Fig 1.
Sja-let-7 derived from S. japonicum worms involves in the activation of HSCs.
(A) Workflow of transwell systems establishment. (B-C) Detection of 8 Sj-miRNAs in the LX-2 cells from the transwell system composed by worms coming from BALB/c mice (n = 3) and KM mice (n = 6) (D-E) Detection of α-SMA, Col1α1 and Col3α1 mRNA expression of the LX-2 cells from the transwell system composed by worms coming from BALB/c mice (n = 3) and KM mice (n = 6). (F-G) Detection of 11 Sj-miRNAs in the LX-2 cells from the transwell system composed by MM worms coming from BALB/c mice (n = 3) and KM mice (n = 6). (H-K) Treatment efficiency analysis and detection of α-SMA, Col1α1 and Col3α1 mRNA expression towards LX-2 cells from the transwell system composed by worms coming from BALB/c mice (n = 3). (L-O) Treatment efficiency analysis and detection of α-SMA, Col1α1 and Col3α1 mRNA expression towards LX-2 cells from the transwell system composed by worms coming from KM mice (n = 3). All graph data are expressed as the mean ± SD of at least three biological replicates per group. *P< 0.05, **P< 0.01, ns, not significant. Abbreviation: Sj: S. japonicum; MM: mated male. Panel A was created with Biorender.com.
Fig 2.
S. japonicum worm-derived EVs transfer sja-let-7 into HSCs and reduced their activation.
(A) TEM analysis for SjEVs. Black arrows indicate the cup-shape EVs. Scale bar, 200 nm. (B) NTA analysis for SjEVs. (C) Microscopy image of PKH67-labelled SjEVs (green) incubated with LX-2 cells for 2 h. White arrows indicate SjEVs labelled with PKH67. Scale bar, 25 μm. (D) Detection of α-SMA, Col1α1 and Col3α1 mRNA expression of the LX-2 cells (n = 3). (E) Detection of sja-let-7 in the LX-2 cells (n = 3). (F) Detection of α-SMA, Col1α1 and Col3α1 mRNA expression of the LX-2 cells (n = 3). All graph data are expressed as the mean ± SD of at least three biological replicates per group. *P< 0.05, **P< 0.01, ns, not significant. Abbreviation: Sj: S. japonicum; TEM: transmission electron microscope; NTA: nanoparticle tracking analyses; NC: negative control.
Fig 3.
Sja-let-7 reduces the activation of HSCs through Col1α2/TGF-β/Smad axis in vivo.
(A-D) Treatment efficiency analysis and detection of α-SMA, Col1α1 and Col3α1 mRNA expression towards LX-2 cells after treated with NC or sja-let-7 mimics for 48 h (n = 3). (E) The binding site of sja-let-7 on the 3′-UTR of M. musculus and Homo sapien Col1α2. (F) Results of the dual-luciferase reporter assay (n = 3). (G-J) Detection of Col1α2, Smad2, Smad3 and Smad7 mRNA expression of the LX-2 cells after treated with NC or sja-let-7 mimics (n = 3). All graph data are expressed as the mean ± SD of at least three biological replicates per group. *P< 0.05, **P< 0.01, ns, not significant. Abbreviation: NC: negative control; UTR: untranslated region; WT: wild type; MUT: mutant.
Fig 4.
FISH analysis on the liver section.
White arrows indicate the cells that co-located with sja-let-7 and Col1α2. Scale bar, 50 μm. FISH: fluorescence in situ hybridization.
Fig 5.
Sja-let-7 attenuates the fibrotic progression of schistosome-induced liver fibrosis.
(A) Establishment timeline of schistosome-induced liver fibrosis in a mouse model. (B) Detection of sja-let-7 expression in the liver and serum (n = 6). (C) Liver histological analysis with H&E staining. Black arrows indicate the egg granuloma. Scale bar, 200 μm. Insets show a higher magnification of the outlined area. Scale bar, 100 μm. (D) Ishak score of liver fibrosis (n = 6). (E) Area of a single granuloma. (F) Percent of egg granuloma areas (n = 6). (G-H) Liver histological analysis with Masson and Sirius red staining. Black arrows indicate the positive staining area. Scale bar, 200 μm. Insets show a higher magnification of the outlined area. Scale bar, 100 μm. (I-J) Positive area of Masson and Sirius red staining (n = 6). All graph data are expressed as the mean ± SD of at least three biological replicates per group. *P< 0.05, **P< 0.01, ns, not significant. Abbreviation: Sj: S. japonicum; NC: negative control; H&E: hematoxylin and eosin.
Fig 6.
Sja-let-7 reduced the expression of fibrotic markers after treated with sja-let-7 agomir.
(A-C) Detection of α-SMA, Col1α1 and Col1α3 mRNA expression in the liver (n = 6). (D) Liver IHC analysis of α-SMA, Col1α1 and Col1α3. Scale bar, 200 μm. Insets show a higher magnification of the outlined area. Scale bar, 50 μm. (E) Immunofluorescence analysis of α-SMA, Col1α1 and Col1α3 after treated with sja-let-7 agomir. White arrows indicate the egg granuloma. Scale bar, 100 μm. Insets show a higher magnification of the outlined area. Scale bar, 50 μm. All graph data are expressed as the mean ± SD of at least three biological replicates per group. *P< 0.05, **P< 0.01, ns, not significant. Abbreviation: Sj: S. japonicum; NC: negative control; IHC: immunohistochemical analysis.
Fig 7.
Sja-let-7 suppressed the schistosome-induced liver fibrosis is mediated via Col1α2/TGF-β/Smad axis.
(A) Polarization microscopy observation of type I and type III collagen fibers. Scale bar, 100 μm. Insets show a higher magnification of the outlined area. Scale bar, 50 μm. (B) Ratio of type I and type III collagen fibers (n = 6). (C) Detection of Col1α2 mRNA expression in the liver (n = 6). (D) Liver IHC analysis of Col1α2. Scale bar, 200 μm. Insets show a higher magnification of the outlined area. Scale bar, 50 μm. (E) Positive area of Col1α2 (n = 6). (F-I) Detection of TGF-β, Smad2, Smad3 and Smad7 mRNA expression in the liver (n = 6). (J) Liver IHC analysis of TGF-β. Scale bar, 200 μm. Insets show a higher magnification of the outlined area. Scale bar, 50 μm. (K) Positive area of TGF-β (n = 6). (L) Liver IHC analysis of p-smad2/3. (M) Positive area of p-smad2/3 (n = 6). All graph data are expressed as the mean ± SD of at least three biological replicates per group. *P< 0.05, **P< 0.01, ns, not significant. Abbreviation: Sj: S. japonicum; NC: negative control; Col I: type I collagen fiber; Col III: type III collagen fibers; IHC: immunohistochemical analysis.
Fig 8.
Graphical abstract of the mechanism.
The S. japonicum worms dwelling in the host portal vein release SjEVs that contain sja-let-7 to suppress the schistosome-induced liver fibrosis via Col1α2/TGF-β/Smad axis. This figure was created with Biorender.com.