Fig 1.
Sch B treatment improves liver function and ameliorates liver fibrosis in S. mansoni-infected mice.
(A) Representative images showing gross pathology of control, infected, or Sch B treated mice. Hepatosplenomegaly was observed in infected mice (liver, yellow arrows; spleen, red arrows). White spots seen on the surface of liver indicate granuloma nodules. Effect of Sch B on (B) body weight changes, (C) liver weight index (calculated by liver weight relative to body weight), and (D) spleen weight index (calculated by spleen weight relative to body weight). All results are shown as mean ± S.D. (n = 11–15). (E) ALT and (F) AST levels measured in the serum. Hemolyzed samples were excluded. All results are shown as mean ± S.D. (n = 6–9). (G) Representative images showing H&E staining of liver sections of the mice. Fibrotic areas were encircled by a yellow dotted line. (H) Fibrosis was evaluated by Ishak fibrosis scoring based on H&E staining. (I) Representative images of Masson’s trichrome staining on liver sections of the mice. Collagen were stained as blue which was encircled by a yellow dotted line. (J) Ishak fibrosis scoring based on Masson’s trichrome staining. (K) Representative images of Sirius red staining on liver sections of the mice. Collagen were stained as red and were encircled by a yellow dotted line. (L) Ishak fibrosis scoring based on Sirius red staining. For quantification in (G, I, and K), the number and the corresponding areas of each circled area were potted directly on the graph. Each dot represents one fibrotic or positively stained area. Quantification or scoring was performed on five (H&E staining and Masson’s trichrome staining) or three (Sirius red staining) slides in each group. 15 random microscopic fields were examined on each slide. Images are shown at 100× magnifications and scale bars correspond to 200 μm. * P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001, and **** P-value < 0.0001 compared with control group; # P-value < 0.05 compared with infected group.
Fig 2.
Sch B downregulates fibrotic genes and protein expression in S. mansoni-infected mice.
(A-H) RNA transcription levels of fibrotic markers including fibronectin, collagen I, α-SMA, TGF-β, TNF-α, MMP-2, MMP-9, and TIMP, measured by qPCR. Data are presented as mean ± S.D. (n = 9–11). (I) Representative western blot images showing protein levels of fibrotic markers. (J-O) Protein expression levels of fibrotic markers, relative to that of α-tubulin. Results are presented as mean ± S.D. (n = 5). (P) MMP-2 and MMP-9 activities were detected by gelatin zymography. The activities were detected as unstained bands on a blue background. M, markers; C, control group; I, infected group; S, Sch B-treated group. Two independent experiments were shown. (Q) The bands were quantified by densitometry using image J. Results are presented as mean ± S.D. (n = 3). * P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001, and **** P-value < 0.0001 compared with control group; # P-value < 0.05, and ### P-value < 0.001 compared with infected group.
Fig 3.
Sch B ameliorates inflammasome activation and inhibits pyroptosis in S. mansoni-infected mice.
(A-E) RNA transcription levels of inflammasomes markers, measured by qPCR. Data are presented as mean ± S.D. (n = 9–11). (F) Representative western blot images showing protein levels of inflammasomes markers. (G-K) Protein expression levels of inflammasomes markers, relative to that of α-tubulin. Results are presented as mean ± S.D. (n = 5). (L) Serum level of lactate dehydrogenase (LDH). Hemolyzed samples were excluded. Results are presented as mean ± S.D. (n = 6–9). * P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001, and **** P-value < 0.0001 compared with control group; # P-value < 0.05, ## P-value < 0.01, and ### P-value < 0.001 compared with infected group.
Fig 4.
Proliferation and inflammasome activation in RAW264.7 cells stimulated by S. mansoni soluble egg antigens.
RAW264.7 cells were treated with indicated concentration of soluble egg antigen (SEA) and Sch B. (A-B) 24- and 48- hour proliferation of RAW264.7 cells, measured by WST-1 assay. (C-G) mRNA expression of inflammasome component. All results are presented as the mean ± S.D. from three independent experiments. * P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001, and **** P-value < 0.0001 compared between groups.
