Adenosine triphosphate (atp)

Male C57BL/6 mice aged 6–8-weeks-old were purchased from Zhejiang Academy of Medical Sciences, Hangzhou, China. Following euthanasia by cervical dislocation, the lack of heartbeat was confirmed in each animal in accordance with the approved Zhejiang Academy of Medical Sciences protocol. BMDMs from the bilateral posterior femur of mice were rinsed using the DMEM (Genom, Hangzhou, China) medium. BMDMs were cultured in DMEM media supplemented with 50 ng/mL mouse recombinant macrophage colony-stimulating factor, 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µM) in a humidified atmosphere containing 5% CO₂ at 37 °C. After 7 days of culture, the cells were divided into different groups as follows: vehicle; DI (0.25 mM, Sigma, USA) + vehicle; Dimethyl sulfoxide (DMSO, Sigma, USA) + LPS (500 ng/mL, for 4 h, Sigma, USA) + ATP (5 mM, for 1 h, Sigma, USA); DI (0.25 mM, pre-treatment for 2 h) + LPS + ATP; N-acetyl-L-cysteine (NAC, 1 mM, Sigma, USA) or ML385(10 µM, Selleck, China), DI (0.25 mM, con-treatment for 2 h) + LPS + ATP. The concentrations were performed as described [18 (link), 19 (link)]. The Ethics committee of the Zhejiang Academy of Medical Sciences approved the experimental protocol. All animal experiments met the ARRIVE guidelines [20 (link)].

A rat renal proximal tubular cell line (NRK-52E) was kindly provided by Xiao-li Nie, a professor of School of Traditional Chinese Medicine, Southern Medical University. The cells were cultured at 37°C with 5% CO₂ in DMEM/basic medium (Gibco BRL, USA) supplemented with 10% fetal bovine serum (Gibco BRL, USA) and 1% penicillin/streptomycin (Gibco BRL, USA). To investigate LPS-induced renal inflammation in NRK-52E cells, cells were stimulated with 5 μg/mL LPS (L4391, Sigma) for 3 h and total protein or RNA was extracted. Besides, to investigate the levels of inflammatory cytokine secretion in the supernatant through an enzyme-linked immunosorbent assay experiment, cells were treated with 5 μg/mL LPS for 24 h. To investigate the anti-inflammatory effect of MCL in vitro, cells were pretreated with different concentrations of MCL (1.25 μM, 2.5 μM, and 5 μM) (Accendatech Co., Ltd., Tianjin, China) for 48 h and then incubated with LPS. To induce NLRP3 inflammasome activation, cells were incubated with LPS and then with 3 mM ATP (S1985, Selleck, USA) for 1 h before the cells were extracted. In some experiments, cells were treated with 1 μM mitoquinone (MitoQ) (MedChemExpress) for 24 h or 1 μM rotenone (MedChemExpress) for 24 h.