| Background and purposeHepatopulmonary syndrome(HPS)is a severe pulmonary complication of liver cirrhosis and is commonly seen in patients with advanced liver disease,with a prevalence of 13% to 57%.Its development can seriously affect the quality life of patients and lead to an increased risk of death.Liver transplantation is currently the only treatment available for HPS,but limited by the shortage of liver sources and the high cost of treatment.Most patients do not have effective treatment.The primary pathogenesis of HPS is expansion of pulmonary microvascular and pulmonary angiogenesis.S100 calcium-binding protein A4(S100A4),which belongs to the S100 family,is a metastasis-associated protein expressed in various cell types and plays an essential role in different types of tumour metastasis by regulating adhesion,cell motility and extracellular matrix remodelling.The currently known pathways in which S100A4 acts are mainly the Wnt/β-catenin pathway.Recent studies have shown that S100A4 can also promote angiogenesis.There are no studies on the effect of S100A4 on angiogenesis in HPS.Niclosamide can inhibit the formation of β-catenin/TCF complex and the transcription of target gene S100A4.Based on this,this study was proposed to explore the possible mechanism of its action in BDL-induced HPS in rats by constructing an animal model with HE staining,Masson staining,protein immunoblotting and fluorescence quantitative PCR combined with selective inhibition of S100A4 by niclosamide.Materials and methods1.Construction of rat BDL modelBile duct ligation(BDL)was used to establish a rat BDL model.The liver and lung tissues were taken for HE and Masson staining after 1,3 and 5 weeks of surgery to observe the pathophysiological changes of liver and lung tissues.2.Detection of S100A4 expression in lung tissues of rats with BDLThe expression of the S100A4 gene in rat lung tissues was detected by quantitative fluorescence PCR;the expression of the S100A4 protein in rat lung tissues was determined by western blot.3.Effect of niclosamide on BDL-induced HPS in ratsGavage with niclosamide suspension was started on day 15 after BDL and continued for one week.Liver and lung tissues were taken at the end of week 5,and whether niclosamide treatment improved liver and lung pathological injury was assessed by comparison of HE staining and Masson staining of paraffin sections.Fluorescence quantitative PCR and western bolt were used to detect the changes in the S100A4 gene and protein expression in the lung tissues of rats after niclosamide treatment.4.Effects of S100A4 on proliferation,migration and tube-formation of Pulmonary Microvascular Endothelial Cells(PMVECs)S100A4-specific small interfering RNA(si RNA)was used to silence the target gene.The effects on cell proliferation were detected by scratch assay,migration ability by transwell assay and angiogenesis ability by tube formation assay.5.Effect of niclosamide on proliferation,migration and tube-formation of PMVECsThe effect of niclosamide on cell proliferation was observed by scratch assay,the effect of niclosamide on cell migration ability was detected by transwell assay,and the effect of niclosamide on cell angiogenesis ability was detected by tube formation assay after treatment with serum from BDL 5W and NIC 5W rats.Results1.A rat BDL model was successfully constructed.2.m RNA and protein expression of S100A4 was significantly increased in the lungs of BDL rats.3.In vivo,niclosamide reduced HPS manifestation in BDL rats.4.In vitro,specific inhibition of S100A4 reduced lung microvascular endothelial cell migration,proliferation,and tube-forming capacity.5.Niclosamide attenuated the migratory,proliferative,and tube-forming capacities of serum-stimulated pulmonary microvascular endothelial cells in BDL 5W rats.ConclusionsS100A4 can promote the disease development of HPS by promoting vascular neogenesis,and niclosamide attenuates the pathological changes of HPS to some extent in rats. |
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