DIM (CHN, ≥98% HPLC, CAS:1968-05-4, chemical structure is shown in Fig. 1A) was purchsed from Sigma-Aldrich (USA). Immunization Grade Bovine type II collagen (C II) was purchased from Chondrex, Inc. (USA). Sodium carboxymethylcellulose (CMC-Na, viscocity: 600-1000 mpa.s, USP) was purchased from Shanghai Macklin Biochemical Co., Ltd. Freund's complete and incomplete adjuvant and ADP were purchased from Sigma-Aldrich (St. Louis, USA). Mice IL-6, TNF-a, and IL-1β ELISA Kits were purchased from Jiangsu Meimian Industrial Co., Ltd (Jiangsu, China). FITC Anti-Mouse CD41 Antibody was purchased from Elabscience (Wuhan, China). APC anti-mouse/rat CD62P Antibody was purchased from BioLegend (San Diego, CA). Fluo-4 Calcium Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China). For Western Blot analysis, primary antibodies included p38 MAPK, JNK, p44/42 MAPK (Erk1/2), AKT, mTOR, p70S6K, 4E-BP1, NF-κB p65, IKKα, IKKβ, IκBα and their corresponding phosphorylation antibody, Phospho-p38MAPK (Thr180/Tyr182), Phospho-JNK (Thr183/Tyr185), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Phospho-Akt (Ser473), Phospho-p70 S6 Kinase (Thr389), Phospho-4E-BP1 (Ser65), Phospho-IKKα/β (Ser176/180), Phospho-IκBα (Ser32), and Phospho-NF-κB p65 (Ser536), which were all purchased from Cell Signaling Technology (USA).
Female DBA/1 mice aged 10-12 weeks (about 20 g/body weight) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. C57BL/6 mice aged 10-12 weeks were purchased from the Experiment Animal Center in Southern Medical University. All mice were fed in well-ventilated cages with free access to commercial diet and tap water. The room housed mice was with fixed temperature (22 ± 2)°C and humidity (50 ± 20%) under standard laboratory conditions of 12 h/12 h light/dark cycles. All the experimental procedures abided by the guidelines of ethical regulations for institutional animal care and use in the Southern Medical University and approved by the Southern Medical University Ethics Committee for Animal Laboratory Research (FD2022027).
CIA model was established by subcutaneous immunization of Freund's complete adjuvant emulsion concluding bovine type II collagen at the root of the female DBA/1 mice tail at first day. After 21 days mice were secondly subcutaneous reimmunized with Freund's incomplete adjuvant emulsion concluding bovine type II collagen [20]. The mice were randomly divided into four groups of six, which were the normal control group, the CIA model group, cigarette smoke exposure CIA model group (for short: smoking group), and cigarette smoke exposure CIA model treated with DIM (for short: DIM group). For smoking and DIM group, the mice were exposed to the active smoke of commercially available cigarettes using an animal smoke modeling system on the day of the first immunization. Expose 4 cigarettes per day (each lasts 4-5 min) and blow air (4 L/min) over the burning cigarette intermittently to simulate the breathing cycle of human smoker and prevents CO asphyxiation. The mice smoked four cigarettes an hour a day, 5 days a week, until the end of the experiment [21, 22]. Meanwhile, immunized mice were administered with oral gavage of 100 μL 1% CMC-Na or DIM 40 mg/kg once per day consecutively.
Mice body weights were measured every 5 days and foot pad thickness were measured every 3 days. According to severity degree inflammatory symptom scores for paw arthritis from CIA mice are measured. Each mouse with four paws had an Inflammatory symptom score of up to 60 [23]. The mice were euthanized at day 80, and bone, spleen, liver and serum samples were collected. The fixed paws were applied to histopathology analysis and bone erosion evaluation by HE staining. Spleen and liver indices were calculated by weighing spleen and liver and fixed for HE staining. The serum samples were applied to the ELISA assay.The whole detail design was presented as Supplementary Fig. 1.
After isoflurane inhalation anesthesia, blood was collected from the orbital venous plexus of experimental mice, and serum was separated by centrifuging at 2000 × g for 10 min. The levels of inflammatory cytokines in serum were respectively detected with the mice IL-6, TNF-α, and IL-1β ELISA Kits (Jiangsu Meimian Industrial Co., Ltd, Jiangsu, China) according to the manufacturer's instructions.
