Mir 145 5p agomir

An equal volume of mir-145-5p agomir or scrambled miRNA agomir (RiboBio, Guangdong, China) was injected in the tail vein of the mice on Day 30. Injected mir-145-5p agomir (n=4) or scrambled miRNA agomir (n=4) in the tail vein of mice at 1-week intervals. Four weeks after injection, the hind feet were used for micro-computed tomography (CT) analysis, and the ankles were used for histopathological analysis.

Male DBA/1 mice (15–20 g; 8 weeks old) were used in the present study. A total of 30 animals were housed at 24±1°C with a 12:12-h light/dark cycle and a relative humidity of 56±5%. Animals were provided access to food and water ad libitum, and were fed a commercial diet according to the guidelines of the Animal Ethical and Welfare Committee. All mice were housed individually. Lyophilized bovine type II collagen (Chondrex, Inc., Redmond, WA, USA) was dissolved overnight at 4°C in 0.05 M acetic acid under constant stirring. Next, the collagen was emulsified using Freund's complete adjuvant (Chondrex, Inc.) to provide a final concentration of 2 mg/ml. Mice were injected in the tail with 0.1 ml of emulsion on day 1 and booted intraperitoneally on day 21 with collagen. On day 30, an equal volume of mir-145-5p agomir or scrambled miRNA agomir (15 nM; Guangzhou RiboBio Co., Ltd.) was injected in the tail vein of mice at 1-week intervals. Four weeks after the first injection, the mice were sacrificed by injecting pentobarbital sodium at a dose of 100 mg/kg, then various indications such as respiration, heartbeat, pupil reflection, and nerve reflex were observed in the mice, and when all the aforementioned reactions disappeared, death was judged. Next, the right hind limb was dissected and synovial tissue was collected for subsequent experiments.

The TNBS-induced colitis model was conducted in male BALB/c mice (week age: 6-8 w; weight: 20-22 g, Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China), and raised under specific pathogen-free conditions in the Animal Experimental Center of our hospital. This colitis mouse model was established following a previously published protocol.¹⁹ (link)^,20 (link) Experimental groups were pre-sensitised with 1% (w/v) TNBS acid (Sigma, St. Louis, MO, USA) for 7 days, and then challenged with 100 μL 2.5% TNBS mixture (one volume 5% TNBS in one volume of absolute ethanol) on day 8 by enema, whereas the control group were administered with same volume of 50% ethanol solution. After another 7 days, all mice were sacrificed for further research. Furthermore, the TNBS-induced colitis model was also established in Cldn8^−/− mice to demonstrate the role of the miR-145/SOX9/CLDN8 pathway in colitis initiation.
MiRNA agomir (RiboBio, Shanghai, China) was administered to TNBS-induced colitis mice to observe the effect of miR-145-5p on regulating the expression of Sox9 in vivo. miR-145-5p agomir and its corresponding negative control at a dose of 10 mg/kg were administered to TNBS-induced colitis mice via intraperitoneal injection on days 8, 9, and 10. In addition, the sequences of miR-145-5p agomir could be required from RiboBio (Shanghai, China).