Fig 5.
Sch B inhibits apoptosis in S. mansoni-infected mice.
(A) RNA transcription levels of apoptotic markers. Data are presented as mean ± S.D. (n = 9–11). (B) Representative western blot images showing protein levels of apoptotic markers. (C) Protein expression levels of apoptotic markers, relative to that of α-tubulin. Results are presented as mean ± S.D. (n = 5). (D) Representative plots showing Annexin V-FITC and PI double staining, measured by flow cytometry. Viable cells are shown in the bottom left quadrant (double negative); early apoptotic cells are shown in the bottom right quadrant (Annexin V positive and PI negative); late apoptotic cells are shown in the upper right quadrant (double-positive). (E) Percentages of viable cells, shown as the mean ± S.D. (n = 5). (F) Percentages of apoptotic cells are shown as the mean ± SD (n = 5). (G) Representative microphotographs of TUNEL staining, shown at 200× and 400× magnifications. Scale bar corresponds to 100 μm. (H) Quantification was performed on three slides in each group. Ten random microscopic fields were counted on each slide. * P-value < 0.05, *** P-value < 0.001, and **** P-value < 0.0001 compared with control group; # P-value < 0.05, ## P-value < 0.01, and ### P-value <0.001 compared with infected group.
Fig 6.
Sch B treatment resolves splenic inflammation and apoptosis in S. mansoni-infected mice.
(A) Representative images showing H&E staining of spleen sections of the mice at 100× and 400× magnification. Tingible body macrophages (yellow arrows) containing apoptotic bodies (blue arrows) were seen in the splenic white pulp of S. mansoni-infected mice. Fewer apoptotic bodies were observed after S. mansoni-infected mice were treated with Sch B. Scale bars correspond to 200 μm. RP, red pulps; WP, white pulps; C, central artery. (B-F) RNA transcription levels of inflammasome markers. (G-J) RNA transcription levels of apoptotic markers. Data are presented as mean ± S.D. (n = 8). (K) Representative plots showing Annexin V-FITC and PI double staining, measured by flow cytometry. (L) Percentages of apoptotic cells are shown as the mean ± S.D. (n = 5). * P-value < 0.05 and ** P-value < 0.01 compared with control group; # P-value < 0.05 compared with infected group.
Fig 7.
Sch B treatment regulates Th1 and Th2 immune responses in S. mansoni-infected mice.
(A, D, G, J, M) Concentrations of IL-2, IFN-γ, IL-4, IL-5, and IL-10 in the serum, measured by ELISA. (B, E, H, K, N) Relative mRNA expression levels of splenic cytokines, measured by qPCR. (C, F, I, L, O) Relative mRNA expression levels of liver cytokines, measured by qPCR. IL-2 and IFN-γ are Th1 cytokines; IL-4, IL-5, and IL-10 are Th 2 cytokines. All results are presented as mean ± S.D. (n = 5–11). * P-value < 0.05, ** P-value < 0.05, *** P-value < 0.001, and **** P-value < 0.0001 compared with control group; # P-value < 0.05 and ## P-value < 0.01 compared with infected group.
Fig 8.
Sch B provides damage to male S. mansoni.
(A-L) Representative SEM images showing the ultrastructural surface of adult S. mansoni worms isolated from the mice. (A-C) Male worms isolated from the control mice. (D-F) Male worms isolated from Sch B-treated mice. Ruptures (yellow arrows) were seen on the tubercles of the body surface. (G-I) Female worms isolated from the control mice. (J-L) Female worms isolated from Sch B-treated mice. No specific morphologic change was observed, compared with control worms. Scale bar, 1.0 mm for A, D, G, J (50× magnification, except for J: 35× magnification); 200 μm for B, E, H, K (200× magnification); 100 μm for C, F, I, L (500× magnification). (M) The number of worms recovered from the mice. (N) The number of eggs counted per gram of mouse liver. (O) The number of eggs counted per gram of mouse feces. Mann-Whitney U test was used to analyzed differences between groups. Data are presented as mean ± S.D. (n = 10). * P-value < 0.05 compared with control group; NS: no significance.