Sinomenine ameliorates collagen-induced arthritis in mice by targeting GBP5 and regulating the P2X7 receptor to suppress NLRP3-related signaling pathways

Abstract

Sinomenine (SIN) is an isoquinoline alkaloid isolated from Sinomenii Caulis, a traditional Chinese medicine used to treat rheumatoid arthritis (RA). Clinical trials have shown that SIN has comparable efficacy to methotrexate in treating patients with RA but with fewer adverse effects. In this study, we explored the anti-inflammatory effects and therapeutic targets of SIN in LPS-induced RAW264.7 cells and in collagen-induced arthritis (CIA) mice. LPS-induced RAW264.7 cells were pretreated with SIN (160, 320, 640 µM); and CIA mice were administered SIN (25, 50 and 100 mg·kg⁻¹·d⁻¹, i.p.) for 30 days. We first conducted a solvent-induced protein precipitation (SIP) assay in LPS-stimulated RAW264.7 cells and found positive evidence for the direct binding of SIN to guanylate-binding protein 5 (GBP5), which was supported by molecular simulation docking, proteomics, and binding affinity assays (K_D = 3.486 µM). More importantly, SIN treatment markedly decreased the expression levels of proteins involved in the GBP5/P2X7R-NLRP3 pathways in both LPS-induced RAW264.7 cells and the paw tissue of CIA mice. Moreover, the levels of IL-1β, IL-18, IL-6, and TNF-α in both the supernatant of inflammatory cells and the serum of CIA mice were significantly reduced. This study illustrates a novel anti-inflammatory mechanism of SIN; SIN suppresses the activity of NLRP3-related pathways by competitively binding GBP5 and downregulating P2X7R protein expression, which ultimately contributes to the reduction of IL-1β and IL-18 production. The binding specificity of SIN to GBP5 and its inhibitory effect on GBP5 activity suggest that SIN has great potential as a specific GBP5 antagonist.

Introduction

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that is mainly characterized by persistent synovial inflammation and articular cartilage damage [1]. The treatment strategy for RA is early detection and control of disease progression. The commonly used drugs include nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), glucocorticoids, and biological agents [2]. However, the long-term use of these drugs often brings a series of toxic side effects. For example, although glucocorticoids and NSAIDs have been proven to effectively relieve pain and inflammation, their side effects cannot be ignored. They are likely to lead to recurrence and aggravation of the disease after long-term use. Methotrexate (MTX), as the preferred drug, usually causes nausea and vomiting, oral ulcers, liver damage, and other side effects [3]. In addition, the common side effects of leflunomide (LEF) include diarrhea, nausea, headache, rash, pruritus, alopecia, hypertension, chest pain, palpitations, infection, and liver function damage [3, 4]. In addition, biomacromolecular anti-RA drugs, such as tumor necrosis factor α (TNF-α) inhibitors, commonly have side effects such as infection and allergy [5, 6]. Therefore, safer and more effective drugs are still needed for the treatment of RA. Notably, traditional Chinese medicine (TCM) such as Sinomenii Caulis, Radix Aconiti, and Tripterygium wilfordii has long been used to treat RA, and under the condition of rational use, it has suitable efficacy and a low incidence of adverse reactions [7].

Sinomenine (SIN) is an isoquinoline alkaloid extracted from Sinomenii Caulis, a traditional Chinese medicine used to treat rheumatic arthralgia. SIN has been proven to have anti-inflammatory [8, 9], analgesic [10, 11], immunosuppressive [7, 12], antitumor [13, 14], and prohistamine release effects [15]. Moreover, clinical randomized controlled trials have shown that SIN has comparable efficacy to methotrexate in treating patients with RA but with fewer adverse effects [16]. In addition, patients treated with SIN plus MTX had lower gastrointestinal side effects and liver toxicity than those treated with MTX plus LEF [17]. These results indicate that SIN has ideal anti-RA treatment efficacy and safety. SIN preparations, such as Zhengqing Fengtongning (ZQFTN) tablets and injections, have been used in the treatment of RA [18, 19]. Studies have shown that SIN could inhibit the inflammatory responses of macrophages and fibroblast-like synoviocytes by acting on the alpha 7 nicotinic acetylcholine receptor (α7nAChR), a key receptor in the cholinergic anti-inflammatory pathway, which reveals a new anti-inflammatory mechanism of SIN [20–23]. SIN could also selectively regulate the methylation of a specific GCG site in the microsomal prostaglandin E synthetase 1 (mPGES-1) promoter region, thereby inhibiting the lipopolysaccharide (LPS)-induced upregulation of mPGES-1 expression and the binding of nuclear factor κB (NF-κB) to the mPGES-1 promoter, resulting in the inhibition of prostaglandin E₂ (PGE₂) generation and the reduction of the pain response [24, 25]. Although α7nAChR and mPGES-1 have been thoroughly studied in the mechanism of action of SIN, there is still insufficient evidence proving that they are the direct binding targets of SIN. Thus, the direct target of SIN in inflammatory diseases still needs to be further explored.

The NLRP3/ASC/Caspase-1/Cleaved caspase-1 signaling pathway is one of the intracellular mechanisms of the inflammatory defense response that plays a role in coordinating the innate immune system response to pathogenic stimuli [26]. NLRP3 is mainly regulated by Guanylate-binding protein 5 (GBP5) and P2X ligand-gated ion channel 7 (P2X7 receptor, P2X7R) [27, 28]. Activation of the NLRP3 signaling pathway drives IL-1β and IL-18 maturation and secretion, which ultimately exacerbate the inflammatory response [29–31]. Previous studies have shown that SIN can reduce LPS-induced macrophage inflammation and the mRNA expression level of P2X7R [32]. However, whether SIN can act on GBP5 and P2X7R to further regulate downstream signaling pathways remains to be investigated.

Thanks to the rapid development of mass spectrometry technology, the discovery of drug targets has evolved from confirmatory studies based on scientific hypotheses to high-throughput target screening at the proteome level. Solvent-induced protein precipitation (SIP) [33, 34] is a novel approach based on the principle of directly using complex cell lysates without modifying the compound of interest, and it relies on the fact that ligand-binding proteins are more resistant to solvent-induced precipitation. It provides an alternative method for studying drug-protein interactions. Due to the different mechanisms of protein denaturation, the SIP method based on organic solvent precipitation stability and the CETSA method based on thermal stability are complementary. In addition, the experiments used to investigate the binding affinity of small molecules to the purified active protein in the absence of other interference can also be applied to visually reflect direct binding [35, 36], which can be considered supplementary evidence for nonmodified target screening.

Herein, the SIP method was first applied to investigate the distribution of potential target proteins of SIN in LPS-stimulated RAW264.7 cells. Then, high-throughput proteomic analysis was employed to quantitatively identify potential target proteins. Using the identified targets, molecular simulation docking, molecule-protein binding affinity assays, and CETSA were used to investigate the direct binding property of SIN to the target protein. Based on the confirmation of the direct target, LPS-stimulated RAW264.7 and collagen-induced arthritis mouse models were applied to evaluate the effects of SIN on the molecular pathways regulated by the target protein.

