| Liver fibrosis is an excessive wound-healing reaction in response to chronic injury, which requires the participation of inflammatory cells, immune cells and hepatic stellate cells(HSCs). Though as a major cause of morbidity and mortality worldwide, there are no special anti-fibrosis drugs in clinical at present. Clonorchiasis is a food-borne parasitic disease caused by Clonorchis sinensis(C. sinensis) and is prevalent in East Asia and Southeast Asia. In China, clonorchiasis also affects people’s quality of life. C. sinensis adults parasitize in intra-hepatic bile duct and result in a range of diseases in liver and bile duct, like liver fibrosis, pyogenic cholangitis and cholangiocarcinoma. Although we have paid more attention to C. sinensis-induced liver fibrosis, the molecular mechanisms remain unclear. Lysophospholipase A(Lyso PLA) is one of the members of the family of phospholipase and exists in many tissue, cells, parasites and bacteria, whose functions are to deacylate lysophospholipids and also plays an important role in pathogenesis of parasites and bacteria infection. C.sinensis Lysophospholipase A(Cs Lyso PLA) has been well expressed and characterized as the members of phospholipase family. In this study, the aim is to investigate the potential role of Cs Lyso PLA in C. sinensis-induced liver fibrosis, elucidate the molecular mechanisms and provide the new theory and targets for the treatment of clonorchiasis.Methods 1. Preparation of anti-serum of Cs Lyso PLA Cs Lyso PLA was emulsified with equivoluminal complete Freund’s adjuvant in primary immunization for one time or with incomplete Freund’s adjuvant in booster immunization at 2-week intervalsfor two times by subcutaneously immunizing SD rats and New Zealand rabbits. Anti-serum was collected before immunization or at two weeks after the last injection. 2. The expression of Cs Lyso PLA in the liver and serum of C.sinensis-infected BALB/c mice BALB/c mice were infected with C.sinensismetacercariae by gavage administration for 7d, 30 d, 60 d, 90 d, 135 d and 180 d. Liver tissue and serum were collected. The liver tissue samples were fixed in formalin, then paraffin-embedded and 4 μm sections cut. The expression of Cs Lyso PLA was detected by immunohistochemistry in liver tissue sections and by ELISA in serum. 3. Injection of Cs Lyso PLA in BALB/c mice via tail vein BALB/c mice were injected with Cs Lyso PLA twice a week for 18 weeks. Liver tissue and serum were collected. The liver tissue samples were fixed in formalin, then paraffin-embedded and 4 μm sections cut or quick-freezed in liquid nitrogen. 4. Inflammatory cells infiltration and collagen deposition in the liverof Cs Lyso PLA-injected BALB/c mice Liver tissue sections were stained with hematoxylin and eosin(H&E) and Masson’s trichrome to qualitatively assess the collagen architecture and the extent of fibrosis. 5. Detection of ALT and AST in the serum of Cs Lyso PLA-injected BALB/c mice The levels of liver injury were measured in the light of aspartate aminotransferase assay kit. Enzyme activities are expressed as units per liter(U/L).6. The expressions of α-SMA and Collagen-I in the liverof Cs Lyso PLA-injected BALB/c mice Total protein lysates were extracted from the liver of C.sinensis-infected BALB/c mice. The expressions of α-SMA and Collagen-I were examined by Western bloting. The liver tissue sections were stained by immunofluorescence for α-SMA. 7. The expression of IL-25 in C.sinensis-infected and Cs Lyso PLA-injected BALB/c mice Serum levels of IL-25 were evaluated based on Mouse IL-17E(IL-25) ELISA Ready-SET-Go!? In liver tissue sections, IL-25 was detected by immunohistochemistry.In total protein lysates of liver, IL-25 was detected by Western bloting. 8. Immunofluorescence double-stain of IL-25 and CD68 in the liverof Cs Lyso PLA-injected BALB/c mice Sections of liver tissue were stained by immunofluorescence for IL-25 and CD68. Images were obtained using Olympus BX63 and cell Sens Dimension. 9. The expression of IL-25 in Cs Lyso PLA-induced RAW264.7 cells RAW264.7 cells were cultured with different concentration of Cs Lyso PLA. The total RNA of RAW264.7 cells was collected by Trizol method. c DNAs were synthesized using a Revert Aid First Strand c DNA Synthesis Kit and amplified on a Bio-Rad CFX96 Real-Time system with SYBR Green I for quantitative analysis. IL-25 were measured and normalized by β-actin expression. Relative multiples of changes in m RNA expression were analyzed by calculating 2-ΔΔCt. The total protein of RAW264.7 cells was obtained by RIPA buffer. Western bloting detected the production of IL-25.10. Cs Lyso PLA stimulates IL-25 expression in RAW264.7 cells via PKA-dependent B-Raf-ERK1/2 signaling pathwayRAW264.7 cells were cultured with Cs Lyso PLA for 15 min, 30 min and 60 min or RAW264.7 cells were pretreated with H-89 and SC79, then cultured with Cs Lyso PLA. The total protein of RAW264.7 cells was obtained by RIPA buffer. Western bloting detected the activation of Braf, ERK1/2 and AKT. The total RNA of RAW264.7 cells was collected by Trizol reagent. IL-25 was measured using Q-PCR and normalized by β-actin expression. 