2-BFI hydrochloride

Cell lines

Human colorectal adenocarcinoma cell lines HT-29 and HCT-116

Preparation Method

Human colorectal adenocarcinoma cell lines HT-29 and HCT-116 were cultured in McCoy’s 5A Medium (modified), with L-Glutamine, supplemented with 10% fetal bovine serum, 100units/mL penicillin and 100μg/mL streptomycin. Cell cultures were incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37℃. 2-BFI hydrochloride was dissolved in water as a 10mM masterbatch and storage at −80℃ refrigerator. HT-29 and HCT-116 cells were seeded in triplicate on 96-well plates at a density of 10×10³cells/100μL. The next day, cells were exposed to 2-BFI (250μM) and/or HCQ for 24h and 48h. The MTT assay was used to measure cell viability according to the commercially available protocol. The generation of intracellular ROS level and apoptosis detection were measured using flow cytometry technique. The Autophagy Kit was used to monitor autophagic flux.

Reaction Conditions

250μM; 24-48h

Applications

Treatment of HT-29 and HCT-116 colorectal cancer cells with 2-BFI hydrochloride significantly potentiated hydroxychloroquine cytotoxicity, elevated ROS, increased early/late apoptosis, and blocked autophagic flux.

Animal models

Male adult Sprague-Dawley rats

Preparation Method

Male adult (2-month-old, 250-300g) Sprague-Dawley rats were housed in a temperature (23±1℃) and humidity (50%) controlled animal facility with 12:12h light–dark cycle. Food and water were available ad libitum. Focal cerebral ischemic stroke model was established by permanent distal middle cerebral artery occlusion (dMCAO) method. Immediately following dMCAO, rats were randomly selected and administered 2-BFI hydrochloride (3mg/kg) or saline intraperitoneally. Rats undergo neurobehavioral tests to evaluate functional outcome 1 and 3 days after ischemia. The modified beam balance test was used to assess the motor balance and coordination in ischemic rats, especially in the hindlimb. The brains of rat were removed 3 days after ischemia, 2mm coronal sections were cut and stained with 2,3,5-triphenyltetrazolium hydrochloride (TTC). Western blotting procedures were performed to assess the profile of mTOR activation. To test the inflammatory response after 2BFI treatment, colorimetric ELISA kits were used to detect the following cytokines in brain protein extract: IL-10,TNF-α, TGF-β.

Dosage form

3mg/kg; i.p.; 3 days

Applications

2-BFI hydrochloride reduced cerebral infarct volume, improved motor deficits in dMCAO rats, inhibited mTOR phosphorylation and shifted cytokine balance toward anti-inflammation.

References:
[1] Kowalski S, Wityk P, Raczak-Gutknecht J, Olszewska A, Zmijewski M, Kocic I. The imidazoline I₂ receptor agonist 2-BFI enhances cytotoxic activity of hydroxychloroquine by modulating oxidative stress, energy-related metabolism and autophagic influx in human colorectal adenocarcinoma cell lines. Eur J Pharmacol. 2025;996:177590.
[2] Cheng Y, Zhang W, Cao W, et al. 2-BFI attenuates ischemic injury by modulating mTOR signaling and neuroinflammation in rats. Neurosci Lett. 2021;750:135766.