Materials and methods

Materials and reagents

Sinomenine (purity ≥99% by HPLC) and sinomenine hydrochloride were kindly provided by Hunan Zhengqing Pharmaceutical Group Co. Ltd. (Hunan, China). LPS (Escherichia coli 055: B5), dexamethasone (DEX), and MTX were obtained from Sigma Chemical Corporation (St. Louis, Missouri, USA). A438079, a specific antagonist of the P2X ligand-gated ion channel 7 receptor (P2X7R), was purchased from Abcam Reagent Company (Cambridge, UK). Bovine type II collagen, complete Freund’s adjuvant, and incomplete Freund’s adjuvant were obtained from Chondrex Inc. (Washington State, USA). DMEM high glucose medium, penicillin & streptomycin solution, and other reagents were purchased from Gibco BRL Corporation (Grand Island, New York, USA). The Mem-PER Plus membrane protein extraction reagent, Protein Extraction Kit, and BCA Protein Quantification Kit were obtained from Thermo Fisher Scientific Co., Ltd. (Massachusetts, USA). A recombinant anti-P2X7R antibody was also purchased from Abcam. An NLR family pyrin domain containing 3 (NLRP3) rabbit mAb, a Na⁺/K⁺-ATPase antibody, an apoptosis-related speck-like protein (ASC) rabbit mAb, a caspase-1 rabbit mAb, a cleaved caspase-1 rabbit mAb, an inducible nitric oxide synthase (iNOS) rabbit mAb, and an anti-rabbit IgG HRP -linked antibody were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). A guanylate-binding protein 5 (GBP5) antibody was purchased from Novus Biologicals Co. (Littleton, Colorado, USA). ELISA kits were purchased from Wuhan Huamei Biological Engineering Co., Ltd (Wuhan, China).

Cell culture and intervention

RAW264.7 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM containing 10% fetal bovine serum (FBS) (Gibco-BRL, Grand Island, NY, USA) and antibiotics at 37 °C in a humidified atmosphere with 5% CO₂. RAW264.7 cells were seeded in 6-well plates at a density of 20 × 10⁴ to 40 × 10⁴ cells/well and incubated for 24 h, pretreated with or without SIN for 1 h, incubated with or without SIN for another 18 h, and costimulated with LPS (200 ng/mL) for 18 h. The intervention doses of SIN, DEX, and A438079, a specific antagonist of P2X7R, were described in previous experiments [9, 37].

Collagen-induced arthritis (CIA) model experiments in DBA/1 mice

Female DBA/1 mice aged 8–9 weeks and weighing 18–22 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) (license number: SCXK (BJ) 2016-0011). They were maintained in the animal facility of the SPF Animal Laboratory of the International Institute of Translational Chinese Medicine, Guangzhou University of Chinese Medicine (Guangzhou, China) (license number: SYXK (GZ) 2019-0144). All animal experiments were approved by the Animal Experiment Ethics Committee of Guangzhou University of Chinese Medicine (Approval number: IITCM-20220304). The mice were randomly divided into a blank control group, a model group, SIN groups (high, medium, and low doses), a methotrexate group, and a P2X7R antagonist A438079 group. On Day 0, an equal volume of Complete Freund’s Adjuvant (CFA) and bovine type II collagen (CII) was mixed and emulsified on ice using an electrically powered homogenizer [38]. The endpoint of emulsification was that the emulsion was taken up into the needle and did not disperse after adding it dropwise to normal saline. The mice were weighed thereafter, and the incidence of arthritis in each group was recorded every two days. On d 21, the mice were subjected to a booster injection of CII with incomplete Freund’s adjuvant (IFA) in the same volume as the Day 0 immunization schedule. From the second immunization, the mice in the model group were given normal saline (po), those in the SIN group were given SIN (25, 50, and 100 mg/kg everyday) by intraperitoneal injection (ip), and those in the MTX group were given MTX 10 mg/kg per week by oral administration (po) [9]. The P2X7R antagonist group was given A438079 5 mg/kg per week (ip) [39]. Paw thickness (at the ankles of the left and right hind paws) was obtained for each mouse by recording the severity of paw swelling using a Vernier caliper. In addition, the arthritis scores of the four feet were assessed every two days during the experiment using the following criteria, and the scores of each foot were summed to give a score for each mouse [11]: Score 0: no signs of erythema and swelling. Score 1: Erythema and slight swelling of the tarsi or ankle joints. Score 2: There is erythema and slight swelling from the ankle to the tarsal joint. Score 3: Erythema and moderate swelling from the ankle to the metatarsal joint. Score 4: erythema and severe swelling including the ankles, feet, and fingers, or stiffness of the limbs. After 30 days of drug administration, the mice were sacrificed, and the plasma and tissue were collected and stored on d 31.

Solvent-induced protein precipitation assay

A solvent-induced protein precipitation (SIP) assay was performed as previously reported, with appropriate modifications according to the specific situation [40, 41]. When the cells grew to 70%–80% confluence, 200 ng/ml LPS was added, and the inflammatory model was established after 18 h of intervention. Cells in the control group and LPS group were collected in 2.0 ml centrifuge tubes and centrifuged at 3000 r/min for 5 min in a 4 °C centrifuge. Then, 0.2% NP-40 lysate was added to the cells and lysed on ice for 40 min after blowing evenly. Then, the cells were centrifuged at 14000 r/min for 10 min at 4 °C, and the supernatant was used as the total protein extract. Protein concentration was measured at a wavelength of 595 nm using Coomassie Brilliant Blue quantification. The protein extract of the model group was then divided into three equal parts, two of which were incubated with 100 μM and 10 mM SIN (dissolved in DMSO), and the other part was incubated with the same amount of DMSO as the control group for 1 h. Subsequently, an organic solvent mixture of acetone/ethanol/acetic acid at a ratio of 50:50:1 was added to each sample to a final concentration of 10% organic solvent. After incubation, the mixtures were equilibrated at 800 r/min for 20 min at 37 °C. The mixtures were then centrifuged at 14000 r/min for 10 min at 4 °C to separate the supernatant, which contained relatively stable proteins without denaturation. A fraction of each sample was used for protein electrophoresis analysis, and the remaining protein solution was stored in an ultralow temperature freezer at −80 °C for subsequent quantitative analysis. After SDS-PAGE, silver staining was performed using a rapid silver staining kit (Biosharp, Hefei, Anhui, China). Finally, the gel was scanned, and the distribution of trend bands was analyzed.

Liquid chromatography-tandem mass spectrometry (LC‒MS/MS) analysis

Samples were subjected to ultrasonication at −80 °C, followed by centrifugation and collection of proteins, and the protein concentration was determined using a BCA kit. An appropriate amount of acetone was added and allowed to precipitate for 2 h at −20 °C. Samples were centrifuged at 4500 × g for 5 min, the supernatant was discarded, and the precipitate was washed twice with precooled acetone. After the precipitate was dried, triethylamine - carbonic acid buffer solution was added to a final concentration of 200 mM, the precipitate was dispersed by sonication, trypsin was added at a ratio of 1:50 (protease: protein, m/m), and enzymolysis was performed overnight. Dithiothreitol was added to achieve a final concentration of 5 mM, and the mixture temperature was lowered to 56 °C and stored for 30 min. Iodoacetamide was then added to a final concentration of 11 mM and incubated at room temperature in the dark for 15 min. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile and was used to dissolve the peptides. Peptides were separated with 6% to 24% solvent B (0.1% formic acid, 100% acetonitrile) on an EASY-nLC 1200 UPLC system (ThermoFisher Scientific, Massachusetts, USA) at a constant flow rate (450 nL/min). Peptides were separated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS) and then injected into a capillary ion source for ionization and analyzed by timsTOF Pro mass spectrometry. The peptide parent ions and their secondary fragments were detected and analyzed by high-resolution TOF technology. The obtained mass spectrometry data were processed using the MaxQuant search engine (v.1.6.15.0). Tandem mass spectrometry was used to search the human SwissProt database (20422 entries) and the reverse bait database. Mass tolerance for precursor ions was set to 20 ppm for the first search, 5 ppm for the main search, and 0.02 Da for fragment ions. The Cys amino group was fixed, while the N-terminal acetylation and the oxidation of Met were variable.

Molecular docking

Molecular docking was performed using AutoDock Vina software. The 3D structure of SIN was downloaded using the PubChem database (http://pubchem.ncbi.nlm.nih.gov/), and the crystal structure of GBP5 was downloaded from the Protein Data Bank (PDB code 7CKF).

