Jagged-1/Notch Pathway and Key Transient Markers Involved in Biliary Fibrosis during Opisthorchis felineus Infection

Abstract

Chronic opisthorchiasis associated with Opisthorchis felineus infection is accompanied by severe fibrotic complications. It is of high practical significance to elucidate the mechanisms of hepatic fibrosis in chronic infection dynamics. The goal of the study is to investigate the temporal profile of key markers and the Jagged1/Notch signaling pathway in the implementation of fibrosis in a chronic O. felineus infection. For the first time, using histological methods and real-time PCR analysis, we demonstrated the activation of the Jagged1/Notch pathway in liver fibrogenesis, including the activation of the Hes1 and Hey1 target genes during experimental opisthorchiasis in Mesocricetus auratus. Cluster analysis followed by regression analysis of key markers during the infection showed that Jagged1 and Mmp9have the greatest contribution to the development of cholangiofibrosis and periductal fibrosis. Moreover, we detected a significant increase in the number of Jagged1-positive cells in the liver of chronic opisthorchiasis patients compared to that of the control group without infection. The results of the study are extremely informative both in terms of investigation both diverse fibrosis mechanisms as well as potential targets in complex antihelmintic therapy.

Keywords: Jagged1/Notch pathway; Opisthorchis felineus; Syrian hamster; extracellular matrix; human; liver fibrosis.

Figure 1

Fibrotic changes in the liver tissue of O. felineus-infected Syrian hamsters. (A) Heat map progression of periductal fibrosis (con_tis_PF) and cholangiofibrosis (con_tis_ChF), the amount of collagen 1a+ fibers in the liver of infected animals (col_I_PF and col_I_ChF, respectively); (B) histopathological changes in Syrian hamster liver, Van Gieson staining (VG) and IHC analysis for collagen 1a (Col1); (C) Col1a gene was normalized to average Gapdh expression. C—control, 10 w, 30 w, 52 w—week p.i. Data are presented as mean ± SEM, # p ≤ 0.05, as compared to the control group.

Figure 2

State of extracellular matrix in chronic opisthorchiasis, associated with O. felineus infection in the Syrian hamster model. (A) A heat map of ECM component changes; (B) IHC study of α-SMA-, Mmp9-, and TIMP1-positive cells (marked by an asterisk) in study dynamics, magnification ×400; (C,D) Acta2, Mmp9 were normalized to average Gapdh expression. C—control, 10 w, 30 w, 52 w—week p.i. Data are presented as mean ± SEM, # p ≤ 0.05, as compared to the control group, * p ≤ 0.05, as compared to the previous study period.

Figure 3

Stellate cells and fibroblasts in the liver tissue of O. felineus -infected Syrian hamsters. (A) Heat map of changes of FSP- and GFAP-positive cell numbers in the dynamics of infection; (B) GFAP- and FSP-positive cells (marked by an asterisk), IHC study, ×400 magnification; (C) Fgf2 gene was normalized to average Gapdh expression. C—control, 10 w, 30 w, 52 w—week p.i. Data are presented as mean ± SEM, # p ≤ 0.05, as compared to the control group.

Figure 4

TGFβ1/SMAD2 signaling pathway in the liver tissue of O. felineus-infected Syrian hamsters. (A) Heat map of changes in TGF-β1- and SMAD2-positive cells in the course of infection; (B) TGF-β1- and SMAD2-positive cells (marked by an asterisk) in the dynamics of infection, IHC study, ×400 magnification; (C) Tgfb1 gene was normalized to average Gapdh expression. C—control, 10 w, 30 w, 52 w—week p.i. Data are presented as mean ± SEM, # p ≤ 0.05, as compared to the control group, * p ≤ 0.05, as compared to the previous period of investigation.

Figure 5

Jagged1/Notch pathway in the liver tissue in chronic opisthorchiasis in Syrian hamsters. (A) Heat map changes in Jagged1-positive cells in infection dynamics; (B) IHC staining for Jagged1, Jagged1+ cells in the area of periductal fibrosis (All) (marked by an asterisk), magnification ×200, Jagged1+hepatocytes in the area of cholangiofibrosis (Hep) (marked by an asterisk), magnification ×400; (C–E) Jag1, Hey1, Hes1 were normalized to average Gapdh expression. C—control, 10 w, 30 w, 52 w—week p.i. Data are presented as mean ± SEM, # p ≤ 0.05, as compared to the control group.

Figure 6

Jagged1/Notch pathway in the liver tissue in chronic opisthorchiasis and chronic cholecystitis in humans. (A) Boxplot of Jagged1-positive cell count data; (B) Jagged1-positive cells in chronic liver injury with and without O. felineus infection, IHC staining, magnification ×200.

Figure 7

Cluster analysis of 11 markers of fibrosis (interaction of factors: “infection × time”) in hamster liver (Mesocricetus auratus) infected with Opisthorchis felineus (from 10 to 52 weeks p.i.).

Figure 8

Multiple regression analysis of periductal and cholangiofibrosis Jagged1 and Mmp9 markers. Gray on the graph indicates the distribution of the standard deviation, showing the scatter of the data. Values for each week are coded with their own color.