Fig 1.
Enhanced expression of ETRs in liver tissues of patients with schistosome-induced hepatic fibrosis.
(A) Paraffin-embedded sections of liver tissues from patients were stained with H&E, Masson’s trichrome and Sirius Red. Scale bar, 200 μm. (B) The positive staining areas for Sirius Red staining were measured using IPP software (n = 4). (C) Representative immunohistochemical staining of ETAR and ETBR. Black arrows indicate the ETRs positive cells. Scale bar, 100 μm. (D-H) Total RNA was extracted for qPCR analysis of ET-1, ETAR, ETBR, Col1α1 and Col3α1 expression levels. Cont (n = 8), CS (n = 10), CHB (n = 15). (I-K) ETAR and ETBR proteins were determined by western blotting. Image density was quantified using Image J analysis and normalized to GAPDH (n = 7). Data are represented as the median and interquartile range. All data are representative of at least two independent experiments. Significance was calculated using Kruskal–Wallis with Dunn’s posttest (B, D-H, J-K). Abbreviation: Cont, control; CS, chronic schistosomiasis; CHB, chronic hepatitis B.
Fig 2.
Enhanced expression of ETRs in spleen tissues of patients with schistosome-induced hepatic fibrosis.
(A) The human spleen tissues were stained with H&E and Masson’s trichrome. Scale bar, 200 μm. (B-G) qPCR analysis of the expression levels of ET-1, ETAR, ETBR, Col1α1, Col3α1 and IL-10 in spleen samples. Cont (n = 9), CS (n = 12), CHB (n = 13). (H) The IL-10 concentration in spleen tissue homogenates was determined by ELISA. Cont (n = 9), CS (n = 12), CHB (n = 13). (I) Representative immunohistochemical staining of ETAR, ETBR, collagen I and collagen III. Black arrows indicate the ETRs, collagen I and collagen III positive cells. Scale bar, 100 μm. (J) Representative immunofluorescence staining of ETAR, ETBR and CD20+ B cells in human spleen tissues. Insets show a higher magnification of the outlined area. Yellow arrows denote positive cells. Scale bar, 50 μm. (K) The fluorescence intensity was measured using IPP software (n = 4). (L-N) ETAR and ETBR proteins were determined by western blotting, quantified using Image J analysis, and normalized to GAPDH (n = 7). Data are represented as the median and interquartile range from two independent experiments. Multiple comparisons were performed by Kruskal–Wallis with Dunn’s posttest (B-H, M-N) or the Mann-Whitney U-test (K). *P < 0.05. Abbreviation: Cont, control; CS, chronic schistosomiasis; CHB, chronic hepatitis B; RP: red pulp; WP: white pulp.
Fig 3.
ETAR and ETBR were correlated with fibrogenesis.
(A-B) Portal vein and spleen thickness diameter in patients who underwent splenectomy. Cont (n = 9), CS (n = 12), CHB (n = 13). (C-D) Correlations between mRNA levels of ETAR and ETBR with portal vein diameter in CS patients (n = 12); (E-F) Correlations between mRNA levels of ETAR and ETBR with spleen thickness diameter in CS patients (n = 12). Data are represented as the median and interquartile range (A-B). Significance was calculated using Kruskal–Wallis with Dunn’s posttest (A-B) or Spearman rank test (C-F). Abbreviation: Cont, control; CS, chronic schistosomiasis; CHB, chronic hepatitis B.
Fig 4.
Analysis of ETRs expression in murine schistosomiasis.
(A) Micrographs of livers and spleens from different weeks after infection. Scale bar, 1 cm. (B) H&E, Masson’s trichrome and Sirius Red staining of liver sections. Scale bar, 200 μm. (C) Collagen content in livers determined as hydroxyproline content (n = 6). (D) Granuloma size measured from H&E-stained liver sections(n = 6). (E) Fibrosis scores measured from Masson’s trichrome staining of liver sections (n = 6). (F) Positive staining areas for Sirius Red were measured using IPP software (n = 6). (G) H&E and Masson’s trichrome staining of spleen sections. Insets show a higher magnification of the outlined area. Black arrows indicate the Trabeculae. Scale bar, 200 μm. (H) Representative immunohistochemical staining for ETAR and ETBR in the infected spleens. Insets show a higher magnification of the outlined area. Black arrows indicate the ETRs positive cells. Scale bar, 100 μm. (I-N) The expression of ET-1, ETAR, ETBR, Col1α1, Col3α1 and IL-10 in spleens during infection was detected by qPCR (n = 3–6). (O) The IL-10 concentration in spleen tissue homogenates was determined by ELISA (n = 6). (P-R) ETAR and ETBR proteins were determined by western blotting, quantified using Image J analysis, and normalized to GAPDH (n = 5). Data are represented as mean ± SEM. All data are representative of at least three independent experiments. Significance was determined by the two-tailed Student’s t test (C-F, I-O, Q-R). *P < 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, compared with 0W samples (C, F, I-O, Q-R) and compared with 4W samples (D-E).
Fig 5.
Endothelin receptor antagonists mediated reduction of hepatocellular damage.