The paws were removed from CIA model mice. After removing the muscle tissue, the paws were fixed in 4% paraformaldehyde, then decalcified with Roles-Bio Quick Decalcifying Solution (Roles-Bio, Guangzhou, China) for 1-2 days at room temperature. After decalcification, the tissues were dehydrated, processed, and then embedded in paraffin. Serial paraffin sections were stained with hematoxylin and eosin (H&E). Inflammatory scores ranging from 0 to 4 were used to evaluate changes such as synovial hyperplasia, inflammatory cells infiltration, pannus formation, and bone erosion [18, 24]. The evaluators did not know the grouping of the samples in advance and adopted the blind evaluation.
The spleen and liver were removed from all the mice and weighed at the end of the experiment. The spleen and liver indices were expressed as the ratio of spleen and liver wet weight to mice body weight (g/g), respectively. That is, organ index = organ wet weight (g)/animal body weight (g) × 100%. After fixed with 4% paraformaldehyde the spleen and liver were embedded into paraffin. The paraffins were cut into slices and stained with H&E for light microscopy. They were observed and scored according to 0-4 from no effect to severe effect [18, 25]. The evaluators did not know the grouping of the samples in advance and adopted the blind evaluation.
Mice were anesthetized and transected a 3 mm tail-tip for inducing tail bleeding assess hemostasis. Following transection, the tail was immersed in normal saline at 37 °C, and the interval time from bleeding to clotting and blood loss were recorded. The ratio of blood loss to mice body weight (μg/g) was calculated to evaluate platelet clotting function [26, 27]. No animal was allowed to bleed for more than 10 min.
After anesthetized mice cardiac blood collection was performed. The whole blood was gently mixed with 4% sodium citrate, then centrifuged at 200 × g for 10 min at 37 °C to obtain platelet-rich plasma (PRP). For avoiding platelet activation during operation, 1 μM prostaglandin E1 (PGE1) was added into PRP and centrifuged again at 800 × g for 10 min at 37 °C. The precipitates platelets were resuspended using HEPES Buffered Tyrode's Solution [28,29,30]. For evaluating platelet activation, the platelets were incubated with FITC-CD41and APC-CD62p for 30 min at 4 °C in dark, followed assay with Guava easyCyte™ Flowcytometry (Luminex, USA). The positive rate of CD62p expression in CD41-positive cells was counted [31, 32].
Reactive Oxygen Species (ROS) released from platelets was detected with ROS Assay Kit. Platelets were obtained from mice of different groups and incubated with 10 μM fluorescence probe 2,7-dichlorofluorescin diacetate (DCFH-DA) at 37 °C for 20 min in dark. Finally, the mean fluorescence intensity of FITC was measured using flow cytometry.
Platelets were obtained from mice of different groups and incubated with enhanced mitochondrial membrane potential assay kit with JC-1 at 37 °C for 20 min in dark. Relative degrees of mitochondrial polarization were quantified by measuring the red-shifted JC-1 aggregates on PE(Yellow-B) channel and the green-shifted monomers on FITC(Green-B) channel with flow cytometry.
CSE conditional medium preparation is described in reference to existing literature and improved [21, 33]. Take a cigarette with certain brand (about 14 mg of tar, 1.0 mg of nicotine in the smoke), pass the smoke into serum-free DMEM culture medium, and make a certain amount of CSE medium. After adjusted the pH value to 7.4 and filtered with 0.22 μM microporous filter for removing bacteria and large particles, 100% CSE stock medium was obtained. In order to maintain the quality and reproducibility of the prepared CSE, normalized 100% CSE medium prepared according to this standard was an absorbance of about 0.4 at 305 nm wavelength.
To evaluate the effect of DIM on the viability of platelet, LDH assay for platelet membrane damage were performed. The platelet suspensions were pre-incubated with DIM (0, 12.5, 25, 50, 100, 150, and 200 μM) for 15 min at 37 °C. LDH release was detected using the LDH Assay Kit (MedChemExpress, USA). The Cytotoxicity (%) was calculated according to kit instruction.
