Glaucocalyxin B inhibits cartilage inflammatory injury in rheumatoid arthritis by regulating M1 polarization of synovial macrophages through NF-κB pathway

The inhibitory effect of Gla B on SMG M1 polarization

Mouse synovial macrophage (SMG) (Procell Life Science and Technology Co., Ltd., Wuhan, China) was cultured in complete medium at 37^oC with 5% CO₂. After cells reached to the logarithmic phase, cell viability was detected by trypan blue staining. Only cell viability over 80% was further incubated. Cells were divided into DMSO, LPS/IFN-γ (L/I) and Gla B groups. DMSO was used as the control group, and 100 ng/ml LPS (Sigma, USA) and 30 ng/ml recombinant IFN-γ protein (KALANG, USA) was used to induce M1 polarization in L/I group. After pretreatment with 5 μM and 15 μM Gla B for 4 h, LPS/IFN-γ was further added to induce M1 polarization.

(1) Detection of the proportion of F4/80⁺CD86⁺M1 cells by flow cytometry [11]: After LPS/IFN-γ treatment to induce M1 polarization for 48 h, SMG cells were centrifuged, washed with pre-cooled PBS twice, fixed with pre-cooled methanol, incubated with FITC-labeled CD86 monoclonal antibody and PE-labeled F4/80 monoclonal antibody (BD company, Massachusetts, USA) (10 μl) in dark for 20min and washed with PBS twice. The sample was finally resuspended in 50 μl liquid and subjected to flow cytometry. The results were shown in %.

(2) Detection cytokine expression by ELISA: After inducing polarization in SMG for 48 h, the supernatant was collected, centrifuged at 3000 r/min for 20 min and subjected to ELISA according to the manufacturer’s instruction (Abcam, Massachusetts, USA). In brief, the levels of M1 cell markers (including TNF-α, IL-1β, IL-6, iNOS and IL-12) were measured and the levels of M2 cell markers (including IL-10 and TGF-β1) were also detected. The standard curve method was used for calculation and the results were expressed in ng/ml.

(3) Detection of the expression changes of CD86 and P65 by immunofluorescence (IF): SMG cells were inoculated, induced by LPS/IFN-γ for 48 h, washed with PBS for three times, fixed with 4% formaldehyde at room temperature for 0.5 h, permeabilized with 0.2% Triton X-100 for 5 min, incubated with monoclonal antibodies against CD86 and P65 (Abcam, Massachusetts, USA) (dilution 1:300) at 4°C overnight, washed with PBS twice, incubated with fluorescent secondary antibody and mounted with 95% glycerol, followed by observation under fluorescence microscope.

(4) Detect the expression of ROS by DCFH-DA probe [12]: The DCFH-DA probe (Beyotime Biotechnology Co., Ltd., Shanghai, China) was used to detect the intracellular ROS. In brief, cells were seeded into a 6-well plate and subjected to drug intervention for 24 h. The medium was discarded. The DCFH-DA probe was diluted with serum-free medium at a ratio of 1:1000. And 1 ml of medium containing DCFH-DA probe was added to each well, followed by incubation for 30 min. After discarding the medium, cells were washed with serum-free medium twice. The cellular staining level was observed under a fluorescence microscope and the absorbance was detected with a fluorescence spectrophotometer.

(5) Detection of protein expression level by Western-Blot: After LPS/IFN-γ induction for 24 h, cells were collected, washed with pre-cooled PBS and lysed with NP-40 lysate (Beyotime Biotech Co., Ltd., Shanghai, China) on ice for 30 min. After centrifugation, the supernatant was collected and subjected to protein quantification by BCA method (Beyotime Biotechnology Co., Ltd., Shanghai, China). After adjusting protein concentration, the protein sample was subjected to SDS-PAGE electrophoresis, transferred to PVDF membrane, blocked with 5% skimmed milk powder for 2 h, incubated with primary monoclonal antibodies against P65 and p-P65 (Abcam, Massachusetts, USA) diluted in TBST, washed with TBST twice, incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (Abcam, Massachusetts, USA) (dilution 1:2000). After incubation, chemiluminescence method was used for visualization and Image Pro-Plus 6.0 software was used to analyze the optical density. GAPDH was used as the internal control, and the result was expressed as comparison of the optical density value between the target protein and the internal control protein.

