Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells

Abstract

Epidemiological and experimental evidence demonstrated that Clonorchis sinensis is an important risk factor of hepatic fibrosis and cholangiocarcinoma. C. sinensis excretory/secretory products (CsESPs) are protein complex including proteases, antioxidant enzymes, and metabolic enzymes, which may contribute to pathogenesis of liver fluke-associated hepatobiliary diseases. However, potential CsESP candidates involved into hepatic fibrosis and cholangiocarcinoma still remain to be elucidated. In the present study, we performed proteomic identification of CsESP candidates capable of binding and activating human hepatic stellate cell line LX-2. Immunofluorescence analysis confirmed the interaction of CsESPs with LX-2 cell membrane. LX-2 cells could be stimulated by CsESPs from 24 h post incubation (p < 0.05). Specifically, 50 μg/ml of CsESPs showed the strongest effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay which could also be demonstrated by flow cytometry analysis (p < 0.01). Furthermore, expression level of human type III collagen in LX-2 cells treated with CsESPs was significantly higher than that in control cells measured by molecular beacon and semiquantitative reverse transcription (RT)-PCR approaches (p < 0.01). Finally, CsESPs before and after incubation with LX-2 cells were subjected to two-dimensional gel electrophoresis (2-DE) analysis and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Nine proteins with abundance change above threefold were Rho GTPase-activating protein, mitochondrial cytochrome c oxidase subunit Va, α-enolase, phospholipase C, interleukin-15, insect-derived growth factor, cytochrome c oxidase subunit VI, DNAH1 protein, and kinesin light chain. Taken together, we identified potential CsESP candidates capable of binding and activating human hepatic stellate cells, providing more direct evidences that are previously unknown to accelerate strategies for C. sinensis prevention.

LncRNAs Act as a Link between Chronic Liver Disease and Hepatocellular Carcinoma

“…Hepatic fibrosis is a major risk factor for HCC, and it is a continuous wound-healing process that leads to the dysregulation of extracellular matrix (ECM) proteins and the distortion of normal liver architecture [9]. Many chronic liver diseases, such as viral hepatitis, alcohol toxicity, drug abuse, metabolic syndrome, and autoimmune hepatitis result in hepatic fibrosis or cirrhosis [10], which is the primary stage of HCC [11]. While extensive studies on the pathogenesis of the development of HCC from hepatic fibrosis have been performed, their regulatory molecular mechanisms are still only partially understood [11].…”

Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma

“…Additionally, researchers have proposed that mechanical damage caused by the activities of the parasites and Cs ESPs excreted and secreted from the liver fluke is the primary pathogenic mechanism [23]. In our previous studies [24], Cs GRN, one of an ingredient of Cs ESPs, was identified and the prokaryotic expression of the recombinant Cs GRN protein was carried out.…”

“…This study observed that Cs GRN, as a Cs ESP like Cs CBs and Cs severin, has also been shown to promote cell migration and invasion. In consideration of the complex constitution of Cs ESPs, including proteases, antioxidant enzymes and metabolic enzymes [24, 50], interactions among these components are required to determine the authentic factors for pathogenesis in the future. We will explore the actions of granulin members with one or more typical domains in C. sinensis to clarify the difference between these molecules in our further studies.…”

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

“…It’s proved that Cs ESPs play an important role in liver fibrosis caused by clonorchiasis. Cell proliferation was detected by methyl thiazolyltetrazolium (MTT) assay when LX-2 cells were incubated with 50 μg/ml of CsESPs [20]. The phospholipase A2 has been well known for its function in liver fibrosis and inhibition of tumor cells.…”