Cellular thermal shift assay

A cellular thermal shift assay (CETSA) was performed as reported previously, with minor modifications [28, 29]. Cells were harvested and washed three times with PBS. The cell pellet was resuspended in PBS supplemented with 1% PMSF (Sigma), and the suspension was frozen-thawed 15 to 20 times in liquid nitrogen and a 37 °C water bath until flocculent precipitation appeared. The suspension was centrifuged at 14,000 × g for 20 min at 4 °C to separate cell lysates from cell debris. Cell lysates were divided into two aliquots using equalization; one was incubated with SIN at a final concentration of 100 μM, and the other was incubated with the same volume of DMSO. After incubation for 30 min at room temperature, the respective cleavage products were evenly divided into six aliquots of 15 μL each and heated at various temperatures (37, 41, 45, 49, 53, and 57 °C) for 20 min followed by 5-10 min of cooling at 4 °C using a Veriti™ 96-Well Fast Thermal Cycler (Thermo Fisher Scientific, Massachusetts, USA). The heated lysates were centrifuged for 20 min under the same conditions to separate the supernatant from the precipitate. The supernatants were analyzed by SDS‒PAGE and Western blotting. The suitable temperature was determined in preliminary experiments. Then, the binding degree of SIN to protein was examined under different concentration gradients (1600, 800, 400, 200, 100, and 0 μM) at the optimal temperature. Lysates were similarly centrifuged to separate the supernatant and precipitate and subjected to Western blot (WB) analysis to identify target proteins.

Construction of recombinant plasmids and expression and purification of GBP5 protein

The coding sequences for human GBP5 (hGBP5) were optimized and synthesized for Escherichia coli expression (Genewiz, New Jersey, USA) according to a previously reported method [40]. The amino acid sequence of the hGBP5 protein was Q96PP8•GBP5_HUMAN. The sequences encoding hGBP5_1–486 were inserted into pSmart-I (Smart-Lifesciences, Jiangsu, China) using the BamHI and XhoI restriction sites. A coding sequence for a small ubiquitin-like modifier (SUMO) (Smart-Lifesciences, Jiangsu, China) with a 6 × His-tag was inserted between the restriction sites NcoI and BamHI at the N-terminus of GBP5 to facilitate protein purification.

The plasmid was transformed into E. coli BL21 (DE3) competent cells, and the cells were plated and cultured overnight at 37 °C. Monoclonal clones were selected and cultured in 5 tubes of LB medium at 37 °C to an OD₆₀₀ of 0.8-1, and IPTG was added to a final concentration of 0.2 mM. After 4 h of culture, the bacteria were centrifuged, and samples were collected and subjected to SDS‒PAGE. IPTG was added to final concentrations of 0.2 and 1 mM after the inoculum was cultured to an OD₆₀₀ of 0.8–1 and then incubated at 37, 26, and 16 °C with a shaking speed of 220 r/min for 4 and 16 h to induce the expression of the fusion protein. The proteins prepared under different conditions were analyzed by SDS‒PAGE. The best-adapted bacterial solution was selected and centrifuged to collect the bacteria, which were lysed in PBS buffer and centrifuged at 14000 r/min for 1 h at 4 °C. The supernatant precipitate was separately sampled for SDS‒PAGE analysis. The optimal expression clone strain was expanded at 37 °C for 1 L to an OD₆₀₀ of 0.8-1 and then harvested after induction at 16 °C for 12 h. After centrifugation at 5000 r/min for 10 min at room temperature, the bacteria were collected, resuspended, and sonicated. The bacterial solution was centrifuged again, and the supernatant was purified by Ni-column affinity chromatography using PBS buffer (pH 7.4), PBS buffer (pH 7.4 with 0, 20, and 50 mM imidazole), and elution buffer (PBS buffer, pH 7.4 with 500 mM imidazole). The eluted samples were dialyzed to a cold bath in buffer (PBS, pH 7.4). Finally, samples were subjected to Coomassie brilliant blue staining after electrophoresis on 4% to 20% reduced SDS‒PAGE gels to detect a molecular weight of 55 kDa, and protein purity was analyzed by ImageJ software. In addition, protein samples were visualized by Western blotting using 4%–20% reduced SDS‒PAGE.

Binding affinity assay

First, GBP5 protein (1 mL, 1.2 mg/mL, 56 kDa) dissolved in PBS was prepared. A vial of Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Massachusetts, USA) was subsequently removed from the refrigerator, and after equilibration to room temperature, water was added to dilute it to 10 mM. Biotin labeling reactions (1 mL GBP5 plus 45 μL Biotin) were followed by the addition of an appropriate volume of 10 mM biotin reagent solution to the protein solution and incubation for 2 h on ice for 30 min at room temperature. Washing was performed three times (more than 4 h each time) with PBS. Enzyme-linked immunosorbent assay (ELISA) with GBP5-Biotin as the antigen coating and streptavidin-HRP (Boster, California, USA) as the primary antibody was used to detect the biotin labeling of proteins.

The ForteBio Octet RED 96 System (Serial Number: FB-50386) (Sartorius, Gottingen, Germany) was used to evaluate the interaction of SIN molecules with GBP5 proteins. A total of 125 μL of 1 mg/ml protein stock solution was added to 1875 μL of PBS buffer and diluted to 62.5 μg/μL. Super Streptavidin (SSA) Biosensors (Sartorius, Gottingen, Germany) were immersed in PBS for 10 min at room temperature before the experiments were performed. Two hundred microliters of the protein diluent was added to a 96-well plate (Sartorius, Gottingen, Germany). The sensor immersed in PBS was placed on the probe shelf, and the protein was observed to bind to the biotin sensor normally. After it was shown that the protein could bind to the sensor, the sensor was immersed in the protein solution for 10 min. SIN was then dissolved in PBST dilution solution containing 0.5% DMSO at final concentrations of 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 mM. Subsequently, 96-well plates were removed, and 200 μL of dilution solution was added according to the experimental design. The sensors with bound proteins and the blank sensors without bound proteins were positioned. Two hundred microliters of diluted drugs with different concentrations were added. After setting the detection program, the instrument detected the affinity of the protein with different concentrations of drugs according to the selected program. After automatic detection, the results were saved and analyzed by Octet BLI Analysis software.

Immunofluorescence (IF) analysis

The cells were first immobilized with 4% paraformaldehyde, followed by cell rupture with Triton X-100. Since P2X7R is a membrane protein, there was no need to break the membrane during staining. The 3% BSA drops were then added at room temperature. The primary antibody (P2X7R, GBP5) was then added to a confocal dish and incubated overnight in a 4 °C refrigerator. On the second day, the corresponding secondary antibody was added and incubated at room temperature. The slides were placed on a decolorizing shaker, shaken with PBS (pH 7.4) and washed. DAPI staining was subsequently dropped onto slides and incubated in the dark at room temperature. The slide was washed and slightly dried and sealed with an anti-fluorescence quenching sealant. After setting the corresponding wavelength range, the fluorescence microscope was used for observation, and finally, images were collected.

Western blot analysis

Total protein was extracted by adding 4 °C precooled RIPA lysis buffer with 1% PMSF to cells and tissues and the protein concentration was quantified by a BCA assay. Samples were separated by SDS‒PAGE, and proteins were transferred to membranes. After blocking with 5% skim milk powder for 1 h at room temperature, primary antibodies (GBP5, P2X7R, NLRP3, Caspase-1, ASC, Cleaved caspase-1, and iNOS, 1:1000) were added and incubated overnight at 4 °C. After washing with TBST, secondary antibody (sheep anti-rabbit IgG, 1:5000) was added and incubated at room temperature for 1 h, followed by color development with the ECL kit and exposure and photography with an automatic exposure instrument. Grayscale analysis of the bands was performed using ImageJ software.

