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Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

ABSTRACT: Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis.Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2.The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 ?g/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2.Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding could provide a promising treatment strategy to interrupt the process of liver fibrosis caused by clonorchiasis.

SUBMITTER: Wu Y

PROVIDER: S-EPMC5353963 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

Wu Yinjuan Y Li Ye Y Shang Mei M Jian Yu Y Wang Caiqin C Bardeesi Adham Sameer A AS Li Zhaolei Z Chen Tingjin T Zhao Lu L Zhou Lina L He Ai A Huang Yan Y Lv Zhiyue Z Yu Xinbing X Li Xuerong X

Parasites & vectors 20170316 1

<h4>Background</h4>Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, ...[more]

PMID: 28302166

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Project description:BackgroundChronic infection with Clonorchis sinensis can cause hepatobiliary fibrosis and even lead to hepatobiliary carcinoma. Epstein-Barr virus-induced gene 3 protein (EBI3) is a subunit of interleukin 35, which can regulate inflammatory response and the occurrence of fibrotic diseases. Previous studies have reported that the expression of EBI3 in the serum of patients with liver cirrhosis is reduced. The present study aims to investigate the biological effects of EBI3 on liver fibrosis caused by C. sinensis and the underlying molecular mechanisms.MethodsWe first established a mouse model of liver fibrosis induced by C. sinensis infection and then measured the serum expression of EBI3 during the inflammatory and fibrotic phase. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed to investigate the potential role of EBI3 in liver fibrosis by regulating the extracellular matrix structural constituent and collagen catabolic process. Recombinant protein EBI3 (rEBI3) was added to hepatic stellate cells (HSCs) in vitro with C. sinensis antigen to explore its function. Finally, the therapeutic effect of rEBI3 was verified by intravenous injection into C. sinensis-infected mice.ResultsThe results showed that the serum expression of EBI3 increased in the inflammatory response phase but decreased in the fibrotic phase. The excretory-secretory products of C. sinensis (Cs.ESP) were able to stimulate HSC activation, while rEBI3 reduced the activation of HSCs induced by Cs.ESP. Also, the protein expression of gp130 and downstream protein expressions of JAK1, p-JAK1, STAT3 and p-STAT3 in HSCs were increased after rEBI3 incubation. Finally, intravenously injected rEBI3 inhibited hepatic epithelial-mesenchymal transition in C. sinensis-infected mice by inhibiting HSC activation and reducing liver injury.ConclusionThis study confirms that rEBI3 can attenuate C. sinensis-induced liver fibrosis by inhibiting HSC activation and may be one of the potential treatments for liver fibrosis.

Project description:BackgroundPhospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined.ObjectivesTo examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils.MethodsPeripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT.ResultsEosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the expression of PLA2G10 by eosinophils.ConclusionsThese results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.

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Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

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Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

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