Cell lines

Rat renal interstitial fibroblasts (NRK-49F) cells

Preparation Method

Rat renal interstitial fibroblasts (NRK-49F) were cultured in DMEM with F12 containing 10% FBS, 1%penicillin and streptomycin in an atmosphere of 5%CO₂, and 95% air at 37oC. Cells were starved for 24h with DMEM containing 0.5% FBS.

Reaction Conditions

Cells were exposed to uric acid (800 μM) for 36h in the presence or absence of 3-Methyladenine (0–10 mM).

Applications

3-Methyladenine could abolish uric acid-induced α-SMA and collagen I expression. Uric acid also triggered a significant up-regulation of LC3II/I and Beclin-1. 3-Methyladenine dose-dependently suppressed these responses. In addition, treatment with 3-MA decreased TGF-βRI expression levels and the ratio of p-Smad3/Smad3 in a dose-dependent manner.

Animal models

male Sprague-Dawley (SD) rats (2-months old; weight 200±20 g)

Preparation Method

Mice were maintained on a 12h light/dark cycle and provided access to food and water ad libitum. All rats were pretrained to swim in the absence of a load for one week. The saline-treatment group was intravenously injected with 500 μL sterile saline by using 261/2 gauge needle via tail vein;3-MA-treatment group was intravenously injected with 500 μL 3-Methyladenine by using a 261/2 gauge needle via the tail vein.

Dosage form

15 mg/kg

Applications

3-Methyladenine could decrease the distortion of myocardium fibers as well as significantly decrease the width of cardiomyocytes of mice. In addition, 3-Methyladenine treatment obviously reduced autophagosome formation in the myocardium. 3-Methyladenine could also prevent the reduction of Bcl-2/Bax in the left ventricle of OE rats.

3-Methyladenineis a classic autophagic agonist and inhibitor. It inhibits phosphatidylinositol 3-kinase (PI3K), which is located upstream of the IGF/PI3K/mTOR/ULK pathway.[1]3-Methyladenineis capable to induce a consistent and abrupt decrease in cell viability across a series of ontologically unrelated human cell lines. In addition,3-Methyladenine-induced cytotoxicity was not driven by the inhibition of the AKT/mTOR axis.^[2]

In vitro study indicated that the inhibition of autophagy by3-Methyladenineabolished uric acid-induced differentiation of renal fibroblasts to myofibroblasts and activation of transforming growth factor-β1 (TGF-β1), epidermal growth factor receptor (EGFR), and Wnt signaling pathways in cultured renal interstitial fibroblasts. Moreover,3-Methyladeninewas effective in attenuating renal deposition of extracellular matrix (ECM) proteins and expression of α-smooth muscle actin (α-SMA) and reducing renal epithelial cells arrested at the G2/M phase of cell cycle.^[3]

In vivo study demonstrated that the administration of3-Methyladenineinhibited Wnt/β-catenin and Notch/Jagged-1 signaling pathways as well as suppresses EGFR/ERK1/2 signaling pathway. Furthermore, 3-MA treatment remarkably inhibited the infiltration of macrophages and lymphocytes as well as release of multiple profibrogenic cytokines/chemokines in the injured kidney.^[3]

References:
[1]. Yang F, et al. Rapamycin and3-MethyladenineInfluence the Apoptosis, Senescence, and Adipogenesis of Human Adipose-Derived Stem Cells by Promoting and Inhibiting Autophagy: An In Vitro and In Vivo Study. Aesthetic Plast Surg. 2021 Jun;45(3):1294-1309.
[2].Chicote J, et al. Cell Death Triggered by the Autophagy Inhibitory Drug3-Methyladeninein Growing Conditions Proceeds With DNA Damage. Front Pharmacol. 2020 Oct 15;11:580343.
[3].Bao J, et al. Pharmacological inhibition of autophagy by 3-MA attenuates hyperuricemic nephropathy. Clin Sci (Lond). 2018 Nov 2;132(21):2299-2322.