Matrine Protects Endothelial Progenitor Cells against Apoptosis and Promotes Their Migration, Invasion, and Tube Formation Abilities via Modulating miR-126b/FOXO4 Axis.

INTRODUCTION AND OBJECTIVE: Endothelial progenitor cells (EPCs) have been proven to exhibit a therapeutic effect in deep vein thrombosis, but are susceptible to microenvironment. Besides, Matrine has promotive effects on EPCs, but its effects on microRNA (miR)-126 remain obscure, which are therefore discussed in the study. METHODS: The cultured EPCs were extracted from Sprague-Dawley rats and identified by immunofluorescence assay. After being treated with Matrine or transfected with miR-126b inhibitor and small interfering RNA targeting forkhead box (FOXO) 4, the viability and apoptosis of EPCs were determined by cell counting kit-8 assay and flow cytometry. The migration, invasion, and tube formation abilities were detected by scratch, Transwell, and tube formation assays. The target genes of miR-126b were predicted by TargetScan, and verified by dual-luciferase reporter assay. The expressions of miR-126b, FOXO4, matrix metalloproteinase (MMP) 2, MMP9, and vascular endothelial growth factor (VEGF) A were determined by quantitative real-time polymerase chain reaction and Western blot. RESULTS: The EPCs were successfully extracted and cultured, as evidenced by positive reaction cluster of differentiation (CD) 34 and CD133. Matrine promoted the viability, migration, invasion, and tube formation while inhibiting the apoptosis of EPCs, and upregulated the expression of miR-126b. Besides, miR-126b inhibitor reversed the effects of Matrine on EPCs and downregulated the expression levels of MMP2, MMP9, and VEGFA. MiR-126b targeted the FOXO4, and siFOXO4 reversed the abovementioned effects of miR-126b inhibitor on EPCs. CONCLUSION: Matrine protects EPCs from apoptosis and promotes their migration, invasion, and tube formation abilities via regulating miR-126b/FOXO4 axis.

Hydrogen sulfide serves as a biomarker in the anterior segment of patients with diabetic retinopathy.

OBJECTIVE: The present study aims to determine hydrogen sulfide (H2S) concentrations of the aqueous humor from patients with diabetic retinopathy (DR) to compare its levels in the anterior segments, also to investigate its effect on the retinal microvascular endothelial cells under high glucose condition. METHODS: AH samples were collected from patients with proliferative diabetic retinopathy (n = 11), non-proliferative diabetic retinopathy (n = 12) and diabetic patients without DR as controls (n = 12). There were 5 patients with PDR received intraocular anti-VEGF injection (Lucentis). Cultured RF/6A cells were grouped into control group, mannitol group, high glucose group and NaHS co-administrated high glucose group. Concentrations of H2S were detected by chemical assay. Cell apoptosis was evaluated by flow cytometry. RESULTS: A significantly higher H2S level was observed in AH samples of PDR patients among other groups. The H2S level of DR group was higher than that of control group. Decreased H2S levels in the AH of post-injected PDR patients were observed compared with their AH samples before the anti-VEGF injection. In cell culture, low concentration of NaHS can reverse high-glucose-induced apoptosis of RF/6A cells. CONCLUSION: Our study revealed increased H2S levels in the anterior segments of different DR patients. The anti-VEGF injection reduced the H2S level in AH from PDR patients. The study suggested that H2S may serve as a biomarker in the progression of PDR. On the other hand, the H2S donor exerted a protective effect on retinal vascular endothelial cells against high-glucose-induced apoptosis.

Chromosome-level assembly and gene annotation of Decapterus maruadsi genome using Nanopore and Hi-C technologies.

Decapterus maruadsi is one of the representative offshore fish in the Western Pacific. Since the last century, it has become a commercially valuable marine fishery species in the Western Pacific region. Despite its high economic value, there is still a lack of high-quality reference genome of D. maruadsi in germplasm resource evaluation research. Here we report a chromosome-level reference genome of D. maruadsi based on Nanopore sequencing and Hi-C technologies. The whole genome was assembled through 169 contigs with a total length of 723.69 Mb and a contig N50 length of 24.67 Mb. By chromosome scaffolding, 23 chromosomes with a total length of 713.58 Mb were constructed. In addition, a total of 199.49 Mb repetitive elements, 33,515 protein-coding genes, and 6,431 ncRNAs were annotated in the reference genome. This reference genome of D. maruadsi will provide a solid theoretical basis not only for the subsequent development of genomic resources of D. maruadsi but also for the formulation of policies related to the protection of D. maruadsi.

Discovery of novel hydroxyamidine based indoleamine 2,3-dioxygenase 1 (IDO1) and thioredoxin reductase 1 (TrxR1) dual inhibitors.

In order to take advantage of both immunotherapeutic and metabolic antitumor agents, novel dual indoleamine 2,3- dioxygenase 1 (IDO1) and thioredoxin reductase 1 (TrxR1) inhibitors were designed. Thioredoxin reductase 1 (TrxR1) is a main ROS modulator within CRC cells. Indoleamine 2,3-dioxygenase (IDO1) is crucial controller for tryptophan (Trp) metabolism that is also important for CRC immunotherapy. Herein, ten compounds 12a-j containing hydroxyamidine scaffold were designed, synthesized and evaluated for inhibitory activities against IDO1/TrxR1 enzyme and CRC cells. Among these compounds, the most active compound 12d (ZC0109) showed excellent and balanced activity against both IDO1 (IC50 = 0.05 µM) and TrxR1 (IC50 = 3.00 ± 0.25 µM) were selected for further evaluation. Compound ZC0109 exhibited good dual inhibition against IDO1 and TrxR1 both in vitro and in vivo. Further mechanistic studies reveal that, through IDO1 and TrxR1 inhibition by ZC0109 treatment, accumulated ROS effectively induced apoptosis and G1/S cell cycle arrest in cancer cells. In vivo evaluation demonstrated excellent anti-tumor effect of ZC0109 with the notable ability of promoting ROS-induced apoptosis, reducing kynurenine level in plasma and restoring anti-tumor immune response. Thus, ZC0109 represents a potential CRC therapy agent for further development.

[Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system].

To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.