Structural Revision of the Stemona Alkaloids Tuberostemonine O, Dehydrocroomines A and B, and Dehydrocroomine.

The structural revision of four Stemona alkaloids from Stemona tuberosa is reported. The misassignment of the tuberostemonine O structure (1) was recognized when a new alkaloid, tuberostemonine P, was isolated and unambiguously assigned structure 1 in this work. Reinvestigation of the spectroscopic data and NMR calculations led to the revised structure 1a for tuberostemonine O. The structural misassignment of dehydrocroomine A as 2 was corrected by reinterpreting the X-ray crystal structure, which was consistent with 2a. The structural reassignments of dehydrocroomine B (3 to 3a) and dehydrocroomine (4 to 4a) were confirmed by X-ray crystallography and NMR calculations, respectively.

Targeting circRNA-MAP4K2 for the treatment of diabetes-induced retinal vascular dysfunction.

Diabetic retinopathy (DR) is an important ocular vascular disease in working-age adults. However, the molecular mechanism underlying retinal vascular dysfunction is still not fully understood in DR. Circular RNAs have been recognized as the crucial regulators in many biological processes and human diseases. Herein, we determined the role of circular RNA-MAP4K2 (cMAP4K2) in diabetes-induced retinal vascular dysfunction. The results showed that high glucose treatment led to increased levels of cMAP4K2 expression in vitro and in vivo. Silencing of cMAP4K2 could reduce endothelial cell viability, proliferation, migration, and tube formation in vitro and alleviate retinal vascular dysfunction in vivo as shown by decreased vascular leakage and inflammation. By contrast, cMAP4K2 overexpression had an opposite effect on retinal vascular dysfunction. Mechanistically, cMAP4K2 acted as miR-377 sponge to affect the biological activity of miR-377, which led to increased expression of vascular endothelial growth factor A (VEGFA). Clinically, cMAP4K2 expression was significantly up-regulated in the clinical sample of DR patients. Collectively, cMAP4K2 is shown as a potential target for the diagnosis and treatment of diabetic retinopathy.

Silencing of circular RNA­ZYG11B exerts a neuroprotective effect against retinal neurodegeneration.

Ischemic retinal diseases are the major cause of vision impairment worldwide. Currently, there are no available treatments for ischemia­induced retinal neurodegeneration. Circular RNAs (circRNAs) have emerged as important regulators of several biological processes and human diseases. The present study investigated the role of circRNA­ZYG11B (circZYG11B; hsa_circ_0003739) in retinal neurodegeneration. Reverse transcription quantitative polymerase chain reaction (RT­qPCR) demonstrated that circZYG11B expression was markedly increased during retinal neurodegeneration in vivo and in vitro. Cell Counting Kit­8, TUNEL and caspase­3 activity assays revealed that silencing of circZYG11B was able to protect against oxidative stress­ or hypoxic stress­induced retinal ganglion cell (RGC) injury. Furthermore, immunofluorescence staining and hematoxylin and eosin staining revealed that silencing of circZYG11B alleviated ischemia/reperfusion­induced retinal neurodegeneration, as indicated by reduced RGC injury and decreased retinal reactive gliosis. In addition, luciferase reporter, biotin­coupled miRNA capture and RNA immunoprecipitation assays revealed that circZYG11B could regulate RGC function through circZYG11B/microRNA­620/PTEN signaling. Clinically, RT­qPCR assays demonstrated that circZYG11B expression was markedly increased in the aqueous humor of patients with glaucoma. In conclusion, circZYG11B may be considered a promising target for the diagnosis and treatment of retinal ischemic diseases.

Comparative Analysis of Differentially Expressed Circular RNAs in Polarized Macrophages.

Macrophage polarization is a process that macrophages exert different functions according to surrounding micro-environment. Macrophages commonly exist in two distinct subsets: classically activated M1 macrophages and alternatively activated M2 macrophages. Circular RNAs (circRNAs) are a novel class of non-coding RNAs generated by back-splicing. Thousands of circRNAs were identified in different cells and tissues. Recent studies have revealed that circRNAs play a crucial role in regulating transcriptional and post-transcriptional gene expression. However, the effects and roles of circRNAs in macrophage polarization have not been well elucidated. Here, circRNAs expression profiles were determined in human THP-1 macrophages incubated in conditions causing activation toward M1 (interferon-γ + LPS) or M2 (interleukin-4) phenotypes. Overall, 9,720 circular RNA were detected from RNA sequencing data. Compared with M2 macrophages, a total of 140 circRNAs were aberrantly expressed in M1 macrophages, including 71 up-regulated circRNAs and 69 down-regulated circRNAs. Quantitative real-time PCR (qRT-PCR) results were generally consistent with the selected differentially expressed circRNAs. Gene Ontology (GO) and KEGG pathway analyses were used to predict biological functions and potential mechanisms of the host linear transcripts of these up-regulated and down-regulated circRNAs. Furthermore, we found that the expression level of circRNA-RNF19B (circRNF19B) in M1 macrophages was significantly higher than that in THP-1 macrophages and M2 macrophages. circRNF19B expression was increased when M2 converted to M1 whereas decreased when M1 converted to M2. Knockdown of circRNF19B following the activation of THP-1 cells using interferon-γ + LPS diminished the expression of M1 macrophages markers and elevated the expression of M2 macrophages markers. In conclusion, these data suggest the involvement of altered circRNAs expression patterns in macrophages exposure to different activating conditions. Circular RNAs may play important roles in regulating macrophage polarization.

