Project description:Synovial fibroblasts were extracted from knee joints of naïve mice(DBA/1J, male 8-12 week old), and passaged 3-4 times. Cells were plated at a concentration of 1 X 106 cells/dish in 60 mm dish in 5 ml of RPMI1640 medium with 1% heat-inactivated FCS at 37°C, 5% CO2. Cells were treated vehicle or stimulated by IL-1beta. In IL-1beta(5ng/ml) stimulated groups, indomethacin (1 μM) and iloprost (1μM) were further added either alone or in combination. After 6 hour stimulation as described above, cells were isolated and frozen by liquid nitrogen, and stored at –80°C before use. Total RNA was extracted by RNAeasy Mini Kit. The RNA sample was labeled for hybridization onto the genechip array according to the standard affymetrix protcols. Keywords = arthritis prostacyclin Keywords: other
Project description:The aim of this study was to explore the role of ARNO, also known as Cyth2, in synovial fibroblasts using NGS-derived transcriptome analysis (RNA-seq). Synovial fibroblasts were isolated from mouse synovium, and stimulated with IL-1beta for 12 hours in healthy cells and cells where Cyth2 expression was knocked-down by silencing RNA. Methods: Synovial fibroblasts were extracted from the synovium of healthy mice and expanded in vitro, RNA was extracted from naïve, IL-1β stimulated and IL-1β stimulated ARNO knock-down synovial fibroblast. Methods: Whole Transcriptome Profiling of synovial fibroblasts were generated by deep sequencing, in triplicate, using Illumina NextSeq™ 500 platform. Libraries were prepared using polyA selection (TruSeq stranded mRNA kit). Methods: The sequence reads that passed quality filters were aligned to mouse reference genome (GRCM38) using Hisat2 version 2.1.0. we mapped about 30 million sequence reads per sample (75 bp, paired-end) Methods: Featurecounts version 1.4.6 was used to quantify reads counts. Data quality control, non-expressed gene filtering, median ratio normalization (MRN) implemented in DESeq2 package and identification of differentially expressed (DE) genes were done using the R bioconductor project DEbrowser. Results: Firstly, We detected differentially expressed (DE) genes among three conditions, which pass the threshold of >4 fold, adjp <0.01. Then, we detected DE genes in IL-1β stimulated ARNO knock-down synovial fibroblast compared to IL-1β stimulated synovial fibroblast, which pass the threshold of >2 fold, adjp <0.01. DE genes reflect decreased cellular inflammatory response after ARNO knockdown Conclusions: Our results reflect an ARNO-mediated inflammatory response in synovial fibroblast, provides new opportunitties for targeting fibroblast in chronic arthritis and joint disease.
Project description:Acetaminophen is a widely used antipyretic and analgesic drug, and its overdose is the leading cause of drug-induced acute liver failure. This study aimed to investigate the effect and mechanism of Lacticaseibacillus casei Shirota (LcS), an extensively used and highly studied probiotic, on acetaminophen-induced acute liver injury. C57BL/6 mice were gavaged with LcS suspension or saline once daily for 7 days before the acute liver injury was induced via intraperitoneal injection of 300 mg/kg acetaminophen. The results showed that LcS significantly decreased acetaminophen-induced liver and ileum injury, as demonstrated by reductions in the increases in aspartate aminotransferase, total bile acids, total bilirubin, indirect bilirubin and hepatic cell necrosis. Moreover, LcS alleviated the acetaminophen-induced intestinal mucosal permeability, elevation in serum IL-1α and lipopolysaccharide, and decreased levels of serum eosinophil chemokine (eotaxin) and hepatic glutathione levels. Furthermore, analysis of the gut microbiota and metabolome showed that LcS reduced the acetaminophen-enriched levels of Cyanobacteria, Oxyphotobacteria, long-chain fatty acids, cholesterol and sugars in the gut. Additionally, the transcriptome and proteomics showed that LcS mitigated the downregulation of metabolism and immune pathways as well as glutathione formation during acetaminophen-induced acute liver injury. This is the first study showing that pretreatment with LcS alleviates acetaminophen-enriched acute liver injury, and it provides a reference for the application of LcS.