Gla B inhibited SMG M1 polarization through NF-κB (P65)

The primer sequences for shRNA-P65 (pAd1566 plasmid) were as follows: the upstream primer: 5′-CACCGGACTACGACCTGAATGCTGTCGAAACAGCATTCAGGTCGTAGTCC-3′; the downstream primer: 5′-AAAAGGACTACGACCTGAATGCTGTCGAAACAGCATTCAGGTC′. The primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Cells were divided into L/I, L/I + shRNA-P65 and L/I + Gla B + shRNA-P65 groups. shRNA lentivirus transfection was transfected with Lipofectamine 2000. The level of P65 was detected after transfection for 48 h. Gal B of 15 μM was used. The proportion of M1 type cells and the markers of M1/M2 type cells were detected according to the above method.

The effect of M1 SMG cells on synovial cell injury

To clarify the injury of M1 SMG on synovial cells, the culture medium from SMG and M1-SMG cells was centrifuged at 3000 r/min for 20 min, which was further used to culture mouse cartilage cells (Procell Life Science and Technology Co., Ltd., Wuhan, China). Control, SMG and M1-SMG groups were set.

(1) Detection of apoptotic level by flow cytometry: Cells were collected, washed with pre-cooled PBS, centrifuged at 3000 rpm/min for 30 min, suspended in binding buffer and staining by using apoptosis detection kit (BD Company, Massachusetts, USA). In brief, cell suspension was incubated with 5 μl of Annexin V-FITC in dark for 5 min, further incubated with 5 μl of PI staining in dark for 5 min, washed with PBS and finally subjected to flow cytometry. Cells with Annexin V-FITC (+) PI (+) and Annexin V-FITC (+) PI (−) were defined as apoptotic cells.

(2) The expression levels of Bcl-2, Bax, and Caspase-3 in cells were detected according to the above method.

(3) The expression level of Caspase-3 in cells was measured according to the above method.

Gla B inhibited cartilage injury and M1 polarization of macrophages in RA mice

A total of 40 BALB/c mice (8–10 weeks old) were randomly divided into Control, RA and Gla B groups. Mice were intraperitoneally injected with collagen type II monoclonal antibody complex and lipopolysaccharide (LPS) to construct RA model. To be specific, mice were intraperitoneally injected with collagen type II monoclonal antibody complex (5 mg/kg/d) for 10 days. Meanwhile, mice were simultaneously intraperitoneally injected with 100 μg LPS on the first and fourth days. Thus, a stable collagen antibody-induced RA was established in approximately two weeks [13]. Mice in the Gla B group were administered with Gla B (10 μg/ml and 20 μg/ml) by gavage, while mice in the Control and RA groups were treated with equal amounts of normal saline.

(1) Arthritis score measures [14]: During the 10-day period of RA modeling and 5 days of continued culture (15 days in total), arthritis score was measured every 3 days (5 times in total). The macroscopic scoring system was used to evaluate joint inflammation in mice. The criteria were as follows: 11–15 points: the entire feet and toes showed severe arthritis; 6–10 points: more than two joints showed severe arthritis; 1–5 points: two joints showed inflammation; 0 point: no arthritis.

(2) ELISA: Mice were kept for 5 days after model construction. After protein extraction of joint tissue in mice, the expression of TNF-α, IL-1β, IL-6 and iNOS was determined.

(3) H and E staining of mouse joint tissue: The joint tissue was fixed with 4% paraformaldehyde (PFA), dehydrated, transparent, embedded in wax and sliced. According to the conventional experimental procedures of H and E staining, the paraffin-embedded sections were deparaffinized, hydrated, stained, dehydrated, permeabilized and mounted, followed by photography under microscope.