Targeting circular RNA-MET for anti-angiogenesis treatment via inhibiting endothelial tip cell specialization.

Endothelial tip cell specialization plays an essential role in angiogenesis, which is tightly regulated by the complicated gene regulatory network. Circular RNA (circRNA) is a type of covalently closed non-coding RNA that regulates gene expression in eukaryotes. Here, we report that the levels of circMET expression are significantly upregulated in the retinas of mice with oxygen-induced retinopathy, choroidal neovascularization, and diabetic retinopathy. circMET silencing significantly reduces pathological angiogenesis and inhibits tip cell specialization in vivo. circMET silencing also decreases endothelial migration and sprouting in vitro. Mechanistically, circMET regulates endothelial sprouting and pathological angiogenesis by acting as a scaffold to enhance the interaction between IGF2BP2 and NRARP/ESM1. Clinically, circMET is significantly upregulated in the clinical samples of the patients of diabetic retinopathy. circMET silencing could reduce diabetic vitreous-induced endothelial sprouting and retinal angiogenesis in vivo. Collectively, these data identify a circRNA-mediated mechanism that coordinates tip cell specialization and pathological angiogenesis. circMET silencing is an exploitable therapeutic approach for the treatment of neovascular diseases.

Differential MicroRNA Expression Pattern in Endothelial Progenitor Cells During Diabetic Retinopathy.

Endothelial progenitor cells (EPCs) are involved in the pathogenesis of microvascular dysfunction in diabetic retinopathy (DR). MicroRNAs (miRNAs) serve as crucial regulators in many biological process and human diseases. Herein, to investigate the expression profile and possible role of miRNAs in EPCs, small RNA sequencing was conducted to identify EPC dysfunction-related miRNAs in DR. A total of 72 miRNAs were differentially expressed in EPCs following high glucose stress. Based on Gene Ontology (GO) analysis, the target genes of differentially expressed miRNAs were targeted to "protein binding," "cell differentiation," and "cytoskeleton." Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that cGMP-PKG signaling pathway was tightly associated with miRNA-mediated EPC function. Furthermore, miR-375-3p was verified to be up-regulated in the clinical samples of DR patients. Inhibition of miR-375-3p protected against hyperglycemic stress- or hypoxic stress-induced EPC injury, which increased the viability, proliferation, migration, and tube formation ability of EPCs and retarded the development of apoptosis. Collectively, this study provides a novel insight into the pathogenesis of EPC dysfunction in DR. miR-375-3p is a potential target for the diagnosis or treatment of DR.

Lycocasuarines I-Q, new Lycopodium alkaloids isolated from Lycopodiastrum casuarinoides.

Lycocasuarines I-Q (1-9), nine new lycodine-type Lycopodium alkaloids were isolated from the aerial parts of Lycopodiastrum casuarinoides (Spring) Holub ex R.D.Dixit. Their structures were unambiguously determined via extensive spectroscopic analyses, including HRESIMS, NMR, and electronic circular dichroism (ECD). The new alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase (AChE). Compounds 3, 5-8 exhibited marginal or weak activities with IC50 values ranging from 97.2 to 171.9 µM.

Lycodine-type alkaloids and their glycosides from Lycopodiastrum casuarinoides.

Thirteen previously undescribed lycodine-type Lycopodium alkaloids, namely, five alkaloids (lycocasuarines D-H) each possessing an uncommon five-membered C ring and eight Lycopodium alkaloid glycosides (casuarinosides A-H), together with a known analog, were isolated from the aerial parts of Lycopodiastrum casuarinoides (Spring) Holub ex R.D.Dixit (Lycopodiaceae). The structures of the compounds were elucidated by extensive spectroscopic analysis, single-crystal X-ray diffraction, and chemical methods. In addition, the acetylcholinesterase inhibitory activity of the isolated compounds was evaluated.