Methyl salicylate 2-O-β-d-lactoside alleviates the pathological progression of pristane-induced systemic lupus erythematosus-like disease in mice via suppression of inflammatory response and signal transduction

Figures & data

Figure 1 Chemical structure of methyl salicylate 2-O-β-d-lactoside.

Figure 2 Sera of BALB/c mice in each group were collected every month after intravenous injection of pristane, and relative levels of the autoantibodies were detected by ELISA.

Notes: (A) Type IgG of anti-DNA; (B) type IgM of anti-DNA; (C) type IgG of anti-Sm; (D) type IgM of anti-Sm; (E) type IgG of antihistone; (F) type IgM of antihistone. The data represent mean ± SEM (n=8–12). *P<0.05, **P<0.01, significant compared with the control group; ^#P<0.05, ^##P<0.01, ^###P<0.001, significant compared with the model group.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean; dsDNA, double-stranded DNA; MSL, methyl salicylate 2-O-β-d-lactoside.

Figure 3 Total IgG and cytokines in peripheral circulatory system.

Notes: Levels of total IgG (A) in the serum collected every month after induction, levels of IL-6 (B) in the serum collected 1 month later, and levels of IL-17A (C), ICAM1 (D), and MCP1 (E) in the serum collected 6 months later were detected by ELISA. Data represent mean ± SEM (n=8–12). **P<0.01, ***P<0.001, significant compared with the control group; ^#P<0.05, ^##P<0.01, significant compared with the model group.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean; MSL, methyl salicylate 2-O-β-d-lactoside.

Figure 4 Behavioral experiments.

Notes: (A) Incidents of spontaneous activity; (B) motor-coordination abilities of the BALB/c mice were assessed by stationary and Rota rod tests. Data represent mean ± SEM (n=8–12). **P<0.01, significant compared with the control group; ^#P<0.05, ^##P<0.01, significant compared with the model group.
Abbreviations: SEM, standard error of the mean; MSL, methyl salicylate 2-O-β-d-lactoside; Rota, rotation.

Figure 5 Splenomegaly and pathological examination.

Notes: (A) Spleens from BALB/c mice in each group; (B) spleen index; (C) spleens stained with H&E for histological examination (red arrow indicates red pulp); (D) spleens stained with CD68 mAb for immunohistochemistry assay (red arrows indicate positive cells). Control group (a, ×400), model group (b, ×400), MSL-L group (c, ×400), MSL-M group (d, ×400), MSL-H group (e, ×400), prednisone group (f, ×400). Data represent mean ± SEM (n=12). ***P<0.01, significant compared with the control group; ^##P<0.01, significant compared with the model group.
Abbreviations: H&E, hematoxylin and eosin; mAb, monoclonal antibody; MSL, methyl salicylate 2-O-β-d-lactoside; SEM, standard error of the mean.

Figure 6 Progression of arthritis in BALB/c mice from each group.

Notes: (A) Apparent phenomena of arthritis: normal foreleg (a), arthritis-like foreleg (b), arthritis-like hind leg (c), normal hind leg (d). (B) X-ray diffraction patterns of arthritis: arthritis-like hind leg (a), normal hind leg (b), arthritis-like foreleg (c), normal foreleg (d); arrowheads indicate the swelling degree of the arthritis. (C) Survival curve of mice without severe arthritis (score ≥3). (D) Hind-paw volumes of BALB/c mice in each group. (E) Arthritis scores of BALB/c mice in each group. (F) X-ray diffraction patterns of hind legs of BALB/c mice in each group (arrows indicate the malformation of the arthritis). Data represent mean ± SEM (n=12). **P<0.01, ***P<0.001, significant compared with the control group; ^#P<0.05, ^##P<0.01, ^###P<0.001, significant compared with the model group.
Abbreviations: SEM, standard error of the mean; MSL, methyl salicylate 2-O-β-d-lactoside.

Figure 7 Examination for renal function and pathology.