(4) Detection of the expression of CD86 and P65 by immunohistochemistry (IHC): The joint tissue was fixed with 4% PFA, embedded in paraffin, sliced, soaked in 1:50 acetone solution for 3 min, dried, soaked in xylene, treated with gradient ethanol, subjected to antigen retrieval in 0.01 mol/L citrate buffer, treated with 3% hydrogen peroxide for 10–15 min to eliminate endogenous peroxidase, blocked with 5% bovine serum albumin (BSA) at 37°C for 15–30 min, incubated with monoclonal antibodies against CD86 and P65 (Abcam, Massachusetts, USA) at 37°C, incubated with the secondary antibody and visualized with DAB. The slices were counterstained with hematoxylin, soaked in gradient ethanol, dehydrated, transparent and sealed with resin.

(5) Safranin O-fast green staining: The paraffin-embedded slices were deparaffinized, sequentially immersed in xylene I for 20 min, xylene II for 20 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 75 % alcohol for 5min and washed with tap water. The slices were stained with Fast green staining for 5–10 min, dried to discard excessive dying solution until the cartilage was colorless, soaked in differentiation solution and washed in tap water. For Safranin staining, the slices were stained with safranin staining solution for 15–30 s and dehydrated quickly with absolute ethanol. The slices were transparent with xylene for 5 minutes, mounted with neutral gum, observed and photographed under microscope.

Molecule-protein docking model of Gla B and P65

The receptor protein, P65 (PDB ID: 2LWW), was retrieved from the Protein Data Bank database. The following parameters were set: the appropriate box center of P65 receptor protein (center_x = 83.126, center_y = 96.417, center_z = 90.612) and box grid parameters (size_x = 50, Size_y = 60, size_z = 52) to include the active pocket sites that small molecule ligands might bind to. Molecular docking was performed between P65 receptor protein and Gla B ligand small molecules (AutoDock Vina 1.1.2). PyMOL was used to visualize the hydrogen bonding between the receptor protein and the small ligand molecule in three-dimensional images. And Ligplus software was utilized to show the hydrophobic interaction between the receptor protein and the small ligand molecule in two-dimensional images.

Validation of the targeted-binding relationship between Gla B and P65 by Pull-down assay

In brief, 15μg of recombinant protein was combined with Biotin-labeled Gla B (Biotin-Gla B). Recombinant protein G magnetic beads were incubated with P65 antibody. After washing with Tris buffer, P65 was detected by Western-Blot accordingly, followed by biotin detection by the horseradish peroxidase-conjugated antibiotin antibody (CST, Boston, USA).

Statistical analysis

The SPSS 20.0 software was used for statistical analysis. The measurement data were expressed as mean ± standard deviation $(\overline{x}\pm \text{ s})$. One-way ANOVA was used for comparison between multiple groups, and SNK test was used for comparison between groups. A P value < 0.05 indicated statistical significance.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethical approval and consent to participate

The study approved with Ethics Committee.

Glaucocalyxin B inhibited SMG M1 polarization

In our study, F4/80 and CD86 were used to label M1 macrophages. LPS and IFN-γ can induce SMG M1 polarization, with the cell proportion over 80%. Gla B treatment could inhibit M1 polarization and the ratio of F4/80 + CD86 + M1 cells was significantly down-regulated, which was lower than L/I (Figure 1A–1B). The detection of M1 cell marker protein found that the levels of TNF-α, IL-1β, IL-6, iNOS and IL-12 were significantly up-regulated after SMG M1 polarization; and Gla B could inhibit the level of cytokines, which was lower than L/I group (Figure 1C–1F, 1I). The expression of M2 cell marker protein (including TGF-β1 and IL-10) was significantly changed, which was independent of Gla B (Figure 1G–1H). As a marker of M1 cell, ROS was up-regulated in L/I (1.98 ± 0.12), which was significantly higher than DMSO (0.07 ± 0.01); and Gla B could inhibit the level of ROS (1.10 ± 0.55) and (0.44 ± 0.07) (Figure 1J).