Reactive oxygen species detection

A reactive oxygen species (ROS) detection kit (Beyotime, Shanghai, China) was used to evaluate the ROS levels in the cells. RAW264.7 cells were loaded with the fluorescent probe DCFH-DA. DCFH-DA was diluted in serum-free medium at a ratio of 1:1000 to give a final concentration of 10 μM. The cell culture medium of the six-well plate was removed, and 2 mL of diluted DCFH-DA was added to each well. They were then placed in a 37 °C cell incubator and incubated for 30 min. Cells were subsequently washed three times with PBS buffer to adequately remove DCFH-DA that did not enter the cells. Finally, the cells were loaded with in situ probes and were analyzed by flow cytometry under the parameters of FSC 110, SSC 275, and FITC 280.

Intracellular calcium ion (Ca²⁺) detection

Intracellular calcium levels was evaluated by the Fluo-4 Direct™ Calcium Assay kit (Thermo Fisher Scientific, Cat. No. F10471). First, a 250 mM stock solution of probenecid was prepared by adding 1 mL of Fluo-4 Direct™ calcium assay buffer to each 77 mg vial of water-soluble probenecid. Then, the 2× Fluo-4 Direct™ calcium reagent loading solution with a final probenecid concentration of 5 mM for the kit was prepared by adding 10 mL Fluo-4 Direct™ calcium assay buffer and 200 μL 250 mM probenecid stock solution to one bottle of Fluo-4 Direct™ calcium reagent. After that, microplates containing cells were removed from the incubator. An equal volume of 2 × Fluo-4 Direct™ calcium reagent loading solution was directly added to wells containing cells in the culture medium. The plate was incubated at 37 °C for 60 min. Fluorescence was measured using a SpectraMax i3X multifunctional microplate reader (Molecular Devices, California, USA) for excitation at 494 nm and emission at 516 nm.

Enzyme-linked immunosorbent assay

ELISA kits were used in our study to determine the levels of inflammatory cytokines, including IL-1β, IL-18, TNF-α, IL-6, and PGE₂ according to the protocol’s guidelines.

Hematoxylin-eosin (H&E) staining

The foot tissue of CIA mice was fixed with 4% paraformaldehyde, decalcified, paraffin-embedded, and sectioned (4 µm). The slides were then stained with hematoxylin for 3 min and eosin 0.5% for 5 min, and sealed with neutral glue. Finally, microscopic examination, image acquisition, and analysis were performed.

Microcomputed tomography (μCT)

Specimens were scanned using a Bruker Micro-CT Skyscan 1276 system (Kontich, Belgium). Scan settings were as follows: voxel size 6.533712 μm, medium resolution, 85 kV, 200 μA, 1 mm Al filter, and integration time 384 ms. Density measurements were calibrated to the manufacturer’s calcium hydroxyapatite (CaHA) phantom. Analysis was performed using the manufacturer’s evaluation software. Reconstruction was accomplished by NRecon (version 1.7.4.2) 2. 3D images were obtained from contoured 2D images by methods based on distance transformation of the grayscale original images (CTvox; version 3.3.0). 3D and 2D analyses were performed using the software CT Analyzer (version 1.20.3.0). The ROI of the region of interest was analyzed using CT Analyzer (software version 1.20.3.0, Bruker, Germany).

Immunohistochemistry (IHC)

Paraffin sections were dewaxed, followed by antigen retrieval in EDTA (pH 9.0) antigenic repair solution. The slices were then placed in a 3% oxygen solution to block endogenous peroxidases. Subsequently, 3% BSA was added and incubated with P2X7R, GBP5, and NLRP3 antibodies at 4 °C overnight, and the corresponding HRP-labeled rabbit IgG antibody was added and incubated on the second day. Then, color was developed using a freshly prepared DAB color-developing solution. The cell nucleus was restained with hematoxylin dye solution, washed with water, and finally dehydrated and sealed. The slides were examined under a microscope, and the images were collected and analyzed.

Statistical analysis

Data are presented as the mean ± SEM. GraphPad Prism 7.0 software (San Diego, California, USA) was used for statistical analysis. One-way analysis of variance (ANOVA) was used to compare the differences among groups. The Bonferroni test was used to compare the data conforming to a normal distribution for homogeneity of variance; otherwise, Tamhane’s T2 test was applied. The nonparametric Kruskal‒Wallis test was used to compare data that did not conform to a normal distribution. Data were considered statistically significant when P < 0.05.

Results

The potential target proteins of SIN were screened by combining SIP with proteomics in LPS-induced RAW264.7 cells

The inflammatory response is one of the main pathological features of RA, and abnormally activated macrophages are particularly critical target cells in the inflammatory mechanism of RA [42]. To investigate the potential target protein for SIN direct binding in macrophages, SIP experiments were first applied to examine protein distribution. SIP is a new proteomics strategy based on energetics, and it is used to analyze the interaction between drugs and their target proteins through quantitative proteomics [34, 43]. The results showed that the molecular weight of proteins that could bind to SIN was mainly between 25 kDa and 100 kDa (Fig. 1a). The molecular weights of the identified proteins were present at different stages and were uniformly distributed (Fig. 1b). The main protein molecular weight distribution was consistent with that in the SIP experiment. The proteins of the LPS, control, and SIN at 100 μM and 10 mM groups were compared with each other in pairs. A total of 475 different proteins were obtained with ratios less than 0.6 and greater than 1.5. These included 355 proteins from LPS versus Control, 16 proteins from SIN at 100 µM versus LPS, 89 proteins from SIN at 10 mM versus LPS, and 15 proteins from SIN at 10 mM versus SIN at 100 µM (Fig. 1c). KEGG enrichment analysis of differentially expressed proteins showed that the SIN-binding proteins were mainly enriched in DNA replication, cell cycle, viral carcinogenesis, and virus infections (Fig. 2a). GO analysis of molecular functions showed that the functions of these proteins mainly included kinase activity, catalytic activity, and ligase activity (Fig. 2b).

Fig. 1. A SIP combined proteomic approach was used to screen for potential target proteins interacting with sinomenine in LPS-induced macrophages.

a For the SIP assay, the total protein of RAW264.7 cells was incubated with SIN in vitro, and the proteins were electrophoresed on SDS‒PAGE gels and directly scanned. The molecular weights of proteins identified by proteomics are shown in b. c The obtained mass spectrometry data were processed using the MaxQuant search engine, and the tandem mass spectra were searched in the human SwissProt database and the reverse bait database.

Fig. 2. A SIP combined proteomic approach was used to screen for potential target proteins interacting with sinomenine in LPS-induced macrophages.

a, b Enrichment analysis was used to analyze the distribution of biological pathways and molecular functions of 105 differential proteins in SIN/LPS.

GBP5 protein was identified as a direct binding target of sinomenine

Proteomic analysis suggested that there might be strong binding between SIN and GBP5 proteins. SIN can bind to the active pocket of GBP5 with a binding energy of −7.72 kcal/mol, which is also the binding pocket of GDP, the endogenous ligand for GBP5, and the binding energy of GDP to GBP5 is −12.5 kcal/mol (Fig. 3a, b). Possible amino acids of the GBP5 protein bound by SIN include ASP-182, LEU-245, SER-69, ARG-48, GLY-50, VAL-67, ALA-68, and SER-69. To further investigate the binding of SIN to GBP5, CETSA, a drug-free drug target assay was performed first. As expected, the possibility of SIN binding to GBP5 was verified by the results of the CETSA experiment, which showed that SIN shifted the Tm50 value of GBP5 by 4.25 ± 0.87 °C and increased the thermal stability of GBP5 (Fig. 3c), indicating that SIN could directly bind to GBP5. Additionally, a binding affinity assay was used to evaluate the binding strength between SIN and the hGBP5 protein. First, the pSmart-I vector was used for protein synthesis by inserting the corresponding nucleic acid sequence of amino acids 1 to 486 between BamHI-XhoI and adding the stop codon (Fig. 4a). GBP5 protein was shown to be successfully expressed after SDS‒PAGE and Coomassie brilliant blue staining (Fig. 4b—i). The molecular weight of the purified protein was approximately 55 kDa, and the purity of the obtained protein reached 95.03% (Fig. 4b—ii). The molecular weight of the refolded protein was approximately 66 kDa, which is consistent with the theoretical value (Fig. 4b—iii). The curve of association and dissociation between SIN and GBP5, as well as their steady-state curves with a K_D(M) value of 3.486 × 10⁻⁶ verified the direct binding between them (Fig. 4c).