After obtained according to previous describe from C57BL/6 mice, the platelets suspension was pre-co-incubated with DIM at 37 °C for 15 min, then co-incubated with agonists, 20 μM ADP (Sigma-Aldrich, USA) and 5%CSE, for 15 min. Samples were incubated with FITC-CD41 and APC-CD62p for 30 min at 4 °C in dark. The CD41 and CD62p positive cells were counted with Flowcytometry. The platelet activation rate was calculated with CD62p positivity ratio in CD41-positive cells.
The platelet suspension was pre-co-incubated with DIM at 37 °C for 15 min, then co-incubated with agonists, 20 μM ADP and 5%CSE, for 15 min. Treated samples were incubated with Flou-4AMand solubility enhancer at 37 °C for 30 min in dark according to the Fluo-4 Calcium Assay Kit (Beyotime Biotechnology, shanghai, China) instruction manual. Then, the platelets were centrifuged at 800 × g for 10 min. The supernatant was discarded and sediment was resuspended using PBS. Green fluorescence from Fluo-4AM was detected by Flowcytometry.
Platelets obtained from C57BL/6 mice was mixed with an equal volume of HEPES Buffered Tyrode's Solution and equally distributed into 96-well microplates. DIM was added and incubated at 37 °C for 15 min. Then, 20 μM ADP and 5%CSE were added and incubated at 37 °C for 15 min. The optical density (OD) of each well at 490 nm was measured using a microplate reader. The OD was then detected again by shaking at room temperature for 30 min. The aggregation rate was calculated [28, 34].
The genes associated with the disease were collected through The National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov). Related genes of RA were collected by using the term "rheumatoid arthritis" and "platelet activation" and followed by filtering into the term "Homo sapiens" from NCBI. To obtain the genes associated with 3,3'-diindolylmethane (DIM), we used three ways, which are NCBI, PharmMapper(http://www.lilab-ecust.cn/pharmmapper/) and the Comparative Toxicogenomics Database (CTD, http://ctdbase.org/), and obtained a comprehensive DIM-related genes data. After drawing the Venn diagram, the common target genes from RA, platelet activation and DIM intersected were imported into STRING database (https://cn.string-db.org/) to query Protein-Protein Interaction Networks (PPIs). The organism was set to Homo sapiens, and only interaction scores >0.4 were selected. Cytoscape software was used to visualize and analyze the order of degree and combined score results. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of hub genes was performed using the ClusterProfiler package in R to the annotated proteins using p < 0.05 as the restriction. Additionally, the background species was defined as "Homo sapiens", and the genes were defined with their official gene symbols. The ggplot2 (3.3.6) package in R (4.2.1) software were used for statistical and visual analysis.
The chemical structure of DIM was downloaded from PubChem website (https://pubchem.ncbi.nlm.nih.gov/). The core domain of target proteins were obtained from PDB database (http://www.rcsb.org/) and removed water and phosphoric acid from proteins by using PyMOL software. Molecular docking was employed by AutoDockTools 1.5.6 software and followed with Vina script was conducted for calculating Molecular binding energy and displaying molecular docking results. The lower scores indicate the better binding affinity. Moreover, Discovery Studio 2019 was conducted for searching dock site and calculating docking LibDockScore. The output molecular docking results were imported into PyMOL software for 3D and 2D molecular docking conformation display for evaluating the reliability of analysis and prediction.
Platelet samples were lysed on ice by RIPA containing phenylmethyl sulfonyl fluoride (PMSF) and phosphorylase inhibitor cocktail. BCA kit was used to measure the protein concentration in the supernatant after lysis. All the protein samples were electrophoresed on SDS-PAGE gels and transferred onto PVDF membranes. The blots were incubated with the primary antibodies overnight at 4 °C. The next day, all blots were incubated with goat anti-mouse IgG or goat anti-rabbit IgG for 1 h at room temperature after PBST washing five times (each time for 5 min). The protein bands were exposed with Clarity™ Western ECL Substrate kit (Bio-Rad Laboratories, Shanghai, China) and measured by ChemiDoc XRS+ System (Bio-Rad Laboratories, Shanghai, China). Finally, the band density was quantified by Image J software.
Data from multiple experiments were presented as the mean ± standard deviation (SD). Statistical and simple linear regression analysis were performed using SPSS27.0 and Graphpad Prism 10.0 for Windows. The statistical difference comparisons (p values) between two groups were calculated using Student's t test and P values between more than three groups were calculated using one-way ANOVA followed by posthoc test. p < 0.05 was considered statistically significant.