IF staining showed that the levels of CD86 and P65 were significantly up-regulated in M1 SMG, which were higher than those in DMSO group. Gla B could inhibit the expression of P65 and CD86, with lower fluorescence intensity than that of L/I group (Figure 2A–2B). Protein detection also revealed that the levels of P65 and p-P65 were up-regulated in M1 SMG, and Gla B could inhibit the expression of P65 and p-P65 (Figure 2C–2D).

The role of NF-κB (p65) in Glaucocalyxin B-induced inhibition of SMG M1 polarization

After P65 silencing by shRNA, SMG M1 polarization was induced, followed by the treatment of 15μM Gla B. As a result, P65 silencing could inhibit SMG M1 polarization, which was independent of Gla B. Gla B had no significant effect in shRNA cells, without difference in the proportion of M1 cells (Figure 3A–3B). The detection of cytokines also revealed no significant differences in the levels of TNF-α, IL-1β, IL-6, iNOS and IL-12 between the groups; and no significant difference in the expression of TGF-β1 and IL-10 (Figure 3C–3I). The detection of ROS revealed no significant difference between L/I + shRNA (0.33 ± 0.08) and L/I + shRNA + Gla B (0.31 ± 0.06), which was lower than the L/I group (1.87 ± 0.43), (Figure 3J).

M1 SMG induced cartilage cell injury

To investigate the possible injury of M1 SMG on cartilage cells, SMG or M1-SMG was co-cultured with cartilage cells. As a result, SMG had no obvious injury on cartilage cells, with a low apoptotic level; M1-SMG could promote cartilage cell injury and increase apoptotic level (Figure 4A–4B). Caspase-3 fluorescent staining showed that Caspase-3 was barely expressed in Control and SMG, while M1-SMG could enhance the expression of Caspase-3 and promote (Figure 4C). Detection of apoptotic proteins also found that M1-SMG promoted the expression of Bax and Caspase-3, with significantly higher relative protein expression level than that of Control and SMG (Figure 4D–4E).

The effect of Glaucocalyxin B on cartilage injury in RA mice

Collagen antibodies and LPS were combined to construct RA mouse model. The clinicopathological score of RA mice was significantly increased over time, which was significantly different from Control. H and E staining and safranin-O-Green staining showed obvious injury in cartilage, obvious inflammatory response in the cartilage tissue and significant apoptosis, showing abnormal characteristics of cartilage tissue. While the tissue structure was intact in Control group, without no obvious inflammation or injury, which were significantly different from RA mice. Gla B intervention could decrease the pathological score of mice. And the score of the same time period was significantly lower than that of RA (Figure 5B). Gla B could also attenuate cartilage injury in RA mice. The staining results showed that Gla B inhibited cartilage inflammation and bone destruction, which was significantly different from RA in pathology (Figure 5A).

The staining of CD86 and P65 showed no obvious CD86 expression, relatively low infiltration level of M1 cells and relatively low level of P65 in Control group. The expression of CD86 and P65 was significantly up-regulated in RA, which was significantly different from Control group; with obvious infiltration of M1 cells. Gla B could inhibit the levels of CD86 and P65, which was significantly different from RA (Figure 6A). The detection of M1 cell marker protein showed that the levels of TNF-α, IL-1β, IL-6 and iNOS were significantly up-regulated in RA, which were higher than those of Control. Gla B inhibited the expression of TNF-α, IL-1β, IL-6 and iNOS, which was significantly lower than that of RA group (Figure 6B–6E).

Validation of Glaucocalyxin B targeting P65

Molecular docking revealed a targeted binding relationship between Glaucocalyxin B and the pocket of P65 protein. The binding of Glaucocalyxin B could inhibit the phosphorylation and isomerization of P65, thereby inhibiting the activation of NF-κB. The results showed that Glaucocalyxin B had hydrogen bonds with Glu, Ser and Met. The binding energy was -8.4 kcal/mol. According to the results of docking, Gla B could bind to the protein pocket of P65 to exert the inhibitory effect of P65.

Pull-down assay also showed that recombinant P65 protein could bind to Gla B, Biotin-labeled Gla B could bind to P65, with positive expression of Biotin. Taken together, the results of molecular docking and pull-down assay showed that Gla B had a binding relationship with P65 (Figure 7).