Fig. 3. GBP5 protein was identified as a direct binding target of sinomenine.

a, b AutoDock Vina software was applied to predict the molecular docking of SIN or GDP with GBP5. c The binding affinity of SIN to GBP5 was evaluated and validated via CETSA assay, and the melting curve was fitted by GraphPad with Boltzmann sigmoidal.

Fig. 4. GBP5 protein was identified as a direct binding target of sinomenine.

a The pSmart-I vector was used, the corresponding nucleic acid sequence of amino acids 1–486 was inserted between BamHI-XhoI, and the stop codon was added, which did not fuse with the C-terminal His tag. b—i The GBP5 protein was detected with 12% reduced SDS‒PAGE and Coomassie brilliant blue staining (Lane M: Protein Marker, Lane 1: induced precipitation, Lane 2: blank precipitation, Lane 3: induced supernatant, Lane 4: blank supernatant, Lane 5: induced whole bacteria, Lane 6: blank whole bacteria). b—ii The purified protein was electrophoresed on a 10% reduced SDS‒PAGE gel with a loading volume of 2 μg and then stained with Coomassie brilliant blue. (Lane M: Protein Marker, Lane 1: GBP5 with Sumo tag, Lane 2: GBP5 without the Sumo tag). b—iii The purified and refolded proteins were detected by 10% SDS‒PAGE and Western blot analysis (Lane M: Protein Marker, Lane 1: GBP5 without the SUMO tag, Lane 2: GBP5 with the SUMO tag). His-tag polyclonal antibody was used as the primary antibody at a dilution of 1:5000. Goat anti-rabbit IgG (H + L)-HRP was used as the secondary antibody, and the dilution was 1:20000. c Binding affinity assays for SIN and GBP5 were performed using the ForteBio Octet RED 96 System. Octet BLI Analysis software was used for data processing. The R² of the saturation curve is 0.978, and the K_D(M) value is 3.486 × 10⁻⁶.

SIN inhibited the GBP5/P2X7R-NLRP3 pathway in LPS-stimulated RAW264.7 cells

GBP5 is abnormally upregulated by dysregulated immune responses under pathological conditions [27, 44, 45]. Studies have shown that GBP5 promotes the assembly of the NLRP3 inflammasome in response to inflammation, as validated in both GBP5 knockout mice and cell culture models [44, 46]. The results showed that SIN markedly inhibited the high expression of iNOS in LPS-stimulated cells, and the difference was obvious at high doses (P < 0.05) (Fig. 6a). It has also been reported that P2X7R regulates the activity of NLRP3 by affecting the intracellular potassium level through its activity. Thus, to investigate whether the mechanism of the anti-inflammatory effect of SIN is related to the regulation of the NLRP3 pathway, we used LPS-induced RAW264.7 cells to study the proteins in this pathway. The expression of GBP5 in the model group was higher than that in the normal cells, as detected by IF and WB (P < 0.05) (Figs. 5a, 6b). Moreover, SIN dose-dependently downregulated the expression of GBP5 in the inflammatory state (P < 0.01). The expression of P2X7R in LPS-induced macrophages was also higher than that in normal cells (Figs. 5b, 6c), and SIN showed a dose-dependent inhibitory effect on the abnormally upregulated expression of P2X7R (P < 0.05). More interestingly, the inhibitory effect of SIN on P2X7R was comparable to that of A438079 at the middle dose (320 μM). The expression level of P2X7R in the DEX group was decreased compared with that in the model group, but the difference was not significantly different (P > 0.05). Moreover, SIN inhibited the high protein expression levels of NLRP3 (Figs. 5c, 6d), ASC, caspase-1, and cleaved caspase-1 in inflammatory cells (P < 0.05) (Fig. 6e, f, g).

Fig. 6. Sinomenine regulated the activation of the GBP5/P2X7R-NLRP3 pathway in LPS-induced RAW264.7 cells.

a–g The cells were also seeded in 6-well plates at a density of 2.0 × 10⁵ cells/well for drug and model intervention. After that, the cells were collected for WB detection, and the gray value of the bands was analyzed by ImageJ software. n = 3, values are presented as the mean ± SEM, ^##P < 0.01, ^###P < 0.001 vs. RAW264.7 cells, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. LPS-stimulated RAW264.7 cells.

Fig. 5. Sinomenine regulated the activation of the GBP5/P2X7R/NLRP3 pathway in LPS-induced RAW264.7 cells.

a–c RAW264.7 cells were seeded in 35 mm confocal dishes at a density of 2.0 × 10⁵ cells/well and cultured to adhere for 12 h before intervention. Subsequently, the medium was changed to a new medium and the drug was added for 1 h in advance, followed by incubation with/without LPS for another 18 h. Cells were stained and analyzed by fluorescence microscopy.

SIN reduced the levels of proinflammatory factors, ROS, and Ca²⁺ in LPS-stimulated RAW264.7 cells

P2X7R acts as a cation-gated channel, and its activation can lead to a large amount of intracellular influx of Ca²⁺ and Na⁺ and efflux of K⁺, thereby affecting mitochondrial function and stimulating it to secrete ROS [41, 47, 48]. Activation of P2X7R leads to a substantial release of PGE₂ and IL-1β and may even cause fever [49]. Our previous study found that P2X7R mRNA levels can be decreased by SIN [50]. In this study, we demonstrated that SIN also obviously downregulated P2X7R protein expression in LPS-stimulated RAW264.7 cells (P < 0.05) (Figs. 5b, 6c). Therefore, we hypothesized that the inhibitory effect of SIN on P2X7R might further induce changes in intracellular Ca²⁺ and ROS levels, which reflected the effect of SIN on mitochondria at the inflammatory level. In addition, it is also necessary to determine whether the regulation of the NLRP3 pathway by SIN affects the extracellular secretion of IL-1β and IL-18 and its regulation of other pro-inflammatory cytokines. We found that the secretion levels of IL-1β, IL-18, IL-6, and TNF-α in the culture supernatant of inflammatory cells were effectively reduced by SIN in a dose-dependent manner (P < 0.05) (Fig. 7c–f). Moreover, SIN also effectively reduced the levels of ROS and Ca²⁺, which supported the inhibitory effect of SIN on the activity of P2X7R under inflammatory conditions (P < 0.05) (Fig. 7a, b, g). Moreover, the P2X7R-specific antagonists A438079 and DEX also effectively decreased the Ca²⁺ level (P < 0.05). DEX inhibited ROS generation (P < 0.05), but A438079 showed no significant difference in ROS inhibition (P > 0.05).

Fig. 7. Sinomenine dose-dependently reduced the levels of proinflammatory factors, ROS, and Ca²⁺ in LPS-induced RAW264.7 cells.

a, b Intracellular ROS levels were determined by flow cytometry. The letter(s) C, M, SL, SM, SH, A, and D denote the control, model, SIN at 160 μM, SIN at 320 μM, SIN at 640 μM, P2X7R antagonist A438079 at 10 μM, and dexamethasone at 0.5 μM groups, respectively. c–f The levels of IL-1β, IL-18, IL-6, and TNF-α in the cell culture supernatant were measured by ELISA kits. g The intracellular Ca²⁺ levels were determined by using a multifunctional microplate reader with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. n = 3, values are presented as the mean ± SEM, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. RAW264.7 cells, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. LPS-stimulated RAW264.7 cells.

