Fig 2: Western blot analysis. (A) Bands indicating the levels of the S1PR1, STAT3 and p-STAT3 proteins in the 5 groups. (B) Fold changes in the level of S1PR1 protein between the 5 groups. (C) Fold changes in the level of STAT3 protein between the 5 groups. (D) Fold changes in the level of p-STAT3 protein between the 5 groups. (E) Relative ratios of the levels of p-STAT3/t-STAT3 proteins. These results revealed that S1PR1 overexpression increased the protein levels of S1PR1, and the levels of p-STAT3 in the S1PR1 overexpression group were lower than those in the RHD group, and the silencing of STAT3 by STAT3-siRNA decreased the level of the STAT3 protein and the total amount of p-STAT3 in valve tissue. Data are presented as the mean ± standard deviation; #P<0.05 compared with the control group; *P<0.05 compared with the RHD group. S1PR1, sphingosine-1-phosphate receptor 1; STAT3, signal transducer and activator of transcription 3; RHD, rheumatic heart disease; t-STAT3, total-signal transducer and activator of transcription 3; p-STAT3, phosphorylated signal transducer and activator of transcription 3.

Fig 3: Inhibition of S1PR2 by JTE013 reduced NETs accumulation. (A) Representative images of CitH3+ (green) MPO+ (red) double-immunofluorescence staining NETs area in mouse TAD lesions from each group. (B) The JTE013 treatment diminished NETs density (n = 4). (C–E) Representative immunoblot and the corresponding analysis of mouse CitH3 and SPHK1 in each group (n = 6). (F,G) JTE013 treatment reduced serum CitH3 and S1P levels (n = 6). Scale: 50 µm. Data are expressed as means ± SD. CitH3, a specific marker of NETs; MPO, a marker for neutrophil abundance.

Fig 4: Immunohistochemical staining for S1PR1, STAT3 and p-STAT3. (A) Immunohistochemical staining for S1PR1, STAT3 and p-STAT3 in valvular tissues; magnification, ×400; scale bar, 50 µm. Arrows indicate positively stained cells. (B) Percentage of positive cells. (C) The IHS. (D) Ratio of p-STAT3/t-STAT3. These results revealed that S1PR1 was expressed at higher levels in the control group and S1PR1 overexpression group than the other 3 groups, and the levels of p-STAT3 in the S1PR1 overexpression group were lower than those in the RHD group, and the silencing of STAT3 by STAT3-siRNA decreased the level of the STAT3 protein and the total amount of p-STAT3 in valvular tissue. Data are presented as the mean ± standard deviation and the median and interquartile range; #P<0.05 compared with the control group. *P<0.05 compared with the RHD group. S1PR1, sphingosine-1-phosphate receptor 1; STAT3, signal transducer and activator of transcription 3; RHD, rheumatic heart disease; IHS, immunohistochemical score; t-STAT3, total-signal transducer and activator of transcription 3; p-STAT3, phosphorylated signal transducer and activator of transcription 3.

Fig 5: Levels of Th17 cell-related transcription factors and cytokines. (A) Immunohistochemical staining for ROR?t, IL-6 and IL-17 in valvular tissues; magnification, ×400; scale bar, 50 µm. Arrows indicate positively stained cells. (B) Percentage of positive cells. (C) The IHS. (D) ELISA of serum IL-6 and IL-17 levels. These results revealed that the levels of ROR?t, IL-6 and IL-17 were reduced in the S1PR1 overexpression group and STAT3-siRNA group. Data are presented as the mean ± standard deviation and the median and interquartile range. #P<0.05 compared with the control group; *P<0.05 compared with the RHD group. ROR?t, retinoic acid-related orphan receptor ?T; IL, interleukin; IHS, immunohistochemical score; RHD, rheumatic heart disease.