Quercetin-3-rutinoside (rutin) is a bioflavonoid that is common in foods. The finding that quercetin-3-rutinoside inhibits protein disulfide isomerase (PDI) and potently blocks thrombosis in vivo has enabled the evaluation of PDI inhibition in multiple animal models of thrombus formation and has prompted clinical studies of PDI inhibition in thrombosis. Nonetheless, how quercetin-3-rutinoside blocks PDI activity remains an unanswered question. Combining NMR spectroscopy, site-directed mutagenesis, and biological assays, we identified H256 as the key residue for PDI interacting with quercetin-3-rutinoside. Quercetin-3-rutinoside inhibited the activity of PDI (WT) but not PDI (H256A). Molecular dynamic simulations indicated that the flavonoid skeleton, but not the rutinoside conjugate, is embedded in the major binding pocket on the b′ domain. Among several quercetin-3-rutinoside analogues tested, only compounds with a phenoxyl group at position 7 showed direct binding to PDI, further supporting our molecular model. Studies using purified coagulation factors showed that quercetin-3-rutinoside inhibited the augmenting effects of PDI (WT), but not PDI (H256A), on tissue factor (TF) activity. Quercetin-3-rutinoside also inhibited chemotherapy-induced TF activity enhancement on endothelial cells. Together, our studies show that residue H256 in PDI and the phenoxyl group at position 7 in quercetin-3-rutinoside are essential for inhibition of PDI by quercetin-3-rutinoside. These results provide new insight into the molecular mechanism by which flavonoids block PDI activity.

No abstract

Long non-coding RNAs (lncRNAs) are ncRNA transcripts >200 nucleotides that are important genetic regulators. LncRNAs can directly regulate mRNA through a lncRNA-mRNA regulatory mode and can also regulate mRNA through competitive binding to micro (mi)RNA, which is generally known as the competitive endogenous RNA (ceRNA) network. The present study evaluated the functional roles and regulatory networks of lncRNAs in chronic glomerulonephritis (CGN). The proliferative ability of mouse glomerular mesangial cells (GMCs) induced by different concentrations of lipopolysaccharide (LPS) was assessed using the Cell Counting Kit-8 assay, and RNA sequencing (RNA-seq) was performed to identify differentially expressed lncRNAs in LPS-induced GMCs. Based on the sequencing results, six lncRNAs were selected for validation using reverse transcription-quantitative PCR (RT-qPCR). Furthermore, the lncRNA-mRNA regulatory network and the lncRNA-miRNA-mRNA ceRNA network were constructed to assess the role and mechanism of CGN-related lncRNAs. To elucidate the biological functions of lncRNAs, Gene Ontology (GO) biological process term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on all mRNAs involved in the lncRNA-mRNA regulatory network and in the ceRNA network. A total of 1,532 differentially expressed lncRNAs, including 594 upregulated lncRNAs and 938 downregulated lncRNAs, were identified using RNA-seq.The results of RT-qPCR validation were consistent with RNA-seq results. An lncRNA-mRNA regulatory network, including 236 lncRNAs and 556 mRNAs, and a ceRNA network, including 6 lncRNAs, 18 miRNAs and 419 mRNAs, were successfully constructed. The GO biological process term enrichment and KEGG pathway enrichment analyses demonstrated that those lncRNAs were often related to inflammatory response and substance metabolism. The present study identified key CGN-related lncRNAs in LPS-induced GMCs, and further demonstrated a global view of the lncRNA-mRNA regulatory network and ceRNA network involved in CGN. These results offered novel insights into the roles of lncRNAs in the pathogenesis of CGN and identified potential diagnostic biomarkers.

Lately, researches on lncRNA-mRNA regulatory network and lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) network have been the hotspots, the present study explored the functional roles and regulatory networks of lncRNAs in chronic glomerulonephritis (CGN). The proliferation ability of glomerular mesangial cells (GMCs) induced by different concentrations of lipopolysaccharide (LPS) were detected with CCK-8 assay and RNA-seq was performed to study differentially expressed lncRNAs in LPS-induced GMCs. According to the sequencing results, 6 lncRNAs were selected for RT-qPCR validation. Furthermore, lncRNA-mRNA regulatory network and lncRNA-miRNA-mRNA ceRNA network were constructed to examine the role and mechanism of proliferation-related lncRNAs. To reveal the biological functions of lncRNAs, GO biological process and KEGG pathway analysis were we performed on all mRNAs involed in lncRNA-mRNA regulatory network and ceRNA network. Finally, 1532 differentially expressed lncRNAs, including 594 upregulated lncRNAs and 938 downregulated lncRNAs were screened out by RNA-seq. The results of RT-qPCR validation were consistent with RNA-seq result. The lncRNA-mRNA regulatory network, including 236 lncRNAs and 556 mRNAs and lncRNA-miRNA-mRNA ceRNA network, including 6 lncRNAs, 18 miRNAs, and 419 mRNAs were successfully constructed. The GO biological process and KEGG pathway analysis demonstrated that those lncRNAs were mainly related to inflammatory response and substance metabolism. This study first identified key proliferation-related lncRNAs in LPS-induced GMCs, and further revealed a global view of lncRNA-mRNA regulatory network and lncRNA-miRNA-mRNA ceRNA network involved in CGN. Our findings offered novel insights into the roles of lncRNAs in the pathogenesis of CGN and provided promising diagnostic biomarkers.