Clonorchis sinensis infection induces hepatobiliary injury via disturbing sphingolipid metabolism and activating sphingosine 1-phosphate receptor 2

Abstract

Clonorchis sinensis (C. sinensis) infection induces severe hepatobiliary injuries, which can cause inflammation, periductal fibrosis, and even cholangiocarcinoma. Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) and its receptor S1P receptors (S1PRs) have been implicated in many liver-related diseases. However, the role of S1PRs in C. sinensis-mediated biliary epithelial cells (BECs) proliferation and hepatobiliary injury has not been elucidated. In the present study, we found that C. sinensis infection resulted in alteration of bioactive lipids and sphingolipid metabolic pathways in mice liver. Furthermore, S1PR2 was predominantly activated among these S1PRs in BECs both in vivo and in vitro. Using JTE-013, a specific antagonist of S1PR2, we found that the hepatobiliary pathological injuries, inflammation, bile duct hyperplasia, and periductal fibrosis can be significantly inhibited in C. sinensis-infected mice. In addition, both C. sinensis excretory-secretory products (CsESPs)- and S1P-induced activation of AKT and ERK1/2 were inhibited by JTE-013 in BECs. Therefore, the sphingolipid metabolism pathway and S1PR2 play an important role, and may serve as potential therapeutic targets in hepatobiliary injury caused by C. sinensis-infection.

Keywords: Clonorchis sinensis; biliary epithelial cells (BECs); hepatobiliary injuries; sphingolipid metabolism; sphingosine 1-phosphate receptor 2.

Figure 1

Lipid metabolism changes after C. sinensis infection. (A) Pie plot of metabolite classification and proportion. (B) Volcano plot for the C. sinensis-infected group vs the control group by metabolomics. (C) Heatmap of hierarchical clustering analysis for the C. sinensis-infected group vs the control group by metabolomics. (D) Boxplot displays a quantitative analysis of metabolite content of LPC for the C. sinensis-infected group vs the control group by metabolomics. (E) Boxplot displays a quantitative analysis of metabolite content of SHexCer for the C sinensis-infected group vs the control group by metabolomics. (F) KEGG Pathway Analysis for the C. sinensis-infected group vs the control group by metabolomics.

Figure 2

C. sinensis infection resulted in activation of sphingolipid metabolic pathways. (A) qRT-PCR assays were performed to measure the mRNA levels of enzymes involved in sphingolipid metabolic pathway, including Sptlc1, Sptlc3, Cers2, Cers4, Smpd2, Smpd3, Smpd4, Samd8, Sgms2, Galc, Gba2, Gla, Ugcg, Asah1, Asah2, Acer2, Acer3, Cerk, Neu2, Neu3, Sgpl1, Sgpp1, Sgpp2, Sphk1, and Sphk2. (B) The results schemed as a diagram. (C) Western blotting analysis of the protein expression levers of Sphk1 and Sphk2 in the liver of control and C. sinensis-infected mice. (D) The gene expression levels of ABC transporter protein. Measurement of the Abcb1a relative mRNA expression in liver tissue of control and C. sinensis infected mice. (E) The concentration of S1P in liver was determined by ELISA in control and C. sinensis-infected mice. Compared with control group, *P<0.05, **P<0.01, ***P<0.001.

Figure 3

C. sinensis infection significantly increased S1PR2 expression. (A) The relative mRNA expression for S1pr1, S1pr2 and S1pr3 in hepatobiliary tissue of control and C. sinensis-infected mice. (B) The relative protein expression for S1PR1, S1PR2 and S1PR3 in liver of control and C. sinensis-infected mice. (C) The expression of S1PR2 in liver of control and C. sinensis-infected mice by immunofluorescence assay. Compared with control group, **P<0.01, ***P<0.001.

Figure 4

Inhibition of S1PR2 alleviated hepatobiliary damage in C. sinensis-infected mice. (A) The images of hepatobiliary injury in control and C. sinensis-infected mice. (B) The liver weight/body weight ratio in control and C. sinensis-infected mice. (C) The serum analysis of AST, ALT, ALP, TBA, DBIL, and TBIL in control and C sinensis-infected mice. (D) The H&E analysis the damage in hepatobiliary tissue of control and C. sinensis-infected mice under 40× and 200× microscope. (E) The Alcian Blue analysis the damage in hepatobiliary tissue of control and C. sinensis-infected mice under 40× and 200× microscope. Compared with indicated groups, *P<0.05, **P<0.01, ***P<0.001.

Figure 5

Inhibition of S1PR2 abated periductal fibrosis in BALB/C mice after C. sinensis infection. (A) The Sirius Red’s staining of the hepatobiliary tissue was carried out in the control and C. sinensis-infected mice under 100× and 200× microscope. (B) The measurement of hydroxyproline content in hepatobiliary tissue of control and C. sinensis-infected mice. (C) The protein expression of alpha-SMA in hepatobiliary tissue of control and C. sinensis-infected mice. Compared with indicated groups, *P<0.05, **P<0.01, ***P<0.001.

Figure 6

The inhibition of S1PR2 alleviated BECs proliferation in mice infected with C. sinensis. (A) The expression of BECs proliferation marker CK19 in infected mice compared to that in control mice. (B) The relative mRNA expression for CK19 in hepatobiliary tissue of control and C. sinensis-infected mice. (C) The protein expression of CK19 in hepatobiliary tissue of control and C. sinensis-infected mice. Compared with indicated groups, *P<0.05, **P<0.01, ***P<0.001.

Figure 7

The inhibition of S1PR2 decreased the production of the pro-inflammatory cytokines after C. sinensis infection. (A–C) Measurement of the pro-inflammatory cytokines IL-1β, TNFα, and IL-6 the relative mRNA expression in hepatobiliary tissue of control and C. sinensis-infected mice. (D-F) Measurement of the pro-inflammatory cytokines IL-1β, TNFα, and IL-6 by ELISA in hepatobiliary tissue homogenate of control and C. sinensis-infected mice. Compared with indicated groups, *P<0.05, **P<0.01, ***P<0.001.

Figure 8

Role of S1PR2 played in CsESPs -induced migration and pro-inflammatory cytokines production of BECs. (A) After CsESPs stimulation, the protein levels of S1PR1, S1PR2, and S1PR3 were detected by western blotting. (B) Effect of JTE-013 on CsESPs/S1P-induced cell migrated in cultured BECs by transwell assay. (C-E) Measurement of the pro-inflammatory cytokines IL-1β, TNFα, and IL-6 the relative protein expression by ELISA in in the BECs culture supernatant. (F, G) BECs were pretreated with JTE-013 (10μM) for 30 minutes, then treated with vehicle control (DMSO), CsESPs (60ug/ml), or S1P (100nM). Protein levels of P-AKT, AKT, P-ERK1/2, and ERK1/2 were determined by western blotting. Compared with indicated groups, *P<0.05, **P<0.01, ***P<0.001.