The NF-κB signalling pathway and TM7SF3 contribute to liver fibrosis caused by secreted phospholipase A2 of Clonorchis sinensis.

BACKGROUND: The NF-κB signalling pathway has been reported to be related to liver fibrosis, and we investigated whether the NF-κB signalling pathway is involved in liver fibrosis caused by secreted phospholipase A2 of Clonorchis sinensis (CssPLA2). Furthermore, expression of the receptor of CssPLA2 on the cell surface of hepatic stellate cells (HSCs) may greatly contribute to liver fibrosis. METHODS: CssPLA2 was administered to BALB/c mice by abdominal injection. The levels of markers of NF-κB signalling pathway activation in mouse liver tissue were measured by quantitative RT-PCR, ELISA and western blot. Additionally, HSCs were incubated with CssPLA2, and an NF-κB signalling inhibitor (BAY 11-7082) was applied to test whether the NF-κB signalling pathway plays a role in the effect of CssPLA2. Then, the interaction between CssPLA2 and its receptor transmembrane 7 superfamily member 3 (TM7SF3) was confirmed by co-immunoprecipitation (co-IP) and GST pull-down. To determine how TM7SF3 influences the ability of CssPLA2 to cause liver fibrosis, a TM7SF3 antibody was used to block TM7SF3. RESULTS: The levels of the NF-ΚB signalling pathway activation markers TNF-α, IL-1ß and phospho-p65 were increased by CssPLA2 in the context of liver fibrosis. In addition, the interaction between TM7SF3 and CssPLA2 was confirmed by co-IP and GST pull-down. When TM7SF3 was blocked by an antibody targeting 1-295 amino acids of TM7SF3, activation of HSCs caused by CssPLA2 was alleviated. CONCLUSIONS: The NF-ΚB signalling pathway is involved in the activation of HSCs by CssPLA2. TM7SF3, the receptor of CssPLA2, plays important roles in liver fibrosis caused by CssPLA2.

[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].

OBJECTIVE: To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. METHODS: Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. RESULTS: The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. CONCLUSION: Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.