Notes: (A, B) Kidneys were stained with H&E and PAS, respectively, for histological examination: control group (a, ×400), model group (b, ×400), MSL-L group (c, ×400), MSL-M group (d, ×400), MSL-H group (e, ×400), and prednisone group (f, ×400). (C) Creatinine levels in serum from each group. (D) Ratio of total protein to creatinine in urine from each group. (E) Deposition of total IgG in kidneys of BALB/c mice in each group was observed under fluorescence microscopy. Images (×200), from top to bottom, represent the nucleus, total IgG, and merged images with the total IgG and the nucleus. The IgG was stained with red fluorescent dye (red, exposure 100 milliseconds), and the nuclei were stained by DAPI (blue, exposure 30 milliseconds). (F) Deposition of C3 in kidneys of BALB/c mice in each group was observed under confocal laser-scanning microscopy. Images (×400), from top to bottom, represent the nucleus, C3, and merged images with the C3 and the nucleus. The C3 was stained with green fluorescent dye and the nuclei stained by DAPI. (G, H) Fluorescence intensity of total IgG and complement C3 in kidneys of BALB/c mice in each group was analyzed and calculated by ImageJ (version Image-Pro Plus 6.0; Media Cybernetics, Inc., Rockville, MD, USA). Data represent mean ± SEM (n=6–8). *P<0.05, ***P<0.001, significant compared with the control group; ^#P<0.05, ^##P<0.01, significant compared with the model group.
Abbreviations: H&E, hematoxylin and eosin; PAS, periodic acid–Schiff; MSL, methyl salicylate 2-O-β-d-lactoside; DAPI, 4′,6-diamidino-2-phenylindole; SEM, standard error of the mean.

Figure 8 Relative expression levels of TNFα (A), IL-6 (B), IL-17A (C), and MCP1 (D) in renal tissues were detected by ELISA.

Notes: Data represent mean ± SEM (n=6–8). *P<0.05, **P<0.01, ***P<0.001, significant compared with the control group; ^#P<0.05, ^##P<0.01, significant compared with the model group.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean; MSL, methyl salicylate 2-O-β-d-lactoside.

Figure 9 Inflammatory pathway in renal tissues of the BALB/c mice in each group.

Notes: (A, D) Western blot assays for JAK–STAT3 and NFκB signaling, respectively. (B, C) Quantitative analysis for Western blot assays of JAK–STAT3 signaling pathways. The bands for p-JAK, p-STAT3, and p-Erk were normalized to total JAK, total STAT3 and total Erk, while the bands for total JAK, total STAT3, and total Erk were all normalized to GAPDH. (E, F) Quantitative analysis for Western blot assays of NFκB-signaling pathways. The bands for p-IKK, p-IκBα, and p-NFκB were normalized to total IKK, total IκBα, and total NFκB, while the bands for total IKK, total IκBα, and total NFκB were all normalized to GAPDH. Data represent mean ± SEM (n=5–6). **P<0.01, ***P<0.001, significant compared with the control group; ^#P<0.05, ^##P<0.01, significant compared with the model group.
Abbreviations: SEM, standard error of the mean; MSL, methyl salicylate 2-O-β-d-lactoside.

Table S1 The information regarding the main antibodies used in this study

Antibody	Dilution	Source
Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb 2697	1:1,000	Cell Signaling Technology (CST)
IKKα (3G12) Mouse mAb 11930	1:2,000	CST
IKKβ (D30C6) Rabbit mAb 8943	1:2,000	CST
Phospho-IκBα (Ser32) (14D4) Rabbit mAb 2859	1:1,000	CST
IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) 4814	1:1,000	CST
Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb 3033	1:1,000	CST
NF-κB p65 (D14E12) XP^® Rabbit mAb 8242	1:2,000	CST
Phospho-Stat3 (Tyr705) (D3A7) XP^® Rabbit mAb	1:1,000	CST
Stat3 (124H6) Mouse mAb #9139	1:2,000	CST
Phospho-Jak1 (Tyr1022/1023) Antibody #3331	1:1,000	CST
Jak1 (6G4) Rabbit mAb #3344	1:2,000	CST
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP^® Rabbit mAb	1:1,000	CST
p44/42 MAPK (Erk1/2) Antibody	1:2,000	CST
GAPDH mouse mAb	1:2,000	Santa Cruz Biotechnology
Anti-CD68 antibody (ab955)	1:200	Abcam
Anti-rabbit IgG, HRP-linked Antibody 7074	1:2,000	CST
Anti-mouse IgG, HRP-linked Antibody 7076	1:2,000	CST
Anti-C3 antibody (ab11887)	1:10	Abcam
Anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor^® 647 Conjugate) #4410	1:1,000	CST
Anti-rabbit IgG (H+L), F(ab′)2 Fragment (Alexa Fluor^® 488 Conjugate) #4412	1:1,000	CST