Establishment of a human TLR4 compound heterozygous knockout hESC line (WAe009-A-N) to model toll-like receptor 4 deficiency by CRISPR/Cas9 system.

Toll-like receptor 4 (TLR4) is a pattern recognition receptor (PRRS) and an important protective immune receptor. TLR4 deficiency can lead to Inflammatory bowel disease. To explore the role of TLR4, we used CRISPR/Cas9 system to produce TLR4 compound heterozygous knockout embryonic stem cells in H9 cell line. The WAe009-A-N has a compound heterozygous 7 bp deletion/8 bp deletion in TLR4 exon 3, which resulted in a frameshift in the translation of TLR4, and TLR4 protein wasn't detectable in this cell line. In addition, the WAe009-A-N with normal karyotype can express pluripotent markers and differentiate into three germ layers in vitro.

Autophagy Ameliorates Reactive Oxygen Species-Induced Platelet Storage Lesions.

Platelet transfusion is a life-saving therapy to prevent bleeding; however, the availability of platelets for transfusion is limited by the markedly short shelf life owing to the development of platelet storage lesions (PSLs). The mechanism of PSLs remains obscure. Dissection of the intracellular biological changes in stored platelets may help to reduce PSLs and improve platelet transfusion efficiency. In the present study, we explore the changes of stored platelets at room temperature under constant agitation. We found that platelets during storage showed an increased reactive oxygen species (ROS) generation accompanied with receptor shedding, apoptosis, and diminished platelet aggregation. ROS scavenger reduced platelet shedding but also impaired platelet aggregation. Autophagy is a conserved catabolic process that sequesters protein aggregates and damaged organelles into lysosomes for degradation and platelets' own intact autophagic system. We revealed that there exist a stable autophagic flux in platelets at the early stage of storage, and the autophagic flux in platelets perished after long-term storage. Treatment stored platelets with rapamycin, which stimulates autophagy in eukaryotic cells, markedly ameliorated PSLs, and improved platelet aggregation in response to extracellular stimuli.

Saccharides from Arctium lappa L. root reduce platelet activation and thrombus formation in a laser injury thrombosis mouse model.

Arctium lappa L., also known as burdock, is a popular medicinal plant in traditional Chinese medicine due to its potential therapeutic properties. Saccharides from Arctium lappa L. root (ALR-S) have been extensively studied for their anti-inflammatory and anti-diabetes effects. Platelets play a pivotal role in thrombosis. The present study describes the effects of ALR-S on platelet activation and thrombosis using a laser injury thrombosis in vivo model. The study also measured the effects of ALR-S on platelet activation by analysing aggregation, ATP release, platelet spreading, adhesion and clot retraction in vitro. Specifically, the effects were ALR-S concentration-dependent inhibition of platelet aggregation and ATP release. Activated platelets pretreated with ALR-S showed diminished CD62P expression levels and fibrinogen binding, as measured by flow cytometry. ALR-S inhibited platelet spreading on fibrinogen and adhesion on collagen under shear. ALR-S attenuated platelet activation by decreasing oxidative stress and thrombus formation. These results demonstrated the antiplatelet effects of ALR-S, suggesting the antithrombotic and cardiovascular protective activities of ALR-S as a functional food.

Asiatic acid alleviates Ang-II induced cardiac hypertrophy and fibrosis via miR-126/PIK3R2 signaling.

BACKGROUND: Cardiac hypertrophy is an independent risk factor of many cardiovascular diseases. Studies have demonstrated that microRNA-126 (miR-126) was involved in angiogenesis during physiological and pathological process. However, its role in cardiac hypertrophy has not been known clearly. Our previous study demonstrated that asiatic acid (AA) has obvious protective effect on cardiac hypertrophy. Here, this study aimed to discover the regulatory role of miR-126 and its mechanism in cardiac hypertrophy, and to determine whether AA's anti-hypertrophy effect is partially miR-126 dependent. METHODS: Male Sprague Dawley rats were AngII infused via osmotic minipumps for 4 weeks and were treated with AA (20 mg/kg/day) by oral gavage. Cardiac hypertrophy was assessed using the echocardiography and histological analysis. In vitro studies,cardiomyocyte and cardiac fibroblasts (CF) were treted with AngII and AngII plus AA. And, the effect of AA on miR-126 and PI3K/AKT signaling pathway was investigated. RESULTS: Treatment of rats with AA decreased the ratio of heart weight to tibia length and hypertrophy markers. In vitro exprements demonstrated that AA significantly attenuated AngII-induced cardiac growth and cardiac fibroblast collagen expression. Moreover, our results found downregulation of miR-126 and activation of PI3K/AKT signaling pathway in AngII infusion induced cardiac hypertrophy model. It was also determined that miR-126 targets PIK3R2 directly. CONCLUSIONS: AA supplementation upregulated the expression of miR-126 and conferred cardio-protection effect against AngII induced cardiac hypertrophy.

Generation of a hiPSC line ZZUNEUi012-A from a healthy female individual.

Induced pluripotent stem cells (iPSCs) have differentiation potential into different somatic cell types in vitro and are a useful tool to investigate pathomechanistic and cellular processes. In this study, we generated human induced pluripotent stem cells (iPSC) ZZUNEUi012-A from an apparently healthy female individual using an integration-free reprogramming method. The generated hiPSC line was pluripotent and had normal karyotype, showed robust expression of pluripotency markers and could differentiate into all three germ layers in vitro.