SIN alleviated arthritis symptoms in type II collagen induced arthritis models in DBA/1 mice

We further investigated the effects of SIN on inflammatory markers in collagen-induced DBA/1 mice. We were also curious to reveal the regulatory effect of SIN on P2X7R in vivo. Given the role of the P2X7R antagonist A438079 in inflammatory diseases [39, 51, 52], it was chosen as one of the positive control drugs in this study, and the other positive control drug was MTX, the currently preferred drug for RA treatment. The immune and drug intervention procedures in mice are shown in Fig. 8a. As shown in Fig. 8b–f, there were marked differences between the model group and the control group in indices such as foot swelling, foot thickness, and inflammation score (P < 0.01), which proved that the animal model was successfully constructed. After the analysis of the weight changes of mice, it was found that the weight of mice in the model group showed a downward trend compared with that in the healthy control group, and the results of the measurement on the last day showed a significant difference (P < 0.01). In addition, SIN, A438079, and MTX alleviated the weight loss of mice in the inflammatory state to a certain extent. There was a significant difference in body weight between the SIN high-dose group and the model group (P < 0.05) (Fig. 8c). In the analysis of other inflammatory indicators, SIN reduced the morbidity of mice in a dose-dependent manner, suppressed the swelling and thickening of the paw, and relieved the joint inflammation score of CIA mice, especially after high-dose administration, with sharp distinctions in all indicators compared with those in the model group (P < 0.01) (Fig. 8d–f). In addition, the inflammation score of the MTX group was lower than that of the model group (P < 0.05) (Fig. 8e). However, the P2X7R antagonist A438079 showed no variation in arthritis indices compared with those in the model group (P > 0.05).

Fig. 8. Sinomenine dose-dependently alleviated arthritis symptoms in CIA mice.

a DBA/1 mice were immunized twice and sacrificed one month after drug intervention, during which inflammatory markers were measured every two days. b The plantar region of the mice was photographed on the day of dissection to record foot swelling. c–f Body weight, arthritis index, inflammation score, and plantar thickness were recorded every two days and then combined for statistical analysis. N = 11, values are presented as the mean ± SEM, ^##P < 0.01, ^###P < 0.001 vs. Control group, ^*P < 0.05, ^**P < 0.01 vs. Model group.

SIN reduced the levels of proinflammatory cytokines and alleviated both joint inflammation and bone destruction in type II collagen induced arthritis model DBA/1 mice

As joints are the most affected sites in RA patients, foot tissues of the CIA model DBA mice were selected as the target tissues for further pathological and pharmacological mechanism studies. We further investigated the effects of SIN on the inflammation levels and pathological changes in the foot tissue and bone microstructure of CIA mice. The results of H&E staining showed that SIN dose-dependently reduced inflammatory infiltration and synovial thickening in the joints of CIA mice (Fig. 9a). In addition, SIN dose-dependently reduced joint inflammatory infiltration and synovial thickening, resulting in higher joint integrity. Moreover, the results of µCT scanning showed that compared with the model group, the SIN group showed no bone erosion of the foot tissue and improved bone density (Fig. 9b, c). Moreover, the levels of IL-1β, IL-18, TNF-α, IL-6, and PGE₂ in the serum of SIN-treated mice were decreased in a dose-dependent manner (P < 0.05) (Fig. 9d–h). MTX also reduced the levels of proinflammatory factors (P < 0.05) while having a protective effect on bone tissue (Fig. 9a–c). When compared with the model group, the A438079 group exhibited reduced IL-6 levels (P > 0.05) (Fig. 9e), but A438079 had little effect on other pro-inflammatory cytokines (P < 0.05), bone pathology, and microstructure.

Fig. 9. Sinomenine reduced the levels of proinflammatory cytokines and alleviated both joint inflammation and bone destruction in CIA mice.

a The foot tissues of CIA mice were fixed and subjected to H&E staining to analyze the pathological changes in joints. b Using the set of projection images as raw material, the Feldkamp algorithm was used to create a dataset stack of cross-sectional images, which was the reconstruction process, and to generate a 3D image of the scanned object. c Projected X-ray images were the “raw material” of the micro-CT imaging process. Parts ①, ②, and ③ represent the transverse, coronal, and sagittal cross-sections, respectively. d–h Serum samples from CIA mice were removed from the −80 °C freezer and assayed for TNF-α, IL-1β, IL-18, IL-6, and PGE₂ levels using ELISA kits. n = 11, values are presented as the mean ± SEM, ^###P < 0.001 vs. Control group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Model group.

SIN inhibited the activity of the GBP5/P2X7R-NLRP3 pathway in the paw tissue of type ǁ collagen-induced DBA/1 mice

To further investigate the in vivo effects of SIN on GBP5, P2X7R, and NLRP3 and their downstream inflammatory responses, the CIA model in DBA/1 mice was used for the in vivo mechanistic study. Immunohistochemistry and Western blotting were used as the main detection methods for protein phenotypes in mouse foot tissues (Fig. 10). Stained tissue slides were observed with a microscope (Fig. 10a), and the percentage of positive cells was quantified with the use of Aipathwell analysis software. The ratio of GBP5-, P2X7R-, and NLRP3-positive cells/area to total cell number/area in the model group was higher than that in the normal control group (P < 0.05) (Fig. 10b–g), which proved that the animal model was successfully established. Moreover, it is noteworthy that SIN dose-dependently decreased the tissue distribution and expression levels of these three proteins. In addition, A438079 inhibited the expression of NLRP3 protein when compared with the model group (P < 0.05) (Fig. 10f–g). MTX also showed a marked inhibitory effect on GBP5, P2X7R, and NLRP3 expression (P < 0.05). Consistent with the results of IHC, the WB results found that SIN dose-dependently downregulated the protein expression of GBP5, P2X7R, NLRP3, ASC, Caspase-1, and Cleaved caspase-1 in the foot tissues (P < 0.05, P < 0.01, P < 0.001) (Fig. 11). Taken together with the previous evidence that SIN reduced serum IL-1β and IL-18 levels in CIA mice, it can be concluded that SIN inhibits the activity of the GBP5/P2X7R-NLRP3 pathway, which may be part of the mechanism underlying the immunosuppressive effect of SIN (Fig. 12).

Fig. 10. Sinomenine inhibited the activity of the GBP5/P2X7R-NLRP3 pathway in the paw tissue of CIA mice.

a Immunohistochemical staining of paw tissue from each dosing group is presented. The large picture is the image magnified 5 times based on the original section, and the small picture in the black box is the image magnified 20 times. b, d, f The proportion of GBP5-, P2X7R-, and NLRP3-positive cells in the foot tissue was analyzed by Aipathwell automated image analysis software, and the positive cell ratio was calculated as the number of positive cells/total cells. c, e, g In addition, the ratio of positive cell area was calculated as positive area/tissue area. n = 11, values are presented as the mean ± SEM, ^#P < 0.05, ^###P < 0.001 vs. Control group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Model group.

Fig. 11. Sinomenine suppressed the activity of the GBP5/P2X7R-NLRP3 pathway in the paw tissue of CIA mice.

a–f The relative protein expression levels of GBP5, P2X7R, NLRP3, ASC, caspase-1, and cleaved caspase-1 in mouse foot tissues after liquid nitrogen grinding, tissue homogenization, and protein extraction were investigated by Western blotting. The letters C, M, SL, SM, SH, MTX, and A denote control, model, SIN (25, 50, and 100 mg/kg everyday), methotrexate at 10 mg/kg, and the P2X7R antagonist A438079 at 5 mg/kg, respectively. n = 3, values are presented as the mean ± SEM, ^##P < 0.01, ^###P < 0.001 vs. Control group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Model group.