Mice were infected percutaneously with 16 S. japonicum cercariae or remained uninfected. At 6 weeks post-infection, all infected mice were treated with praziquantel to kill the parasites and then received either endothelin receptor antagonists or vehicle and were necropsied at 12 weeks post-infection. (A) Time schedule for parasite infection and administration of anti-parasite drug or endothelin receptor antagonists and sample withdrawal. (B) Macrograph of livers and spleens from uninfected mice, infected mice and infected mice treated with endothelin receptor antagonists. Scale bar, 1 cm. (C) H&E, Masson’s trichrome and Sirius Red staining of liver sections. Scale bar, 200 μm. (D-E) Serum ALT and AST levels were measured (n = 5–7). (F-G) Liver and spleen indexes were determined (n = 7). (H) Measurement of hepatic portal venous pressure in vivo by RM6240BD. (I) Statistical analysis of hepatic portal vein pressure (n = 7). (J) Analysis of the portal vein diameter in vivo (n = 7). (K) Collagen content in livers determined as hydroxyproline content (n = 6). (L) Granuloma size measured based on H&E staining of liver sections (n = 7). (M) Fibrosis scores measured based on Masson’s trichrome staining of liver sections (n = 7). (N) The positive staining areas for Sirius Red were measured using IPP software (n = 7). (O) Representative immunohistochemical staining for ETAR, ETBR, collagen I and collagen III in infected livers. Black arrows indicate the ETRs, collagen I and collagen III positive cells. Scale bar, 100 μm. (P-T) qPCR analysis of the expression levels of ET-1, ETAR, ETBR, Col1α1 and Col3α1 in liver samples (n = 6). (U-W) ETAR and ETBR proteins were determined by western blotting. 1, uninfected; 2, infected; 3, infected + vehicle; 4, infected + BQ-123; 5, infected + BQ-788; 6, infected + BQ-123 + BQ-788. Image density was quantified using Image J analysis and normalized to GAPDH (n = 5). Data are represented as mean ± SEM of three independent experiments. Multiple comparisons were performed by one-way ANOVA with Tukey’s correction for comparison between two groups (D-G, I-N, P-T, V-W). Abbreviation: ALT: alanine aminotransferase; AST: aspartate aminotransferase.
Fig 6.
Endothelin receptor antagonists attenuated schistosomiasis-induced splenic fibrosis in mice.
(A) H&E and Masson’s trichrome staining of spleen sections. Scale bar, 200 μm. (B) Representative immunohistochemical staining for ETAR and ETBR in infected spleens. Insets show a higher magnification of the outlined area. Black arrows indicate the ETRs positive cells. Scale bar, 100 μm. (C-H) The expression levels of ET-1, ETAR, ETBR, Col1α1, Col3α1 and IL-10 in spleens were determined by qPCR (n = 7). (I) The IL-10 concentration in spleen tissue homogenates was determined by ELISA (n = 6). (J-L) ETAR and ETBR proteins were determined by western blotting. 1, uninfected; 2, infected; 3, infected + vehicle; 4, infected + BQ-123; 5, infected + BQ-788; 6, infected + BQ-123 + BQ-788. Image density was quantified using Image J analysis and normalized to GAPDH (n = 5). Data are represented as mean ± SEM of three independent experiments. Multiple comparisons were performed by one-way ANOVA with Tukey’s correction for comparison between two groups (C-I, K-L).
Fig 7.
Endothelin receptor antagonists inhibited B cell activation.
(A) Representative immunofluorescence staining of ETAR, ETBR and CD20+ B cells in mouse spleen tissues. Insets show a higher magnification of the outlined area. Yellow arrows denote positive cells. Scale bar, 50 μm. (B) The fluorescence intensity was measured using IPP software (n = 4). (C-J) CD19+ MACS-isolated mouse splenic B cells were restimulated with SEA (20 μg/ml) for 2 days. (C-D) Representative FACS plots and a summary of the intracellular IL-10 expression of B cells after addition of Brefeldin A to the last 4 hours of the culture (n = 7). (E-F) Mean fluorescence intensity of CD40 and CD86 expression (n = 7). (G-J) IL-10, IgG, IgM and TGF-β1 concentration in culture supernatants as determined by ELISA (n = 6–8). (K-N) SEA-restimulated B cells were co-cultured for 4 days with CD25- depleted CD4 T cells. (L-M) The frequency of CD25+Foxp3+ Treg cells after co-culture, as shown in a representative FACS plots and summarized (n = 7). (N) IL-10 concentration in culture supernatants after co-culture (n = 3). Data are represented as mean ± SEM of three independent experiments. Significance was calculated using the two-tailed Student’s t test (B) or one-way ANOVA with Tukey’s correction for comparison between two groups (D-J, M-N). ns, not significant; **P< 0.01.
Fig 8.
Endothelin receptor antagonists decreased CD4+CD25+Foxp3+ T cells in mouse spleen tissues.
(A) Representative immunohistochemical staining for Foxp3 in infected livers and spleens. Insets show a higher magnification of the outlined area. Black arrows indicate the Foxp3+ positive cells. Scale bar, 100 μm. (B-C) The frequency of CD25+Foxp3+ Treg cells in mouse splenocytes, as shown in a representative FACS plots and summarized (n = 7). Data are represented as mean ± SEM of three independent experiments. Multiple comparisons were performed by one-way ANOVA with Tukey’s correction for comparison between two groups (C).
Table 1.
Patients’ demographic characteristics.
Table 2.
Primer sequences used in this study.