11. The effect of IL-25 in LX-2 cells LX-2 cells were treated with IL-25. The total RNA of LX-2 cells were collected by Trizol reagent. The m RNA expression of α-SMA was measured using Q-PCR and normalized by β-actin expression. The total protein of LX-2 cells was obtained using RIPA buffer. The production of α-SMA was detected by Western bloting. The expression of Collagen-I was measured by immunofluorescence in the slides of LX-2 cells. The migration of LX-2 cells was examined by the Transwell plate with 8.0 μm pore polycarbornate membrane insert. The migration cells numbers were counted by using a light microscope and enumerate the number of stained cells in five random fields. 12. The effect of Cs Lyso PLA in TGF-β1-induced LX-2 cells LX-2 cells were pretreated with Cs Lyso PLA, and then stimulated by TGF-β1. The total RNA of LX-2 cells were collected by Trizol reagent. The m RNA expression of α-SMA, MMP2 and Collagen-I were measured by Q-PCR and normalized with β-actin expression. The total protein of LX-2 cells was extracted by RIPA buffer. Western bloting detected the expression of α-SMA and the activation of Smad3, JNK2 and ERK1/2. The expression of α-SMA was also measured by immunofluorescence in the slides of LX-2 cells. The proliferation of LX-2 cells was investigated using CCK-8 reagent. The migration of LX-2 cells was examined by the Transwell plate with 8.0 μm pore polycarbornate membrane insert.Results1. The Cs Lyso PLA in the liver of C.sinensis-infected BALB/c micewas increased gradually and reduced since 90 d, which was consistent with the expression in serum. 2. H&E staining of liver showed the significant infiltration of inflammatory cells and Masson’s trichrome staining revealed massive deposition of collagen, which were around in veins and gradually extended to liver parenchyma. The activities of AST and ALT in serum of Cs Lyso PLA-injected mice were significantly higher than that of in control mice. 3. The α-SMA and Collagen-I protein in the liver of Cs Lyso PLA-injected mice was increased compared with the control mice detected by Western bloting and immunofluorescence. 4. The concentration of IL-25 in the serum of C.sinensis-infected mice was significantly elevated, reached the peak on 60 d, and gradually reduced up to 180 d. Although there were no significant differences between Cs Lyso PLA-injected mice and the control mice, the concentration of IL-25 in the serum of Cs Lyso PLA-injected mice had the potential trend toward higher level than the control mice. In contrast, the expression of IL-25 was stronger in the liver of Cs Lyso PLA-injected mice measured by Western bloting. In addition, immunohistochemistry also showed IL-25 expression in the liver of two different treatments. 5. Immunofluorescence staining showed co-staining of IL-25 and CD68 in macrophages. 6. The m RNA expression of IL-25 was upregulated in RAW264.7 cells. The protein expression of IL-25 was also enhanced dramatically compared with the control.This was achived via PKA-dependent B-Raf-ERK1/2 signaling pathway. 7. Both the m RNA expression and protein expression of α-SMA were increased after stimulated by IL-25 in LX-2 cells compared with the control. Immunofluorescence staining showed the higher expression of Collagen-I in LX-2 cells. Moreover, IL-25 increased the migration of LX-2 cells. 8. Cs Lyso PLA inhibited the m RNA expressions of α-SMA, MMP2 and Collagen-I in TGF-β1-pretreated LX-2cells. Western bloting also revealed the inhibition of α-SMA expression and the activation of Smad3, JNK2 and ERK1/2. In addition, TGF-β1 could promote the proliferation and migration of LX-2 cells, which were inhibited by the treatment of Cs Lyso PLA.Conclusions On one hand, Cs Lyso PLA induces liver fibrosis by stimulating macrophages to secrete IL-25. IL-25 is a critical downstream mediator of Cs Lyso PLA-induced liver fibrosis by activating HSCs. On the other hand, Cs Lyso PLA inhibits the activation effects of TGF-β1 on HSCs by attenuating the activation of Smad3, JNK2 and ERK1/2. Cs Lyso PLA whether to play the role of promoting or inhibiting fibrosis may be related to C. sinensis infection period, the intensity of infection and recurrent infection status, and other factors related to the host immune function. |
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