Fig. 12. Schematic diagram of the molecular mechanism of SIN.

SIN downregulates the expression of P2X7R in inflammatory cells and directly binds to the GBP5 protein, thereby inhibiting the downstream NLRP3/ASC/Caspase-1/Cleaved caspase-1 pathway and ultimately reducing the secretion of IL-1β and IL-18. Moreover, SIN regulates intracellular calcium levels by inhibiting P2X7R, thus affecting the release of ROS by mitochondria. This mechanism diagram was made using the Figdraw platform of the Home for Researchers website, and the export ID is PTSTlbbb4b.

Discussion

GBP5 is a direct protein target of sinomenine

Different from chemical proteomics, which must be chemically derived from drug molecules, nonchemical modification methods can achieve high-throughput, large-scale, and unbiased drug modification-free target screening at the proteome level by combining cutting-edge quantitative and qualitative proteomics [53]. In recent years, researchers have developed a variety of unmodified methods for studying drug-protein interactions, such as cell thermal displacement analysis (cellular thermal shift assay, CETSA) [43, 54], stability of proteins from rates of oxidation (SPROX) [55, 56], drug affinity responsive target stability assay (DARTS) [57], chemical denaturant and protein precipitation (CPP) [58], and thermal proteomic analysis (thermal proteome profiling, TPP) [54, 59, 60]. The targets of FK506, rapamycin, and resveratrol were successfully identified by using these methods. In the SPROX method, researchers assess the thermodynamic stability changes after protein‒ligand binding by measuring the oxidation rate of methionine-containing residues as a function of chemical denaturant concentration, thereby revealing the target protein [56]. In addition, DARTS uses the principle that proteins are more resistant to proteolysis when bound to drugs [61]. In addition, CPP is based on the principle that chemical denaturants induce protein precipitation to identify the protein targets of drugs [58]. As an emerging method for monitoring target contacts, CETSA reveals drug targets by measuring their denaturation [62]. Although these methods are suitable tools for validating drug targets, they are not suitable for discovering unknown drug targets at the proteomic level. This deficiency was remedied by the development of the TPP method, which uses quantitative proteomics methods to quantify proteins in soluble fractions instead of having to use Western blotting as a readout and allows the identification of ligand-induced changes in protein stability at the proteomic level [63, 64]. The above methods are based on the principle of protein stability after ligand induction, but some proteins are not sensitive to oxidative denaturation, protease hydrolysis, and thermal denaturation after binding with a ligand. Some proteins, such as BCR-ABL, do not respond to thermal denaturation after binding with dasatinib [59], indicating that different methods were necessary to identify the true targets. Therefore, a new target screening method was developed, the SIP assay, which is used to precipitate proteins by reducing permittivity and competing for protein water, as opposed to thermal denaturation-induced protein precipitation [34]. SIP is based on the principle of greater tolerance to organic solvents after ligand-binding proteins. In addition, the SIP method obtained proteome coverage similar to the traditional bottom-up experiments, but was superior to the SPROX method. Two model drugs, MTX and SNS-032, were verified and evaluated by the SIP method, and the known targets were successfully revealed. In addition, three known protein targets of the heat shock protein 90 family, NADH dehydrogenase subunits NDUFV1 and NDUFAB1, were identified [34]. However, there are still some shortcomings in these methods of target protein screening with pharmacological modifications [65]. First, the binding between the ligand drug and the protein may cause structural changes in other proteins, resulting in false-positives. Second, interference with nonspecific proteins is also a problem faced by nonchemical modification methods. In addition, some target proteins with low abundance are easily covered by target proteins with high abundance and cannot be detected.

Herein, the approach we have adopted to find drug targets is a combined SIP and proteomics strategy. The SIP method was used to investigate whether the incubation of SIN with total protein extracted from inflammatory macrophages resulted in differential binding and the general distribution of these differentially bound proteins, and proteomic analysis was used to analyze the binding proteins qualitatively and quantitatively. To intuitively analyze the binding energy of SIN and GBP5, molecular simulation was used to predict the binding energy of SIN and GBP5. In addition, the binding energy of GBP5 and its ligand GDP was also evaluated. The excessive immune response in RA patients often leads to disorders of energy metabolism, and purine metabolism is increased in RA [66, 67]. GDP is a product of GTP metabolism and acts as a ligand for GBP5 [35]. Although the binding energy of SIN to GBP5 (−7.72 kcal) is lower than the binding energy of GDP with GBP5, it has some instability since GDP is an endogenous energy metabolite [68]. In fact, in addition to performing molecular docking to predict the binding mode as well as the binding energy, we also applied molecular dynamics simulations to dynamically analyze the binding of SIN to the GBP5 protein. The molecular dynamics results are not presented in this article. These results showed that the top ten amino acids with the highest binding potential energy of SIN on GBP5 are not identical to those on the active pocket position of GBP5. The amino acids that may effectively bind to SIN according to molecular docking simulations are ASP-182, LEU-245, SER-69, ARG-48, GLY-50, VAL-67, and ALA-68, while the top ten amino acids according to the molecular dynamics simulations include LYS-232, LYS-231, LYS-230, PHE-228, PRO-229, LEU-176, PRO-174, HIS-143, THR-149, and ARG-205. This result suggested that the binding of SIN to GBP5 more likely affects the stability of the protein rather than its activity.

While proteomics has demonstrated the potential of SIN binding to target proteins, more analytical strategies are needed to rule out the possibility of false-positives and confirm the authenticity and credibility of the results. Due to the different principles of protein denaturation, the SIP method based on organic solvent precipitation stability and the CETSA method based on thermal stability have suitable complementarity. With these in mind, we applied other protein-molecule interaction assays, including affinity assays and CETSA, to collect more supporting evidence. The results of CETSA showed that the heat shift value of SIN was 4.25 ± 0.87, and the binding showed a dose-dependent increasing trend, which also supported the results of quantitative proteomics. The results of this binding are similar to the trend found by Wang et al. in the direct binding of jujuboside A to FAT atypical cadherin 4 using CETSA [43]. Different from nonmodified proteomics methods such as SIP and CETSA, the binding affinity assay we used directly synthesizes and purifies the hGBP5 protein expressed in E. coli, links it to biotin, and specifically connects it to SSA biosensors. The probe can detect the intensity of the binding signal with SIN molecules at a certain protein concentration and evaluate the binding affinity between them. This method is free from the influence of living cells and can intuitively reflect the binding signal between proteins and small molecules [69, 70]. Thus, this approach provides structural evidence for the direct binding of SIN to the GBP5 protein. The binding affinity assay using the ForteBio Octet system showed that the signal was effective, and the K_D(M) value was approximately 3.486 × 10⁶, indicating that there was good binding affinity between SIN and GBP5. In summary, the above molecular target identification experiments all indicated the direct binding of SIN to the GBP5 protein. Although we have verified the direct binding of SIN to GBP5, the exact binding site has not been confirmed, so verification using site mutation experiments is still needed.

