding energies to AKT were calculated and are listed in Supplementary Table 4. Wogonin, berberine and palmatine were predicted to bind more strongly to AKT (< ‒5 kcal/mol), and the docking results are shown in Supplementary Figure 7.

Discussion

The goal of RA treatment is to achieve remission.⁴ Despite infections and other side effects, the combination treatment strategy can successfully mitigate arthralgia and deformity in most patients with RA. This challenge has been proposed for some patients whose symptoms have not fulfilled the diagnostic criteria or whose disease progression is difficult to ameliorate with existing drugs.⁴ TCM has been used to treat patients with RA and pre-RA in China for thousands of years. As a classic formula used in clinical treatment, SMS provides new insights into intervention targets in RA progression.

Inflammation and bone destruction are the most important pathological changes associated with RA. In this study, H&E staining and toluidine blue staining indicated that a high dose of SMS could alleviate inflammatory cell infiltration and cartilage destruction in the ankle joints of patients with RA. Micro-CT confirmed the bone-protective effects of SMS. Notably, SMS has been shown to exert osteogenic effects on osteoblasts in vitro, which further accounts for the bone protection induced by SMS in RA.⁶¹

Neutrophils play important roles in the development of autoimmune diseases. Activated neutrophils in rheumatoid arthritis drive inflammation by producing various cytokines, ROS, and NETs.⁴⁵ NETs significantly enhance the inflammatory response in RA by exposing autoantigens to adaptive systems and by inducing the production of IL-6, IL-8, chemokines and adhesion molecules.⁶² The PAD4-mediated citrullination of histone proteins is an important component of NETs. In murine sepsis models, Cl-amidine, a PAD inhibitor, inhibits NET formation and increases the survival rate.⁶³ In patients with relatively high levels of ACPAs, elevated levels of hypoxia-inducible factor 1α (HIF-1α) and PAD4 were observed, along with cellular protein citrullination.⁶⁴ SMS significantly inhibited PAD4 expression in mice with AA and in neutrophils, accompanied by decreased NET formation. The regulatory effect of PAD4 could result from compounds in the SMS decoction. Given that PAD4 is a promising target for RA intervention, some studies have been carried out to identify natural inhibitors of TCM.^65–67 Systematic analysis of the effective components of SMS deposits and their potential targets has also been conducted using advanced approaches that require thorough integration and validation.^31,34,40

ROS exacerbate RA by activating immune cells, worsening inflammation, and inducing osteoclasts.⁶⁸ ROS also facilitate the formation of NETs.⁶⁹ Excessive accumulation of ROS disrupts cellular homeostasis, leading to oxidative stress, mitochondrial dysfunction, and the induction of autophagy.⁷⁰ In this study, SMS strongly suppressed ROS production in local joints in vivo and inhibited ROS production by neutrophils in vitro. However, the latest research on bone marrow-derived neutrophils in CIA mice using single-cell sequencing technology has proposed that ROS play bidirectional roles in different subsets of neutrophils during immune priming.⁷¹ Neutrophil heterogeneity may also affect the roles of NETs formed by different disease subsets in diseases,⁷² leaving more sophisticated questions for researchers to resolve.

The PI3K signalling pathway is one of the essential signalling pathways regulating cell growth, proliferation, motility, metabolism and survival.^73,74 Activation of the PI3K/AKT signalling pathway in neutrophils leads to ROS generation and NET formation.^75,76 The RA-related sleep deprivation likely exacerbates the oxidative stress, leading to the production of ROS and the activation of AKT signalling.⁷⁸ Inhibition of the PI3K/AKT signalling pathway has been reported to strongly alleviate RA by suppressing the excretion of inflammatory cytokines and delaying the apoptosis of fibroblasts,^79,80 which is consistent with our results. SMS can reduce ROS production and phosphorylation in the PI3K/AKT pathway both in vivo and in vitro.

This SMS decoction is a traditional prescription containing various ingredients. The active compounds remain to be investigated. Besides, how the autophagy contributes to NET formation is still confusing. As previously reported, the induction of apoptosis contributes to decreased autophagy and NET formation.^57,77 Further research is needed to elucidate these underlying mechanisms of SMS.

Conclusion

SMS offers the potential advantage over conventional therapies through multi-target regulatory mechanisms. Our study demonstrated that SMS had significant anti-inflammatory and immunomodulatory effects through the AKT/ROS/autophagy axis in an experimental RA model and in neutrophils.