Inhibition of the NLRP3-related signaling pathway by directly targeting GBP5 and regulating P2X7R is a novel mechanism of sinomenine

The abnormal upregulation of GBP5 expression is caused by dysregulated immune responses under pathological conditions, such as synovial tissues affected by RA [71] and some human malignancies, such as gastric adenocarcinoma [72], myeloid carcinoma [73], and glioblastoma [74]. In addition, it is also closely related to inflammatory bowel diseases [28] and sepsis-associated liver injury [27]. After confirming the in vitro intermolecular interaction results, we further sought to verify the regulation of GBP5 by SIN at the molecular level. Our study showed that SIN downregulated the expression of GBP5 protein both in vitro and in vivo. Among the many known inflammasome complexes, the most extensively studied is the NOD-like receptor family, which includes the pyrin domain of NLRP3 [75]. When activated by infection or cellular stress, NLRP3 forms a complex with ASC containing the caspase recruitment domain, which then triggers the cleavage of pro-caspase-1 into caspase-1 [76]. Ultimately, it stimulates the maturation and secretion of the proinflammatory cytokines IL-1β and IL-18, and it promotes the recruitment of inflammatory cells to target cells and organs. Studies have shown that the knockdown of GBP5 inhibits the activation of the NLRP3 inflammasome and the secretion of IL-1β and IL-18, thereby slowing the development of lupus nephritis [77]. Interestingly, our data demonstrated that SIN blocked the downstream NLRP3/ASC/Caspase-1/Cleaved caspase-1 pathway, which ultimately resulted in a decrease in secreted IL-1β/IL-18 levels. In addition, studies have suggested that the binding of small molecules to proteins may affect the degradation of this protein and thus play a role in the regulation of relevant signals. For example, arnicolide D, the main component of coriander, can bind proto-oncogene tyrosine kinase protein and may promote the degradation of this protein in prostate cancer cells [78]. Our results also showed that SIN can directly bind to the GBP5 protein and downregulate its protein expression, but GBP5 mRNA levels and GBP5 protein activity were not evaluated. Therefore, we cannot confirm at this time whether SIN only affects the degradation of GBP5 at the protein level, thus reducing its detected expression level. Alternatively, the direct binding of sinomenine to GBP5 may lead to a decrease in protein activity, triggering a downstream cascade of effects. Thus, these scientific questions still need to be explored. In addition, GBP5 is an interferon-induced protein, and upregulated expression of GBP5 in inflammatory macrophages is often associated with increased IFN-γ levels. However, IFN-γ levels have not been measured [79], so further studies are needed.

P2X7R is an ATP-gated receptor that participates in the inflammatory response mainly by activating NLRP3 activity and prompting the maturation and nonclassical secretion of proinflammatory factors [80, 81], playing a key role in immunity and autoimmunity [82]. P2X7R is widely expressed in almost all tissues in the body, covering various cells, including monocytes/macrophages [83], T cells [84], fibroblasts [85], mast cells [86], microglia [87], epithelial cells [88], and endocrine and exocrine pancreatic cells [89]. The monocyte-macrophage cell line is the most distributed among the cells of the immune inflammatory system [90]. Increasingly abundant research in this field has revealed that P2X7R may be a critical regulator and potential target for bone and joint diseases [91]. Our study first demonstrated that SIN decreased the expression levels and distribution of P2X7R in RAW264.7 cells and substantially inhibited P2X7R protein expression in CIA mice. This regulatory effect of SIN on P2X7R was accompanied by a dose-dependent decrease in intracellular Ca²⁺ levels and further reduction of mitochondrial ROS production. This finding demonstrated that SIN may also regulate mitochondrial function in inflammatory cells, but this notion has not yet been studied. The effect of SIN on inflammatory cells and tissues at low doses was similar to that of A438079, a specific antagonist of P2X7R. The effect of A438079 on NLRP3 and its downstream cascade was obvious in cells, but in contrast, the anti-inflammatory and bone tissue-protective effects in animals were not different from those in the model group. However, it was different from the previous results of A438079 in CIA mice [39]. This result is undoubtedly an interesting phenomenon for the antagonist itself, but the underlying mechanism remains to be explored. In addition, immunohistochemical staining results suggested that GBP5, P2X7R, and NLRP3 proteins were highly expressed in bone marrow-associated cells in the inflammatory state, but the distribution of immunopositive cells in the synovial cells was not significant. This result demonstrated an interesting phenomenon, which may be explained by the fact that the bone marrow contains bone marrow-derived macrophages as well as pro-monocytes and that GBP5 is precisely the target protein that was screened from inflammatory RAW264.7 cells. However, whether these proteins are highly expressed in synovial cells in the inflammatory state remains to be further explored and validated. Overall, both GBP5 and P2X7R have cascading effects on the downstream NLRP3 signaling pathway, which leads us to question whether P2X7R and GBP5 are related to each other. This hypothesis also needs further investigation. In summary, according to literature reports and analysis of our experimental data, NLRP3 is coregulated by GBP5 and P2X7R in the inflammatory environment. The regulation of SIN on this pathway mainly depends on directly binding with GBP5 and inhibiting its expression and partly depends on inhibiting the expression of P2X7R.

As shown in Fig. 12, our study demonstrated that SIN bound directly to GBP5, thereby inhibiting its activity, and coregulated P2X7R to suppress the downstream NLRP3-induced inflammatory pathway. Since no GBP5 antagonist has been reported, the direct binding specificity of SIN to GBP5 and its inhibitory effect on GBP5 activity demonstrated its great potential as a GBP5 antagonist. These pharmacological results combined with our previous molecular interaction studies suggest that GBP5 is a potential target of SIN in inflammatory cells as well. Although this study has revealed new anti-inflammatory targets and mechanisms of SIN to a certain extent, there are still many deficiencies and limitations in our study. For example, although we have verified the direct binding of SIN to GBP5, the exact binding site has not been confirmed. GBP5 is a guanylate-binding enzyme, and SIN can downregulate its expression after binding to it. However, due to the lack of existing activity detection kits, no further tests have been conducted on the effect of SIN on its enzyme activity, and it is still unknown whether SIN affects the functional activity of GBP5. Moreover, the effect of SIN on the protein stability of GBP5 also needs to be further explored. When performing the selection of cell types, LPS-stimulated RAW264.7 cells instead of human-derived cells were chosen for proteomic target screening, which may indicate that the differential targets obtained may not match exactly with the actual targets in clinical cases and may add to the controversy that GBP5 could be a therapeutic target of SIN in inflammatory diseases such as RA. Moreover, the molecular mechanism of this study has not been verified in cell or animal models with gene knockout or overexpression, which makes the chain of evidence for the regulation of the molecular mechanism of SIN on GBP5 targets incomplete.

Conclusion

Herein, for the first time, we innovatively proved that SIN could efficiently and directly bind GBP5 using proteomics analysis and molecule-protein interaction analysis. By using comprehensive pharmacological evaluation, our data revealed GBP5 as a potential target for the anti-inflammatory effects of SIN. The results showed that SIN inhibited the GBP5/P2X7R-NLRP3 signaling pathway to attenuate inflammation both in vivo and in vitro. Our study also provides evidence for GBP5 as a therapeutic target for inflammatory diseases such as RA. It is worth noting that this study provides a new molecular basis for the use of SIN in the treatment of inflammation-related diseases, including RA, and has provided a reference for the application of SIN in autoimmune diseases, cancer, and other inflammation-related diseases.

Author contributions

JML: Methodology, Data curation, Formal analysis, Investigation, Validation, and Visualization, Writing original draft. HSD: Methodology, Data curation, Formal analysis, Investigation, Validation, and Visualization. YDY: Methodology, Data curation, Formal analysis, Investigation, Validation, and Visualization. JQH: Methodology, Data curation, Formal analysis, Investigation, Validation, and Visualization. WTW: Methodology, Data curation, Formal analysis, Investigation, Validation, and Visualization. YD: Writing—review and editing. PXW: Writing—review and editing. Liang Liu: Writing—review and editing. ZQL: Writing—review and editing. YX: Conceptualization, Writing—review and editing. Lin-Lin Lu: Conceptualization, Resources and Supervision, Writing—review and editing. HZ: Conceptualization, Funding acquisition, Resources and Supervision, Writing—review and editing. All authors approved the final manuscript.

Competing interests

The authors declare no competing interests.